The present invention relates to new metal carbonyl complexes of curcumin derivatives, their synthesis and their uses in therapeutic application.

Carbon monoxide (CO) is an odorless and colorless gas having an affinity for hemoglobin (Hb) 210 to 250 times higher than the one of oxygen leading to the predominant formation of carboxy-hemoglobin (COHb) instead of oxy-hemoglobin. When high quantities of CO are inhaled resulting in 20 to 30% of COHb formation, the oxygen delivery in tissues is prevented, giving a general hypoxia.

Toxicological studies of this gas have concluded that an air quantity of CO as low as 0.4% can be lethal in less than one hour (Otterbein, L. E. ; Choi, A. M. K. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000, 279, 1029-1037).

To the opposite side, CO at very low concentrations can have benefitial effects on cellular and physiological functions.

CO production in mammalian organisms including humans can be achieved by heme catabolism. After having been knocked, the skin presents a hematoma and turns to black or purple indicating the bonding of oxygen to the heme. The hematoma colour turns then to green when the heme oxygenase (HO-1) catalyzes the heme oxidation, liberating an equimolar quantity of biliverdin, iron and CO. In mammals, biliverdin is then reduced in bilirubin by biliverdin reductase giving a yellow color to the hematoma.

In response to inflammation, it has been demonstrated that CO is released in the human body in such a way that the 6 cm ³ usually exhaled per day by a healthy human are significantly increased in pathological states. CO comes into the cell and controls physiological processes such as platelet aggregation (Brune, B.; Ullrich, V. Mol. Pharmacol. 1987, 32, 497-504), vasodilatation (Ramos, K. S.; Lin, H.; McGrath, J. J. Biochem. Pharmacol. 1989, 38, 1368-1370) or apoptosis (Fang, J.; Akaike, T.; Maeda, H. Apoptosis 2004, 9, 27-35).

Furthermore HO-1 /CO system is a part of the defense against cell stress, including heavy metals, reactive oxygenated species, lipopolysaccharides (LPS) and other anti-infammatory processes. The relevance of the HO-1 /CO system on human health has been highlighted by the first case of HO-1 deficiency in 1999 (Yachie, A.; Toma, T.; Mizuno, K.; Okamoto, H; Shimura, S.; Ohta, K.; Kasahara, Y.; Koizumi, S. Exp. Biol. Med. 2003, 228, 550-556; Ohta, K; Yachie, A.; Fujimoto, K; Kaneda, H.; Wada, T.; Toma, T.; Seno, A.; Kasahara, Y.; Yokoyama, H.; Seki, H.; Koizumi, S. Am. J. Kidney Dis. 2000, 35, 863-870), who died at 6 years old due to a growth delay, anemia, thrombocytosis, hyperlipidemia, leukocytosis, elevated heme sera levels and low bilirubin sera levels, confirming the vital role of HO-1 /CO system.

Thus the HO-1 /CO system has become a major concern at the end of 1990 and a therapeutic effect of CO has been contemplated by targeting its release in a secure and controlled way.

Motterlini et al. has first described CO releasing molecules (CO-RM) liable to deliver CO in a controlled manner in biological systems (Motterlini, R.; Clark, J. E.; Foresti, R.; Sarathchandra, P.; Mann, B. E.; Green, C. J. Cir. Res. 2002, 90, E17-E24). Recent reviews have resumed the medical potential of this new class of compounds (Mann, B. E.; Motterlini, R. Chem. Commun. 2007, 4197-4208 ; Motterlini, R. Biochem. Soc. Trans. 2007, 35, 1142-1146 ; Alberto, R; Motterlini, R. Dalton Trans. 2007, 1651 -1660.

Nevertheless, in order to transform this class of products into drugs for treating specific vascular and inflamatory pathological states, new CO-RM having an improved efficacy and safety must be developped.

