An N-Acetyl Cysteine Ruthenium Tricarbonyl Conjugate Enables Simultaneous Release of CO and Ablation of Reactive Oxygen Species

We have designed and synthesised a [Ru(CO)3Cl2(NAC)] pro-drug that features an N-acetyl cysteine (NAC) ligand. This NAC carbon monoxide releasing molecule (CORM) conjugate is able to simultaneously release biologically active CO and to ablate the concurrent formation of reactive oxygen species (ROS). Complexes of the general formulae [Ru(CO)3(L)3]2+, including [Ru(CO)3Cl(glycinate)] (CORM-3), have been shown to produce ROS through a water–gas shift reaction, which contributes significantly, for example, to their antibacterial activity. In contrast, NAC-CORM conjugates do not produce ROS or possess antibacterial activity. In addition, we demonstrate the synergistic effect of CO and NAC both for the inhibition of nitric oxide (formation) and in the expression of tumour-necrosis factor (TNF)-α. This work highlights the advantages of combining a CO-releasing scaffold with the anti-oxidant and anti-inflammatory drug NAC in a unique pro-drug.

Abstract: We have designed and synthesised a [Ru(CO) 3 Cl 2 (NAC)] pro-drug that features an N-acetyl cysteine (NAC) ligand.T his NAC carbon monoxider eleasing molecule (CORM) conjugate is able to simultaneously release biologicallya ctive CO and to ablate the concurrent formation of reactive oxygen species (ROS). Complexes of the general formulae [Ru(CO) 3 (L) 3 ] 2 + ,i ncluding [Ru(CO) 3 Cl(glycinate)]( CORM- 3), have been shown to produce ROS through aw ater-gass hift reaction, which contributes significantly,f or example, to their antibacterial activity.I nc ontrast, NAC-CORM conjugates do not produce ROS or possess antibacterial activity.I na ddition, we demonstrate the synergistic effect of CO and NAC both for the inhibition of nitric oxide (formation)a nd in the expression of tumour-necrosis factor (TNF)-a.This work highlights the advantages of combining aC O-releasing scaffold with the anti-oxidant and anti-inflammatory drug NAC in au nique pro-drug.

(CNR) 3 ]a nd
[Ru(CO) 3 Cl 2 (thiogalactopyranoside)] complexes. [7,8] The two most studied CORMsd erive from a{ Ru II (CO) 3 }s caffold:t he DMSO-soluble [{Ru(CO) 3 Cl 2 } 2 ]( CORM-2) and its water-soluble derivative fac-[Ru(CO) 3 Cl(k 2 -H 2 NCH 2 CO 2 )] (CORM-3). [9] Once characterised as fast CO releasers, it is has now been shown that these CORMsa re unable to transfer CO to deoxymyoglobin (deoxy-Mb) as previously accepted. [10] This correlatesw ell with the absence of CO in the headspace of solutionso f CORM-3 and other [Ru(CO) 3 Cl 2 (L)] (L = ligand) complexes, as determined by gas-chromatography (GC) methods. [11,12] Yet, these two CORMs have attractedg reat interest due to their considerable biological effects in animal modelso fd isease withouti ncreasing carboxyhemoglobin (CO-Hb)l evels in circulation. For instance, CORM-3 has been shown to protect against myocardial infarction and heart failure [13,14] as well as to help conservation of tissues for transplantation, [15] while CORM-2 was able to protect allogeneic aortic transplants in mice. [16] Recent studies have demonstrated the importance of the natureo ft he ancillary ligand(s) in complexes of the general formulae [Ru(CO) 3 (L) 3 ] 2 + for their stabilityi na queous media and subsequently on their CO release profile, cytotoxicity and anti-inflammatory properties. [17,18] In addition,t he reactivity of CORM-3 and [Ru(CO) 3 Cl 2 (thiazole)] was characterised in the presence of proteins, such as lysozyme, bovine serum albumin (BSA) or human transferrin. [11,12,18,19] It has been observed that the products resulting from the hydrolytic decomposition of CORM-3 and [Ru(CO) 3 Cl 2 (thiazole)] reactr apidly with histidine residues on proteins to generate protein-Ru adducts bearing two or one CO ligands, respectively.F urthermore, it has been [ [20,21] Finally,d uring the hydrolytic decomposition process, many of these CORMs generater eactive oxygen species (ROS) through aw ater-gas shift reaction (WGSR;F igure 1A)t hat can also contributet ot heir