Acyloxybutadiene iron tricarbonyl complexes as enzyme-triggered CO-releasing molecules (ET-CORMs)

Article abstract of DOI:10.1002/anie.201006598

(Chemical Equation Presented) Molecular hazardous materials transport: Enzyme-triggered CO-releasing molecules (ET-CORMs) offer new options for the delivery of CO. The cleavage of dienylester iron tricarbonyl complexes by an esterase under mild oxidative

Full text of DOI:10.1002/anie.201006598

Communications
DOI: 10.1002/anie.201006598
Carbon Monoxide Release
Acyloxybutadiene Iron Tricarbonyl Complexes as Enzyme-Triggered
CO-Releasing Molecules (ET-CORMs)**
Steffen Romanski, Birgit Kraus, Ulrich Schatzschneider, Jꢀrg-Martin Neudꢀrfl,
Sabine Amslinger,* and Hans-Gꢁnther Schmalz*
Like nitric oxide (NO), carbon monoxide (CO) is an
important yet only recently recognized biological signaling
molecule.[1] CO is actually constantly produced in small doses
in our body in the course of heme degradation by the heme-
oxygenase (HO) enzymes. It exhibits cytoprotective, anti-
inflammatory, vasodilatory, and other effects, which are
important for instance in our bodyꢀs response to injuries.[2]
Despite these beneficial biological properties, the application
of CO as a therapeutic agent is still in its infancy.[3] Not
surprisingly, the use of gaseous CO is risky and strongly
limited by the high affinity of CO towards hemoglobin and
the resulting systemic effects on oxygen transport and low
bioavailability.[4]
A promising strategy to circumvent these problems and to
deliver controlled amounts of CO directly to a tissue is the use
of CO-releasing molecules (CORMs). As pioneers in this
field, Motterlini and co-workers have identified a series of
transition-metal carbonyl complexes that fulfill this func-
tion.[5] While his first CORMs, such as [Mn2(CO)10], needed
UVactivation, the dinuclear ruthenium complex 1 (CORM-2)
liberates CO upon ligand exchange with DMSO
(Scheme 1).[6] The related mononuclear glycinato complex 2
(CORM-3) is better soluble in water and releases CO under
physiological conditions.[7]
These and several other CORMs[8] were tested in various
biological assays, and promising activities (for example
vasodilatory, anti-inflammatory, renoprotective, anti-ische-
mic, and anti-apoptotic effects) were documented, and
preclinical studies are in progress.[5] Nevertheless, the search
for new CORMs still remains a challenging task as the
existing compounds suffer from serious limitations. For
example, CO release from CORM-3 is very fast (t1/2
ꢀ 1 min) and unspecific,[9] which hampers the delivery of
controlled amounts of CO to a target tissue. An approach to
overcome this problem could be the use of stable molecules as
precursors that are converted into CORMs by means of a
trigger.[10] One possibility to achieve this is the pH-dependent
CO liberation from a boranocarbonate (CORM-A1)[11] or
amino derivatives thereof.[12] Another approach is the photo-
induced CO release of transition-metal carbonyl complexes
with UV-absorbing organic ligands.[13]
As a new concept, we introduce acyloxybutadiene–iron
tricarbonyl complexes as enzyme-triggered CO-releasing
molecules (ET-CORMs). The idea resulted from an earlier
observation that dienol–iron tricarbonyl complexes like 4 are
very labile and readily decompose already under slightly
oxidative conditions (presumably via a 16-VE intermediate of
type 5). We now envisioned that such complexes could
potentially act as CORMs, provided that they can be
generated under physiological conditions from stable precur-
sors. An appealing possibility would be the use of dienylester
complexes of type 3, which are expected to be sufficiently
stable. However, once such complexes have entered a cell
they may be cleaved by intracellular esterases. The oxidative
decomposition of the resulting dienol–iron tricarbonyl com-
plexes 4 would then be linked to the release of three
molecules of CO (Scheme 2).
Scheme 1. Selected carbonyl complexes frequently used as CO-releas-
ing molecules in biological studies.
