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  • Generation of Carbon Monoxide Releasing Molecules (CO-RMs) asDrug Candidates for the Treatment of Acute Liver Injury: Targetingof CO-RMs to the LiverAna R. Marques, Lukas Kromer, David J. Gallo, Nuno Penacho, Sandra S. Rodrigues,

    Joao D. Seixas,, Goncalo J. L. Bernardes,, Patrcia M. Reis,, Sherrie L. Otterbein, Rachel A. Ruggieri,

    Ana S. G. Goncalves, Ana M. L. Goncalves, Marta N. De Matos, Isabel Bento, Leo E. Otterbein,,

    Walter A. Blattler, and Carlos C. Romao*,,

    Alfama Inc., Taguspark, nucleo central 267, 2740-122 Porto Salvo, Portugal, and 100 Cummings Center, Beverly, Massachusetts01915, United StatesInstituto de Tecnologia Qumica e Biologica da Universidade Nova de Lisboa, Av. da Republica, EAN, 2780-157 Oeiras, PortugalHarvard Medical School, Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts02215, United States

    *S Supporting Information

    ABSTRACT: The discovery of the biological effects of carbonmonoxide (CO) in recent years strongly suggests that COcould find applications as a therapeutic agent. CO is a highlytoxic gas when used at industrial doses, due in part to itsbinding affinity to hemoglobin. Since hemoglobin binds COwith the highest affinity in vivo, it also constitutes a majorbarrier to the delivery of CO to tissues in need of therapy. Amethod of delivering CO that can bypass hemoglobin is theuse of pro-drugs or CO carriers, called CO-releasing molecules (CO-RMs) that become activated and release CO in tissues inneed of treatment. Organometallic carbonyl complexes are best suited to play the role of CO carriers, and indeed the natural COcarrier molecules hemoglobin and myoglobin belong to this class of chemical compounds. Here we describe the preparation ofnovel molybdenum CO-RMs of general formula Mo(CO)3(CNCRRCO2R)3 (R, R = H, Me, iPr, CH2Ph, CO2Li,CH2CH2, CH2(CH2)3CH2; R = H, Li), which present favorable druglike characteristics, have low toxicity, anddemonstrate specific CO delivery to the liver in the treatment of acetaminophen (APAP)-induced acute liver failure in mice.

    INTRODUCTIONThree of the most toxic gases, nitric oxide (NO), carbonmonoxide (CO), and hydrogen sulfide (H2S), are produced inanimals as biologically active factors. They act as cellularregulators of diverse physiological and pathological processes,such as vasodilation, inflammation, endothelial and lunginjuries, asthma, and others, as summarized in two recentreviews.1,2 Similar regulatory pathways have been a fertile fieldfor drug development, and the NO, CO, and H2S pathwaysshould be no exception. Indeed NO has been used to treatangina pectoris in the form of nitrate drugs for more than 100years and is still today one of the most commonly used drugs totreat this disease. Nitrates such as nitroglycerine aremetabolized by mitochondrial aldehyde dehydrogenases andrelease the active agent NO in this process. Thus, nitrates areNO-releasing molecules or NO-RMs,3 in analogy to the CO-releasing molecules or CO-RMs, which have been proposed asdrugs through the pioneering work of our collaborating groupsled by Roberto Motterlini and Brian Mann.4,5 CO-RMs arecarriers of CO or pro-drugs that upon transformation releaseCO as the active principle. The natural carrier of CO in animals

    is the heme of hemoglobin, which reversibly binds CO throughits iron center, thus forming an organometallic carbonylcompound named carboxyhemoglobin (COHb). COHb ridsthe body of the endogenously produced CO by tightly bindingit and carrying it to the lung, where it is exchanged for oxygen,thus preventing the accumulation of toxic levels of CO intissues.6 Endogenous CO is produced in the first enzymaticstep of the normal catabolism of heme. The enzyme hemeoxygenase (HO), which is present in a constitutive (HO-2) andan inducible (HO-1) isoform, opens the porphyrin ring byoxidatively releasing the -methine bridge as CO.7 Theinducible isoform HO-1 is expressed at sites of injury andrapidly destroys the otherwise toxic heme, which is released as aconsequence of cell death. The reaction produces Fe2+, CO,and biliverdin, which is then reduced to bilirubin. Researchperformed over the last two decades has clearly demonstratedthat CO is more than a mere degradation product; it serves