Curcumin [l ,7-bis(4-hydroxy-3-methoxyphenyl)-l,6-heptane-3,5-dione] is the major yellow pigment extracted from turmeric, the powdered rhizome of the perennial herb Curcuma longa L. Comsumption of curcumin has been associated with a plethora of beneficial effects on human health, among which the anti-inflammatory and cancer chemopreventive activities are predominant. In particular, curcumin has been shown to have cytoprotective effects by inducing HO-1 in vascular endothelium (Motterlini, R; Foresti, R; Bassi, R.; Green, C. J. Free Radical Biol. Med. 2000, 28, 1303-1312), neuronal cells (Scapagnini, G; Foresti, R; Calabrese, V.; Giuffrida Stella, A. M.; Green, C. I; Motterlini, R. Mol. Pharmacol. 2002, 3, 554-561), renal ephytelial cells (Balogun, E.; Hoque, M.; Gong, P.; Killeen, E.; Green, C. J.; Foresti, R; Alam, J.; Motterlini, R. Biochem. J. 2003, 371, 887-895), and human monocytes (Rushworth, S. A.; Ogborne, R. M.; Charalambos, C. A.; O'Connell, M. A. Biochem. Biophys. Res. Comm. 2006, 341, 1007-1016). However, curcumin presents a poor bioavailability and efficacy in vivo, as only negligible amounts of curcumin were found in the blood and plasma of rats after oral administration of curcumin (lg/kg), indicating only poor absorption from the gut (Wahlstrom B., Blennow, G. Acta Pharmacol. Toxicol. 1978, 43, 86-92).

One of the aims of the present invention is to provide new metal complexed curcumin-CO derivatives active in vascular and inflammatory diseases

Another aim of the invention is to provide new metal complexed curcumin-CO derivatives having a lower toxicity and an improved bioavailability compared with curcumin.

Another aim of the invention is to provide pharmaceutical compositions comprising metal complexed curcumin-CO derivatives.

The present invention relates to a curcumin derivative having the following general formula (I):

wherein:

- Rl, R2, R7 and Rl 1 represent independently from each other:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to CIO, PEG, PEG-X-CO-, wherein X = O, NH, NH(CH ₂ ) ₂ or CH ₂ ,

and, Ra, Rb, Rc, Rd and Re represent independently from each other:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to

CIO,

Ri is any lateral chain of an amino acid,

Rii is OH or a peptidic chain comprising from 1 to 10 residue(s).

- R3 represents:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to

Ra, Rb, Rc, Rd and Re are as defined above,

Rf represents : H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to C10,

- R4, R5, R6, R8, R9 and R10 represent independently from each other:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to CIO, OR, SR, Br, CI, F, I,

wherein R represents: H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to CIO,

said curcumin derivative being complexed with at least one, preferably 1 to 5, in particular one or two identical or different metal carbonyl of formula (II):

M _x (CO) _y (II), wherein M is a metal selected from the list consisting of Co, Fe, Mn, Ru, Rh, Ni, Mo, V and Cr, and,

x = 1 and y = 2, 3, 4 or

x = 2 and y = 6,

for the manufacture of a drug intended for the treatment of cardiovascular and inflammatory diseases such as myocardial ischemia and heart diseases, rheumatoid arthritis, acute and chronic skin wound (wound healing), psoriasis, diabetes, diabetic nephropathy, metabolic syndrome, sickle-cell disease, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, neuropathic pain, hypertension, pulmonary arterial hypertension, septicemia, septic shock, haemorrhagic shock, cancer and chronic obstructive pulmonary disease

provided that said complexation is different from the complexation with the two carbonyl groups of said curcumin derivative.

By curcumin derivative is meant a compound having a structure based on curcumin:

and having the substitutions disclosed for Rl to Rl 1.

As curcumin comprises a β-diketo group, it is in equilibrium with the keto-enol form:

and thus, the compounds of formula (I) also covers the keto-enol form that could be on one or the other side of both carbonyl or a mixture of both when the compound of formula (I) does not have a symetry axe, i.e. when it bears at least one substituant on a phenyl group that is different form its counterpart on the other phenyl group, except when R3 is linked by a double bond. When the compound does not have a symetry axe, the carbon bearing R3 become of course asymetric and therefore, the compound of formula (I) corresponds to a racemic mixture, the enantiomer (R) or (S), or a non equal mixture of both enantiomers giving an optically active compound.

By linear alkyl group from CI to CIO is meant a group such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl.

By branched alkyl group is meant an alkyl group as defined above bearing substituants selected from the list of linear alkyl groups defined above, said linear alkyl group being also liable to be branched.

Both linear and branched alkyl definitions applies to the entire specification.

By cycloalkyl group from C3 to CIO is meant a group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. Such groups can also be substituted by a lienar or branched alkyl group as defined above.

The definition of cycloalkyl group applies also to the entire specification.

B PEG is meant a molecule of the following structure:

Polyethylene glycols

(PEG)

wherein n is an integer comprised from 4 to 1000, preferably 4 to 100, more preferably

17

R represent H or linear or branched alkyl C1-C6.

