[(η-C5H4R)Fe(CO)3]+, R = (CH2)nCO2Me (n = 0-2), and CO 2CH2CH2OH: A new group of CO-releasing molecules

Article abstract of DOI:10.1039/b704832g

A new group of CO-releasing molecules, CO-RMs, based on cyclopentadienyl iron carbonyls have been identified. X-Ray structures have been determined for [(η-C₅H₄CO₂Me)Fe(CO)₂X], X = Cl, Br, I, NO₃, CO₂Me, [(η-C₅H ₄CO₂Me)Fe(CO)₂]₂, [(η-C ₅H₄CO₂CH₂CH₂OH)Fe(CO) ₂]₂ and [(η-C₅H₄CO ₂Me)Fe(CO)₃][FeCl₄]. Half-lives for CO release, ¹H, ¹³C, and ¹⁷OC NMR and IR spectra have been determined along with some biological data for these compounds, [(η-C ₅H₄CO₂CH₂CH₂OH)Fe(CO) ₃]⁺ and [{η-C₅H₄(CH ₂)_nCO₂Me}Fe(CO)₃]⁺, n = 1, 2. More specifically, cytotoxicity assays and inhibition of nitrite formation in stimulated RAW264.7 macrophages are reported for most of the compounds analyzed. [(η-C₅H₅)Fe(CO)₂X], X = Cl, Br, I, were also examined for comparison. Correlations between the half-lives for CO release and spectroscopic parameters are found within each group of compounds, but not between the groups. The Royal Society of Chemistry.

Full text of DOI:10.1039/b704832g

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PAPER
www.rsc.org/dalton | Dalton Transactions
[
[
(g-C H R)Fe(CO) X], X = Cl, Br, I, NO , CO Me and
5
4
2
3
2
+
(g-C H R)Fe(CO) ] , R = (CH ) CO Me (n = 0–2), and
5
4
3
2 n
2
CO CH CH OH: a new group of CO-releasing molecules†‡§
David Scapens, Harry Adams, Tony R. Johnson, Brian E. Mann,* Philip Sawle, Rehan Aqil,
2
2
2
a
a
a
a
b
c
c
b
Trevor Perrior and Roberto Motterlini
Received 30th March 2007, Accepted 21st May 2007
First published as an Advance Article on the web 19th September 2007
DOI: 10.1039/b704832g
A new group of CO-releasing molecules, CO-RMs, based on cyclopentadienyl iron carbonyls have
been identified. X-Ray structures have been determined for [(g-C
Br, I, NO , CO Me, [(g-C CO Me)Fe(CO) , [(g-C CO CH
(g-C CO Me)Fe(CO) ][FeCl
5
H
4
CO
2
Me)Fe(CO)
2
X], X = Cl,
3
2
5
H
4
2
2
]
2
5
H
4
2
2
CH
2
OH)Fe(CO)
2
]
2
and
1
13
17
[
5
H
4
2
3
4
]. Half-lives for CO release, H, C, and OC NMR and IR
spectra have been determined along with some biological data for these compounds,
+
+
[
(g-C
5
H
4
CO
2
CH
2
CH
2
OH)Fe(CO)
3
] and [{g-C
5
H
4
(CH
2
)
n
CO
2
Me}Fe(CO)
3
] , n = 1, 2. More
specifically, cytotoxicity assays and inhibition of nitrite formation in stimulated RAW264.7
macrophages are reported for most of the compounds analyzed. [(g-C
were also examined for comparison. Correlations between the half-lives for CO release and
spectroscopic parameters are found within each group of compounds, but not between the groups.
5
H
5
)Fe(CO)
2
X], X = Cl, Br, I,
Introduction
CO-releasing molecules (CO-RMs) is highly attractive from a
pharmaceutical point of view as the total quantity of CO being
administered can be tightly controlled by weight and a solution of
the molecules can be given where required to avoid the exposure
of the whole body to CO.
Carbon monoxide is a signalling molecule produced in mam-
mals by the oxidation of heme by heme oxygenase. It is anti-
inflammatory, protects against ischemia-reperfusion injury, pre-
vents endothelial cell apoptosis, produces vasodilatation and
protects against graft rejection. The biological role of CO is
being investigated in animal models of disease for its possible
Early attempts to devise CO-releasing molecules involved
6
[
Fe(CO)
5
], [Mn(CO)
5
]
2
and [Ru(CO)
3
Cl
2
]
2
.
[Fe(CO) ] and
5
[Mn(CO)
5
2
] do not dissolve in water and photolysis was used to
1
use in a number of medical applications. These include but are
liberate the CO. These problems coupled with the high toxicity
of [Fe(CO) ] resulted in a search for water-soluble CO-releasing
2
not restricted to organ transplantation, cardiopulmonary bypass
5
3
4
surgery, and angioplasty. More recently, administration of CO
gas to animals has been shown to reverse established pulmonary
molecules which were stable in solid form, but released CO when
◦
introduced into biological fluid at 37 C. [Ru(CO)
3
Cl
2
2
] partially
5
hypertension.
met these problems. It had to be initially dissolved in DMSO
in order to use it subsequently in aqueous solutions but in the
From the studies conducted so far, the general therapeutic
approach is that CO would be provided as an additive to the
air being breathed. However, due to the high toxicity of CO,
great care is necessary to control the dose and protect the people
administering and receiving the gas. Hence the use of solid
process it lost a variable quantity of CO to give a mixture of
6
[
Ru(CO)
3
Cl
2
(DMSO)] and [Ru(CO)
2
Cl
2
(DMSO) ] (isomers).
2
[
Ru(CO)
3
Cl was modified to give [Ru(CO)
2
]
2
3
Cl(glycinate)]
7
which is now well established as a CO-releasing molecule. It has
been used to provide CO for a wide range of biological applications
such as protection of the heart against myocardial infarction and
the kidney against both ischaemia-induced acute renal failure,
and the toxic effects of cisplatin.
8
a
Department of Chemistry, University of Sheffield, Sheffield, UK S3 7HF.
9
E-mail: b.mann@sheffield.ac.uk; Fax: +44 (0)114 2738673; Tel: +44
(
0)114 2229332
10
b
Vascular Biology Unit, Department of Surgical Research, Northwick
Park Institute for Medical Research, Harrow, Middlesex, UK E-mail:
r.motterlini@imperial.ac.uk; Fax: +44 (0)208 8693538; Tel: +44 (0)208
2−
More recently, [H
3
BCO
2
]
(CORM-A1) has also been shown
to provide CO which produces vasodilatation in isolated vessels
11
8
693181
and promotes a reduction in mean arterial pressure in vivo.
c
NCE Discovery Ltd., 418 Cambridge Science Park, Cambridge, UK CB4
In this report, the first viable CO-releasing molecules based on
0
PZ. E-mail: t.perrior@ncediscovery.com; Fax: +44 (0)1223 433 181;
12
iron compounds are identified after an initial report in 2001.
Tel: +44 (0)1223 433 188
4
A number of iron(0) compounds of the type [(g -substituted
†
Based on the presentation given at Dalton Discussion No. 10, 3rd–5th
September 2007, University of Durham, Durham, UK.
CCDC reference numbers 640137–640144. For crystallographic data in
CIF or other electronic format see DOI: 10.1039/b704832g
Electronic supplementary information (ESI) available: [(g-
pyrone)Fe(CO) ] have been reported in 2005 and 2006 as CO-
3
‡
releasing molecules. They release CO slowly and were published
13
without patent protection.
§
The known compound, [(g-C
5
H
5
)Fe(CO)
3
]Cl, is water-soluble
C
5
H
4
CO
2
CH
2
CH
2
OH)Fe(CO)
2 2
] cis–trans ratio in solution, cytotoxicity
Fe(CO) Br] in murine RAW264.7 macrophages,
◦
and slowly releases CO to myoglobin at 37 C with a half-
of [(g-C
5
H
4
CO
2
)CH
3
2
12
X-ray crystal data. See DOI: 10.1039/b704832g
life of 69 min. It produces vasodilatation when added to
4
962 | Dalton Trans., 2007, 4962–4973
This journal is © The Royal Society of Chemistry 2007

View Article Online
a rat aorta pre-contracted using phenylephrine. Unfortunately,
after CO is released, the compound precipitates, probably due
to the formation of a neutral compound which is insoluble
in water. As these types of compounds are likely to be used
therapeutically in the cardiovascular system, the precipitate could
block micro-arteries leading to medical complications. In order
to avoid this problem, substituents have been introduced into
the cyclopentadienyl ring and the resulting compounds tested
for CO release, water solubility and biological activity. In this
+
paper, [{g-C
with [(g-C
CO (CH
5
H
4
(CH
2
)
n
CO
2
Me}Fe(CO)
3
] , n = 0, 1, 2, along
5
H
4
CO Me)Fe(CO)
2
2
X], X = Cl, Br, I, NO
3
, and [{g-
+
C
5
H
4
2
2
)
2
OH}Fe(CO)
3
] are examined as CO-releasing
1
3
17
molecules. Parameters such as d( CO), d( OC) and m(CO) are
examined as predictors of the rate of CO loss.
Fig. 1 The X-ray crystal structure of [(g-C
5 4 2 2 2
H CO Me)Fe(CO) ] . Se-
lected bond lengths [ A˚ ]:- Fe1–Fe1 2.525(3), Fe1–C1 1.762(10), Fe1–C2
1
2
.920(8), Fe1–C3 2.108(8), Fe1–C4 2.143(8), Fe1–C5 2.143(9), Fe1–C6
.136(8), Fe1–C7 2.077(8). Thermal ellipsoids are shown at 50%.