[*] Dipl.-Chem. S. Romanski, Dr. J.-M. Neudꢀrfl, Prof. Dr. H.-G. Schmalz
Department fꢁr Chemie, Universitꢂt zu Kꢀln
Greinstrasse 4, 50939 Kꢀln (Germany)
E-mail: schmalz@uni-koeln.de
To probe this concept, we first had to synthesize some
potentially suitable acyloxydiene–iron tricarbonyl complexes.
As such we selected complexes rac-8, rac-10, and rac-12,
which were prepared from cyclohexenone 6 (Scheme 3).[14]
The synthesis of rac-8 and rac-10 started with the kinetic
deprotonation of 6 (LDA, THF, À788C) and trapping the
intermediate dienolate with either acetic anhydride (to give 7)
or pivalic chloride (to give 9). Thermal treatment of the
obtained dienes with [Fe2(CO)9] in diethyl ether gave rise to
the complexes rac-8 and rac-10 in good yield. The synthesis of
complex rac-12 (the isomer of rac-8) was initiated by
Dr. S. Amslinger
Institut fꢁr Organische Chemie, Universitꢂt Regensburg
Universitꢂtsstrasse 31, 93053 Regensburg (Germany)
E-mail: sabine.amslinger@chemie.uni-regensburg.de
Dr. B. Kraus
Lehrstuhl fꢁr Pharmazeutische Biologie
Universitꢂt Regensburg (Germany)
Prof. Dr. U. Schatzschneider
Institut fꢁr Anorganische Chemie, Universitꢂt Wꢁrzburg (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(FOR 630) and the Fonds der Chemischen Industrie (doctorate
stipend to S.R. and a Liebig fellowship to S.A.)
thermodynamically controlled deprotonation of
6 with
LiHMDS in the presence of 1.5 equiv of TPPA followed by
acetylation of the resulting dienolate. Complexation of 11
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201006598.
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Figure 1. Structure of complex rac-12 in the crystalline state. C gray,
H white, Fe orange, O red.
Scheme 2. Proposed mode of action of enzyme-triggered CO-releasing
molecules (ET-CORMs) of type 3.
pounds rac-8, rac-10, rac-12, and rac-13 was investigated
under the conditions of the myoglobin assay later used to
detect the CO release (see below). Typically, a solution of a
complex in a small amount of DMSO was mixed with a
solution of an esterase in phosphate buffer, and the con-
version was monitored by means of HPLC on a chiral
stationary phase (or GC in the case of rac-8). This also
allowed us to detect kinetic resolution as an unambiguous
proof of an enzymatic process.[17] After testing a variety of
commercially available esterases, we found suitable esterases
for all ester substrates (Table 1). The reaction of complexes
Scheme 3. Synthesis of complexes rac-8, rac-10, and rac-12. a) LDA,
THF, À788C, then Ac2O or PivCl; b) [Fe2(CO)9], Et2O, 408C, 20 h;
c) LiHMDS, TPPA, THF, À788C, then Ac2O. LDA=lithium diisopropyl-
amide, LiHMDS=lithium hexamethyldisilazide, PivCl=pivaloyl chlo-
ride, TPPA=trispyrrolidinophosphoric acid triamide.
Table 1: Identification of suitable esterases for the enzymatic hydrolysis
of acyloxydiene–iron tricarbonyl complexes as detected by kinetic
resolution.
then afforded rac-12. Furthermore, the diacetoxydiene com-
plex rac-13,[15] derived from dimedone as a nontoxic precur-
sor, and the methoxy-substituted complex rac-14,[16] as an
esterase-insensitive reference sample, were synthesized by
known procedures (Scheme 4).
Enzyme[a]
rac-8
rac-10
rac-12
rac-13
rac-14
PLE
LCR
À
À
+
+
+
À
À
À
+
+
[a] PLE: pig-liver esterase, LCR: lipase from Candida rugosa.
rac-12 and rac-13 with PLE was clearly associated with kinetic
resolution (70% to over 90% ee of the remaining complex at
80% conversion). For complexes rac-8 and rac-10, a lipase
from Candida rugosa (LCR) proved to be superior to PLE.
Not unexpectedly, the pivalate rac-10 reacted significantly
slower than the acetoxydiene complexes rac-8, rac-12, and
rac-13. The methoxy complex rac-14 did not react at all under
these conditions, as expected.
