DOI: 10.1002/chem.

201403901

Full Paper

& Drug Design

Design and Synthesis of New Hybrid Molecules That Activate the

Transcription Factor Nrf2 and Simultaneously Release Carbon
Monoxide
Jayne Louise Wilson,[a] Sarah Fayad Kobeissi,[a] Souhila Oudir,[b] Benjamin Haas,[a]
Brian Michel,[c] Jean-Luc Dubois Rand,[d] Anthony Ollivier,[b] Thierry Martens,[b]
Michael Rivard,[b] Roberto Motterlini,*[a] and Roberta Foresti*[a]

Abstract: The transcription factor Nrf2 and its downstream

target heme oxygenase-1 (HO-1) are essential protective systems against oxidative stress and inflammation. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), actively contribute to this protection, suggesting
that exploitation of these cellular systems may offer new
therapeutic avenues in a variety of diseases. Starting from
a CO-releasing compound and a chemical scaffold exhibiting
electrophilic characteristics (esters of fumaric acid), we

Introduction
The transcription factor Nrf2 is a crucial initiator of the cellular
stress response as it coordinates the induction of antioxidant
and detoxification genes that repair damage and restore cellular homeostasis.[1] As part of this response, heme oxygenase1 (HO-1) plays a prominent role by utilizing heme to produce
CO, biliverdin/bilirubin and iron, important signaling and protective molecules against oxidative stress and inflammation.[2, 3]
CO gas administered at low doses (250 ppm) has been shown
to recapitulate many of the beneficial effects of HO-1 in
[a] Dr. J. L. Wilson, S. Fayad Kobeissi, Dr. B. Haas, Dr. R. Motterlini,+
Dr. R. Foresti+
Inserm, Unit 955, Equipe 3 and Facult de Mdicine
Universit Paris-Est Crteil, 8 Rue du General Sarrail
94000 Crteil (France)
E-mail: roberto.motterlini@inserm.fr
roberta.foresti@inserm.fr
[b] Dr. S. Oudir, Dr. A. Ollivier, Prof. T. Martens, Dr. M. Rivard
Institut de Chimie et des Matriaux Paris-Est
UMR 7182 CNRS-Universit Paris-Est Crteil
94320 Thiais (France)
[c] Dr. B. Michel
Department of Chemistry, University of California Berkeley
419 Latimer Hall, Berkeley CA 94720 (USA)
[d] Prof. J.-L. Dubois Rand
AP-HP, Hpital Henri Mondor-A. Chenevier, Service Hospitalier
8 Rue du General Sarrail, 94000 Crteil (France)
[+] These authors equally contributed to this work.
Supporting information for this article (including additional experimental
details) is available on the WWW under http://dx.doi.org/10.1002/
chem.201403901.
Chem. Eur. J. 2014, 20, 14698 14704

report the synthesis of hybrid molecules that simultaneously

activate Nrf2 and liberate CO. These hybrid compounds,
which we termed HYCOs, release CO to myoglobin and activate the CO-sensitive fluorescent probe COP-1, while also
potently inducing nuclear accumulation of Nrf2 and HO-1 expression and activity in different cell types. Thus, we provide
here the first example of a new class of pharmacologically
active molecules that target the HO-1 pathway by combining an Nrf2 activator coordinated to a CO-releasing group.

