Chemistry and catalysis advances in organometallic chemistry and catalysis

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41.3.3.2 The Search for Other Potential CORMs

550

METAL CARBONYLS FOR CO-BASED THERAPIES: CHALLENGES AND SUCCESSES

Mo

CO

[NEt

]

CORM-3

CORM-2

ALF062

ALF186

ALF492

Figure 41.2

Experimental CORMs tested in animal models of disease.

the basis of an educated guess and produced very encouraging results [67]. CORM-2 is a vasodilator of rat aortic rings

ex vivo, and decreases the blood pressure of rats when administered in vivo. This last property was assigned to CO activity

because it was not elicited by the CO-free control [RuCl

(DMSO)

]. Although it has become the most popular CORM in the

literature [62, 66], the pharmacology of CORM-2 remains uncharacterized. It breaks its dimeric structure in DMSO to form

a mixture of fac-[Ru(CO)

Cl

2

(OSMe

)] and cis-cis-trans-[Ru(CO)

(SOMe

)

], none of which was studied separately.

Notwithstanding, the coordination of the successful [Ru

(CO)

] fragment to biological ligands that favor water solubility

led to the very important glycinato derivative fac-[Ru(CO)

(

-O

CCH

)] (CORM-3) [68].

During this initial discovery process, Motterlini and coworkers [67] searched for a simple methodology to quantify the

ability of a given MCC to release CO and act as a potentially useful CORM. Their choice fell on the so-called Mb assay.

The method comprises the addition of the candidate MCC to a solution of deoxy-Mb generated in situ from Mb and excess

dithionite. The reaction medium remains anoxic for circa 2 h in which time CO may be transferred from the CORM to

the hemeprotein. The amount of carboxymyoglobin (COMb) formed is quantiﬁed by UV–vis spectroscopy following the

replacement of the absorption of deoxy-Mb at 555 cm

−1

by the new absorptions of COHb at 541 and 578 cm

−1

. The

characterization and ranking of the CO-releasing activity of different CORMs can be made through the comparison of the

value of their half-life, t

(1/2)

, the time taken to form 0.5 equiv of COMb. This deﬁnition allows the comparison of CORMs

that deliver different numbers of CO ligands per mole.

According to the Mb assay, CORM-3 was described as a fast CO releaser because it formed 1 equiv of COMb within

minutes (t

(1/2)

= 2.3 min) [68, 69]. Solutions of CORM-3 left aging for circa 18–24 h fail to carbonylate deoxy-Mb and are

considered to contain an inactive decomposition product of CORM-3, named iCORM-3, which still contains cis-[Ru(CO)

]

fragments (IR evidence). CORM-3 became a reference CORM [66] producing impressive biological results in vitro, ex vivo,

and in vivo with remarkable therapeutic efﬁcacy in several major indications such as transplant [70, 71], organ preservation

[72], myocardial infarct [73], and rheumatoid arthritis [74, 75]. The therapeutic action of CORM-3 was always assigned

to CO in view of the inefﬁcacy of the negative control iCORM-3. So, a small t

1/2

in the Mb assay became the hallmark

of an active CORM and made the Mb assay an almost mandatory test for the identiﬁcation of CORMs. However, in spite

of recent improvements [76], the interpretation and use of this assay has to be seriously revised because it was found that

the fast transfer of CO from CORM-3 to deoxy-Mb results from the reaction of CORM-3 with dithionite and sulﬁte ions

present in the test, which displace CO from the Ru center. In fact, both CORM-2 and CORM-3 are unable to carbonylate

puriﬁed, dithionite-free deoxy-Mb or Hb [77]. Therefore, the early classiﬁcation of CORM-3 as a fast CO releaser has to

be abandoned since it is actually a no-releaser in this Mb assay. If we further consider that no CO gas can be detected in

the headspace of aqueous solutions of CORM-3 and its analogs [78, 79], and the fact that an organometallic ﬂuorescent

scavenger detects CO in cells treated with CORM-3 [80], we realize that its chemistry is not straightforward. However,

its therapeutic activity remains undisputed and its mode of action deserves to be explained. To this end, the ﬁrst step is to

consider its aqueous solution chemistry [81]. CORM-3, and indeed all other [Ru

(CO)

] type CORMs studied so far [79,

82], react with water at pH

> 4 to release H

and form species such as [Ru(CO)

(COOH)L

]

−

(water–gas shift reaction). At

THERAPEUTIC DELIVERY OF CO

551

physiological pH (7.4), these metallacarboxylates release CO

(GC detection; eventually H

also) [78] and form many other

species, depending on the nature of the ancillary ligands L [high performance liquid chromatography mass spectrometry

(HPLC-MS) evidence] [78, 79]. This chemical lability is also mirrored in the wide variation of the t

1/2

of CORM-3, which

is highest in saline (0.9% aqueous NaCl) and shortest in plasma [69]. This chemistry explains that CO

is released during

the preparation of iCORM-3 instead of CO [78], and explains the very rapid formation of adducts of CORM-3 and other

related complexes with proteins (serum albumin, transferrin, Hb, Mb, and lysozyme) which bear the cis-[Ru(CO)

(H

2

]

or [Ru(CO)(H

]

fragments as ascertained by electrospray ionization mass spectrometry (ESI-MS), Fourier transform

infrared spectroscopy (FTIR), and X-ray crystallography [78, 79].