The esterase-triggered CO release from the complexes
(triggered by enzymatic hydrolysis) was then assessed by
means of the myoglobin (Mb)-based assay established by
Motterlini et al.[6] In this assay, the conversion of deoxymyo-
globin (deoxy-Mb; generated in situ by reduction of Mb with
dithionite) to carbonmonoxy myoglobin (MbCO) is deter-
mined spectrophotometrically (500–750 nm). In accordance
with the proposed mechanism (Scheme 2), a clean CO release
was detected from rac-12 (Figure 2) and rac-13 in the
presence of PLE, and from rac-8, rac-10, and rac-12 in the
presence of LCR. Under the same conditions, no CO release
(or very slow CO release in case of rac-13) was detected in the
absence of an esterase.[18] Thus, the postulated enzyme-
triggered CO release was unambiguously demonstrated.[19]
Scheme 4. Structure of complexes rac-13 and rac-14.
All of the complexes thus prepared proved to be
reasonably air-stable and were fully characterized by
common spectroscopic methods. The structural assignments
were further confirmed by an X-ray crystal structure analysis
of rac-12 (Figure 1), which also revealed the conformational
preferences of this compound in the solid state.
While we expected the acetates rac-8, rac-12, and rac-13 to
be readily cleaved by esterases, such as pig-liver esterase
(PLE), we considered the pivalate rac-10 to be less reactive
towards hydrolysis (also under non-enzymatic conditions),
thus making this complex a particularly meaningful probe for
esterase activation.
In preparation for testing the ET-CORM properties of the
complexes, we first had to identify suitable esterases for the
individual complexes. The enzymatic ester cleavage of com-
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Prior to performing the iNOS inhibition assays, we
assessed the potential cytotoxicity of the ET-CORMs in two
different ways using the murine macrophage cell line
RAW267.4 in which iNOS can be easily induced by lipo-
polysaccharide (LPS).[24] The so-called MTT test was used to
measure the viability of cells when exposed to the com-
plexes.[25] Furthermore, the number of intact cells was
determined photometrically by staining the cell nuclei with
crystal violet dye. Table 2 shows the concentrations of ET-
CORMs that caused inhibition of 50% (IC50) and 20% (IC20)
in the two assays.
Table 2: Cytotoxicity data of ET-CORMs.[a]
MTT
Crystal violet
Compound
IC50 [mM]
IC20 [mM]
IC50 [mM]
IC20 [mM]
Figure 2. Detection of enzyme-triggered CO release by monitoring the
rac-8
67.1Æ3.6
28.1Æ0.6
73.6Æ6.9
>100
47.0Æ2.7
95.2Æ3.8
>100
28.6Æ7.2
>100
14.4Æ3.5
37.7Æ1.0
>100
changes of the Q-band region of the UV/Vis spectra of reduced horse
skeletal muscle myoglobin (76 mm) in the presence of complex rac-12
(1.3 equiv) and PLE (ca. 0.01 equiv) in phosphate buffer solution
(0.1m, pH 7.4).
[a]
[a]
rac-10
rac-12
rac-13
rac-14
39.6Æ0.9
54.3Æ1.0
>100
11.2Æ2.2
21.8Æ0.6
>100
[a] Determined by MTT and crystal violet assay using RAW264.7 cells
stimulated with LPS (10 ngmLÀ1). [b] An unusual color was observed
with rac-10 in the MTTassay that precluded the correct assessment of the
cell viability.
Having successfully characterized our ET-CORMs in vi-
tro, we next turned our attention to probe the biological
potential of these compounds in a cellular assay. An
important biological target that can be addressed by CO is
the pro-inflammatory acting enzyme inducible nitric oxide
synthase (iNOS). Nitric oxide synthases (NOS) produce NO
and l-citrulline from l-arginine and molecular oxygen using
NADPH as a co-substrate. NOS possess a tightly bound heme
cofactor that is needed for their oxygenase activity
(Figure 3).[20] iNOS itself is the inducible form of NOS and
plays an important role in cellular defense mechanisms
against bacteria, viruses, parasites, and tumors.[21] With
respect to iNOS, CO is able to act in two different ways. It
can prevent the dimerization of the inactive monomeric iNOS
protein to the enzymatically active dimer. Secondly, iNOS
activity can be suppressed by binding of CO to the iron center
of heme (Fe protoporphyrin IX) to generate the inactive
complex carbonmonoxy-iNOS (Figure 3).[23]
For the pivalate rac-10, no significant toxicity was found in
the crystal violet assay at the concentrations tested. The
methoxy-substituted complex rac-14 also did not display any
toxicity up to 100 mm. In contrast, the acetates rac-8, rac-12,
and rac-13 gave IC20 values in a range 11–28 mm in the MTT
test and a slightly wider range 14–38 mm in the crystal violet
staining.