Results and discussion
At first sight, the synthesis of compounds of the type, [(g-
C
5
H
4
R)Fe(CO)
3
]X, should be easy. There are numerous ex-
14
amples of substituted ferrocenes, but a literature search for
+
examples of the cation [(g-C
5
H
4
18
R)Fe(CO)
3
]
only yielded R =
and OH. However, there is much more
literature regarding the synthesis of the dimeric compounds [(g-
1
5
16
17
Me, Et, CHPh ,
2
1
9
C
5
H
4
R)Fe(CO)
this work, the previously reported [(g-C
is used as a starting material to yield water-soluble CO-
releasing molecules. [(g-C CO Me)Fe(CO) I] has been reported
2
]
2
containing functionality on the Cp ring. In
20
5
H
4
CO
2
Me)Fe(CO)
2 2
]
5
H
4
2
2
20
previously.
The preparation of [(g-C
thesis of C CO Me from [C
5
H
4
CO
2
Me)Fe(CO)
] and ClCO
2
]
2
involves the syn-
−
5
H
5
2
5
H
5
2
Me. C CO Me
5
H
5
2
readily dimerises by the Diels–Alder reaction so it is reacted
immediately with [Fe (CO) ] to give the product. The use of
Fe (CO) ] rather than the published route using [Fe(CO) ] is
preferred as in our hands the synthesis proved to be more
reliable. [(g-C CO Me)Fe(CO) was used to synthesise
(g-C CO Me)Fe(CO) Me, Cl, Br, I and NO
X], X = CO
and [(g-C CO Me)Fe(CO) ] . Transesterification was used to
convert [(g-C CO Me)Fe(CO) to [(g-C CO CH CH OH)-
Fe(CO) to enhance the water solubility. Two routes
were used to convert [(g-C R)Fe(CO) to [(g-C R)Fe-
] : (i) protonation of [(g-C CO Me)Fe(CO) (CO Me)]
R)Fe(CO) with oxidising agents
Fe] in the presence of CO.
Me substituent with
2
9
[
2
9
5
Fig.
Fe(CO)
.5224(10), Fe1–C1 1.7633(19), Fe1–C3 1.920(2), Fe1–C4 1.9234(18),
Fe1–C5 2.145(2), Fe1–C6 2.1434(18), Fe1–C7 2.0988(19), Fe1–C8
.0915(19), Fe1–C9 2.1302(19), Fe2–C2 1.760(2), Fe2–C3 1.9336(18),
Fe2–C4 1.914(2), Fe2–C10 2.0926(19), Fe2–C11 2.121(2), Fe2–C12
2.1293(19), Fe2–C13 2.1227(18), Fe2–C14 2.1135(19). Thermal ellipsoids
are shown at 50%.
2
The X-ray crystal structure of [(g-C
5 4 2 2 2
H CO CH CH OH)-
5
H
4
2
2 2
]
2 2
]
. O7 and O10 are disordered. Selected bond lengths [ A˚ ]:- Fe1–Fe2
[
5
H
4
2
2
2
3
2
+
5
H
4
2
3
5
H
4
2
2
]
2
5
H
4
2
2
2
2
2
1,22,23
2 2
]
5
H
4
2
]
2
5
H
4
+
(
CO)
and (ii) reaction of [(g-C
such as SO Cl or [(g-C
3
5
H
4
2
2
2
5
5
H
4
2 2
]
+
coordinated to the oxygen(s) of the bridging carbonyl(s). The
2
2
5
H
)
2
cis-stereochemistry may be favoured by hydrogen bonding over a
In order to compare the effect of the CO
2
+
distance of 1.947 A˚ across the molecule between the RCH O(7A)H
an alkyl one, [{g-C
5
H
4
(CH
2
)
n
CO
2
Me}Fe(CO)
3
] , n = 1, 2, were
2
substituent on one cyclopentadienyl and the O(8)=C(OMe)R on
the other. This interaction is weak as O(7)H is disordered between
two positions, one which hydrogen-bonds with a 51% occupancy
and the atom in the other position does not. This hydrogen-
bonding interaction is also small in solution as it makes no
measurable contribution to the solution cis–trans ratio determined
from IR spectra or the activation energy for carbonyl exchange of
the bridge and terminal carbonyls in the cis-isomer via the trans-
also synthesised.
X-Ray crystal structures
The X-ray crystal structures of many of the compounds were deter-
mined. The X-ray crystal structure of [(g-C CO Me)Fe(CO)
Fig. 1) is similar to those determined previously for many related
compounds. Thecyclopentadienyls aretransacross the plane of the
5
H
4
2
2
]
2
,
(
24,25
Fe
ray crystal structure of [(g-C
the cyclopentadienyls cis across the plane of the Fe
2
(l-CO)
2
moiety as is commonly found.
In contrast, the X-
OH)Fe(CO) shows
(l-CO) moiety
Fig. 2) which is less common excluding those where bonding
isomer. There is also disorder at the CH
The X-ray crystal structures of [(g-C
(Fig. 3), [(g-C CO Me)Fe(CO) Br] (Fig. 4), [(g-C
Fe(CO) I] (Fig. 5), [(g-C CO Me)Fe(CO) (NO
[(g-C CO Me)Fe(CO) (CO Me)] (Fig. 7) and [(g-C
Me)Fe(CO)
2
OH group.
CO Me)Fe(CO)
CO Me)-
)] (Fig. 6),
CO -
5
H
4
CO CH CH
2
2
2
2
]
2
5
H
4
2
2
Cl]
2
2
5
H
4
2
2
5
H
4
2
(
2
5
H
4
2
2
3
26
between the two cyclopentadienyl rings forces a cis-geometry.
5
H
4
2
2
2
5
H
4
2
In three of the cases of cis-geometries, there are Lewis acids
3
][FeCl
4
] (Fig. 8) are very similar to those reported
This journal is © The Royal Society of Chemistry 2007
Dalton Trans., 2007, 4962–4973 | 4963

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Fig. 5 The X-ray crystal structure of [(g-C
5 4 2 2
H CO Me)Fe(CO) I]. Se-
lected bond lengths [ A˚ ]:- Fe1–C1 1.776(5), Fe1–C2 1.793(5), Fe1–C3
5 4 2 2
Fig. 3 The X-ray crystal structure of [(g-C H CO Me)Fe(CO) Cl].
Fe1A–C2A 1.788(2), Fe1A–C1A 1.792(2), Fe1A–C3A 2.081(2), Fe1A–
C4A 2.090(2), Fe1A–C5A 2.125(2), Fe1A–C6A 2.1201(19), Fe1A–C7A
2
2
.086(4), Fe1–C4 2.098(4), Fe1–C5 2.116(4), Fe1–C6 2.130(4), Fe1–C7
.106(4), Fe1–I1 2.6046(9). Thermal ellipsoids are shown at 50%.
2
1
.0829(19), Fe1A–Cl1A 2.2899(6), Fe1B–C1B 1.799(2), Fe1B–C2B
.791(2), Fe1B–C3B 2.086(2), Fe1B–C4B 2.085(2), Fe1B–C5B 2.112(2),
Fe1B–C6B 2.122(2), Fe1B–C7B 2.0919(18), Fe1B–Cl1B 2.2894(6). Ther-
mal ellipsoids are shown at 50%.
Fig. 6 The X-ray crystal structure of [(g-C
5 4 2 2 3
H CO Me)Fe(CO) (NO )].
Selected bond lengths [ A˚ ]:- Fe1–C1 1.791(3), Fe1–C2 1.819(3), Fe1–C3
2
2
.110(3), Fe1–C4 2.095(2), Fe1–C5 2.116(2), Fe1–C6 2.111(3), Fe1–C7
.105(3), Fe1–O7 1.9729(17). Thermal ellipsoids are shown at 50%.
Fig. 4 The X-ray crystal structure of [(g-C
5 4 2 2
H CO Me)Fe(CO) Br].
Selected bond lengths [ A˚ ]:- Fe1–C2 1.783(7), Fe1–C1 1.789(7), Fe1–C3
into account. When all the compounds are taken together, the
evidence for localisation is strong. The CO R group is nearly
2
2
.071(6), Fe1–C4 2.086(6), Fe1–C7 2.094(6), Fe1–C5 2.109(6), Fe1–C6
.133(6), Fe1–Br1 2.4248(10). Thermal ellipsoids are shown at 50%.
2
coplanar with the C H ring with the O=C–C–C torsion angle,
5
4
◦
◦
A, being close to 0 or 180 . The (RO
2
C)C–(cyclopentadienyl
previously for similar compounds. The structures of [(g-C
Fe(CO)
5
H
5
)-
◦
ring centroid)–Fe–X torsion angle B, is near 60 in most cases,
27
28
29
2
Cl], [(g-C
)Fe(CO) I], R
)Fe(CO) (NO
5
Me
5
)Fe(CO)
2
Cl], [(g-C
5
Ph
5
)Fe(CO)
2
Br],
showing that the preferred conformation has the (RO
approximately bisecting the (OC)–Fe–X angle. In the case of [(g-
CO H)Fe(CO) Me] it approximately bisects the (OC)–Fe–
CO) angle. Only in the case of the dimer does it approximately
eclipse an OC–Fe bond. For [(g-C CO Me)Fe(CO) (NO )],
the nitrate O6 is only 2.854 A from the carboxy carbon, C8.