By amino acid is meant a natural amino acid or an unatural amino acid and the absolute configuration of the carbon bearing Ri can be (R), (S), a mixture of both, or (R,S).

By "peptidic chain" is meant a chain comprising one to ten residue(s) or amino acid(s) such as defined above.

Thus, the peptidic chain cam comprise one, two, three, four, five, six, seven, eight, nine or ten amino acids.

The triazol function of the group bearing Ri can be under a salt form, the salt being any pharmaceutically acceptable salt obtained by reaction of an inorganic acid, an organic acid or a halogenoalkyl, on an amino group to give a quaternary ammonium. When Ri is a lateral chain of a basic amino acid and thus bearing an amino group, it can also be under a salt form.

Examples of inorganic acid allowing to obtain pharmaceutically acceptable salts include, without being limited to them, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, formic acid, monohydrogenocarbonic acid, phosphoric acid, monohydrogenophosphoric acid, dihydrogenophosphoric acid, perchloric acid, sulfuric acid, monohydrogenosulfuric acid.

Examples of organic acid allowing to obtain pharmaceutically acceptable salts include, without being limited to them, acetic acid, lactic acid, propionic acid, butyric acid, isobutyric acid, palmitic acid, malic acid, glutamic acid, hydroxymalic acid, malonic acid, benzoic acid, succinic acid, glycolic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, salicylic acid, benzenesulfonic acid, /7-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, hydroxynaphthoic acid.

As the amino acid also bears an acid group, and as one or two of the aromatic groups can be a phenol, they can also be under a pharmaceutically acceptable salt form.

The salt can be obtained with organic or mineral bases, to give for instance to alkali metal salts such as, lithium, sodium, potassium salts.

As an example, see Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19.

When R3 is linked with a double bond, when the compound of formula (I) bears at least one substituant on a phenyl group that is different form its counterpart on the other phenyl group, the double bond can be under E or Z form or a mixture of both.

The expression "....being complexed with at least one ... metal carbonyl of formula (Π) means that the metal carbonyl is not covalently bound to the curcumin derivative but coordinated to a group liable to coordinate a metal carbonyl such as the R3 group which bears an alkyne, an alkene, aromatic, a cycloalkene such as a cyclohexadienyl or a cyclopentadienyl-group, or the Rl and/or R2 and/or R7 and/or Rl 1 group(s) when they correspond to an alkyne or diene, allowing the release of the metal carbonyl from the curcumin derivative once introduced in a mammal.

Each group liable to coordinate a metal carbonyl (referred here as a coordinating group) is able to coordinate a single metal carbonyl except when the coordinating group is an alkyne and the metal carbyl corresponds to Co ₂ (CO)6 wherein two metal carbonyls can be coordinated.. Thus it can exist one, two, three, four or five coordinating groups on the curcumin derivative and at the most five metal carbonyls on said curcumin derivative.

As more than one coordinating group, identical or different, can be present on the same curcumin derivative, more than one metal carbonyl could be present on said curcumin derivative, depending on the number of coordinating groups present on said curcumin derivative. Nevertheless, it could exist more coordinating groups on the curcumin derivative than metal carbonyl present, that is one or more of the coordinating group does not bear at all a metal carbonyl.

Thus, one, two, three, four or five metal carbonyls M _x (CO) _y , identical or different can be present at the same time on the curcumin derivative provided that the number of coordianting groups is equal to (1, 2, 3, 4 or 5 respectively) or is higher than the number of M _x (CO) _y (at least 2 coordinating groups for 1 M _x (CO) _y , at least 3 coordinating groups for 2 M _x (CO) _y , at least 4 coordinating groups for 3 M _x (CO) _y , 5 coordinating groups for 4 M _x (CO) _y ).

When more than one M _x (CO) _y is present on a curcumin derivative, each single M _x (CO) _y is always associated to the same coordinating group.

Advantageously, one or two M _x (CO) _yj identical or different, is(are) complexed to the curcumin derivative.

One of the advantages of the invention is therefore to provide a drug liable to separate into the mammal organism in two constituants:

1) on one hand, the curcumin derivative liable to activate HO-1 expression and thus to generate endogenous CO;

2) on the other hand, exogenous CO, liable to maximise the effet of endogenous CO by supplying the mammal organism with an exogenous CO coming from the metal carbonyl that release itself CO in the mammal organism by dissociation of the metal carbonyl giving the metal and exogenous CO liberated.