    Special Issue: Organometallics in Biology and Medicine

    Received: April 30, 2012Published: July 2, 2012

    Article

    pubs.acs.org/Organometallics

    2012 American Chemical Society 5810 dx.doi.org/10.1021/om300360c | Organometallics 2012, 31, 58105822

    pubs.acs.org/Organometallics

  • important biological functions under various physiological andpathological conditions (summarized in ref 1). Further, it hasbeen demonstrated in animal models that administration of COexogenously could replicate the salutary effects attributed toHO-1 induction, and this has led to the evolution of CO as apotential therapeutic.8 Inhaled CO gas results in global vasculardistribution of CO and presents a formidable barrier to tissuedistribution and penetration due to the hemoglobin of redblood cells. Therefore, novel ways for delivering CO locally tothe sites of injury needed to be developed.9 In analogy withnature, where CO is produced locally at the sites of injurythrough the induction of HO-1, we sought to treat liver injurywith CO-RMs with preferential distribution to the liver, whereCO release is driven by metabolism of the parent compound.Organometallic carbonyl compounds are a well-known

    chemical class of metal complexes that are used primarily asprecursors to other organometallic compounds and/orcatalysts.10 Carbon monoxide binds as a ligand to mosttransition metals in low oxidation states. Consequently, mostorganometallic carbonyl complexes are sensitive to oxidation byoxygen and reaction with water and, as such, are not stableunder ambient conditions (air and humidity) and are thusincompatible with biological systems. Therefore, classicalorganometallic carbonyl chemistry proceeds largely in drysolvents and under inert atmospheres, which are essentiallyorthogonal to biological systems. The main exception relates tothe [MI(CO)3L3] (M = Tc, Re) complexes, designed to be usedas radiopharmaceuticals for diagnostic and therapy proce-dures.11 These stable complexes do not release CO and areexcreted in essentially their intact form (see ref 12 andreferences therein). CO-RMs for pharmaceutical uses need toovercome these limitations and demonstrate sufficient stabilityin water and air to serve as drugs in pill form or in formulationsfor intravenous or topical applications and, as such, be able torelease CO at treatment sites. Furthermore, the stabilizing non-CO ligands (ancillary ligands) have to be of small molecularsize to ensure that the corresponding CO-RMs have areasonable size for drug use (MW < 500600 g/mol).13Using the principles derived for the design of pharmaceutical

    CO-RMs briefly discussed above and detailed in a recentreview,9 we sought to generate druglike CO-RMs withimproved characteristics over the first published and widelyused water-soluble CO-RM, tricarbonylchloro(glycinato)-ruthenium(II) or CORM-3.14 While CORM-3 had demon-strated biological activity in several in vitro and in vivo biologicalsystems (reviewed in refs 1 and 15), its chemical stability, orrather instability, in aqueous solutions excluded it frompharmaceutical uses.16,17

    We started by evaluating and choosing an appropriate metaland its ancillary ligands. We initially considered eight transitionmetals (V, Cr, Mn, Fe, Co, Mo, Ru, Re) as possible COcarriers. Half the metals (V, Cr, Mn, Co) were rapidlyeliminated for known toxicities or for specific chemicallimitations: i.e., known compound instabilities. Other metals(Fe, Ru, Re) were also eliminated because our initial efforts toprepare complexes using these metals and a variety of ancillaryligands always resulted in complexes that lacked the adequateprofiles of stability and CO release in air and/or in aqueoussolutions. Therefore, we decided to develop molybdenum-based CO-RMs of the structure Mo0(CO)nL6n, where we soonfound species displaying drug-like characteristics.In fact, a systematic evaluation of several chemical classes of

    ligands L compatible with zerovalent molybdenum carbonyl

    complexes revealed several Moligand combinations withadequate stability profiles in air and aqueous solutions.However, several of these more stable classes of complexes,such as those bearing -diimine ligands, did not permitsufficient stability for intravenous drug formulations, a requisiteproperty of drugs destined for critical care settings, such asthose targeted in our acute liver failure development program.Molybdenum carbonyl complexes with water-soluble phos-phine ligands were often above the ideal molecular weightrange for drugs and were often toxic, particularly on cumulativeadministrations. Beck and his co-workers had described theformation of molybdenum tricarbonyl complexes with lithiumisocyanoacetate ligands that had a small molecular size andappeared to be soluble and stable in water under ambientconditions.18 We therefore explored derivatives of isocyanoa-cetate ligands for the formation of molybdenum carbonyl CO-RMs for the treatment of liver diseases. The aim was togenerate compounds that preferentially distributed to the liverafter intravenous (iv) administration and were bioactivated inthe liver, therefore delivering CO to this organ with a significantdegree of specificity.All CO-RMs were evaluated in a murine model of

    acetaminophen-induced acute liver injury. Three main reasonscontributed to this choice: (i) there is a strong medical need foradditional treatment modalities for this acute disease,19,20 (ii)the injury is believed to have a large inflammatory component21

    and strong anti-inflammatory activity has been demonstratedfor CO in various animal models of disease,22 and (iii) i

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