2
C)C group
3
0,31,32,33
[
[
(g-C
(g-C
5
R
5
2
5
= any substituent including H,
34
5 5 3 5
5
H
5
2
3
)], and [(g-C
R
)Fe(CO)
], R
= any sub-
C
5
H
4
2
2
2
3,33,35
stituent including H,
selected bond lengths are collected in Table 1.
have been reported previously. Some
(
5
H
4
2
2
3
There are some examples of X-ray crystal structures of
˚
the [(g-C
ing H, in the literature.
are [(g-C
g)Fe(CO)
5
R
4
CO
2
Me)Fe(CO)
2
X], R = any substituent includ-
This causes the (RO
torsion angle B to be close to 46 .
2
C)C–(cyclopentadienyl ring centroid)–Fe–X
3
6,37,38
The closest literature examples
◦
38
5
H
4
CO
2
H)Fe(CO)
2
CH
3
]
and [(g-C
5
H
5
)
2
Ti{(O
2
CC
5
4
H -
37
2
CH
2
Ph}
2
].
[
(g-C
5
H
4
CO
2
R)Fe(CO)
2
]
2
, R = Me, CH
2
CH OH
2
The introduction of the CO
2
R substituent into the cyclopenta-
dienyl ring appears to induce some bond localisation into the
ring (Table 1). For most of the compounds individually, the
differences in C–C bond lengths are not sufficiently different
to prove the localisation when the standard deviation is taken
It is well established that [(g-C
5
R
5
)Fe(CO)
2
]
2
, R
5
= any substituent
including H, exists in solution as interconverting cis- and trans-
39,40,41
isomers.
On account of the hydrogen bonding seen in the
X-ray crystal structure of [(g-C CO CH CH OH)Fe(CO) ] ,
5
H
4
2
2
2
2
2
4
964 | Dalton Trans., 2007, 4962–4973
This journal is © The Royal Society of Chemistry 2007

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Table 1 Selected bond lengths and angles for the RO
MeO CC )Fe(CO) ][FeCl
]. A is the torsion angle between the O=C and C
ring and the Fe–X bond taking X as (g-MeO CC )Fe(CO) in the case of the dimer. The atom numbering in Table 1 follows that in the diagram
with the substituent of the iron being used to differentiate between C and C and C and C
2
CC
5
H
4
group in [(g-MeO
2
CC
5
H
4
)Fe(CO)
2 2 2 5 4 2
] , [(g-RO CC H )Fe(CO) X], and [(g-
7
6
1
2
1
2
5
H
4
3
4
5 4
H (C –C –C –C ) ring and B is the torsion angle between C -centroid of
C
5
H
4
2
5
H
4
2
2
5
3
4
1
2
˚
2
3
˚
3
4
˚
4
5
˚
5
1
˚
1
6
˚
6
7
˚
◦
◦
R
Me
CH
X
Dimer
Dimer
C –C /A
1.417(13)
1.424(2)
C –C /A
1.418(13)
1.416(3)
1.413(3)
1.409(3)
1.399(3)
1.410(9)
1.385(7)
1.421(4)
1.402(2)
C –C /A
1.418(14)
1.405(3)
1.411(3)
1.435(3)
1.439(3)
1.434(9)
1.436(7)
1.434(4)
1.437(2)
C –C /A
1.407(12)
1.420(3)
1.431(3)
1.406(3)
1.409(3)
1.391(8)
1.411(7)
1.413(3)
1.412(2)
1.412(2)
1.406
C –C /A
1.439(11)
1.425(2)
1.411(3)
1.436(3)
1.440(3)
1.449(8)
1.452(6)
1.429(4)
1.422(2)
C –C /A
1.467(12)
1.473(2)
1.476(3)
1.478(3)
1.472(3)
1.477(8)
1.464(6)
1.479(3)
1.482(2)
C –O /A
1.214(11)
1.2058(17)
1.203(2)
1.206(2)
1.209(2)
1.204(7)
1.198(6)
1.206(3)
1.202(2)
A/
B/
15.51
5.11
179.87
6.59
3.44
4.41
4.41
12.58
1.80
10.09
2.12
5.67
117.22
106.49
72.30
65.31
59.21
70.21
68.83
45.93
68.76
—
2
2
CH OH
1
.428(3)
1.423(3)
.432(3)
1.435(9)
1.446(6)
1.426(3)
1.438(2)
Me
Cl
1
Me
Me
Me
Me
Me
H
Cp
Br
I
NO
CO
CO
Me
CH
3
2
Me
1.4201(18) 1.4181(19) 1.432(2)
1.425
1.433
1.4372(18) 1.4913(18) 1.2026(17)
38
1.404
1.387
1.385
1.419
1.420
1.417
1.429
1.412
1.415
1.462
1.484(8)
1.477(8)
1.2472(19)
1.211(7)
1.206(7)
178.36
64.59
75.47
37,a
2
Ti
2
Ph
1.379
1.403
1
.441
3.72
a
[
Cp
2
Ti{(O
2
CC
5
H
4
)Fe(CO)
2
CH
2
Ph} ].
2
5 4 2 3 4
Fig. 8 The X-ray crystal structure of [(g-C H CO Me)Fe(CO) ][FeCl ].
Selected bond lengths [ A˚ ]:- Fe1–C1 1.8189(14), Fe1–C2 1.8242(15),
Fe1–C3 1.8085(14), Fe1–C4 2.0991(13), Fe1–C5 2.0941(13), Fe1–C6
5 4 2 2 2
Fig. 7 The X-ray crystal structure of [(g-C H CO Me)Fe(CO) (CO Me)].
Selected bond lengths [ A˚ ]:- Fe1–C1 1.7689(19), Fe1–C2 1.7662(18),
2
.1099(13), Fe1–C7 2.0963(13), Fe1–C8 2.1042(13), Fe2–Cl1 2.1839(4),
Fe1–C3 2.1028(19), Fe1–C4 2.0939(18), Fe1–C5 2.1150(17), Fe1–C6
Fe2–Cl2 2.1928(4), Fe2–Cl3 2.1843(4), Fe2–Cl4 2.2026(4). Thermal ellip-
soids are shown at 50%.
2
.1052(17), Fe1–C7 2.1111(18), Fe1–C10 1.9593(17). Thermal ellipsoids
are shown at 50%.
C=O found in the crystal structure does not result in an enhanced
concentration of the cis-isomer.
the ratio of cis- and trans-isomers has been determined using m(CO)
39
intensities to check if there is any preference for the cis-isomer
It has been previously observed that [(g-C
5
H
5
)Fe(CO)
2
] shows
2
1
3
‡
◦
13
and by C NMR measurements of DG for exchange between the
at low temperatures, (e.g. −60 C) two CO signals for the cis-
41,42
bridging and terminal carbonyls.
IR spectroscopy using m(CO) intensities was used to determine
the cis–trans ratio of the isomers of [(g-C CO R)Fe(CO)
R = Me, CH CH OH, as previously described. The results are
given in Table S1§. The results show that on going from R = Me
to CH CH OH and in comparison with [(g-C )Fe(CO) , the
presence of the CH CH OH group makes no significant difference
isomer due to the bridging and terminal carbonyls and one
1
3
averaged CO signal for the trans-isomer. On warming, the signals
5
H
4
2
2
]
2
,
broaden and average due to carbonyl bridge-opening coupled with
39
41,42
‡
2
2
Fe–Fe bond rotation.
DG was determined for bridge–terminal
carbonyl exchange in CD
2
Cl
2
for cis-[(g-C
5
H
4
CO
2
R)Fe(CO)
2
]
2
,
‡
−1
2
2
5
H
5
2
]
2
R = Me, CH
2
CH
2
OH, DG 244 = 51 ± 3 kJ mol for R = CH
3
‡
−1
2
2
and DG 258 = 54 ± 3 kJ mol for R = CH
2
CH
2
OH. These
‡
−1
in the ratio, showing that the hydrogen bond between the OH and
values of DG do not differ significantly from 51 ± 3 kJ mol
Dalton Trans., 2007, 4962–4973 | 4965
This journal is © The Royal Society of Chemistry 2007

View Article Online
previously reported for cis-[(g-C
5
H
5
)Fe(CO) ] , once again
2
2
confirming that any interaction between the OH and C=O in
42
[
(g-C
5
H
4
CO
2
CH
2
CH
2
OH)Fe(CO)
2
2
] is negligible.
CO loss in solution
As the [(g-C CO
0
/+
5
H
4
2
R)Fe(CO) X] compounds were made as
2
CO-releasing molecules for possible pharmaceutical use, the CO
release was measured by using electronic spectroscopy to monitor
◦
the formation of carboxymyoglobin from myoglobin at 37 C. The
technique works well for about the first 60 min but after this time,
the results become suspect. The result is that half-lives for CO loss
of up to about 200 min are reasonably accurate but longer times
have less accuracy. The electronic spectra were measured between
Fig. 10 A plot of CO released from 40 lM [(g-C
to myoglobin at 37 C as a function of time and the first order rate fit.
5
H
4
CO
2
Me)Fe(CO)
2
Br]
◦
5
00 and 600 nm and fitted as a weighted sum of the spectra
of myoglobin and carboxymyoglobin. Allowance was made for
−
4
turbidity by applying a k correction to the spectrum. The spectra
were fitted in Excel using the Solver routine.
A typical set of electronic spectra for [(g-C
5
H
4
CO Me)-
2
Fe(CO) Br] are shown in Fig. 9 and the corresponding plot of
2
CO concentration against time with a first order rate fit is shown
in Fig. 10.