Surprisingly, the HO-1 expression activation by the curcumin derivative, when non complexed, is higher than HO-1 expression activation of curcumin alone in a range from about 2 to more than 4, in particular about 4, as determined by a heme oxygenase activity test described in the example part. Another advantage of the invention is to provide compounds presenting a decrease of cell toxicity with respect to non complexed curcumin alone or non complexed curcumin derivative as determined by LDH release and a cell viability tests (described in the example part).

Still another advantage of the invention is to provide compound having a bioavailability higher than curcumin alone.

It should be noted that said complexation does not occur with the two carbonyls of the curcumin derivative, said carbonyls being under keto form or keto-enol form, but occurs only with R3 radicals, or with the Rl and/or R2 group(s) when they correspond to an alkyne or diene.

In an advantageous embodiment, the metal M of formula (Π) is selected from the list consisting of Co, Mn, Ru, Rh, Ni, Mo, V.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, wherein said metal carbonyl is delivered by one of the following means:

a. dissociation of the metal carbonyl from the curcumin derivative on contact with a solvent or a tissue, organ or cell, liberating further CO,

b. increasing of the heme oxygenase expression producing further CO,

In this embodiment, CO can be provided either by an exogenous way coming from the metal carbonyl or by an endogenous way resulting from HO-1 expression activation.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, wherein said metal carbonyl is delivered by both said dissociation of the metal carbonyl and said increasing of the heme oxygenase expression, for delivering an increased level of CO with respect to said dissociation or increasing of the heme oxygenase expression taken alone.

In this embodiment, CO is provided by the exogenous way from metal carbonyl and the endogenous way by HO-1 expression activation.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, wherein said curcumin derivative presents synergistic heme oxygenase expression and activity with respect to a mixture of non complexed curcumin and the metal carbonyl or non complexed curcumin derivatives and the metal carbonyl respectively, or each non complexed curcumin or the metal carbonyl taken alone.

In the present invention, it has been surprisingly found that a simple mixture of each elements, i.e. curcumin or curcumin derivative mixed with a metal carbonyl does not give the same result as with complexed curcumin derivatives relative to the HO-1 expression and thus, a synergy between the curcumin derivative and the metal carbonyl once they are complexed occurs.

It has also been surprisingly found that a metal carbonyl alone causes a decrease of the HO-1 expression compared with a control, curcumin or a non complexed curcumin derivative, while when it is complexed to a curcumin derivative, it causes a synergistic increase of HO-1 expression compared with control, curcumin or non complexed curcumin derivative.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, for the delivery of carbon monoxide to a physiological target.

The complexed curcumin derivative once administered is absorbed by a mammal and goes through the blood circulation to a physiological target wherein it dissociates into curcumin derivative, activating HO-1 expression and releasing endogenous CO, and metal carbonyl which further dissociates into metal and exogenous CO, allowing to deliver CO at the physiological target.

By the expression "physiological target" is meant a tissue, organ or cell.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, wherein said curcumin derivative is complexed with two metal carbonyls of formula (II).

In this embodiment, two M _x (CO) _y are present on the curcumin derivative and are similar or different. Therefore, at least two groups, identical or different, liable to coordinate a metal carbonyl, are present on said curcumin derivative.

In an advantageous embodiment, the present invention relates to a curcumin derivative complexed with two metal carbonyls of formula (II), defined above, selected from the compounds of formula (I) wherein two substituents chosen among the five following substituents: Rl , R2, R3, R7 and Rl 1 , are selected from the list consisting of:

- Rl, R2, R7 and Rl l :

Ra

and complexed to an identical or different metal carbonyl, the three other substituents being as defined in formula (I), with the proviso that they are not complexed to a metal carbonyl of formula (II).

In this embodiment, more than one group, identical or different, liable to coordinate a metal carbonyl, are present on said curcumin derivative but only two of them are coordinated with a metal carbonyl.

Ra, Rb, Rc, Rd, Re and Rf are as defined above.

It must be noted that when said two substituents chosen among the five Rl, R2, R3, R7 and Rl 1 substituents correspond to said alkynes or alkenes above defined, then the three other substituents can be as defined above for the formula (I).

The presence of two metal carbonyls allows to deliver more exogenous CO and thus more

CO (endogenous and exogenoeus) than a curcumin derivative bearing only one metal carbonyl depending on the disease to treat.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, wherein said curcumin derivative is complexed with one metal carbonyl of formula (II).