models of cardiovascular dysfunction[4] and inflammatory conditions such as sepsis and inflammatory bowel disease.[5, 6]
Because of the interest in the therapeutic potential of HO-1/
CO, our group developed compounds (CO-releasing molecules:
CO-RMs) that liberate controlled amounts of CO and exert
a wide array of pharmacological effects related to CO release.[712] With few notable exceptions,[13] the majority of CORMs described in the literature are metal carbonyls containing
either Ru, Fe, Mn, Co, and Mo.[8, 1417] Indeed, the fact that CO
preferentially binds to transition metals renders metal carbonyls ideal vehicles to transport and deliver CO to biological
systems.[18]
We have also independently worked on substances of plant
origin that increase HO-1 expression. Curcumin was the first
chemical found to possess this property[19] and our group was
the first to show that curcumin-mediated HO-1 induction requires activation of Nrf2.[2, 20] The list of Nrf2/HO-1 activators
has grown over time to include several hundred compounds,
with dimethyl fumarate being one of the most recently identified.[21] Dimethyl fumarate is a derivative of the citric acid cycle
metabolite fumarate, which affords cardio-protection through
Nrf2/HO-1[22] and recently emerged in a screening approach as
one of the strongest HO-1 inducers exhibiting a low toxicity
profile.[23] In addition, fumaric acid esters have been used successfully for the treatment of psoriasis and dimethyl fumarate
was recently approved as a drug to treat multiple sclerosis, an
autoimmune disease characterized by chronic inflammation.[24]
Because of their mechanism of action, Nrf2/HO-1 activators require time to mount the cellular stress response, resulting in
a delayed, albeit essential, beneficial effect. We reasoned that

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new molecules, possessing the ability to rapidly release CO
and simultaneously stimulate a longer-lasting Nrf2-dependent
protective proteome, could provide a synergistic effect compared to CO-RMs or Nrf2-activating agents alone (Scheme 1).

Scheme 2. Preparation of HYCO-1 and HYCO-2. a) Propargyl bromide, K2CO3,

acetone; reaction conducted at 40 8C; b) [Co2(CO)8], CHCl3, under argon; RT.

sensitive to CO that was recently developed as a new chemical

tool to detect CO in aqueous solutions and living cells.[29] Addition of 50 mm HYCO-1 or HYCO-2 to a solution of COP-1 elicited
a time-dependent increase in fluorescence, indicating the formation of a carbonylation product that was induced by CO liberated from the compounds (Figure 1 a). CO release by

Scheme 1. Schematic diagram representing the dual bioactive property of

HYCO-1 and its pharmacological action.

Therefore, we designed and synthesized new chemical entities composed of an Nrf2 activator bound to a CO-releasing
molecule, focusing our strategy on the promising results
obtained with dimethyl fumarate as a HO-1 inducer. Here, we
report on the chemistry and biological characterization of
HYCOs (from HYbrid CO-RMs) that act as dual activity
molecules by promoting Nrf2/HO-1 activation and liberating
CO.

Results and Discussion

A key feature of Nrf2 activators is the electrophilic nature of
the a,b-unsaturated carbonyl group (Michael acceptor functionality),[25] which was important to preserve in the synthesis
of the new hybrids. Concerning the CO-releasing moiety, we
chose alkyne-dicobalt hexacarbonyl complexes[26] as good candidates to be associated with the Michael acceptor because
they are readily synthesized, easy to handle and suitable for
biological applications.[27] This led to the synthesis of two propargyl esters of fumaric acid (compounds 1 and 2). The structure of these fumaric acid derivatives allowed the preparation
of two hybrids: HYCO-1, a non-symmetric compound carrying
one [Co2(CO)6] moiety; and HYCO-2, a symmetric molecule
bearing two bimetallic fragments.
We first prepared 1 by treating fumaric acid mono ethyl
ester with propargyl bromide under basic conditions in acetone, giving rise to the expected monopropargyl ester
(Scheme 2). The preparation of the corresponding hybrid commenced with complexation of 1 with dicobalt octacarbonyl in
CHCl3 under an inert atmosphere. The resulting HYCO-1 was
obtained after purification by flash chromatography on silica
gel (cyclohexane/ethyl acetate; 90:10 as eluent). The oily material was solidified in hexane at low temperature, giving HYCO1 as a red solid. Starting from the dipropargyl ester 2,[28] we
synthesized with a comparable procedure the symmetric
hybrid HYCO-2, obtained as a red powder after purification on
silica gel (cyclohexane/ethyl acetate; 95:5).
Having obtained the first HYCOs, we initially tested whether
they released CO by using COP-1, a selective fluorescent probe
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Figure 1. CO release from HYCOs detected using the COP-1 fluorescent