Fig. 11 A plot of half-lives for CO-loss against the average m(CO)
+
for ꢀ, [(g-C
CH CO Me, 4, (CH
, Br, 7, I, 8, NO , 9; ᭺, [(g-C
5
H
4
R)Fe(CO)
3
] , R = H, 1, CO
2
Me, 2, CO
2 2 2
CH CH OH, 3,
2
2
2
)
2
CO
2
Me, 5; ꢀ, [(g-C
5
H
4
CO Me)Fe(CO)
2
2
X], X = Cl,
6
3
5
H
5
)Fe(CO)
2
X], X = Cl, 10, Br, 11, I, 12.
+
Note that the average m(CO) is calculated for [(g-C
5
H
4
R)Fe(CO)
3
] giving
a double weighting to the lower frequency E symmetry band. Trend lines
are included for the three groups of compounds.
Fig. 9 The changes in the electronic spectrum of myoglobin as CO is
the two sets of compounds. This is illustrated for m(CO) in Fig. 11,
◦
released from [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
Br] at 37 C as a function of time.
1
3
17
d( CO) in Fig. 12 and d( OC) in Fig. 13. The correlation with
r(Fe–C) is relatively poor probably due to the errors in determining
both t1/2 and r(Fe–C).
An extra spectrum is included. The curve marked Mb–CO is due to the
myoglobin after saturation with CO gas. The spectra are shown after
baseline correction.
The graphs show some correlation between t1/2 and m(CO),
1
3
17
The half-lives (t1/2) for CO loss are collected in Table 2 along with
d( CO), d( OC) within a group of compounds, but no correlation
1
3
17
m(CO), d( CO), d( OC) and r(Fe–C). Within each group of com-
overall.
+
pounds, [(g-C
5
H
4
R)Fe(CO)
3
]
and [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
X]
When the data for the series [(g-C
with those from [(g-C CO Me)Fe(CO)
Me substituent results in m(CO), r(CO) and d( OC)
5
H
5
)Fe(CO)
2
X] is compared
there is an approximate correlation between t1/2 and m(CO),
5
H
4
2
2
X], the introduction
1
3
17
17
d( CO), d( OC) and r(Fe–C) but there is no correlation between
of the CO
2
17
13
Table 2 The half-lives for CO loss, m(CO), d( O), d( C), and r(Fe–CO)
−
1
17
13
˚
Compound/Ion
t1/2/min
m(CO)/cm
d( O) (ppm)
d( C) (ppm)
Average r(Fe–CO)/A
+
[
[
[
[
[
[
[
[
[
[
[
[
(C
(C
(C
(C
(C
(C
(C
(C
(C
(C
(C
(C
5
5
5
5
5
5
5
5
5
5
5
5
H
H
H
H
H
H
H
H
H
H
H
H
5
4
4
4
4
4
4
4
4
5
5
5
)Fe(CO)
3
]
69
42
62
225
285
63
38
48
170
350
200
150
2125, 2077
2132, 2089
2132, 2089
2121, 2065
2116, 2065
2064, 2022
2060, 2018
2050, 2010
2076, 2036
2054, 2008
2051, 2005
2041, 1997
388.2
399.2
391.2
—
202.4
202.7
200.6
204.7
203.7
210.5
210.8
212.0
209.0
212.4
212.5
213.5
1.816
1.8172
—
—
—
+
CO
CO
CH
CH
2
3
Me)Fe(CO) ]
+
2
CH
CO
CH
2
CH
Me)Fe(CO)
CO Me)Fe(CO)
OH)Fe(CO) ]
2 3
+
2
2
2
3
]
+
2
2
3
]
—
CO
CO
CO
CO
2
2
2
2
Me)Fe(CO)
Me)Fe(CO)
Me)Fe(CO)
Me)Fe(CO)
2
2
2
2
Cl]
Br]
I]
386.4
385.7
384.9
393.7
383.1
382.3
381.2
1.793
1.786
1.785
1.805
(NO
3
)]
2
7
1
)Fe(CO)
)Fe(CO)
)Fe(CO)
2
2
2
Cl]
Br]
I]
1.771
—
3
1.781
4
966 | Dalton Trans., 2007, 4962–4973
This journal is © The Royal Society of Chemistry 2007

View Article Online
13
Fig. 12 A plot of half-lives for CO-loss against the d( CO) for
+
ꢀ
, [(g-C
CH CO Me, 4, (CH
Cl, 6, Br, 7, I, 8, NO , 9; ᭺, [(g-C
Trend lines are included for the three groups of compounds.
5
H
4
R)Fe(CO)
3
] , R = H, 1, CO
2
Me, 2, CO
CO Me)Fe(CO)
X], X = Cl, 10, Br, 11, I, 12.
2
CH
2
CH
2
OH, 3,
2
2
2
)
2
CO
2
Me, 5; ꢀ, [(g-C
5
H
4
2
2
X], X =
3
5
H
5
)Fe(CO)
2
Fig. 14 Cell viability in murine RAW264.7 macrophages exposed for
2
5 4 2 2
4 h to 10, 50 and 100 lM [(g-C H CO Me)Fe(CO) Br]. The percentage
of viable cells was assessed using an Alamar Blue assay.
where it can be seen that even at 100 lM there is no reduction in
activity.
The release of lactic dehydrogenase (LDH) in the medium of
the cells was used as a test for cytotoxicity. A reference for 100%
cytotoxicity was generated by adding Triton to the cells. The results
for [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
Br] are shown in Fig. S1§, where it
can be seen that even at 100 lM there is no detectable toxicity.
One of the key roles of CO in vivo is its ability to reduce
inflammation caused by over-production of NO in macrophages.
The production of NO can be estimated by determining nitrite
formation and this is shown in Fig. 15. Lipopolysaccharide (LPS)
is used to stimulate NO production from macrophages and the
effectiveness of the CO-RM in reducing NO and hence nitrite
formation is determined. Examination of Fig. 15 shows that
addition of [(g-C H CO Me)Fe(CO) Br] to produce a 10 lM
solution has no effect while both the 50 and 100 lM solutions have
a slight effect in inducing nitrite production. After the addition of
LPS to induce nitrite formation, a 10 lM solution has no effect
while both the 50 and 100 lM solutions have a major significant
effect in reducing the amount of nitrite formed.
1
7
Fig. 13 A plot of half-lives for CO-loss against the d( OC) for
+
ꢀ
, [(g-C
CH CO Me, 4, (CH
Cl, 6, Br, 7, I, 8, NO , 9; ᭺, [(g-C
Trend lines are included for the three groups of compounds.
5
H
4
R)Fe(CO)
3
] , R = H, 1, CO
2
Me, 2, CO
CO Me)Fe(CO)
X], X = Cl, 10, Br, 11, I, 12.
2
CH
2
CH
2
OH, 3,
X], X =
2
2
2
)
2
CO
2
Me, 5; ꢀ, [(g-C
5
H
4
2
2
3
5
H
5
)Fe(CO)
2
5
4
2
2
1
3
increasing while d( CO) decreases. This is consistent with the
CO Me substituent withdrawing electron density from the metal
weakening the M–CO bond.
For the cations, [(g-C R)Fe(CO)
2
+
5
H
4
3
] , t1/2 decreases as m(CO)
increases towards the value of free CO. The data are consistent
with a weakening in the Fe–CO bond paralleling the ease of loss
of CO as might be expected for a dissociative reaction. The reverse
is found for [(g-C
5
H
5
)Fe(CO)
2
X] and [(g-C
5
H
4
CO
2
Me)Fe(CO) X].
2
As m(CO) increases and the Fe–CO bond lengthens, t1/2 gets longer.
These results show that within a series of compounds, m(CO),
1
3
17
d( CO) and d( OC) could be of value in predicting which
compounds may release CO rapidly. The prediction is not easy
as the effect of substituents may differently affect the ground and
transition states. Caution is necessary as the number of compounds
is limited.
Biological activity
The compounds were tested for biological activity using murine
RAW264.7 macrophages exposed to three different concentrations
of the CO-RM (10, 50 and 100 lM). After 24 h treatment, three
tests were performed on the macrophages.
Fig. 15 Nitrite levels, an index of NO formation, in the media of
murine RAW264.7 macrophages exposed for 24 h to 10, 50 and 100 lM
−1
[
(g-C
5
H
4
CO
2
Me)Fe(CO)
2
Br]. 1 lg mL LPS was used to induce nitrite
Alamar blue was used to test the redox activity of the cells.
5 4 2 2
formation and to test the effectiveness of [(g-C H CO Me)Fe(CO) Br] in
The results for [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
Br] are shown in Fig. 14,
suppressing nitrite formation.