biological activity,a s is the case forCORM-3 bactericidal killing activity. [22] Herein, we sought to use the knowledge derived from extensives tudies of the stability,C Or eleasea nd biological activity of many CORMs of general formulae [Ru(CO) 3 (L) 3 ] 2 + to design aC ORM conjugate bearing al igand that would deliver biologically active CO and scavenge the ROS knownt ob e formed during the CO release process form [Ru(CO) 3 (L) 3 ] 2 + complexes.T he ligand we chose to introduce into such aC ORM conjugate was the drug N-acetyl cysteine (NAC) (Figure 1B), ap otent anti-oxidanta nd scavenger of hydroxyl radicals that has excellent anti-inflammatory activity. [23] We envisioned that the simultaneous releaseo fC Oa nd NAC could result in an enhanced anti-inflammatory activity,w hile NAC could also abolish any ROS formed during CO release. An identical strategy by combiningt wo drugs with complementary activities,t hat is, the conjugation of cisplatin and aspirin, resulted in as ynergistic effect towards the killing of cancerc ells. [24,25] We startedb ys ynthesising the NAC-CORM conjugate complex through reaction of the commercially availableC ORM-2 dimer with NAC ( Figure 1B). Thereaction occurs in acoordinating solvent( MeOH)t hat generates the solvated species [Ru(CO) 3 Cl 2 (HOMe)] prior to NAC substitution. [26] Analysis of the off-white powder isolated gives as toichiometry that matches the adduct [Ru(CO) 3 Cl 2 (NAC)],w hich can also be ad imer or highero ligomer (see Supporting Information). The FTIR spectrum presents the usual n CO stretching band pattern corresponding to the fac-M(CO) 3 fragment (see Supporting Information):asharp, strong vibration at 2126 cm À1 and av ery strong, broader band at 2062 cm À1 .T his indicatest hat the fac-Ru(CO) 3 arrangement remained intact in the product and no nucleophilic addition to ac oordinated CO has taken place. [18] The C=Os ignal from the amide is observed at 1749cm À1 .W es uggest that the NAC ligand binds the {Ru(CO) 3 Cl 2 }m oiety through the SH group, as documented in other [Ru II (L) n (SHR)] and [Ru II (L) n (SH 2 )] complexes. [27,28] However,t he n SH stretching vibration in the region around 2500 cm À1 is abroad peak probably reflecting hydrogen-bond type interactions in an on-monomeric structure of higher complexity.T he 1 HNMR spectrum of the NAC-CORM complex was acquiredi nC D 3 OD and D 2 O (see Supporting Information). The spectra in both solvents are very similarr evealing as urprisinglyg ood stability in aqueous solution.NoS HorN Hs ignals are observed. However,the spectra of pure NAC in the same solvents do not show the SH proton and the NH protonh as only av ery weak signal (see Supporting Information). The absence of the signals of the CH and CH 2 protons of free NAC in both NAC-CORM spectrai ndicates that there is no ligand dissociation or contamination with excesso fu nreacted NAC. Integration of the two close CH 3 signals in the acetyl region and the two close signals in the CH 2 regiong ives ar atio of 3:2p rotons, suggestingt he presence of either isomers or ac omplex oligomeric structure with magnetically non-equivalent NAC ligands. The CH 2 protons are deshielded relative to free NAC, whereas the CH protons are shielded, butare strongly split and could not be clearly assigned (see Supporting Information). Regardless of structural details,t hese data are in good agreement with FTIR and analytical data that confirm that the complex NAC-CORM contains the intact fac-Ru II (CO) 3 fragment coordinated to the NAC ligand,t hus carrying both the CO delivery and anti-oxidant functions.
We began by determining the rate of CO releaseo ft he aqueous soluble NAC-CORM conjugate to the headspaceo f aphosphate-bufferedsaline (PBS) pH 7.4 solution at room temperaturea nd in the dark, using gas chromatography (GC) with  [a] CORM-3w as synthesised as previously described. [30] [b] Equivalents of CO and CO 2 released in the headspace of ac losed vial after incubation of CORMsi nH 2 Oa fter 24 hat room temperature undern itrogen and in the dark, as determined by GC-TCD.