The interaction with proteins such as serum albumin and transferrin also explains why CORM-3 does not raise the values

of COHb in systemic circulation at therapeutic doses, which is one of the most welcome characteristics of the in vivo therapy

with CORM-3 [73, 83]. In fact, after the loss of one CO as CO

, the resulting cis-Ru

(CO)

fragments are very slow CO

releasers, similar to iCORM-3, which is an unidentiﬁed mixture containing such species [78].

These results suggest that CORM-3 is transported in circulation as a serum albumin or transferrin adduct that slowly

decomposes to release CO without sudden bursts. This kind of mechanism is supported by the observation of steady, low

levels of CO in the treatment of mice with CORM-3 in a cardiovascular model [83]. One important consequence of this

mechanism is that it lacks tissue speciﬁcity. Actually, CORM-3 is active in a broad variety of indications affecting different

tissues and organs [62, 66]. Interestingly, the methylthiogalactose ligand was able to convey a reasonable liver speciﬁcity

to complex ALF492 (Fig. 41.2), which became much more efﬁcacious then CORM-3 in the protection of mice in a model

of cerebral malaria [82].

CO gas protection in this model had been reported but at the cost of very high levels of systemic COHb. Most importantly,

though, this work showed that ALF492 also targets the pharmacological expression of HO-1. Such induction of HO-1 had

already been shown for CORM-2 and CORM-3 [84], and may be a component of the therapeutic efﬁcacy of these and

many other CORMs through the already mentioned CO/HO-1 forward–feedback loop. To complicate things further, both

CORM-2 and CORM-3 (and the Mo(0) complexes described next) have been shown to produce ROS species in aqueous

solution, thereby raising the difﬁculty in the interpretation of their biological action [25, 85].

The Mo

0

(CO)

n

Derivatives ALF062, ALF186

At Alfama, the initial search for metal-based CORMs was supported by

quantiﬁcation of the release of CO to the headspace of the solutions of the compounds in biological media, such as RPMI-

1640 (Roswell Park Memorial Institute-1640), PBS (Phosphate Buffer Saline), HEPES (2-[4-(2-hydroxyethyl)piperazin-1-

yl]ethanesulfonic acid), saline, or plasma. The Mb assay was not used because it was thought important to check the proﬁle

of the complexes in aerobic conditions, which are absent in the Mb assay. This search revealed that the overwhelming

majority of the 18-electron metal carbonyl complexes tested (

>500) had a reasonable stability to air and water, opening

many windows for their use as CORMs. This meant that many MCCs when incubated in biological media under normoxic

conditions released CO at reasonably controlled rates. Lack of aqueous solubility was found to be the main barrier to their

development but this can be overcome through the use of appropriately functionalized ligands.

In order to capitalize on the existing data on CO gas therapy, Alfama’s initial choice of experimental CORMs fell on

compounds that delivered physically detectable amounts of free CO to the biological subject, either in vitro or in vivo. For

in vitro applications, CO release was quantiﬁed in the headspace of samples by the gas chromatography-reducing compound

photometer (GC-RCP) by using a technique implemented by Vreman [86]. For in vivo applications, CO release was controlled

by %COHb in circulation, measured by oximetry, or by CO in tissues, measured through GC-RCP [86]. The benchmarking

compound in this strategy was the water-soluble fac-[Mo(CO)

(histidinate)]Na (ALF186; Fig. 41.2).