This journal is © The Royal Society of Chemistry 2007
Dalton Trans., 2007, 4962–4973 | 4967

View Article Online
+
Table 3 Tests of biological activity of [{g-C
0–2, [(g-C CO CH CH OH)Fe(CO)
X], X = Cl, Br, I, NO and [(g-C )Fe(CO)
the cell viability, cell toxicity and [NO formation by murine RAW264.7
5
H
4
(CH
3
2
)
n
CO
] , [(g-C
X], X = Cl, Br, I on
2
Me}Fe(CO)
3
] ,
replacing it with another NMR tube containing the same solvent
and a thermocouple attached to a Comark Evolution N9009
thermometer. Elemental analysis was carried out on a Perkin
Elmer 2400 CHNS/O Series II Elemental analyser. IR spectra
were recorded on a Bruker TENSOR 27 FT-IR spectrometer
or a Perkin Elmer PARAGON 1000 FT-IR spectrometer. Mass
spectrometry was carried out on either a Waters LCT Electrospray
+
n
=
5
H
4
2
2
2
5
H
4
CO Me)Fe-
2
(
CO)
2
3
5
−
H
5
2
2
]
monocyte macrophages. The * convention is explained below and is based
on more * indicate the better the outcome. NA is not available
−
Compound/ion
2
Cell viability Cytotoxicity [NO ]
+
[
[
[
[
[
[
[
[
[
[
[
(C
(C
(C
(C
(C
(C
(C
(C
(C
(C
(C
5
5
5
5
5
5
5
5
5
5
5
H
H
H
H
H
H
H
H
H
H
H
4
4
4
4
4
4
4
4
5
5
5
CO
CO
CH
CH
CO
CO
CO
CO
)Fe(CO)
)Fe(CO)
)Fe(CO)
2
Me)Fe(CO)
3
]
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
NA
***
***
*
*
*
*
**
**
**
**
NA
*
±
+
TOF Mass spectrometer (ES ) or a VG Autospec Magnetic Sector
2
CH
CO
CH
2
CH
Me)Fe(CO)
CO Me)Fe(CO)
2
OH)Fe(CO)
3
]
+
2
2
2
3
]
Mass spectrometer (EI and FAB). X-Ray data collected were
measured on a Bruker Smart CCD area detector with Oxford
Cryosystems low temperature system. The release of CO from
metal carbonyl complexes was determined spectrophotometrically
by measuring the conversion of deoxymyoglobin (deoxy-Mb) to
+
2
2
3
]
2
2
2
2
Me)Fe(CO)
Me)Fe(CO)
Me)Fe(CO)
Me)Fe(CO)
2
2
2
2
Cl]
Br]
I]
3
(NO )]
2
2
2
Cl]
Br]
I]
6
carbonmonoxy myoglobin (MbCO) as previously described. The
electronic spectra were measured between 500 and 600 nm at time
intervals of 0, 5, 10, 15, 20, 25, 30 and 60 min and fitted as a
weighted sum of the spectra of myoglobin and carboxymyoglobin.
*
*
Greater than 50% cell viability or less than 50% cell toxicity at 10 lM
CO-RM concentration or greater than 50% nitrite production inhibition
at 100 lM CO-RM concentration.** Greater than 50% cell viability or
nitrite production inhibition, or less than 50% cell toxicity at 50 lM CO-
RM concentration.*** Greater than 50% cell viability or less than 50%
cell toxicity at 100 lM CO-RM concentration or greater than 50% nitrite
production inhibition at 10 lM CO-RM concentration.
−
4
Allowance was made for any turbidity by applying a k correction
to the spectrum. The spectra were fitted using Excel and Solver.
The resulting concentrations were then fitted as first-order kinetics.
20,45
C
5
H
4
CO
2
Me
The biological test results are reported in Table 3. All the
compounds tested show excellent cell viability and cytotoxicity
profiles. The compounds also reduce nitrite formation with
the neutral compounds having limited ability while the cations
tested have good ability. The presence of the ester group on the
cyclopentadienyl ring provides a site to add different side chains
to control water/lipid solubility and to include groups to direct
the compounds to specific sites in the body.
A solution of LiCp was prepared by the addition of 156.3 mL
(
0.25 mol) of n-BuLi (1.6 M in hexanes) to 20.65 mL (0.25 mol)
of freshly cracked cyclopentadiene in 280 mL of dry THF at
◦
−
78 C, under argon. After complete addition, the reaction
was allowed to warm slowly to room temperature and then
stirred for a further hour. During this time a white precipitate is
◦
formed. Following this, the reaction was recooled to −78 C and
1
9.3 mL (0.25 mol) of methyl chloroformate added dropwise. This
resulted in complete disappearance of the white precipitate and the
formation of a yellow–orange coloured solution. After warming
to room temperature and then stirring for a further hour, a white
precipitate was produced (LiCl). 500 mL of water was added and
the two layers separated. The aqueous layer was washed with two
Conclusions
The introduction of a substituent into the cyclopentadienyl
+
ring of [(C
5
H
4
CO
2
R)Fe(CO)
3
] and [(C
5
H
4
CO
2
R)Fe(CO) X] can
2
be used to control the rate of CO release. For example, the
introduction of a CO R group into the cyclopentadienyl group
of [(g-C CO R)Fe(CO) X] increases the rate of CO loss to
100 mL portions of diethyl ether, and then the combined organic
2
extracts were washed with five 250 mL portions of water then once
5
H
4
2
2
◦
with saturated brine. It was then dried (MgSO
4
at 0 C for 45 min)
myoglobin. At the three concentrations tested (10, 50 and 100
lM), no significant reduction in cell viability or toxicity was
found for any of the compounds tested. All the compounds show
some activity in inhibiting nitrite formation by LPS-stimulated
macrophages. Further biological testing is required to assess their
usefulness in a wider range of biological applications. Within a
small group of compounds, d( CO), d( OC) and m(CO) correlate
with the rate of CO loss but there is little correlation between the
groups of compounds.
and then the solvent removed on a rotary evaporator to give a
yellow oil. This was used without further purification.
[
(g-C
5
H
4
CO
2
Me)Fe(CO)
2 2
]
1
3
17
All of the substituted cyclopentadiene synthesised in the above
reaction was refluxed with 20 g of [Fe (CO) ] in 200 mL of
2
9
deoxygenated heptane (argon purge), under argon for 24 h.
◦
Following this it was cooled to −18 C overnight and then the
purple crystalline precipitate collected on a sinter and washed with
several portions of pentane. The heptane supernatant was recycled
Experimental
Starting materials and reagents were purchased from Aldrich or
in repeats of the preparation and in this way yields can vary from
43
44
1
Acros. [Fe
2
(CO)
9
]
and [(C
5
H
5
)Fe(CO)
2
X], X = Cl, Br, I, were
4.5 to 6 g (9.57 to 12.8). M
r
= 469.99. H NMR (400 MHz,
synthesised according to the literature. Solvents were dried using
a Grubbs system (column chromatography), including DCM,
THF, toluene, diethyl ether, hexane and petroleum ether. NMR
experiments were carried out on a Bruker AVANCE 500, a
Bruker AMX400 NMR spectrometer or a Bruker AC 250 NMR
spectrometer. The temperature of the sample was measured by
CD
2
Cl
2
): d 3.96 (s, Me, 3H), 5.03 (br, Cp, 2H), 5.29 (br, Cp, 2H).
1
3
◦
C NMR (100.6 MHz, CD
2
Cl
2
, −29 C): d 52.6 (Me), 88.4 (Cp),
91.1 (Cp), 92.6 (ipso Cp), 164.7 (C=O), 208.7 (CO), 265.9 (CO).
−
1
IR (CH
2
Cl ) m(cm ): 2010 (s), 1972 (m), 1724 (m). Mass spec.
2
+
(m/z): 470 (M ). Elemental: Fe
2
C
18
H
14
8
O found (calc) C: 45.60
(46.00), H: 2.79 (3.00).
4
968 | Dalton Trans., 2007, 4962–4973
This journal is © The Royal Society of Chemistry 2007

View Article Online
−
1
[
(g-C
5
H
4
CO
2
Me)Fe(CO)
2
(CO
2
Me)]
m(cm ): 2132(s), 2088(s), 1744(m). Elemental: FeC10
H
7
O
5
BF
4
found (calc) C: 34.12 (34.34), H: 1.93 (2.02).
A solution of [Fe(CpCO
2
Me)(CO)
2
]
2
(2 g, 4.26 mmol) in THF
3
(
30 mL) was added to a sodium amalgam (10 cm mercury;
[
(g-C
5
H
4
CO
2
Me)Fe(CO)
2
Cl]
0
1
.8 g (34.8 mmol) sodium). The mixture was stirred vigorously for
h. The resulting brown solution of Na[(g-C CO Me)Fe(CO) ]
5
H
4
2
2
500mg (1.06mmol) of [(g-C H CO Me)Fe(CO) ] was dissolvedin
5
4
2
2 2
was transferred via syringe into a second flask. The solution was
14 mL of dry THF, under argon. A solution of 127 mg (1.06 mmol)
◦
cooled to −78 C and methyl chloroformate (0.657 mL) was added
of SOCl in 5 mL dry THF was then added drop-wise with
2
dropwise. The reaction mixture was stirred at room temperature
for 3 h, during which time the reaction was monitored by IR
spectroscopy. Removal of the solvent gave an oily residue which
was redissolved in DCM, filtered and washed through Celite. The
solvent was removed from the filtrate and an oily red residue was
obtained. The product was dissolved in the minimum amount of
stirring. After complete addition, stirring was continued for a
further 25 min. Following this, IR showed that there was still
some starting material present. Hence a dilute THF solution of
SOCl was prepared and aliquots of this were added. The reaction
2
was stirred until complete being monitored by IR spectroscopy.
The solvent was removed on a rotary evaporator and the residue
columned on silica gel, initially prepared in petroleum ether and
eluted as a red band with diethyl ether. The solvent was removed
on a rotary evaporator and the product recrystallised from diethyl
ether–petroleum ether. 252 mg (0.932 mmol) of a red crystalline
r
◦
hexane and left to recrystallise at −78 C. A brown–yellow solid
was obtained. Further crystallisation was carried out using DCM–
hexane mixture which yielded a yellow crystalline solid. 802 mg
(
2.73 mmol) of product was obtained. M
r
= 294.04. Yield 32%.