[ c] Cytotoxicity of CORMs was tested in RAW264.7 cells (MTT assay;2 4hincubation; IC 50 ). athermalconductivity detector (TCD, see the Supporting Information).S imilart oa nalogous compounds of the formulae [Ru(CO) 3 (L) 3 ] 2 + ,u nder these conditions CO could not be detected in the headspace of the solution (Table 1). Instead, CO 2 was slowly produced as the result of the extremelyf acile attack of HO À at coordinated CO, followed by the water-gas shift reaction shown in the second step of the scheme in Figure 1A. [11,29] Also, similart oC ORM-3, NAC-CORM didn ot raise the percentageo fC O-Hb, when incubated in sheep blood at 37 8C, as measured by oximetric quantification (data not shown). Finally,w ee valuated the cytotoxicity of NAC-CORM in RAW264.7c ellsb yu sing the MTT assay.I tw as found that NAC-CORM is not toxic up to aconcentration of 100 mm (Table 1). In 2012, Chang and co-workers introduced the coat protein COP-1 as aC Ospecific organometallic probe that turns fluorescence on upon as elective reactionw ith CO through ap alladium-mediated carbonylation reaction. [31] Importantly,t hey also showedt hat the fluorescenceo fC OP-1 in buffer is turned on either by CO gas or by CORM-3. Again similarly to the case of CORM-3 we observed that in PBS (pH 7.4) the NAC-CORM complex triggeredarobust fluorescence turn-onr esponse;atenfold increasew ithin 120 min in comparison with the controlas olution of COP-1 (Figure 2A). In the absence of CO, COP-1 is only weakly fluorescent. [25] When comparedw ith CORM-3, NAC-CORM showed as lower CO releasek inetics as detected by CO reaction with COP-1,i np articular during the first 10 min of incubation with the fluorescent CO-selective probe (Figure 2A). However,a fter 60 and 120 min no significant differences were detected with as imilarm aximum fluorescenceo bserved for both CORMs.
Next we used confocal microscopy to visualise changesi n CO levelsi nH eLa cells after incubation with NAC-CORM. HeLa cells were incubated in the absence (control) or presence of 50 mm NAC-CORM, and then treated with COP-1 and as ignificant increase in intracellular fluorescence for cells incubated with NAC-CORM over the control was observed ( Figure 2B and Figure 1i nS upporting Information). In order to turn fluorescence on CO must be transferred from the coordination sphere of Ru to that of Pd. This can happen if the Ru complexes decompose and the CO liberated to the mediumi sc aptured by COP-1, or if COP-1 reacts directly with some Ru-CO speciese xchanging CO. Previous evidencep oints to the preferred decompositiono f{ Ru II (CO) 3 }-containing complexes according to the scheme in Figure 1A.P roteins make adducts with [Ru(CO) 2 (H 2 O) 3 ] 2 + species, some of which are activeC O delivery species. [11,12,18,20,21] However,i ti sn ot difficult to admit that the very labile coordination sphere of [Ru(CO) 2 (H 2 O) n ] 2 + type species may facilitate reactionw ith COP-1 and CO transfer.F luorescence will be turned on and increasea sl ong as more [Ru(CO) 2 (H 2 O) n ] 2 + species are formed. This process will consumea ll COP-1,w hichi st he limiting reagent, while the decomposition of the CORM will proceed independently.I ndeed, the longeri tt akes to achieved ecomposition and formation of the [Ru(CO) 2 (H 2 O) n ] 2 + type species, the longer it will take for fluorescencet oa ppear.T he fact that NAC-CORM is actually slower than CORM-3 to generate fluorescence at early incubation times is not unexpected, sincet he stability of [Ru II (CO) 2 (CO 2 H)Cl 2 (L)] À present in the first equilibrium of Figure 1A,d epends on the nature of L. [18] In this case NAC-CORM is more stable in aqueous solution than CORM-3, according to the 1 HNMR data. If this process takes place intracellularly,t he fluorescencer eveals that the CORM has been taken-up by the cell and has startedt ol iberate CO. Part of this CO will be scavenged by COP-1 and the restwill eventually trigger the desired biological effects.