probe and myoglobin assays. a) Increase in fluorescence over time after addition of 50 mm HYCO-1 to 2 mm COP-1 in DPBS (lex = 475 nm, emission:
490630 nm). Spectra represent data collected at 0, 5, 15, 30, 45, and 60 min
after addition of HYCO-1; b) Formation of Mb-CO following addition of
20 mm HYCO-1 to 100 mm deoxymyoglobin in PBS. The increase in absorbance at 540 and 576 nm is indicative of Mb-CO formation over time; c) Rates
of CO release by HYCOs assessed by the myoglobin assay; d) Rates of CO release by HYCOs in comparison with CORM-A1 assessed with COP-1. Inactive
CORM-A1 (iCORM-A1), which was depleted of CO, is shown as a negative
control.[13]

HYCOs was confirmed using a myoglobin assay, which measures spectrophotometrically the conversion of deoxymyoglobin to carbon-monoxy myoglobin (Mb-CO) over time[7, 30] (Figure 1 b). When 20 mm of the compounds were added to
100 mm deoxymyoglobin in phosphate buffered saline (PBS),
CO release by HYCO-1 was faster compared to HYCO-2. In fact,
after 60 min incubation HYCO-1 and HYCO-2 liberated 26.0
and 12.9 mm CO, respectively (Figure 1 c).
However, Mb-CO formation promoted by HYCO-1 reached
a plateau after 120 min, whereas Mb-CO levels continuously increased over time with HYCO-2 (Figure S1, see the Supporting
Information). Thus, after 450 min, HYCO-2 liberated approxi-

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mately double the amount of CO compared with HYCO-1, consistent with the presence of two [Co2(CO)6] groups. It is interesting to note that with COP-1 the rate of CO release by
HYCO-1 is similar to the rate obtained with CORM-A1,[13]
a well-established boranocarbonate that spontaneously liberates CO under physiological conditions with a half-life of approximately 21 min (Figure 1 d).
Cyclic voltammetry was used to assess the stability of HYCO1 and HYCO-2 and further compare their ability to release CO.
HYCO-1 was shown to exhibit an irreversible oxidation peak at
1.05 V (vs. SCE) attributed to the oxidation of cobalt(0) (Figure 2 a and 2 b) and involving two electrons (Figure S2, the
Supporting Information). This result confirmed the highly irreversible oxidation of alkyne hexacarbonyl dicobalt complexes
previously described.[31] By increasing the number of scans, the
appearance of a second irreversible peak at 1.35 V (vs. SCE)
was observed and attributed to the oxidation of CO released
from HYCOs to form CO2 in the presence of water, in agreement with the well-documented process of CO adsorption on
a platinum surface[32, 33] (Figure 2 c2 e).
Interestingly, the intensity as well as the potential of the
peak attributed to cobalt remained constant over the number
of scans. This clearly confirms: 1) The stability of HYCO-1 in so-