1
H NMR (400 MHz, d
Cp), 3.8 (s, 3H, Me), 3.5 (s, 3H, Me). C NMR (100.62 MHz,
-acetone): d 51.5 (CH ), 51.7 (CH ), 86.4 (ipso Cp), 87.4 (b-
Cp), 90.7 (a-Cp), 164.6 (Cp C=O), 195.7 (Fe C=O), 212.7 (CO).
6
-acetone): d 5.75 (s, 2H, Cp), 5.25 (s, 2H,
solid was obtained. M = 270.45. Yield 44%. X-Ray quality
1
3
◦
1
crystals were grown from diethyl ether at −18 C. H NMR
(400 MHz, CD Cl ): d 3.93 (s, Me, 3H), 5.18 (br, Cp, 2H), 5.70
d
6
3
3
2
2
1
3
(br, Cp, 2H). C NMR (100.62, CD Cl ): d 52.7 (Me), 83.0 (Cp),
2
2
−
1
17
IR (CH
(
2
Cl
2
) m(cm ): 2044 (s), 1993 (s), 1727 (m). Mass spec.
84.5 (Cp ipso), 91.6 (Cp), 164.4 (C=O), 210.5 (CO). O NMR
+
−1
m/z): 317 (M + Na ). Elemental: FeC11
H
10
O
6
found (calc) C:
(54.2 MHz, CD Cl ): d 386.4 (CO). IR (CH Cl ) m(cm ): 2064 (s),
2
2
2
2
+
+
+
4
5.01 (44.93), H: 3.33 (3.43).
2022 (s). Mass spec. (m/z): 270 (M ), 242 (M − CO), 214 (M −
2
CO). Elemental: FeC
9
H
7
4
O Cl found (calc) C: 39.77 (39.97), H:
2
.34 (2.61), Cl: 12.94 (13.11).
[
(g-C
5
H
4
CO
2
Me)Fe(CO)
3
][FeCl ]
4
4
00 mg (0.851 mmol) of [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
]
2
was dissolved
Cl in
[(g-C
5
H
4
CO
2
Me)Fe(CO)
2
Br]
in 20 mL of benzene, under argon. A solution of SO
2
2
4
00 mg (0.851 mmol) of [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
] was dis-
2
benzene was then added dropwise with stirring. This resulted
in the immediate formation of a yellow precipitate. The reaction
was followed by IR spectroscopy, and when there was no more
of the dimer starting material present, addition was ceased. The
resulting precipitate was collected on a sinter and then washed
with benzene and a little cold DCM. It was then recrystallised
from DCM (i.e. sample dissolved in boiling DCM and then cooled
solved in 20 mL of DCM, under argon. A solution of 150 mg
0.936 mmol) of Br in 5 mL of DCM was then added drop-wise
(
2
with stirring. After complete addition, stirring was continued for
a further 30 min, after which time the reaction was shown to
be complete by IR spectroscopy. The reaction solution was then
transferred to a separating funnel and more DCM was added. It
was washed with three portions of deoxygenated Na
and once with deoxygenated water. Then it was dried (MgSO
filtered, and the solvent removed on a rotary evaporator to give a
red–brown solid. A silica gel column was prepared in petroleum
ether (40–60 C). The product was introduced as a solution in a
little DCM and eluted with petroleum ether containing increasing
amounts of diethyl ether to (2 : 3) as a dark red band. Removal
of solvent gave the product as a dark red solid, which was dried
2
S
2
O
3
(aq.)
),
◦
to −18 C overnight). The resulting yellow crystals were isolated,
4
washed with diethyl ether and then dried under vacuum. 101 mg
(
0.219 mmol) of product obtained. M
r
= 460.66. Yield 26%. X-Ray
quality crystals were obtained from a dilute solution in MeCN–
◦
◦
1
diethyl ether–pentane at −18 C. H NMR (400 MHz, CD CN): v.
3
1
3
broad due to paramagnetic counter ion. C NMR (100.62 MHz,
CD CN): d 60.6 (CH ), 91.3 (ipso Cp), 97.2 (Cp), 99.8 (Cp), 161.7
C=O), 202.7 (CO). O NMR (54.2 MHz, CD
3
3
1
7
(
3
CN): d 399.2 (CO).
in vacuo. 268 mg (0.851 mmol) of product obtained. M
Yield 50%. X-Ray quality crystals were grown from a diethyl ether
r
= 314.90.
−
1
IR (MeCN) m(cm ): 2132 (s), 2089 (vs), 1745 (m). Mass spec.
+
+
+
(
m/z): 263 (M ), 235 (M − CO), 207 (M − 2CO). Elemental:
Fe Cl found (calc) C: 26.14 (26.07), H: 1.62 (1.53), Cl:
0.80 (30.78).
◦
1
solution at −18 C. H NMR (400 MHz, CD
H), 5.19 (br, Cp, 2H), 5.70 (br, Cp, 2H). C NMR (100.62 MHz,
CD Cl ): d 52.7 (Me), 83.2 (Cp), 84.0 (Cp ipso), 90.8 (Cp), 164.3
C=O), 210.8 (CO). O NMR (54.2 MHz, CD
2
2
Cl ): d 3.90 (s, Me,
2
C
10
H
7
O
5
4
1
3
3
3
2
2
1
7
(
2
Cl
2
): d 385.7 (CO).
−
1
[(g-C
5
H
4
CO
2
Me)Fe(CO)
3
][BF
4
]
IR (CH
2
Cl
2
) m(cm ): 2060 (s), 2018 (s). Mass spec. (m/z): 314
+
+
+
(
M ), 286 (M − CO), 258 (M − 2CO). Elemental: FeC
9
H
7
O
4
Br
Two drops of acid (HBF
lution of [(g-C CO Me)Fe(CO)
4
) were added to a vigorously stirred so-
CO Me] (80 mg, 0.272 mmol)
found (calc) C: 34.68 (34.33), H: 2.14 (2.24), Br: 25.16 (25.37).
5
H
4
2
2
2
in THF (10 mL). A white–yellow precipitate was formed instanta-
neously, this solid was filtered off using a sinter and washed twice
20
[
(g-C
5
H
4
CO
2
Me)Fe(CO) I]
2
with anhydrous diethyl ether. 77 mg (0.221 mmol) of product was
800mg (1.70mmol) of [(g-C
40 mL of DCM, under argon. A solution of 497 mg (1.96 mmol) of
5
H
4
CO
2
Me)Fe(CO)
2
]
2
was dissolvedin
1
obtained. M
r
= 349.81. Yield 81%. H NMR (400 MHz, CD
3
OD):
Cl
d 6.55 (s, 2H, Cp), 6.0 (s, 2H, Cp), 3.9 (s, 3H, OMe). IR (CH
2
2
)
I
2
in 20 mL of DCM was then added drop-wise with stirring. After
This journal is © The Royal Society of Chemistry 2007
Dalton Trans., 2007, 4962–4973 | 4969

View Article Online
−
1
complete addition, stirring was continued for a further 3 h, after
which time reaction was shown to be complete by IR spectroscopy.
The reaction solution was then transferred to a separating funnel
and more DCM was added. It was washed with three portions of
164.3 (C=O), 208.1 (CO), 267.7 (CO). IR (CH
(s), 1975 (m), 1785 (s), 1721 (m). Mass spec. (m/z): 531 (MH ).
2
Cl
2
) m(cm ): 2012
+
Elemental: Fe
(3.42).
2
C
20
H O10 found (calc) C: 44.83 (45.32), H: 3.37
18
deoxygenated Na
It was then dried (MgSO
a rotary evaporator to give a black solid. This was dried under
vacuum. 1.03 g of product obtained. M
= 361.90. Yield 84%.
X-Ray quality crystals were obtained from a diethyl ether solution
2
S
2
O
3
(aq.) and once with deoxygenated water.
4
), filtered, and the solvent removed on
[(g-C H CO CH CH OH)Fe(CO) ][PF ]
5
4
2
2
2
3
6
5
6
00 mg (0.943 mmol) of [(g-C
15 mg (1.86 mmol, 0.985 eq.) of ferrocenium hexafluorophos-
5
H
4
CO
2
CH
2
CH
2
OH)Fe(CO)
2
] and
2
r
◦
1
phate were placed in a Schlenk tube under a CO atmosphere.
70 mL of a CO-saturated DCM–THF mixture (2 : 1) was then
added and the reaction stirred for 2.5–3 d in the dark with periodic
bubbling of CO through the solution. A yellow precipitate started
to form and precipitation was completed by addition of diethyl
ether (150 mL). After stirring for 10 min the product was collected
on a sinter, and washed several times with diethyl ether. It was
then extracted through the sinter with acetone and the solvent
removed from the filtrate. Washing the residue with diethyl ether
at −18 C. H NMR (400 MHz, CD
br, Cp, 2H), 5.72 (br, Cp, 2H). C NMR (100.62 MHz, CD
d 52.6 (Me), 83.7 (Cp), 89.9 (Cp), 164.2 (C=O), 212.0 (CO).
2
Cl ): d 3.90 (s, Me, 3H), 5.13
2
1
3
(
2
Cl
2
7
):
O
1
−
1
NMR (54.2 MHz, CD
2
Cl
2
): d 384.9 (CO). IR (CH
2
Cl
2
) m(cm ):
+
+
2
3
050 (s), 2010 (s). Mass spec. (m/z): 362 (M ), 334 (M − CO),
+
06 (M − 2CO). Elemental: FeC
9
H
7
O I found (calc) C: 29.86
4
(
29.87), H: 1.71 (1.95), I: 35.06 (35.07).