In this embodiment, one M _x (CO) _y is present on the curcumin derivative. Therefore, at least one coordinating group is present on said curcumin derivative.

In an advantageous embodiment, the present invention relates to a curcumin derivative complexed with one metal carbonyl, selected from the compounds of formula (I) wherein one substituent chosen among the five following substituents: Rl, R2, R3, R7 and Rl l is selected from the list consisting of: - Rl, R2, R7 and Rl l :

and complexed to a metal carbonyl, the four other substituents being as defined above, with the proviso that they are not complexed to a metal carbonyl of formula (II).

In this embodiment, one or more than one coordinating group(s), identical or different, is(are) present on said curcumin derivative but only one of them is coordinated with a metal carbonyl.

Ra, Rb, Rc, Rd, Re and Rf are as defined above.

It must be noted that when said one substituent chosen among the five Rl, R2, R3, R7 and Rl l substituents corresponds to said alkynes or alkenes above defined, then the four other substituents can be as defined above for the formula (I).

The presence of one metal carbonyls allows to deliver less exogenous CO and thus less CO (endogenous and exogenoeus) than a curcumin derivative bearing only one metal carbonyl depending on the disease to treat.

In an advantageous embodiment, the present invention relates to a curcumin derivative defined above, said curcumin derivative being complexed with at least one, in particular one or two metal carbonyl of formula (II):

M _x (CO) _y (II),

wherein M is a metal selected from the list consisting of Fe, Co and, x = 1 and y = 2 or 3, or

x = 2 and y = 6,

for the manufacture of a drug intended for the treatment of cardiovascular and inflammatory diseases such as myocardial ischemia and heart diseases, rheumatoid arthritis, acute and chronic skin wound (wound healing), psoriasis, diabetes, diabetic nephropathy, metabolic syndrome, sickle-cell disease, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, neuropathic pain, hypertension, pulmonary arterial hypertension, septicemia, septic shock, haemorrhagic shock cancer, chronic obstructive pulmonary disease,

provided that said complexation is different from the complexation with the two carbonyls group of said curcumin derivative.

In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative and at least one of them is coordinated with a metal carbonyl.

The metal of said metal carbonyl is Fe, Co, Ru, Mn, Re, V or a combination thereof.

Avantageously, one or two metal carbonyls chosen among Fe, Co, Ru, Mn, Re or V are complexed to the curcumin derivative and when two of them are complexed, they can be identical or different.

Avantageously, one or two metal carbonyls chosen among Co, Ru, Mn, Re, V are complexed to the curcumin derivative and when two of them are complexed, they can be identical or different.

As an example, the two metal carbonyls can be Fe(CO)3, Co ₂ (CO)6 or both.

In another aspect, the present invention relates to a complexed curcumin derivative having the formula (III):

R7 R4 0 0 R8 R1 1

wherein n is at least one, preferably 1 to 5, in particular 1 or 2, and when n>l , then the metal carbonyls can be identical or different,

wherein the curcumin derivative, defined above, is the following:

- Rl, R2, R7 and Rl 1 represent independently from each other:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to CIO, PEG, PEG-X-CO-, wherein X = O, NH, NH(CH ₂ ) ₂ or CH ₂ ,

Ra, Rb, Rc, Rd and Re represent independently from each other:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to

CIO,

Ri is any lateral chain of an amino acid.

Rii is OH or a peptidic chain comprising from 1 to 10 residue(s). - R3 represents:

H, a linear or branched alkyl group from CI to CIO, a cycloalkyl group from C3 to CIO,

Ra, Rb, Rc, Rd, Re and Rf are as defined above, Rf represents : H, a linear or branched alkyl group from CI to CI O, a cycloalkyl group from C3 to CI O,

- R4, R5, R6, R8, R9 and RIO represent independently from each other:

H, a linear or branched alkyl group from CI to CI O, a cycloalkyl group from C3 to CI O, OR, SR, Br, CI, F, I,

wherein R represents: H, a linear or branched alkyl group from CI to CI O, a cycloalkyl group from C3 to CIO,

and wherein the metal of the metal carbonyl of formula (M _x (CO) _y ) _n , is as defined above and selected from the list consisting of :

Co, Fe, Mn, Ru, Rh, Ni, Mo, V and Cr, and,

x = 1 and y = 2, 3, 4 or

x = 2 and y = 6,

provided that said complexation is different from the complexation with the two carbonyl groups of said curcumin derivative.