ALF186 is stable in air as a solid and in anaerobic aqueous solution, but releases CO in aerobic solutions. When dissolved

in whole sheep blood in vitro, it almost instantaneously releases its full load of CO (3 equiv), which can be easily measured

by the value of %COHb read in a standard oximeter. Analysis of the same blood by GC-RCP conﬁrmed this quantitative

release of CO. When administered to mice, the total load of CO is rapidly released producing a peak of systemic %COHb at

circa 10 min post injection, which is dose dependent and highly reproducible. When this peak is attained, the mean arterial

blood pressure (MABP) of the animals has a signiﬁcant drop. After circa 2–3 h, the mice have exhaled all CO in circulation

and returned their %COHb and MABP close to the original basal levels (Alfama, unpublished work). This kind of CO

delivery partially mimics CO inhalation replacing the gas by a bolus of “solid CO,” which results in a much faster increase

of systemic %COHb. The dose of ALF186 needed to deliver a desired amount of CO to the organism can be accurately

calculated, and the time evolution of CO in circulation known beforehand. Oral administration leads to a CO peak at circa

1 h post administration, and higher doses are needed to reach a targeted %COHb value in comparison to the intraperitoneal

(ip) or intravenous (iv) administration. This compound proved to be a very powerful experimental tool and showed dose-

dependent therapeutic efﬁcacy for CO in a variety of animal models of disease. Interestingly, in several cases, the efﬁcacy

552

METAL CARBONYLS FOR CO-BASED THERAPIES: CHALLENGES AND SUCCESSES

of CORM-3 and ALF186 were mutually exclusive. For instance, ALF186 inhibits indomethacin-induced stomach ulcers,

while CORM-3 does not in the same model [87]. ALF186 rescues the liver in a mouse model of acetaminophen-induced

acute liver failure, yet CORM-3 is ineffective (Alfama, unpublished work). The opposite is observed in the prevention of

myocardial infarction injuries, where CORM-3 is very effective [73] and ALF186 is not. Examples were found where both

CORM-3 and ALF186 work and where none of them works.

ALF062, our code number for one of the oldest known Mo

carbonyl complexes, [Mo(CO)

Br][NEt

] [88], is a rather

lipophilic CORM that can be administered orally in olive oil and gave very good results in the treatment of adjuvant-induced

arthritis [89] in a rat model or as bactericide [90]. In both cases, CORM-3 is also effective [74, 90].

Summary

This historical overview shows that the chemical space of organometallic carbonyls is a suitable source of

therapeutically effective CORMs. However, the criteria that inform the search for useful CORMs have to be broad and

carefully checked to avoid instances of compounds such as CORM-3 being excluded in GC or Mb assays or of spontaneous

2

-activated CO releasers being excluded at the start. Ultimately, in vivo (or surrogate) experiments are decisive owing to

their prodrug characteristics.

Most importantly, it may be remarked that in spite of their therapeutic efﬁcacy, none of the compounds in Fig. 41.2 can

be considered a drug because of their lack of pharmacological proﬁle and druglike properties.

41.3.3.2

The Search for Other Potential CORMs

The very intense and fruitful research effort supported by the

experimental CORMs attracted other research groups to the ﬁeld and new molecules emerged. These new CORMs can be

divided according to the type of trigger that initiates CO release: chemical reactions (substitution, pH changes, oxidation),

photochemical reactions, and enzymatic reactions.

Chemically Triggered CO Release and CORMs

Most compounds described in this section are summarized in the structures

depicted in Figs. 41.3 and 41.4. The former belong to well-known structural motifs of organometallic chemistry such as

the [Fe

(CO)

(

-diene)], [(CpR)Fe(CO)

L]

0/

, [(CpR)Mo(CO)

L]

0/

(CpR

= substituted Cp, indenyl; L = 2e neutral or

negative ligand), [M(CO)

L]

0/

−

= Cr, Mo, W; L = 2e neutral or negative ligand or Fischer carbene), [μ

-(RC

≡CR )

(CO)

], and [Mn(CO)

]

. Compounds in Fig. 41.4 belong to a family of octahedral complexes stabilized by

pentadentate N5 ligands.

In order to characterize the compounds and rank their potential as CORMs, most authors carried on the in vitro “standard

set” of tests (SST) inaugurated with the development of CORM-2 and CORM-3. None of these compounds was tested

in vivo in any animal model of disease. The SST comprises the analysis of the CO-release proﬁle through the Mb assay

1/2

), the determination of the cytotoxicity (generally on RAW264.7 macrophages) through the LDH (lactate dehydrogenase)

assay, the determination of the cell viability through the Alamar blue test, and the determination of the iNOS inhibition by

measuring the amount of inhibition of NO induced by the CORM in a cell culture of RAW246.7 macrophages stimulated

with the proinﬂammatory lipopolysaccharide (LPS). Inhibition of NO production in the latter test is considered a sign of

the anti-inﬂammatory activity of the CORM. The SST was often accompanied by the determination of the vasodilatory

properties of the CORM through its effect on the relaxation of isolated aortic rings precontracted with ephedrine [67].

The two last types of activities are typical for CO gas and are expected to be replicated by a CORM. In this context, the

comparison of the CO-releasing proﬁles is given by the value of the CO-release half-life (t

1/2

) of the CORM.