Our data shows that the new water-soluble NAC-CORM conjugatei sa ble to generate levels of CO in solution that are comparable to those produced with CORM-3, although with slowerk inetics. The slower kinetics are likely driven from an increased stabilityp rovidedb yt he RuÀSb ond present in NAC-CORM that makes the hydrolysis and subsequent CO release slower. This is of particulari mportance for in vivo CO delivery applications, for which ac ontrolled CO release profile is re- Figure 2. A) Comparison of CO release fromN AC-CORM and CORM-3:COr elease measurement using COP-1, read from 490 to 650 nm, followingexcitation (l ex = 475 nm). Photoemission spectraw ere taken at 5t o3 0s,1 0, 30, 60 and 120 min after the additiono f1mm COP-1 to 50 mm of NAC-CORM and CORM-3,r espectively,inP BS pH 7.4 at 37 8C. B) Confocalmicroscopy images for cellularC Or eleaseinu ntreated (control) and treated HeLa cells (50 mm NAC-CORM). After an initial 30 min treatment with NAC-CORM,1mm COP-1 was addeda nd following 30 min incubationp eriod pictures were taken. In each panel, the left picture showsn uclear staining using Hoechst 33342 (blue) and the picturetothe right shows COP-1 turn-on response to CO (green). quired. Incidentally,S -bound adducts [Ru(CO) 3 Cl 2 (SÀR)] were shown to be amongt he most stable speciest oh ydrolysis and CO loss in as eries of [Ru(CO) 3 Cl 2 (L)] adducts with different C, N, O, and Pd onors. [18] In this studyw es et ourselves to develop a[ Ru(CO) 3 (L) 3 ] 2 + complex that would releaseC Oa nd at the same time carry al igand that would ablate ROS produced during CO release, while enhancing the anti-inflammatory properties of CO. After providing clear data for CO releaseb oth in solution and cells, we examined the levels of endogenously formed ROS in E. coli cells treated with either NAC-CORM or CORM-3f or 2h (Figure 3A). The fluorescence intensities (FI) are represented as the subtraction of untreated culturesf rom cultures exposed to either NAC-CORM or CORM-3n ormalised in relation to the OD600 nm of the respective culture. The data reveals as ignificant increaseo fR OS content in cells exposed to CORM-3, but not in those exposed to NAC-CORM. After 2h,c ells treated with 100 mm of NAC-CORM displayed only 25 %o ft he ROS levels induced by CORM-3. This is in accordance with previous data showingt hat the ROS generated from the hydrolytic instabilityo fC ORM-2 could be abolished to similar levels of untreated cells by co-incubation with the ROS scavenger glutathione. [22] The generation of ROS has been shown to also contribute to the observed potent bactericidal activity of CORM-3. [22] Thus, we decided to perform ad irectc omparison of the antibacterial activity of NAC-CORM and CORM-3 to assess the effect of the presence of the anti-oxidant NAC ligand in bacterial survival (Figure3B). We observed that unlike CORM-3, treatment with NAC-CORM did not produce any significant effect on bacterial survival ( Figure 3B). Ourd ata suggests that the presence of the ROS scavenger NAC ligand in the conjugate is able to ablate the ROS formed during CO release that are important for the bactericidale ffect of CORMso ft he formulae [Ru(CO) 3 (L) 3 ] 2 + .
CO released from CORM molecules has been extensively demonstrated to possess anti-inflammatory properties. [1] NAC-CORM was designed to not only scavenge the ROS originated during the CO releasing process, but also to enhancet he antiinflammatory properties of CORMs. First, we tested the effect of NAC-CORM and CORM-3 on the production of NO from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Remarkably, NAC-CORM was ablet or educe nitrite levels in the culture by 84 %r elative to control cells ( Figure 4A). This showsa ne nhanced reduction of nitrite levels compared to CORM-3 (58 %) and NAC (60 %) alone. In addition, we also tested the effect of NAC-CORM in the expression levels of tumour necrosis factor (TNF)-a,akey marker of inflammation progression. Both CO and NAC have been reported to influence the expression levels of TNF-a. [2,23] Treatmento ft he adenocarcinomac ell line Caco-2 with NAC-CORM showedasynergistic effect promoting as ubstantial inhibition of the expression of endogenous TNFa at 4a nd 12 hw hen comparedw ith both CORM-3 andN AC alone at the same concentration (150 mm), as measured by enzyme-linked immunosorbent assay (ELISA) ( Figure 4B). This data provides strong evidence for the synergistic effect of both CO and NAC deliveredb yt he NAC-CORM conjugate here reported.
In summary,w eh ave produced and characterised a[ Ru II (CO) 3 Cl 2 (NAC)] complex that simultaneously delivers CO and abolishes ROS formation. Unlike other CORMso ft he general formulae [Ru(CO) 3 (L) 3 ] 2 + ,s uch as CORM-2 and CORM-3, NAC-CORM favourably reduces the levels of ROS that derive from the hydrolytic instability of such complexes in water.I n addition, our studies using the CO-selective probe COP-1 showed evidencet hat NAC-CORM is more stable compared to CORM-3, as evidenced by as lower CO release kinetics in aqueous solution. Importantly,t he NAC andC Od elivered after hydrolytic decompositiono ft he NAC-CORM complex act synergistically showing an enhanced anti-inflammatory activity,a s demonstrated by both nitrite reduction and inhibition of expression of TNF-a.C ollectively,o ur data suggestsc ombining of CO releasing motifs based on metal carbonyl scaffolds with ligands that may act synergistically to elicit an enhanceda nti-inflammatory response.  The National NMR Facility,s upported by FundaÅ¼op ara aC iÞncia eaTe cnologia (RECI/BBB-BQB/0230/2012). G.J. L.B. is aR oyal SocietyU niversity Research Fellow.