Knowing the cytotoxicity profiles of the different com-
plexes (Figure 4, left side), the stage was set for the
investigation of the influence of the ET-CORMs on nitric
oxide production by iNOS. For this purpose, the generation of
NO was determined indirectly by measuring the accumula-
tion of nitrite in the cell-culture medium using a microplate
assay based on the Griess test.[26] We only used concentrations
of ET-CORMs below the IC20 values
(Table 2) to avoid pseudo positive
results caused by cytotoxic effects.
Amongst the five ET-CORMs inves-
tigated, the diacetate rac-13 was the
most active inhibitor of NO production
in LPS-stimulated RAW267.4 cells.
Treatment of cells with rac-13 at a
concentration of 15 mm resulted in up
to (68 Æ 6)% suppression of LPS-
induced NO formation relative to con-
trol cells that were only treated with
LPS. At 20 mm (a value that is close to
the cytotoxic range), no further increase
Figure 3. Regulation of transcription and protein activity of inducible nitric oxide synthase
(iNOS) by iron species and CO.[22] The gene transcription of iNOS can be induced for example,
by lipopolysaccharide (LPS), interferon g (IFN-g), oxidative stress/reactive oxygen species
(ROS), interleukin 1b (IL-1b), or tumor nekrosis factor a (TNF-a). This occurs by activation of
nuclear factor kB (NF-kB). The activation of NF-kB can be prohibited for example by
glucocorticosteroids, antioxidants, or transforming growth factor 1b (TGF-b1).
of activity was observed. Overall,
rac-13 caused a significant NO inhibi-
tion already at 5 mm ((33 Æ 6)% inhib-
ition). Compared to rac-13, complex
rac-12 exhibits very similar activity,
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exhibited a stronger activity, as for example
cyclopentadienyl-based iron carbonyls ([(g-
C5H4CO2Me)Fe(CO)2Br])[28] or pyrone-based
iron tricarbonyl complexes.[29] Although this
compound displays a certain cytotoxicity, it is
to the best of our knowledge the most potent
CORM ever studied in this type of assay
(30% NO inhibition at 5 mm).[30]
The concept introduced herein offers
promising new options for the development
of a new generation of CORMs, thus allowing
a controlled and possibly even tissue-selective
CO delivery. The short and flexible synthesis
will at least allow the variation of the dieny-
lester ligand thus enabling us to tune the
pharmacological and biological properties
(for example water solubility) in the future.
Received: October 20, 2010
Published online: February 14, 2011
Keywords: carbonyl ligands · CO release ·
.
drug delivery · enzyme catalysis · inhibitors
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Figure 4. Results of the in vitro assays performed with RAW264.7 cells stimulated with
10 ngmLÀ1 of LPS. Left: MTT tests after an incubation time of 24 h at different
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toxicity, however, did not allow measurements above 10 mm.
Interestingly, its isomer rac-8 was found to be less active
((16 Æ 10)% reduction of NO generation at 25 mm). The
esterase-insensitive reference sample rac-14 and the pivalate
rac-10, which was also expected to be less prone to hydrolysis,
did not significantly suppress NO production at concentra-
tions of up to 50 mm (see the Supporting Information,
Figure S18D and E).[27]
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In conclusion, we have shown that acyloxycyclohexa-
diene–iron tricarbonyl complexes are a novel class of power-
ful CO-releasing molecules (ET-CORMs), which are acti-
vated by enzymatic cleavage of the ester functionality. The
proposed trigger mechanism was substantiated by CO-release
experiments (myoglobin assay) and by a cellular assay based
on iNOS inhibition as a meaningful biological response to
CO. The best of the compounds prepared to date (rac-13)
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Communications
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activation of nuclear factor-kB (NF-kB). The activation of NF-
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antioxidants or transforming growth factor-1b (TGF-b1).
