Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme

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Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme Oxygenase-1 and Release Carbon Monoxide in vitro and in vivo Aniket Nikam, Anthony Ollivier, Michael Rivard, Jayne-Louise Wilson, Kevin Mebarki, Thierry Martens, Jean-Luc Dubois-Rande, Roberto Motterlini, and Roberta Foresti J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01509 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 6, 2016

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Journal of Medicinal Chemistry

Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce

Heme Oxygenase-1 and Release Carbon Monoxide in vitro and in vivo Aniket Nikam,†,‡ Anthony Ollivier,∞ Michael Rivard,∞ Jayne Louise Wilson,†,‡ Kevin Mebarki,∞ Thierry Martens,∞ Jean-Luc Dubois-Randé,§ Roberto Motterlini,†,‡,* and Roberta Foresti†,‡,* †

Inserm, U955, Equipe 12, Créteil, 94000, France

‡

University Paris Est Créteil, Faculty of Medicine, Créteil, 94000, France

∞

University Paris Est, ICMPE (UMR 7182), CNRS, UPEC, F-94320 Thiais, France AP-HP, Henri Mondor Hospital, Service Hospitalier, Créteil, 94000, France

§

ABSTRACT: The Nrf2/heme oxygenase-1 (HO-1) axis affords significant protection against oxidative stress and cellular damage. We synthesized a series of cobalt-based hybrid molecules (HYCOs) that combine an Nrf2 inducer with a releaser of carbon monoxide (CO), an anti-inflammatory product of HO-1. Two HYCOs markedly increased Nrf2/HO-1 expression, liberated CO and exerted anti-inflammatory activity in vitro. HYCOs also up-regulated tissue HO-1 and delivered CO in blood after administration in vivo, supporting their potential use against inflammatory conditions.

terized by chronic inflammation and oxidative stress.15-18 In addition, new approaches to activate Nrf2 are being investigated by focusing on molecules that disrupt Keap1Nrf2 binding via non-covalent mechanisms19 or that enhance Nrf2 expression via epigenetic modifications.20;21 Our group is exploring an alternative strategy that aims to harness in one prototypical molecule the beneficial properties of Nrf2 activators with those of HO-1. For this endeavor we have taken advantage of CO-releasing molecules (CO-RMs), which we identified as pharmacological compounds that deliver CO to biological tissues and mimic in many respects the salutary effects of HO-1.4;7;22 The premise is that a new molecule, containing an Nrf2 inducer bound to a CO-RM, will provide greater tissue protection by first limiting damage through CO delivery and subsequently promoting the endogenous upregulation of Nrf2-dependent defensive genes and proteins, a process that takes several hours due to transcription and translation processes. With this concept in mind, we recently reported the synthesis and preliminary biological characterization of two hybrid molecules exhibiting the dual ability to activate the Nrf2/HO-1 cytoprotective pathway and to release controlled amounts of CO.23 The design of these molecules, termed “HYCOs”, was based on the known ability of fumaric esters, such as DMF, to induce HO-1 via activation of the transcription factor Nrf2.12;24 Our efforts led to the preparation of 1 (HYCO-1) and 2 (HYCO-2), two (alkyne)dicobalt hexacarbonyl complexes bearing a propargyl ester of fumaric acid

INTRODUCTION Cells employ several inducible protective pathways in order to combat oxidative stress, inflammation and other harmful conditions. The nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream genes are major players in this protective response1;2 and heme oxygenase-1 (HO-1), an inducible enzyme that converts the substrate heme to the biologically active molecules biliverdin, iron and carbon monoxide (CO), significantly contributes to this effect owing to its antioxidant and anti-inflammatory actions.3;4 In particular, CO has been studied for its antiapoptotic and signaling roles as well as for preventing cardiovascular and inflammatory damage.5-7 Biliverdin and its reduced form bilirubin are also renowned for their antioxidant and immunomodulatory activities.8 A variety of organic scaffolds of natural origin have been investigated for their property to activate Nrf2. Typical examples include sulforaphane, curcumin or carnosol,9-12 which stimulate Nrf2 activation by preventing the interaction of Nrf2 and binding covalently to Kelch-like ECHassociated protein 1 (Keap 1), a cytosolic Nrf2 repressor that maintains low levels of the transcription factor in unstressed conditions.13 In view of the importance of Nrf2, efforts are under way to discover pharmacological agents that target this system.4;14 Indeed, interesting Nrf2 inducers such as dimethylfumarate (DMF) and bardoxolone methyl as well as sulforaphane rich-broccoli extracts are finding their application in the clinic in diseases charac-