In the family of [Fe

(CO)

(

-2-pyrone)] complexes, the least cytotoxic compound, CORM-F3, was also the one that

showed the more favorable CO-release t

1/2

, anti-inﬂammatory, and vasodilatory properties. The trigger of this CO release

is not ascertained and the substitution pattern at the 2-pyrone ring controls the rate of CO release [91, 92].

The substitution at the Cp ring in the [(CpR)Fe(CO)

family of complexes controls both the slow CO-release t

1/2

and solubility [93]. The related [Fe(

5

-C

)(CO)

complexes are very slow CO releasers, clearly more toxic, and do

not show any improvement of the CO release rate, which could be expected through the action of the “indenyl effect” [94].

The [CpFe(CO)

(

κ

-2-pyrone)]

complex behaved as a nonreleaser in the Mb assay but the Mo analog behaved as the

a very rapid CO releaser, with tolerable cytotoxicity and a strong vasodilatory activity. However, the difference of CO

release rates between both compounds remained unexplained [95]. The cobalt alkyne-bridged carbonyl complexes [

2

-

(RC

≡CR )Co

(CO)

] are too lipophilic to enable extracting safe conclusions as to their real value as CORMs [96], and

spurred the mechanistic studies conducted with several M(CO)

(amino ester) and Mo(CO)

(

=CRR ) (examples in Fig. 41.3)

in aqueous PBS solutions [97] and [M(CO)

−

= Cr, Mo, W; X = Cl, Br, I) [98], which brought forward some

speciﬁcities of the behavior of MCCs in water.

The complex [Mn(CO)

{S

2

CNMe(CH

}] was studied in some depth. The complex releases only 0.33 equiv of CO

after 4 h at 37

◦

C in PBS [99]. Kinetic studies, including substitution by

CO, and computational calculations consider that

THERAPEUTIC DELIVERY OF CO

553

(OC)

CORM-F3

OR'

L = CO, Cl, Br, I, NO

; R

′ = CH

, CH

′ R′′ O

′′′

′

′′

′′′

-ReCORM-2

CO

CO

O

Figure 41.3

Organometallic CORM candidates chemically triggered for CO release.

the loss of CO is dissociative and that the unsaturated 16-electron intermediate [Mn(CO)

CNMe(CH

}] efﬁciently

recaptures CO in the absence of competing nucleophiles or CO scavengers. The fact that this release is dramatically extended

to 3.2 equiv of CO in the Mb assay is interpreted by the authors as the result of the capture of CO by deoxy-Mb. However,

use of dithionite-free deoxy-Mb decreases this CO release to circa 1.8 equiv [77]. DFT calculations support the dissociative

mechanism proposed for a series of Mn(CO)

(S,S) type complexes, including the one above [100]. Thus, this CORM is a

potential prodrug activated by a well-deﬁned thermally induced CO dissociation. Although it has not been tested in vivo,

the above characteristics suggest that it will have a CO-release proﬁle similar to that of ALF186 in terms of the rate of

systemic COHb formation. Thermally induced CO release can be a very useful tool for biological studies, namely, those

made in vitro and ex vivo in the absence of the scavenging power of blood. A very well characterized family of such CO

donors has been developed on the basis of pentadentate polypyridine ligands such as those in Fig. 41.4 [101].

The lability of the CO is controlled by the type of N-donor function that occupies its trans position (this N-donor is

circled in the scheme). The design can place CO trans to a negative carboxamido-N (PaPy3

−

; strong

σ -donor), an imine-N

(SBPy3; moderately

π-accepting), and a tertiary amine-N (Tpmen; weak σ -donor) center, respectively. The SBPy3 and

Tpmen complexes spontaneously release CO for H

O in aqueous buffer, independently of the presence of O

and effectively

relax mouse aortic muscle rings in a dose-dependent manner. These very clean tests show that sGC is not involved in

the relaxation mechanism but the BK

2

+

channels are. This contrasts with the [Mn(CO)

CNMe(CH

}] complex

where sGC is considered to be implied in the vasodilation process [99]. However, both studies exclude the participation of

the ATP-dependent K

channels in the vasodilation. These [Fe(N5)(CO)]

compounds (N5

= SBPy3, Tpmen) are the ﬁrst

ones with a really stable negative CORM control molecule. So, comparing the results obtained with [Fe(N5)(CO)]

+

and

[Fe(N5)(H

O)]

will be a very useful way of determining the genuine effects of CO delivery in the presence of CORMs

and their genuine iCORM counterparts, in the absence of other physical (light, heat) or chemical difference. In all other

compounds reported, namely, the experimental CORMs, the control complexes are undeﬁned mixtures of metal-containing

species, such as iCORM-3, and may produce rather strange results and dose-dependent outcomes [85]. Mo(0) complexes

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