Complexed curcumin derivatives of formula (III) can comprise up to five metal carbonyls identical or different as five substituents of the curcumin derivative can represent a group liable to coordinate a metal carbonyl.

Examples of metal carbonyl, without being limited to them, are the following:

Fe(CO) ₃ , Mo(CO) ₃ , Co ₂ (CO) ₆ , Mn(CO) ₃ , Cr(CO) ₃ , Ru(CO) ₃ , Ru(CO) ₂ , Rh(CO) ₃ ,

Ni(CO) ₃ , Mo(CO) ₃ , V(CO) ₄ , Cr(CO) ₃

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative defined above, wherein n = 1 and having the following formula (TV):

wherein at least one substituent chosen among the five substituents Rl, R2, R3, R7 and Rl 1 are selected from the list consisting of the following alkynes and dienes:

- R1, R2, R7 and Rl l

R4, R5, R6, R8, R9, Rl 0, being as defined above,

Ra, Rb, Rc, Rd, Re and Rf are as defined above,

with the proviso that when more than one substituent chosen among Rl, R2, R3, R7 and Rl l correspond to said alkyne or said diene, only one of said substituent is complexed to a metal carbonyl of formula (II).

It must be noted that when said at least one substituent chosen among the five Rl, R2, R3,

R7 and Rl 1 substituents corresponds to said alkynes or alkenes above defined (i. e. coordinating groups), then the four, three, two or one other of the five substituents can be as defined above for the formula (I), that is coordinating groups or not.

In an advantageous embodiment, the present invention relates to complexed curcumin derivatives defined above, having the following formula V:

wherein Rl , R2 is CH3, or

and R4, R5, R6, R7, R8, R9, RIO, Rl l being as defined O

above,

In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative but only one of them is coordinated to a metal carbonyl corresponding to Fe(CO)3.

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative of formula V defined above, having the following formulas:

It should be noted that compounds V-1 and V-2 have a symetry axe.

In an advantageous embodiment, the metal carbonyl present in compounds of formula (V, V-1, V-2 and V-3): Fe(CO) ₃ is replaced by Ru(CO) ₃ to give compounds of formula (V, V'-l, V-2 and V-3). In an advantageous embodiment, the present invention relates to complexed derivatives defined above, having the following formula Va:

and R4, R5, R6, R7, R8, R9, RIO, Rl 1 being as defined above,

In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative but only one of them is coordinated to a metal carbonyl corresponding to Mn(CO)3.

In an advantageous embodiment, the present invention relates to complexed curcumin derivatives defined above, having the following formula Vb:

and R4, R5, R6, R7, R8, R9, RIO, Rl 1 being as defined above, In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative but only one of them is coordinated to a metal carbonyl corresponding to Re(CO)3.

In an advantageous embodiment, the present invention relates to complexed curcumin derivatives defined above, having the following formula Vc:

wherein Rl, R2 is CH3, or

and R4, R5, R6, R7, R8, R9, RIO, Rl 1 being as defined above,

In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative but only one of them is coordinated to a metal carbonyl corresponding to V(CO)3.

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative defined above having the following formula (VI):

wherein Rl, R2, R4, R5, R6, R7, R8, R9, Rl 0, Rl 1 being as defined above, In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative but only one of them is coordinated to a metal carbonyl corresponding to Co ₂ (CO)6.

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative of formula (VI) defined above, having the following formula:

In this embodiment, one group liable to coordinate a metal carbonyl, in R3 position, is present on said curcumin derivative and is coordinated to a metal carbonyl corresponding to Co ₂ (CO) ₆ .

It should be noted that compounds VI-1 has a symetry axe.

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative defined above, having the following formula (VII):

wherein Rl, R3, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In this embodiment, one or more than one group, identical or different, liable to coordinate a metal carbonyl, is(are) present on said curcumin derivative but only one of them is coordinated to a metal carbonyl corresponding to Co ₂ (CO) ₆ . In an advantageous embodiment, the present invention relates to a complexed curcumin derivative of formula (VII) defined above, having the following formula:

In this embodiment, one group in Rl or R2 position, liable to coordinate a metal carbonyl, is present on said curcumin derivative and is coordinated to a metal carbonyl corresponding to Co ₂ (CO) ₆ .