lution; 2) That the release of CO, characterized by its oxidation

peak, occurs consecutively to the oxidation of cobalt(0), and
3) That the release of CO occurs rapidly under oxidative conditions. HYCO-2 exhibited a similar behavior with an oxidation
peak at 1.05 V (vs. SCE) involving four electrons due to the oxidation of cobalt(0) (Figure 2 b and the Supporting Information,
Figure S2). The similar potential required for the oxidation of
cobalt in the two HYCOs confirms the absence of electronic
communication between the two [Co2(CO)6] moieties of HYCO2.[34] The single four-electron oxidation detected with HYCO-2
indicates simultaneous oxidation of the two non-conjugated
[Co2(CO)6]. Again, a second peak attributed to the oxidation of
CO was observed with HYCO-2 at 1.35 V (vs. SCE) (Figure 2 c
and 2 e). The peak intensities revealed that HYCO-2 liberates
approximately double the amount of CO compared with
HYCO-1, in a ratio of 1.8 to 1 (Figure S3, the Supporting Information). Thus, cyclic voltammetry validates both the expected
differences between HYCO-1 and HYCO-2 based on the
number of [Co2(CO)6] moieties, as well as the results of the myoglobin assay.
The second property envisioned for HYCOs was the ability
to activate the Nrf2/HO-1 axis. By using Western blot analysis,
we showed that exposure of BV2 microglia cells to HYCO1 (20 mm) for 2 h strongly promoted the nuclear accumulation of Nrf2 (Figure 3 a). After 6 h of treatment, HYCO-1 induced a concentration-dependent
increase in HO-1 protein expression and heme oxygenase activity (Figure 3 b and 3 c). To investigate
whether the compounds hydrolyze in the culture
medium prior to entering the cell, we also tested the
effect of the hydrolysis product of HYCO-1, fumaric
acid monoethyl ester. We found that no change in
heme oxygenase activity was observed over a 6 h
period (Figure S4 a, the Supporting Information), confirming previous data in other cell types[35] and suggesting that HYCO-1 and HYCO-2 are taken up by
cells as intact molecules.
The effects of HYCO-1 on Nrf2 expression and HO1 activity were observed also in H9C2 rat cardiomyocytes (Figure S5 a5 c, the Supporting Information)
and murine RAW 264.7 macrophages (Figure S6 a, the
Supporting Information), indicating the reproducibility of this response in different cell types. Compound
1 was less potent than HYCO-1 (Figure 2 a) and the
cobalt carbonyl alone ([Co2(CO)8]) could not be
tested due to its instability in DMSO. In contrast,
HYCO-2 was less effective, both as an Nrf2 activator
and as a HO-1 inducing agent (Figures S7 a, S7 b, S8 a,
and S8 b, the Supporting Information), suggesting
that the coordination of the second [Co2(CO)6] to the
scaffold of compound 1 affects its capability to activate the Nrf2/HO-1 axis. We would not expect the
electrophilic tone of HYCO-2, which is required to acFigure 2. CO release from HYCOs assessed by cyclic voltammetry. Cyclic voltammetry
(200 mV s 1) of a) HYCO-1 (1 mm), b) HYCO-2 (1 mm), c) CO alone, d) HYCO-1 alone (scan
tivate Nrf2, to be reduced by the second [Co2(CO)6].
6) and in the presence of CO (scan 7 and 8), and e) HYCO-2 alone (scan 6) and in the
However, the steric hindrance of HYCO-2 due to the
presence of CO (scan 7 and 8). Conditions : Pt electrode, 0 8C, dimethylformamide/water
second [Co2(CO)6] may prevent either its entrance
(20/0.05) as solvent, NEt4BF4 as supporting electrolyte (50 mm).

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Conclusion

Figure 3. Nrf2 accumulation, HO-1 expression and heme oxygenase activity in BV2 cells
treated with HYCO-1. a) Nuclear Nrf2 2 h after incubation of cells with 20 mm HYCO-1; b)
and c) HO-1 protein expression and heme oxygenase activity at 6 h after exposure to
HYCO-1. [*] = p < 0.05 versus 0 mm HYCO-1; [] = p < 0.05 versus 20 mm compound 1.