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as an alkyne ligand (Scheme 1).23 Notably, we found that 1 is more potent in decreasing inflammatory markers in cells in vitro compared to the parent Nrf2 activator. The fact that the Nrf2/HO-1 inducing ability is not limited to fumaric esters prompted us to prepare a series of new Cobased carbonyl hybrids, bearing as alkyne ligands various Nrf2/HO-1 inducers.

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assay showed over the course of 120 min that 3 and 5 release the highest amount of CO, 6 exhibits intermediate release while 4 and 7 liberate the smallest amount of CO (Figure 2A and Figure S1 in Supporting Information). Scheme 1. Synthesis of HYCOs by coordinating cobalt a carbonyls to diverse Nrf2 activators

Here we report the synthesis and in vitro characterization of diverse Co-based hybrids and further examine the capacity of selected compounds to up-regulate tissue HO-1 and deliver CO in vivo.

RESULTS AND DISCUSSION Synthesis of compounds and design rationale. 3 (HYCO-4), 4 (HYCO-5), 5 (HYCO-8), 6 (HYCO-9) and 7 (HYCO-10) were prepared from the corresponding free alkynes 8, 9, 10, 11 and 12, according to the standard procedure (Scheme 1)25. The choice of structure for the free alkynes 8-12 was suggested by previous work describing (pro)electrophiles such as Michael acceptors or phenol derivatives as Nrf2/HO-1 inducers. The structure of free alkyne 8, precursor of 3 and deriving from chromone, was chosen due to the presence of Michael acceptor functionality and inspired by flavones, natural compounds bearing a chromone nucleus and known as Nrf2/HO-1 activators.14;26;27 Free alkyne 9, precursor of 4, derives from 3,3’diindolylmethane and, while not displaying Michael acceptor functionality in its structure, it stimulates Nrf2 via epigenetic modification of the Nrf2 promoter.20;21 Free alkynes 10 and 11, precursors of 5 and 6, respectively, were inspired by the antioxidant t-butylhydroquinone, which can be oxidized into quinone, thus acquiring the electrophilic character needed for activation of Nrf2.28 Free alkyne 12, precursor of 7 and dimethylated derivative of caffeic acid, is a Michael acceptor and is also susceptible to oxidation into quinone.29 In addition to their structural analogy with authentic Nrf2/HO-1 activators, alkynes 8-12 are readily obtained after propargylation of available compounds and consequently useful for the preparation of (alkyne)dicobalt hexacarbonyl complexes. All these elements provided the rationale for choosing alkynes 8-12 for the synthesis of novel Co-based HYCOs. As evident in Scheme 1, one [Co2(CO)6] moiety was coordinated to each Nrf2 activator with the exception of 4, a symmetric molecule bearing two bimetallic fragments.

a

b

Reagents and conditions: (i) Co2(CO)8, RT, CHCl3. 1 and 2 23 were synthesized as previously described.

A similar trend was confirmed with the COP-1 probe (Figure 2B and Figure S2 in Supporting Information). Therefore, 3, 5, 6 and 7 liberated varying levels of CO despite the presence of one [Co2(CO)6] in all the compounds. Although these differences are not easily explained, we suggest that the amount of CO released may be dependent on the stability of the compounds, at least in a cuvette in vitro. Moreover, the small quantity of CO liberated from 4 is counterintuitive as the compound contains two [Co2(CO)6].