[
(g-C
5
H
4
CO
2
Me)Fe(CO)
2
(NO
3
)]
gave a dark yellow solid product which was dried in vacuo. 485 mg
1
(
1.11 mmol) of product was obtained. M
NMR (400 MHz, CD CN): d 3.85 (br, CH
(br, Cp, 2H), 6.41 (br, Cp, 2H). C NMR (100.6 MHz, CD CN):
r
= 437.99. Yield 60%. H
3
00 mg (0.638 mmol) of [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
2
] and 228 mg
3
2
), 4.38 (br, CH ), 5.84
2
(
1.34 mmol) of AgNO were stirred together in 15 mL of acetone
3
1
3
◦
3
at 30 C, under argon. The reaction was monitored by IR
spectroscopy and after 1.5 h the reaction was shown to be
complete. The solution was filtered through Celite and then the
solvent removed on a rotary evaporator to give a red oily residue.
Recrystallisation was attempted from DCM–hexane but this did
not work. The compound was introduced in DCM to a silica gel
d 59.4 (CH
2
), 68.4 (CH ), 84.0 (ipso Cp), 89.7 (Cp), 93.6 (Cp),
2
1
7
1
61.2 (C=O), 200.6 (CO). O NMR (54.2 MHz, CD
3
CN): d 391.2
CN) m(cm ): 2132 (s), 2089 (vs), 1743 (m). Mass
−
1
(CO). IR (CH
3
+
+
spec. (m/z): 293 (M ), 209 (M − 3CO). Elemental: FeC11
H
9
O
6
PF
6
found (calc) C: 30.53 (30.16), H: 1.91 (2.07).
◦
column, prepared in petroleum ether (40–60 C). Elution with
petroleum ether–diethyl ether (1 : 1) gave a very small amount of
a yellow band. The product was eluted as a bright red band with
diethyl ether. Removal of solvent on a rotary evaporator, washing
with petroleum ether and drying in vacuo gave the desired solid
product. 78 mg of a bright red solid was obtained (0.263 mmol).
Methyl iodoacetate
The following procedure is based on a modified literature
46
method. A mixture of methyl bromoacetate (20.00 g, 12.4 mL,
31 mmol) and sodium iodide (25.10 g, 167 mmol, 1.28 eq.) in
1
acetone (90 mL) was stirred at room temperature for 15 h and
M
r
= 297.00. Yield 21%. X-Ray quality crystals were obtained
from a diethyl ether solution at −18 C. H NMR (400 MHz,
CD Cl ): d 3.93 (s, CH ), 5.21 (br, Cp, 2H), 5.78 (br, Cp, 2H).
NMR (100.62 MHz, CD Cl ): d 53.0 (Me), 82.7 (Cp), 83.9 (Cp
ipso), 91.5 (Cp), 164.0 (C=O), 209.0 (CO). O NMR (54.2 MHz,
◦
then heated at 50 C for 2 h. The reaction mixture was then cooled
◦
1
to ambient temperature, filtered to remove sodium bromide and
the solid was washed with diethyl ether (2 x 50 mL). The filtrate
was concentrated in vacuo, diluted with diethyl ether (100 mL)
and the organic layer was washed with water (2 x 50 mL), brine
1
3
2
2
3
C
2
2
1
7
−
1
CD
Elemental: FeC
2.38), N: 4.66 (4.72).
2
Cl
2
): d 393.7 (CO). IR (CH
2
Cl ) m(cm ): 2076 (s), 2036 (s).
2
(
50 mL), dried (anhydrous sodium sulfate) and evaporated to give
9
H
7
NO found (calc) C: 35.73 (36.40), H: 2.44
7
methyl iodoacetate (18.69 g, 93.46 mmol, 72%) as a dark red oil.
(
1
H NMR (400 MHz, CDCl
ICH ).
3
) d 3.73 (s, 3H, OCH ), 3.68 (s, 2H,
3
2
[
(g-C
5
H
4
CO
2
CH
2
CH
2
OH)Fe(CO)
2
]
2
Methyl 3-iodopropionate
1
.01 g (2.16 mmol) of [(g-C
5
H
4
CO
2
Me)Fe(CO)
2
2
] and 15 mg
(
1
0.375 mmol) of NaH (60% disp. in mineral oil) were stirred in
8 mL of ethylene glycol at 55 C overnight, under argon. DCM
46
Using a modified Finkelstein procedure, methyl 3-iodopro-
pionate (23.10 g, 108 mmol, 90%) was prepared from methyl 3-
◦
and deoxygenated water were added and the two layers separated.
The aqueous layer was washed with DCM and then the combined
DCM extracts were washed three times with deoxygenated water
and once with saturated brine. It was then dried (MgSO ) and the
solvent removed on a rotary evaporator. The resulting solid was
washed with several portions of diethyl ether. 1.00 g (1.89 mmol)
of a dark purple solid was obtained. M
The sample can be recrystallised from DCM–hexane but the yield
is lowered to 62%. H NMR (400 MHz, CD
1
bromopropionate (20.00 g, 120 mmol) and sodium iodide (22.98 g,
53 mmol, 1.28 eq.) in acetone (80 mL) as an orange oil. H NMR
1
1
(400 MHz, CDCl
3
) d 3.71 (s, 3H, OCH
3
), 3.31 (s, 2H, J = 7.2 Hz,
4
ICH
2
), 2.97 (s, 2H, J = 7.2 Hz, CH CO
2
2
).
Methyl cyclopenta-1,3-dienylacetate and methyl
r
= 530.04. Yield 88%.
47,48
cyclopenta-1,4-dienylacetate
1
2
Cl
2
): d 3.02 (s, OH,
A solution of methyl iodoacetate (18.50 g, 92.5 mmol) in anhy-
drous tetrahydrofuran (60 mL) was added dropwise to a 2.0 M
solution of sodium cyclopentadienide (46.3 mL, 92.5 mmol) in
tetrahydrofuran over 15 min under nitrogen at −78 C. The
H), 4.00 (br, CH
2
, 2H), 4.50 (br, CH
2
, 2H), 5.06 (br, Cp, 2H),
1
3
◦
5
.39 (br, Cp, 2H). C NMR (100.62 MHz, CD
), 67.2 (CH
2
Cl
2
, −17 C):
◦
d 60.9 (CH
2
2
), 88.4 (Cp), 91.1 (Cp), 91.6 (Cp ipso),
4
970 | Dalton Trans., 2007, 4962–4973
This journal is © The Royal Society of Chemistry 2007

View Article Online
◦
resulting reaction mixture was stirred for a further 3 h at −78 C
(Cp C), 89.7 (Cp CH), 89.4 (Cp CH), 52.5 (OCH
IR (solid) m(cm ) 1979 (s), 1946 (s), 1759 (s), 1737 (s).
3
), 32.4 (CH ).
2
−
1
and then warmed to room temperature, filtered and the resulting
solid was washed with diethyl ether (200 mL). The combined
organics were concentrated in vacuo. The crude oil was purified
by flash chromatography on silica using 10% ethyl acetate in
iso-hexane to afford methyl 3-cyclopenta-1,3-dienylacetate (1-
alkylCp) and methyl 3-cyclopenta-1,4-dienylacetate (2-alkylCp)
[Fe(C H CH CH CO Me)(CO) ]
5
4
2
2
2
2 2
Using the same procedure as for [Fe(C
above, [Fe(C CH CH CO Me)(CO)
0%) was prepared as maroon crystals from a mixture of methyl
5
H
4
CH
2
CO
2
Me)(CO)
2
]
2
5
H
4
2
2
2
2 2
]
(12.75 g, 24.20 mmol,
5
(
2.37 g, 17.3 mmol, 19%) as yellow liquids in an approx. 1 : 1 ratio.
1
3-cyclopenta-1,3-dienylpropionate and methyl 3-cyclopenta-1,4-
dienylpropionate (1.2 : 1, 14.50 g, 96 mmol) and diiron nonacar-
bonyl (34.90 g, 96 mmol) in degassed heptane (350 mL). H NMR
H NMR (500 MHz, CDCl
3
) d 6.53 (m, 1H, Cp CH), 6.46 (m,
H, Cp CH), 6.37 (m, 2H, Cp CH), 6.23 (m, 1H, Cp CH), 3.72 (s,
H, OCH ), 3.71 (s, 3H, OCH ), 3.47 (m, 2H, CH ), 3.44 (m, 2H,
), 3.04 (m, 2H, CH ), 3.02 (m, 2H, CH ).
2
3
CH
1
3
3
2
(
500 MHz, CD
CH), 3.71 (s, 6H, OCH
NMR (500 MHz, CD
2
Cl , RT) d 4.67 (s, 4H, Cp CH), 4.56 (s, 4H, Cp
2
2
2
2
1
3
), 2.80 (s, 4H, CH
2
), 2.66 (s, 4H, CH ). H
2
◦
2
Cl
2
, −30 C) d 4.67 (br, 4H, Cp CH), 4.57
Methyl 3-cyclopenta-1,3-dienylpropionate and methyl
(
br, 4H, Cp CH), 3.67 (s, 6H, OCH
3
), 2.76 (s, 4H, CH
2
), 2.68 (4H,
48
3-cyclopenta-1,4-dienylpropionate
1
◦
CH
2
). H NMR (500 MHz, CD
), 2.68 (s, 8H, CH
, RT) d 172.7 (ester C=O), 105.6 (Cp C), 88.3 (Cp CH),
2
Cl
2
, −50 C) d 4.58 (br, 8H, Cp
1
3
CH), 3.65 (s, 6H, OCH
CD Cl
7.4 (Cp CH), 51.5 (2 × OCH
NMR (126 MHz, CD Cl
, −30 C) d 272.9 (bridging CO), 210.8
(terminal CO), 173.1 (ester C=O), 105.2 (Cp C), 87.9 (Cp CH),
87.0 (Cp CH), 52.1 (OCH ), 34.3 (CH ), 22.5 (CH ). IR (solid)
m(cm ) 1976 (s), 1937 (s), 1788 (s), 1715 (s).