into cells or the chemical reaction that stimulates the Nrf2

pathway.
We then evaluated the cytotoxicity by assessing lactate dehydrogenase activity levels 24 h after exposure of BV2 cells to
increasing concentrations of HYCO-1, compound 1, HYCO-2,
and compound 2. We found that HYCO-1 significantly increased cell damage at 50 and 100 mm and compound 1 at
100 mm (Figure 4 a). HYCO-2 exhibited less toxicity and only at
100 mm, whereas compound 2 was significantly toxic at 50 and
100 mm (Figure S7 c, the Supporting Information). Similar results were observed for HYCO-1 and compound 1 in H9C2 cardiomyocytes, whereas RAW macrophages were more resistant
(Table S1, the Supporting Information). Overall, prolonged exposure to HYCOs was well tolerated by different cell types at
low micromolar concentrations (< 50 mm). This is in line with
previous findings obtained with other electrophilic compounds, including curcumin, sulforaphane, and isothiocyanate
cysteine conjugates.[19, 23, 36] Importantly, HYCOs are effective on
the Nrf2/HO-1 axis in the non-toxic concentration range.
Finally, we examined the anti-inflammatory activity of nontoxic concentrations of HYCOs (520 mm). We found that in
BV2 cells challenged with lipopolysaccharide (LPS) for 24 h to
stimulate an inflammatory response[23] , HYCO-1 markedly decreased LPS-mediated nitrite accumulation, an index of nitric
oxide production and inflammation. This effect was more
potent than that elicited by compound 1, the fumarate derivative, at the same concentrations (Figure 4 b), suggesting that
the [Co2(CO)6] group contributes to the reduction in nitrite
levels. Importantly, compound 1 decreased nitrite production
more effectively than dimethyl fumarate, a known Nrf2 activator and an approved drug[24, 37] (Table 1). Furthermore, in support of the hypothesis that HYCOs are entering the cells intact,
we found that the hydrolysis product of HYCO-1, fumaric acid
monoethyl ester, does not significantly alter nitrite levels
(Figure S4 b, the Supporting Information).
HYCO-1 and compound 1 also reduced nitrite production in
RAW macrophages (Figure S6 b, the Supporting Information),
although these results are confounded by an underlying cytotoxicity at higher concentrations of the compounds. HYCO-2
and compound 2 also decreased nitrite production in BV2 microglia in a concentration-dependent manner; however, in this
case compound 2 was more effective than the hybrid molecule
(Figure S7 d, the Supporting Information).
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We report herein a new class of hybrid molecules,

consisting of an electrophilic fumarate ligand coordinated to a CO-releasing molecule, which are able to
activate the Nrf2/HO-1 axis in cells as well as liberate
CO. Coordination of two [Co2(CO)6] groups appears
to restrict, rather than enhance, the pharmacological
action of these new hybrids, indicating the importance of taking steric hindrance into consideration
when designing future molecules. It is interesting to
note that different laboratories have recently synthe-

Figure 4. Cytotoxicity and nitrite assays in BV2 cells treated with HYCO1 and compound 1. a) Cells were exposed to increasing concentrations of
HYCO-1 or compound 1 for 24 h and lactate dehydrogenase activity was
measured in the supernatant as an index of cytotoxicity; b) Nitrite production in BV2 cells challenged for 24 h with 0.5 mg mL 1 LPS in the presence or
absence of 5, 10, 20 mm HYCO-1 or compound 1. [*] = p < 0.05 versus 0 mm
HYCO-1 or compound 1; [] = p < 0.05 versus LPS alone; [] = p < 0.05 versus
compound 1.

sized compounds by coordinating a CO-RM moiety to ligands

with the specific purpose of rendering them more biologically
relevant and to modulate the release of CO through diverse
mechanisms.[3840] The synthesis of HYCOs, however, is not an
attempt to produce a more biologically compatible CO-RM,
but is instead based on a completely different perspective: To

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(15 mL). The suspension was stirred at 40 8C for 17 h, then cooled
at room temperature and filtered. The filtrate was concentrated in
vacuo and the resulting crude product was purified by silica gel
column chromatography eluting with cyclohexane/EtOAc (90:10)
to obtain compound 1 (217 mg, 1.18 mmol) as a colorless liquid in
75 % yield. 1H NMR (400 MHz, C6D6): d = 6.81 (d, J = 15.8 Hz, 1H),
6.76 (d, J = 15.8 Hz, 1H), 4.31 (d, J = 2.5 Hz, 2H), 3.85 (q, J = 7.1 Hz,
2H), 1.98 (t, J = 2.5 Hz, 1H), 0.85 ppm (t, J = 7.1 Hz, 3H); 13C NMR
(100 MHz, C6D6): d = 164.31, 163.81, 134.60, 132.35, 77.51, 75.41,
61.07, 52.32, 13.93 ppm; (see the Supporting Information, Figure S9
for NMR spectra); IR (neat): n = 3282 (m), 2985 (m), 2131 (w) 1718
(s), 1646 (w), 1446 (w), 1369 (m), 1292 (s), 1256 (s), 1223 (m), 1140
(s), 1096 (m), 1030 (m), 977 (m), 945 cm 1 (w); elemental analysis
calcd (%) for C9H10O4 (182.17): C 59.34; H 5.53; found: C 58.90; H
5.59.