A

Release of CO from HYCOs in vitro and in cells. We first used a hemoglobin assay, where changes in the spectra of deoxyhemoglobin to carboxyhemoglobin (HbCO) in the presence of HYCOs are indicative of CO release from the compounds.30 CO detection was also performed with the fluorescent probe COP-1, which has been recently developed to measure CO in solutions and in cells.23;31 Both methods were performed in a cuvette where HYCOs were incubated directly with either deoxyhemoglobin or COP-1. From these measurements we determined that all compounds liberated CO, albeit in different amounts and with different kinetics. In particular, the hemoglobin

B

Figure 2. Detection of CO release from compounds 3-7 over 120 min using a hemoglobin-based assay (A) or the COsensitive fluorescent probe COP-1 (B).

We reported in a recent article that 2, another symmetric molecule bearing two [Co2(CO)6] (Scheme 1), releases CO with a slower kinetic compared to 1 (Scheme 1), a similar compound coordinated to a single [Co2(CO)6].23 Consider-

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ing the behavior of 2 and 4, both being a dual [Co2(CO)6] hybrid molecule, we can infer that the presence of two [Co2(CO)6] significantly slows down the liberation of CO.

3 or 6 h but significantly raised its levels 24 h after treatment (Figure 4). This effect was compared to that of DMF, which has been already shown to induce GSH synthesis.32

We then assessed with COP-1 whether HYCOs delivered CO to cells and observed no increase in intracellular CO when BV2 microglia were treated with 10 µM HYCOs (Figure S3, Supporting Information). We have evidence, however, that CO accumulates in different cell types, including BV2, when CO-RMs and HYCOs containing other metals are used (unpublished observations). In the case of CORM-3, the intracellular delivery of CO using COP-1 has also been documented in human kidney cells.31 Thus, these results suggest that either HYCOs release insufficient amounts of CO to enable the detection of fluorescence in cells by COP-1 or the up-take of these compounds into cells is restricted. The former hypothesis is the most likely, as illustrated below by the results showing the effect of HYCOs on Nrf2/HO-1 expression and blood HbCO in vivo.

Figure 4. Cellular reduced glutathione measured after exposure of BV2 cells to 10 µM compounds 3-7 and DMF (n=3).

These data are very interesting when viewed from a recent perspective proposed by Satoh and Lipton,28 who suggested that drugs developed as Nrf2 activators should be evaluated for their two opposing actions: cytoprotective versus cytotoxic effects. The cytoprotective effects are due to the extent of activation of Nrf2-dependent genes while cytotoxicity involves GSH depletion. Thus, according to these authors, small activators that cause a strong Nrf2-dependent response without markedly depleting GSH content are promising compounds in terms of tolerability. As a consequence of this reasoning, the fact that the HYCOs studied herein induce the antioxidant and protective protein HO-1 and do not cause a decrease in GSH but actually increase it over time are desirable features. Nevertheless, HYCOs may still exert cytotoxic effects independent of GSH depletion that need to be ascertained. It is known that reactive oxygen species (ROS) triggered by electrophiles and CO are important signaling mediators for cellular adaptation to stress.4 Thus, we assessed whether HYCOs influence ROS production and investigated whether this mechanism contributes to Nrf2/HO-1 induction. 3 and 7, the most potent inducers of Nrf2/HO-1, increased ROS levels and pre-incubation of cells with the antioxidant N-acetylcysteine (NAC) abolished this effect (Figure 5A).

Nrf2, HO-1 expression and glutathione levels following treatment with HYCOs. As shown in Figure 3A, 3 and 7 were the most potent Nrf2 activators after treatment of cells with the compounds for 2 h.

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Figure 3. (A) Nuclear expression of Nrf2 2 h after exposure of BV2 cells to 10 µM compounds 3-7; (B) HO-1 protein expression measured in BV2 cells 6 h after treatment with 10 µM compounds 3-7. The lower panels in (A) and (B) represent the densitometric analysis of 3 independent Western blots.