3
2
). C NMR (126 MHz,
A 2.0 M solution of sodium cyclopentadienide in tetrahydrofuran
2
2
(
105 mL, 210 mmol) was added dropwise over 15 min to a
1
3
8
3
), 34.3 (CH
2
), 22.5 (CH
2
).
C
stirred solution of methyl 3-iodopropionate (45.00 g, 210 mmol) in
anhydrous diethyl ether (280 mL) and anhydrous tetrahydrofuran
◦
2
2
◦
(
200 mL) under nitrogen at −78 C. The resulting reaction mixture
◦
◦
3
2
2
was stirred at −78 C for 2 h and then stored at −20 C for a
further 15 h. The resulting red suspension was quenched with 1 M
ammonium chloride solution (800 mL), and the organic phase
was extracted with diethyl ether (5 x 400 mL). The combined
organic layer was washed with 1 M ammonium chloride solution
−
1
[
Fe(C
5
H
4
CH
2
CO
2
Me)(CO)
3
][BF ]
4
The following procedure is based on a modified literature
(
2 x 500 mL) and dried (anhydrous sodium sulfate), filtered
22
method. Ferrocinium tetrafluoroborate (274 mg, 1.00 mmol,
and concentrated in vacuo. The crude oil was purified by flash
chromatography on silica using 5% ethyl acetate in iso-hexane to
afford methyl 3-cyclopenta-1,3-dienylpropionate (1-alkylCp) and
methyl 3-cyclopenta-1,4-dienylpropionate (2-alkylCp) (14.72 g,
2
eq.) was added to [Fe(C
5
H
4
CH
2
CO
2
Me)(CO)
2
] (250 mg,
2
0
.502 mmol) under nitrogen. An anhydrous mixture of degassed
DCM–THF (33 mL; 2 : 1) was added and CO was then bubbled
through the resulting reaction mixture for a period of 15 min. The
reaction mixture was then stirred under a CO atmosphere. After 18
and 24 h, CO was passed through the reaction mixture for 10 min.
In total, the reaction mixture was stirred under a CO atmosphere
for 36 h after which the reaction flask was flushed with nitrogen.
The reaction mixture was then concentrated in vacuo and the
resulting black solid was washed with degassed diethyl ether (5 ×
9
6.7 mmol, 46%) as yellow liquids in a 1.2 : 1 ratio. Major isomer
1
(
1-alkylCp): H NMR (400 MHz, CDCl
3
) d 6.40 (m, 1H, Cp-
H3), 6.25 (m, 1H, Cp-H4), 6.02 (m, 1H, Cp-H2), 3.66 (s, 3H,
OCH
3
), 2.93 (dd, 2H, J = 3.7 and 1.9 Hz, Cp-H5), 2.71 (m, 2H,
1
CH CO
2
2
), 2.55 (m, 2H, CpCH
2
). Minor isomer (2-alkylCp): H
NMR (400 MHz, CDCl
Cp-H4), 6.16 (m, 1H, Cp-H1), 3.66 (s, 3H, OCH
J = 2.9 and 1.5 Hz, Cp-H5), 2.71 (m, 2H, CH CO
CpCH ).
3
) d 6.40 (m, 1H, Cp-H3), 6.39 (m, 1H,
), 2.88 (dd, 2H,
), 2.55 (m, 2H,
3
2
0 mL), after which the product was extracted with degassed DCM
2
2
(
5 × 20 mL). The combined organic layers were concentrated
2
in vacuo to give an orange solid which was then washed with
degassed dichloromethane (20 mL). The resulting yellow solid
was dissolved in acetone (20 mL), filtered and the solvent removed
[
Fe(C
5
H
4
CH
2
CO
2
Me)(CO)
2 2
]
in vacuo to give the title compound as a yellow solid (66.4 mg,
A mixture of methyl 3-cyclopenta-1,3-dienylacetate (1-alkylCp)
and methyl 3-cyclopenta-1,4-dienylacetate (2-alkylCp) (2.00 g,
1
0
.183 mmol, 36%). H NMR (500 MHz, CD
3
COCD , RT, low
3
concentration sample) d 6.25 (t, 2H, Cp CH), 6.08 (t, 2H, Cp
1
4.59 mmol) in degassed heptane (55 mL) was added to diiron
1
CH), 3.87 (s, 2H, CH
CD COCD , RT, high concentration sample) d 6.22 (br, 2H, Cp
CH), 6.06 (br, 2H, Cp CH), 3.86 (s, 2H, CH ), 3.77 (s, 3H, OCH ).
2
), 3.77 (s, 3H, OCH
3
). H NMR (500 MHz,
nonacarbonyl (5.31 g, 14.59 mmol) under nitrogen at room
temperature. The resulting reaction mixture was heated to reflux
3
3
2
3
◦
at 110 C and stirred for 18 h, then cooled to ambient temperature
13
◦
C NMR (126 MHz, CD
3
COCD
3
, −30 C) d 204.7 (terminal CO),
at which point precipitation of maroon crystals was observed.
The solution was further cooled in the freezer for 1 h and then
filtered through a sinter funnel. The crystals collected were washed
thoroughly with degassed hexane (4 x 50 mL). The crystals were
dissolved in degassed dichloromethane (4 x 50 mL) and the solvent
1
(
70.9 (ester C=O), 106.4 (Cp C), 92.9 (Cp CH), 89.7 (Cp CH), 53.4
−1
OCH
Fe(C
Using the ferrocinium oxidation procedure given above,
3
), 32.1 (CH
2
). IR (solid) m(cm ) 2121 (s), 2065 (s), 1737 (s).
[
5
H
4
CH CH CO
2
2
2
Me)(CO)
3
]BF ]
4
22
was concentrated in vacuo to yield the iron sandwich complex
1
(
2.67 g, 5.36 mmol, 30%) as maroon crystals. H NMR (500 MHz,
[Fe(C CH CH CO Me)(CO) ][BF ] (95.0 mg, 0.251 mmol,
15%) was prepared as a yellow solid from [Fe(C CH
CH CO Me)(CO) (900 mg, 1.71 mmol) and ferrocinium
5
H
4
2
2
2
3
4
CD
2
Cl
2
, RT) d 4.76 (br, 4H, Cp), 4.69 (br, 4H, Cp), 3.74 (s, 6H,
5
H
4
2
-
1
3
◦
OMe), 3.56 (s, 4H CH
d 272.5 (bridging CO), 210.3 (terminal CO), 171.2 (C=O), 98.0
2
). C NMR (126 MHz, CD
2
Cl
2
, −30 C)
2
2
]
2 2
tetrafluoroborate (933 mg, 3.42 mmol, 2 eq.) under a carbon
This journal is © The Royal Society of Chemistry 2007
Dalton Trans., 2007, 4962–4973 | 4971

View Article Online
−
1
monoxide atmosphere for 36 h. IR (solid) m(cm ) 2059 (s), 2009 (s),
Acknowledgements
1
1
735 (s). H NMR (400 MHz, CD
sample) d 6.15 (t, 2H, Cp CH), 6.07 (t, 2H, Cp CH), 3.67 (s,
H, OCH ), 2.94 (t, 2H, CH ), 2.80 (t, 2H, CH , signal overlaps
with H O signal). H NMR (500 MHz, CD COCD , RT, high
concentration sample) d 6.13 (br, 2H, Cp CH), 6.05 (br, 2H, Cp
3
COCD
3
, RT, low concentration
This work was supported by a EPSRC DTA studentship (DS) and
PDRA (TRJ) and Hemocorm Ltd.
3
3
2
2
1
2
3
3
1
3
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CH), 3.67 (s, 3H, OCH
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), 2.93 (t, 2H, CH
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), 2.82 (t, 2H, CH ). C
2
◦
3
COCD
3
, −30 C) d 203.7 (terminal CO),
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005, 38, 127.
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1
1
5
0
The assays were performed following the published procedure.
Murine RAW264.7 macrophages were purchased from the Eu-
ropean Collection of Cell Cultures (Salisbury, Wiltshire, UK)
and cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, 2 mM L-glutamine,
−
1
−1
1
00 units mL penicillin and 0.1 mg mL streptomycin. Cultures
◦
were maintained at 37 C in a 5% CO
2
humidified atmosphere
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0% confluence. Macrophages were exposed for 24 h to LPS (1 lg
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−
1
mL ) in the presence or absence of CO-RMs (10, 50 and 100 lM)
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1
4 R. C. Kerber, Comprehensive Organometallic Chemistry II, ed.
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51
as previously described. The measurement of this parameter is
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with sodium nitrite (0 lM to 300 lM in cell culture medium).
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carried out according to the manufacturer’s instructions (Serotec,
UK) as previously reported. The assay is based on the detection
of metabolic activity of living cells using a redox indicator which
changes from an oxidised (blue) form to a reduced (red) form. The
intensity of the red colour is proportional to the metabolism of the
cells, which is calculated as the difference in absorbance between
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Dalton Trans., 2007, 4962–4973 | 4973