Table 1. Comparison of the anti-inflammatory potency of dimethyl fumarate, compound 1 and HYCO-1.[a]
Compound (5 mm)

Dimethyl fumarate
Compound 1
HYCO-1

Nitrile levels [mm]

LPS

LPS + compound

86.3  2.9
86.7  1.6
78.0  1.3

69.2  1.0[b]
43.2  1.0[b, c]
28.6  1.3[bd]

% decrease
versus LPS
19.8
50.1
63.3

[a] Nitrite levels were measured in cells following 24 h incubation with

0.5 mg mL 1 lipopolysaccharide (LPS) alone or in the presence of 5 mm dimethyl fumarate, compound 1 or HYCO-1. All values (mean  SEM) are
shown as percentage of LPS alone. [b] p < 0.05 versus LPS alone; [c] p <
0.05 versus dimethyl fumarate; [d] p < 0.05 versus compound 1.

obtain a hybrid molecule that targets and maximizes the activity of the HO-1 pathway. The results presented here on HYCO1 and HYCO-2 are guiding our current efforts on the synthesis
of additional HYCOs for which other electrophilic ligands, and
transition-metal carbonyls with diverse CO-releasing properties,
are being considered. HYCOs represent a promising and
unique tool for the development of therapeutic molecules that
exploit, in a comprehensive fashion, the Nrf2/HO-1/CO protective cellular machinery. Since dimethyl fumarate was recently
approved as a drug to treat multiple sclerosis[24] , our data suggest that the coordination of fumaric ester derivatives to
a metal carbonyl can be used as a scaffold for the synthesis
and characterization of more potent anti-inflammatory drugs.

Experimental Section
Synthesis and characterization of new compounds
All reactions were performed under an atmosphere of argon. Solvents and reagents were used without further purification. Silica
gel 60A (SDS) was used for column chromatography and E. Merk
silica gel plates 60 F254 (0.25 mm thick) were used for analytical
thin layer chromatography. TLC were observed under UV at
254 nm and revealed using KMnO4 reagent with heating. Dicobalt
octacarbonyl was purchased from Strem. Fumaric acid monoethyl
ester and acetone were purchased from Acros Organic. Potassium
carbonate and ethyl acetate (EtOAc) were purchased from VWR
Chemicals. CHCl3 and cyclohexane were purchased from Carlo
Erba. Propargyl bromide was purchased from Alfa Aesar. Dipropargyl fumarate, compound 2, was prepared according to the literature procedure and spectral properties were in accordance with
those reported.[28] 1H and 13C spectra for characterization of new
compounds were collected in C6D6 (Euriso-top) at 20 8C and were
recorded on a Bruker Avance-II 400 MHz spectrometer. All chemical
shifts are reported in parts per million and the residual solvent
peak from C6D6 at d = 7.16 ppm was used as a reference. Splitting
patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet. IR spectra were recorded at 20 8C on
a Bruker Tensor 27 FTIR spectrometer. Elemental analyses were performed using a Perkin-Elmer 2400 apparatus at the Institut de
Chimie des Substances Naturelles (Gif sur Yvette, France).
Synthesis of compound 1: Propargyl bromide solution 80 wt % in
toluene (529 mL, 4.75 mmol, 3.0 equiv) and potassium carbonate
(262 mg, 1.90 mmol, 1.2 equiv) were added to a solution of fumaric
acid monoethyl ester (228 mg, 1.58 mmol, 1.0 equiv) in acetone
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Synthesis of HYCO-1: Dicobalt octacarbonyl (218 mg, 0.64 mmol,