All HYCOs tested, with the exception of 4, also significantly up-regulated HO-1 protein expression after 6 h incubation (Figure 3B). Apart from inherent differences in their potency to induce the Nrf2/HO-1 axis, which may be again related to stability of the compounds and their ability to disrupt the Keap1-Nrf2 protein interaction, these data clearly show that most of the HYCOs studied here enter the cell, even though we cannot detect an accumulation of intracellular CO using the COP-1 probe. Activation of Nrf2 leads to transcription of many antioxidant and detoxification genes, including those involved in the synthesis of glutathione, the most important and abundant cellular thiol protecting against oxidative stress. Therefore, we also assessed the production of reduced glutathione (GSH) in cells incubated with HYCOs. We found that all HYCOs, excluding 4, did not change GSH at

A

B Nrf2 Lamin A/C HO-1 β-actin

Figure 5. ROS production (A) and Nrf2/HO-1 expression (B) in BV2 cells exposed to 10 µM compounds 3 and 7 in the presence or absence of NAC (n=3-5).

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injury despite 3 liberating significantly more CO than 7. Among the Co-based HYCOs synthesized, 4 turned out to be the most puzzling molecule as its predicted biological activities, i.e. CO liberation and Nrf2/HO-1/GSH modulating properties, were absent at the concentrations tested. This might explain why the compound did not exert antiinflammatory activity in BV2 cells challenged with lipopolysaccharide (Figure 6B). In contrast, all other HYCOs, and especially 3 and 7, markedly decreased the production of nitrite, a standard index of inflammation. In the case of 20 µM 5, we interpret the strong reduction in inflammation mostly due to its profound toxic effect. Taking into consideration the cytotoxic and biological profiles of the compounds tested, we selected 3 and 7 for preliminary evaluation of their in vivo pharmacological actions.

However, NAC did not reduce the activation of Nrf2/HO-1 mediated by 3 and 7 (Figures 5B and S4, Supporting Information), indicating that ROS are not implicated in the induction mechanism and suggesting that the properties of the alkyne part of the HYCOs are mostly responsible for this effect. Cellular toxicity and anti-inflammatory action of HYCOs. Increasing concentrations of HYCOs from 1 to 10 µM caused little release of lactate dehydrogenase (LDH), an index of cell injury, from BV2 cells exposed to the compounds for 24 h (Figure 6A). Twenty µM 3, 6 and 7 increased cell toxicity to 20, 47 and 33%, respectively. At 20 µM 4 was virtually without effect while 5 elicited 100% toxicity.

A 3

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Assessment of tissue HO-1 induction and blood HbCO levels in vivo after HYCOs administration. Compounds 3 and 7 increased HO-1 expression in mouse lung and liver 6 h after administration by oral gavage (Figures 7A, B and C).

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[µM]

Figure 6. (A) BV2 cell damage determined by lactate dehydrogenase (LDH) release after exposure to increasing concentrations (0, 1, 2.5, 5, 10 and 20 µM) of compounds 3-7. (B) Nitrite production following incubation of BV2 cells with lipopolysaccharide (LPS) in the presence of increasing concentrations of compounds 3-7. (n=3 independent experiments).

Figure 7. (A) Liver and lung HO-1 expression 6 h after oral gavage of mice with 3 and 7 (3 mg in sesame oil). Densitometric analysis of 3 independent Western blots from liver (B) or lung samples (C). (D) Carboxy hemoglobin (HbCO) after treatment of mice with 3 and 7 (n=3 animals).