[23] R. N. Watts, P. Ponka, D. R. Richardson, Biochem. J. 2003, 369,
429 – 440.
[24] Cytotoxicity is an important issue when dealing with CO-
releasing molecules. The toxicity of CO, which leads to its deadly
effect in mammals, is due to its high affinity to the prosthetic
heme groups of hemoglobin and myoglobin, resulting in
hypoxia, together with binding of CO to cytochrome c oxidase
in mitochondria. Thus, the mitochondrial electron transport
chain is inhibited and a breakdown of the energy supply of the
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[14] Cyclohexenone-derived complexes may also be considered as
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complexes of nontoxic natural products containing a cyclo-
hexenone substructure.
ˇ
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[15] A. Bohꢁc, M. Lettrichovꢁ, P. Hrnciac, M. Hutta, J. Organomet.
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[25] This assay is based upon the conversion of the yellow MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
by mitochondrial dehydrogenases into a violet formazan dye,
which can be quantified photometrically.
[18] In a control experiment, it was confirmed that compound rac-14
did not show CO release under the conditions of the myoglobin
assay in the presence of PLE or LCR.
[26] Supernatants of cells were mixed 1:1 (v/v) with Griess reagent
(1% sulfanilamide, 0.1% naphthylethylenediamine dihydro-
chloride in 2% phosphoric acid) and the absorbance was
measured at 560 nm. Nitrite content was quantified by using
sodium nitrite as standard. See: E. Park, M. R. Quinn,
J. Leukocyte Biol. 1993, 54, 119 – 124.
[27] The ether rac-14 showed a significant iNOS inhibition of (11 Æ
3)% at a concentration of 100 mm. This effect most likely results
from some esterase-independent decomposition reaction.
[28] D. Scapens, H. Adams, T. R. Johnson, B. E. Mann, P. Sawle, R.
Aqil, T. Perrior, R. Motterlini, Dalton Trans. 2007, 4962 – 4973.
[29] CORM-active pyrone–iron tricarbonyl complexes can also be
regarded as “dienylester complexes”; however, they do not need
esterase activation. See: P. Sawle, J. Hammad, I. J. S. Fairlamb, B.
Moulton, C. T. OꢀBrien, J. L. Lynam, A. K. Dhume-Klair, R.
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[30] Potential effects of iron that could act on the transcriptional level
of iNOS have not yet been addressed and will be included in
further studies. It was shown that free cellular iron can lead to a
decrease in iNOS protein expression by an inhibition of gene
transcription of iNOS; see: G. Weiss, G. Werner-Felmayer, E. R.
Werner, K. Grꢃnewald, H. Wachter, M. W. Hentze, J. Exp. Med.
1994, 180, 969 – 976.
[19] The presence of small amounts of oxygen is required for the CO
release, and the rate of CO release depends on the concentration
of esterase (enzymatic hydrolysis) and of dithionite added
(redox potential). While some oxygen is essential for the
oxidative decomposition (Scheme 2), the assay does not tolerate
higher O2 concentrations (re-oxidation of the CO-binding FeII
form of Mb). Under the assay conditions, typical times for the
release of 0.5 mol of CO per mol of ET-CORM are as follows:
56 min for rac-8 (0.09 equiv of LCR), 183 min for rac-12
(0.01 equiv of PLE) and 86 min for rac-13 (0.01 equiv of PLE).
Owing to the complexity of the parameter space, such values do
not allow a quantitative comparison of the ET-CORMS or any
predictions of their biological performance.
[20] W. K. Alderton, C. E. Cooper, R. G. Knowles, Biochem. J. 2001,
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[21] H. Kleinert, P. M. Schwarz, U. Fꢂrstermann, Biol. Chem. 2005,
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[22] Exceptionally high NO quantities account for cell damage,
which is a crucial factor in the pathophysiology of human
diseases. The gene transcription of iNOS can be induced for
example, by lipopolysaccharide (LPS), interferon-g (IFN-g),
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