1.0 equiv) was added to a degassed solution of compound
1 (116 mg, 0.64 mmol, 1.0 equiv) in CHCl3 (5 mL). The reaction mixture was stirred at room temperature for 17 h and then concentrated in vacuo. The resulting crude product was purified by silica gel
column chromatography eluting with cyclohexane/EtOAc (90:10)
to obtain HYCO-1 (229 mg, 0.49 mmol) as a dark-red gummy solid
in 76 % yield. The oily material was solidified in hexane at low temperature ( 18 8C), giving HYCO-1 as a dark-red solid. 1H NMR
(400 MHz, C6D6): d = 7.05 (s, 2H), 5.31 (s, 1H), 4.95 (s, 2H), 3.85 (q,
J = 6.9 Hz, 1H), 0.84 ppm (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
C6D6): d = 199.35, 164.41, 134.93, 132.71, 88.80, 71.95, 65.26, 61.21,
13.89 ppm; (see the Supporting Information, Figure S10 for the
NMR spectra); IR (neat): n = 3090 (m), 2987 (m), 2940 (m), 2098 (w),
2053 (m), 1999 (s), 1721 (m), 1646 (w), 1542 (w), 1467 (w), 1445
(w), 1394 (w), 1368 (m), 1351 (m), 1288 (m), 1254 (m), 1219 (m),
1147 (m), 1097 (w), 1032 (m), 978 cm 1 (m); elemental analysis
calcd (%) for C15H10Co2O10 (468.10): C 38.49; H 2.15; found: C 38.48;
H 2.24.
Synthesis of HYCO-2: Compound 2 (50 mg, 0.26 mmol, 1.0 equiv)
in CHCl3 (5 mL) and dicobalt octacarbonyl (300 mg, 0.88 mmol,
3.4 equiv) were added to a degassed solution of dipropargyl fumarate. The reaction mixture was stirred at room temperature for
18 h and then concentrated in vacuo. The resulting crude product
was purified by silica gel column chromatography eluting with cyclohexane/EtOAc (95:5) to obtain HYCO-2 (150 mg, 0.20 mmol) as
a dark-red powder in 76 % yield. 1H NMR (400 MHz, C6D6): d = 7.15
(s, 2H), 5.30 (s, 2H), 4.93 ppm (s, 4H); 13C NMR (100 MHz, C6D6): d =
199.34, 164.20, 133.82, 88.67, 71.93, 65.47 ppm; (see the Supporting Information, Figure S11 for NMR spectra); IR (neat): n = 3086 (s),
2916 (m), 2851 (m), 2098 (w), 2017 (s), 1714 (m), 1582 (w), 1537
(w), 1437 (w), 1353 (m), 1288 (m), 1153 (m), 1054 (w), 982 (m),
942 cm 1 (m); elemental analysis calcd (%) for C22H8Co4O16 (764.02):
C 34.58; H 1.06; found: C 34.61; H 1.01.

Cell culture
Cells were grown in an atmosphere of 5 % CO2 at 37 8C in either
75 cm2 flasks, 100 mm diameter Petri dishes, 6- or 24-well plates
containing medium supplemented with 10 % fetal bovine serum
(Lonza) and penicillin (100 U mL 1)/streptomycin (100 mg mL 1; Life
Technologies). BV2 mouse microglial cells were kindly donated by
Professor Rosario Donato (University of Perugia) and Dr. Adjanie
Patabendige (Institute of Infection & Global Health, Liverpool), and
grown in RPMI-1640 containing 2 g L 1 glucose and supplemented
with 0.3 g L 1 l-glutamine, as previously described.[41] RAW 264.7
macrophages (ATCC, Teddington, Middlesex, UK) and H9C2 cardiomyocytes (ATCC, Teddington, Middlesex, UK) were grown in Dul-