These data highlight the following points: 1) HYCOs up to 10 µM are relatively safe for further biological characterization in this cell system; 2) the hydroquinone derivatives 5 and 6 appear to be the most cytotoxic, and the presence of the OH increases the toxic nature of the compound; 3) the similar toxicity of 3 and 7 at 20 µM may be due to the Michael acceptor functionality, which is a chemical feature common to both HYCOs; 4) the [Co2(CO)6] moiety does not appear to be the major cause of LDH release as all HYCOs contain at least one [Co2(CO)6] and 4, with two [Co2(CO)6], does not produce any toxicity; 5) the toxicity is not linked to the amount of CO released by the compounds since 3 and 5 release similar amounts of CO but 3 is much less toxic. Moreover, 3 and 7 cause similar cell

Compound 7 appeared more powerful than compound 3 even though they stimulated similar Nrf2 activation and HO-1 expression in BV2 cells. Although an exact correspondence between in vitro and in vivo data cannot be expected, this may also indicate different absorption patterns of 3 and 7 in vivo. HO-1 expression was not detected in the brain and only occasionally in the heart (data not shown). In parallel to this effect we measured a consistent elevation of HbCO in blood 6 h after treatment of animals with these compounds (Figure 7D). Higher levels of HbCO were achieved with 3 compared to 7, in accordance with the results obtained in vitro using the hemoglobin assay and the COP-1 probe. Because of this difference, and

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Journal of Medicinal Chemistry yield. 1H NMR (400 MHz, C6D6) δ 8.22 (d, J = 7.5 Hz, 1H), 7.19 (m, 1H), 7.13 (m, 1H), 6.96 (t, J = 7.2 Hz, 1H), 6.79 (t, J = 7.1 Hz, 1H), 5.33 (s, 1H), 4.98 (s, 2H). 13C NMR (100 MHz, C6D6) δ 199.25, 177.22, 160.38, 156.12, 151.64, 134.31, 126.08, 125.79, 125.11, 118.51, 115.59, 87.65, 72.08, 66.86. Anal. Calcd for C19H8Co2O10 (514.13): C, 44.39; H, 1.57. Found: C, 44.49; H, 1.57.

the fact that 7 stimulated higher tissue HO-1 expression than 3, we presume that the rise in HbCO at 6 h is mostly due to CO released by the [Co2(CO)6] moiety of HYCOs and is not yet a reflection of CO derived from heme oxygenase activity.

CONCLUSION We have synthesized novel Co-based hybrids using different Nrf2 inducers and find that they release CO and activate the Nrf2/HO-1 axis with various potencies. The lack of biological activity of 4, similar to that observed previously for the symmetric compound 2,23 strengthens the idea that coordination of two [Co2(CO)6] to Nrf2 activators causes steric hindrance that severely limits the potential pharmacological properties of these molecules. 3 and 7, the HYCOs with the best combined profile from the in vitro characterization, are also shown to be active in vivo as they induce tissue HO-1 and increase HbCO when administered orally to mice. As diversification of the metal fragment is also of interest in the design of hybrids between Nrf2 activators and CO-releasers, future work will focus on the preparation of other metal carbonyl complexes based on our portfolio of CO-RMs. The comparison of the biological activities exerted by different classes of these hybrids will help to identify the most promising molecules to be used against inflammatory conditions.

(Hexacarbonyldicobalt)3,3'-methanediylbis[1-(prop2-yn-1-yl)-1H-indole] (4). To a degassed solution of 9 (166 mg, 0.51 mmol) in CHCl3 (0.1 M) was added dicobalt octacarbonyl (2.0 equiv). After 18 h of stirring at 20 °C, the reaction mixture was concentrated in vacuo. The resulting crude product was purified by flash silica gel column chromatography eluting with cyclohexane/EtOAc (98.5/2.5 → 95/5) to give 4 (210 mg, 0.23 mmol) as a dark red solid in 46% yield. 1H NMR (400 MHz, C6D6) δ 7.73 (d, J = 7.7 Hz, 2H), 7.30 (m, 2H), 7.23-7.16 (M, 4H), 6.66 (s, 2H), 5.35 (s, 2H), 4.68 (s, 4H), 4.28 (s, 2H). 13C NMR (100 MHz, C6D6) δ 199.39, 136.74, 129.12, 125.82, 122.25, 120.34, 119.80, 115.82, 109.68, 91.61, 73.24, 47.83, 21.66. Anal. Calcd for C35H18Co4N2O12 (894.26): C, 47.01; H, 2.03; N, 3.13. Found: C, 47.07; H, 1.92; N, 3.14. (Hexacarbonyldicobalt)-4-(prop-2-yn-1-yloxy)phenol (5). To a degassed solution of 4-propargyloxyphenol33 10 (64 mg, 0.43 mmol) in CHCl3 (0.1 M) was added dicobalt octacarbonyl (1.0 equiv). After 18 h of stirring at 20 °C, the reaction mixture was concentrated in vacuo. The resulting crude product was purified by flash silica gel column chromatography eluting with cyclohexane/EtOAc (17/3) to give 5 (107 mg, 0.25 mmol) as a dark red solid in 57% yield. 1H NMR (400 MHz, C6D6) δ 6.66 (d, J = 8.1 Hz, 2H), 6.45 (d, J = 8.2 Hz, 2H), 5.40 (s, 1H), 4.62 (s, 2H). 13C NMR (100 MHz, C6D6) δ 199.97, 152.48, 150.83, 116.28, 115.94, 90.55, 71.97, 68.59. Anal. Calcd for C15H8Co2O8 (434.09): C, 41.50; H, 1.86. Found: C, 41.77; H, 1.72.

EXPERIMENTAL SECTION Synthesis and characterization of HYCOs. Free alkynes 8-12 were prepared according to standard procedures. Preparation of compounds 8, 9 and 12 (precursors of 3, 4 and 7) are reported in the Supporting Information. Compounds 10 and 11 were prepared according to published reports33;34 and spectral properties were in accordance with those reported. 1H and 13C spectra for characterization of new compounds were collected in CDCl3 or C6D6 (Euriso-top) at 20 °C and were recorded on a Bruker Avance-II 400 MHz spectrometer. All chemical shifts are reported in parts per million. The residual solvent peak from CDCl3 at 7.26 ppm or from C6D6 at 7.16 ppm was used as a reference. Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Elemental analyses were performed using a Perkin-Elmer 2400 apparatus at the “Institut de Chimie des Substances Naturelles” (Gif sur Yvette, France). The purity of all compounds was assessed by elemental analysis; the maximum error obtained is less than 0.3% by element.

(Hexacarbonyldicobalt)-1-methoxy-4-(prop-2-yn-1yloxy)benzene (6). To a degassed solution of propargyl para-anisyl ether34 11 (288 mg, 1.78 mmol) in CHCl3 (0.1 M) was added dicobalt octacarbonyl (1.0 equiv). After 18 h of stirring at 20 °C, the reaction mixture was concentrated in vacuo. The resulting crude product was purified by flash silica gel column chromatography eluting with cyclohexane/EtOAc (90/10) to give 6 (705 mg, 1.57 mmol) as a dark red powder in 88% yield. 1H NMR (400 MHz, C6D6) δ 6.75 (s, 4H), 5.42 (s, 1H), 4.67 (s, 2H), 3.33 (s, 3H). 13C NMR (100 MHz, C6D6) δ 199.83, 154.92, 152.66, 115.92, 115.04, 90.55, 72.03, 68.59, 55.23. Anal. Calcd for C16H10Co2O8 (448.11): C, 42.88; H, 2.25. Found: C, 42.80; H, 2.16.