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beccos modified Eagles medium (DMEM) containing glucose
(4.5 g L 1) and either Glutamax (Life technologies) or l-glutamine
(0.6 g L 1), respectively, and were cultured as previously described
by our group.[25, 42]

Detection of CO release using the COP-1 fluorescent probe

To assay the CO release from compounds, a 2 mm solution of COP1 was prepared in DPBS ( Ca, Mg; Gibco Cell Culture, Life Technologies) in a cuvette. The solution was maintained at 37 8C in
a spectrofluorometer chamber throughout the assay using
a JASCO temperature controller (details of spectroscopy equipment can be found in the Supporting Information). The fluorescence intensity of COP-1 in buffer was measured to obtain a background fluorescence spectrum. Spectra were then recorded immediately after addition of 50 mm HYCO-1, HYCO-2, CORM-A1 or
iCORM-A1 and 15, 30, 45, and 60 min thereafter. A peak in the
spectra at 507 nm is indicative of the reaction between CO and the
probe. The mean fluorescence intensity (MFI) at each time-point
was calculated using the fluorescence intensity values recorded at
507 nm.

Heme oxygenase activity assay

Heme oxygenase activity was determined by a spectrophotometric
assay that measures the formation of bilirubin as a difference in
absorbance between 464 and 530 nm (extinction coefficient for bilirubin 40 m 1 cm 1).[43] BV2 microglia, RAW 264.7 macrophages,
and H9C2 cardiomyocytes were incubated with increasing concentrations of compounds for 6 h. Following incubation, cells were
harvested in ice cold PBS (pH 7.4) containing 2 mm MgCl2 and
stored at 80 8C. Cell samples were incubated in the dark at 37 8C
for 1 h in a reaction mix containing PBS/MgCl2, 20 mm hemin (Frontier Scientific, Newark, DE) as a heme oxygenase substrate, rat liver
cytosol as a source of biliverdin reductase, 2 mm glucose-6-phosphate, 0.5 U mL 1 glucose-6-phosphate dehydrogenase, and
0.8 mm NADPH. The reaction was terminated by addition of 1 mL
pure chloroform and a solution containing only bilirubin was extracted by vortex-centrifugation cycles. Absorbance values corresponding to the formation of bilirubin were obtained using an Ultraspec 500 pro visible spectrophotometer (GE Healthcare Life Sciences) and the data presented as p mol bilirubin mg 1 protein h 1.
Protein concentrations were measured using a Pierce BCA Protein
Assay kit (Thermo Scientific).

Determination of nitrite production

BV2 microglia and RAW 264.7 macrophages were treated with
0.5 mg mL 1 or 10 ng mL 1 lipopolysaccharide (LPS), respectively, in
the presence or absence of increasing concentrations of compounds. At the end of the incubation, nitrite levels were measured
using the Griess method.[23, 44] Briefly, cell plates were centrifuged at
10 000  g for 5 min and the cell-free supernatant was incubated
with an equal volume of Griess reagent (2 mm N-(1-naphtyl)ethylenediamide, 30 mm sulfanilamide, and 140 mm o-phosphoric acid)
for 10 min at room temperature. The absorbance was measured at
560 nm. Concentrations of nitrite (mm) were calculated from a standard curve of sodium nitrite.

Acknowledgements
This work was supported by a Blanc II International Grant from
the Agence Nationale de la Recherche (MITO-CO), the Fonds
Chem. Eur. J. 2014, 20, 14698 14704

www.chemeurj.org

National de la Recherche Luxembourg, a Multidisciplinary

Grant from UPEC, ClinServ International Lebanon and the AREMCAR Foundation and a Marie Curie Intra-European Fellowship
(to Dr. Jayne Louise Wilson). The authors thank Jonathan Ward
(University of York) for helping with the analysis of myoglobin
assay data. COP-1 was kindly provided by Prof. Christopher
Chang from the University of California, Berkeley.
Keywords: biological activity drug design enzymes
fluorescent probes heme proteins
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Received: June 10, 2014

Published online on September 15, 2014

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