(Hexacarbonyldicobalt)prop-2-yn-1-yl 4-oxo-4Hchromene-2-carboxylate (3). To a degassed solution of 8 (157 mg, 0.69 mmol) in CHCl3 (0.1 M) was added dicobalt octacarbonyl (1.0 equiv). After 18 h of stirring at 20 °C, the reaction mixture was concentrated in vacuo. The resulting crude product was purified by flash silica gel column chromatography eluting with cyclohexane/EtOAc (90/10) to give 3 (252 mg, 0.49 mmol) as a dark red solid in 71%

(Hexacarbonyldicobalt)-prop-2-yn-1-yl (2E)-3-(3,4dimethoxyphenyl)prop-2-enoate (7). To a degassed solution of 12 (239 mg, 0.97 mmol) in CHCl3 (0.1 M) was added dicobalt octacarbonyl (1.0 equiv). After 18 h of stirring at 20 °C, the reaction mixture was concentrated in vacuo. The resulting crude product was purified by flash silica gel column chromatography eluting with cyclohex-

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ane/EtOAc (80/20) to give 7 (464 mg, 0.87 mmol) as a dark red powder in 90% yield. 1H NMR (400 MHz, C6D6) δ 8.03 (d, J = 15.4 Hz, 1H), 6.87 (d, J = 6.6 Hz, 1H), 6.79 (s, 1H), 6.59 (d, J = 15.7 Hz, 1H), 6.34 (d, J = 7.0 Hz, 1H), 5.41 (s, 1H), 5.20 (s, 2H), 3.26 (s, 6H). 13C NMR (100 MHz, C6D6) δ 199.63, 166.57, 152.37, 150.27, 146.36, 122.81, 115.07, 111.71, 110.64, 90.55, 71.98, 64.33, 55.33, 55.26. Anal. Calcd for C20H14Co2O10 (532.19): C, 45.14; H, 2.65. Found: C, 45.18; H, 2.66.

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and nitrite production, detection of CO release from the HYCOs using either COP-1 or a carbonmonoxy hemoglobin (HbCO) assay, detection of intracellular CO, ROS production, assessment of blood HbCO levels in vivo and statistical analysis. Synthesis of compounds 8, 9, 10 and NMR spectra of 8, 9, 10 and all HYCOs. Absorption spectra of HbCO in vitro. Fluorescent spectra of COP-1 following reaction with HYCOs. This material is available free of charge via the Internet at http://pubs.acs.org.

Materials for biological studies. All chemicals of analytical grade were purchased from Sigma-Aldrich, unless otherwise indicated. COP-1, synthesized as described previously,31 was prepared in DMSO as a stock solution (1 mM) and aliquots stored at -20 °C until use.

AUTHOR INFORMATION Corresponding Author *Phone, +33 (0) 149813637. E-mail: [email protected] or [email protected].

Author Contributions

Cell culture and experimental protocols. BV2 mouse microglial cells were grown in RPMI-1640 containing 2 g/L glucose and supplemented with 0.3 g/L L-glutamine, as previously described.23;35 To determine HO-1 protein expression, BV2 cells were seeded in 6-well-plates and incubated with 10 µM HYCOs for 6 h and cell lysate prepared as described23. To assess the nuclear translocation of Nrf2 in BV2 microglia, cells were seeded in Petri dishes and incubated with 10 µM HYCOs for 2 h. Isolation of nuclear fractions was performed using a Nuclear Extract Kit from Active Motif as per manufacturer’s instructions and stored at -80 °C. Nrf2 and HO-1 expression were also assessed in cells pre-treated with 1 mM NAC for 18 h prior to incubation with 3 and 7.

All authors have given approval to the final version of the manuscript. *These authors contributed equally to the work.

Funding Sources This work was supported by an International Grant from the Agence National de la Recherche (MITO-CO), a Multidisciplinary Grant from UPEC, a Marie Curie Intra-European Fellowship, SATT IdF Innov and the AREMCAR Foundation.

ACKNOWLEDGMENT The authors thank Dr Brian Michel and Prof. Christopher Chang from the University of California, Berkeley, for kindly providing COP-1. We are grateful for the technical assistance of personnel in the flow cytometry platform of Inserm U955.

Animal studies. Male C57 BL/6J mice were used for all experiments (Janvier Labs, France). Animals received at 8 weeks of age were placed on a standard diet and allowed to acclimatize for 2 weeks on a 12 hr light/dark cycle. All experiments were performed in compliance with INSERM guidelines for the use of animals and approved by the institutional review board at Paris-Est Créteil Val de Marne University (Project number: A94028245). Mice were administered, by oral gavage, with a single dose of the following: 1) 200 µl of sesame oil (vehicle, control group); 2) 3 (3 mg in sesame oil), and; 3) 7 (3 mg in sesame oil). Mice were sacrificed 6 hr after treatment by cervical dislocation. Livers, lungs, hearts and brains were excised and snap frozen for western blot analysis. Tissue samples were homogenized in lysis buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0). Lysates were centrifuged for 20 min at 15,000 g at 4 °C and supernatants were collected and stored at -80 °C for further analysis. Blood was also collected 6 h after treatment for the assessment of carbonmonoxy hemoglobin (HbCO) levels according to a method previously described (see Supporting Information).

ABBREVIATIONS CO-RMs, CO-releasing molecules; DMF, dimtheylfumarate; HO-1, heme oxygenase-1; HYCOs, hybrids between Nrf2 activators and cobalt-based CO-RMs, Nrf2, nuclear factor erythroid 2-related factor 2.

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ASSOCIATED CONTENT Supporting Information. Methods used for the biological characterization of HYCOs including: cytotoxicity assay, Western blot, determination of cellular glutathione

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Table of Contents artwork (TOC)

CO release

500

In vivo

60

400 40

Time (min)

300 30

*

520

540

560

580

Wavelength (nm)

HO-1 Nrf2 C HYCOs

5

*

0

5

*

4 3

*

2 1 0

H YC O -4 H YC O -1 0

500

10

H YC O -4 H YC O -1 0

0

0

Lung

10

HbCO (%)

15

HYCOs

20

100

C O N

200

HO-1

In vitro

CO releaser

C O N

Nrf2/HO-1 activator

HYCO-10

HYCO-4

HYCO

Fluorescence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 16

8 ACS Paragon Plus Environment

Page 9 of 16


Figure 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 16

Figure 2 A

B


Page 11 of 16

Figure 3

A

B

Nrf2

HO-1

Lamin A/C

β-actin

CON 3

4

5

6

7

CON 3

HO-1 exprression (fold incrrease)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47



4

5

6

7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 16

Figure 4


Page 13 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


A

B Nrf2 Lamin A/C HO-1 β-actin


Figure 5


Figure 6

A 3

LDH release (%)

120

4

5

6

7

*

100 80 60

* *

40

* *

20

0 1 2. 5 5 10 20

0 1 2. 5 5 10 20

0 1 2. 5 5 10 20

0 1 2. 5 5 10 20

0 1 2. 5 5 10 20

0

[µM]

B 3 60

4

*

*

5

6

*

* †

40

† †

† †

7

*

† †

†

†

† †

†

20 †

†

†

[µM]


1 2. 5 5 10 20

1 2. 5 5 10 20 CO LPN S

1 2. 5 5 10 20 CO LPN S

1 2. 5 5 10 20 CO LPN S

0 CO LPN S 1 2. 5 5 10 20 CO LPN S

Nitrite [M ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 14 of 16

Page 15 of 16

A

Figure 7

B 3

Liver

HO-1 β-actin

1

HO-1 β-actin CON CON 3

3

7

0

7

CON

D

(fold increase)

C

*

Liver

2

Lung

HO-1 expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47



3

7


In vivo

CO release

500 60

400 40

Time (min)

300 30

*

520

540

560

580

Wavelength (nm)

HO-1 Nrf2 C HYCOs

5

*

0

5

*

4 3

*

2 1 0


H YC O -4 H YC O -1 0

500

10

H YC O -4 H YC O -1 0

0

0

Lung

10

HbCO (%)

15

HYCOs

20

100

C O N

200

HO-1

In vitro

CO releaser

C O N

Nrf2/HO-1 activator

HYCO-10

HYCO-4

HYCO

Fluorescence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 16 of 16

Diverse Nrf2 Activators Coordinated to Cobalt Carbonyls Induce Heme

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