The compound VI- 1 presenting a symmetry axe because R4 and R8, R7 and Rl 1, and R6 and RIO are similar, then the substitution in Rl or R2 gives the same curcumin derivative VII-l .

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative defined above, wherein n = 2 and having the following formula VTII:

wherein at least two substituents chosen among the five substituents Rl, R2, R3, R7 and Rl 1 are selected from the list consisting of the following alkynes and dienes:

- R1, R2, R7 and Rl l

R4, R5, R6, R8, R9, RIO, being as defined above,

Ra, Rb, Rc, Rd, Re and Rf are as defined above,

with the proviso that when more than two substituents chosen among Rl, R2, R3, R7 and Rl l correspond to said alkyne or said diene, only two of said substituent is complexed to a metal carbonyl of formula (II).

It must be noted that when said at least two substituents chosen among the five Rl , R2, R3, R7 and Rl 1 substituents correspond to said alkynes or alkenes above defined, then the three, two or one other of the five substituents can be as defined above for the formula (I).

In an advantageous embodiment, the present invention relates to a complexed curcumin derivative of formula VIII defined above, having the following formula VIII-l :

wherein Rl, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-2:

wherein Rl, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-3 :

wherein R3, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-4:

wherein R3, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to complexed derivative of formula VIII defined above, having the following formula VIII-5:

wherein R2, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-6:

whereinR2, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VHI-7:

wherein R3, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-8:

wherein R3, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above. In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-9:

wherein R2, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-10:

wherein Rl, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VIII-l 1 :

wherein R2, R4, R5, R6, R7, R8, R9, R10, Rl 1 are as defined above. In an advantageous embodiment, the present invention relates to a complexed derivative of formula VIII defined above, having the following formula VHI-12:

wherein R3, R4, R5, R6, R7, R8, R9, RIO, Rl 1 are as defined above.

In another aspect, the present invention relates to a pharmaceutical composition for the delivery of carbon monoxide to a physiological target, comprising a complexed curcumin derivative defined above, in association with a pharmaceutically acceptable vehicle.

The pharmaceutically vehicle is well known from a man skilled in the art.

The curcumin derivative makes available CO by to ways:

one way by HO-1 expression activation of curcumin derivative and delivery of endogenous CO,

another way by metal carbonyl which makes available CO suitable for physiological effect by at least one of the following means:

a) CO derived by dissociation of the metal carbonyl due to the presence of a solvent in the composition is present in the composition in dissolved form, for instance,

b) on contact with a solvent such as water, in particular with contact with an aqueous physiological fluid such as blood or lymph, the metal carbonyl releases CO,

c) on contact with a tissue, organ, or cell the metal carbonyl releases CO; in this case, the metal carbonyl does not dissociate in contact with a solvent.

d) on irradiation the metal carbonyl releases CO; the composition must be irradiated before administration to produce for example a solution of dissolved CO or be irradiated in situ after administration to provide controlled localized release administration.

In an advantageous embodiment, the present invention relates to a pharmaceutical composition defined above, for an administration by an oral, intravenous, subcutaneous, nasal, inhalatory, intramuscular, intraperitoneal, sublingual, topical or suppository route.

In an advantageous embodiment, the present invention relates to a pharmaceutical composition defined above, wherein said administration is oral and the complexed curcumin derivative is at a dose comprised from about 10 mg/kg to about 80 mg/kg.

Below 10 mg/kg the dose is too low to deliver a sufficient quantity of CO to the physiological target. Above 80 mg/kg, toxicity hazards occur.

In an advantageous embodiment, the present invention relates to a pharmaceutical composition defined above, wherein said administration is intravenous and the complexed curcumin derivative is at a dose comprised from about 500 μg/kg to about 8000 μg/kg.

Below 500μg/kg the dose is too low to deliver a sufficient quantity of CO to the physiological target. Above 8000 μg/kg, toxicity hazards occur.

In another aspect, the present invention relates to a process of treating a viable mammalian organ previously isolated from a mammal comprising contacting said organ with a complexed curcumin derivative defined above.

In this aspect, the complexed curcumin derivative is prescribed in treatment, for instance by perfusion, of a viable mammalian organ extracorporeally, for example, during storage and/or transport of an organ for transplant surgery. For this purpose, the complexe curcumin derivative is in a dissolved form, preferably in an aqueous solution. The viable organ may be any tissue containing living cells, such as heart, a kidney, a liver, a skin or muscle flap... DESCRIPTION OF THE FIGURES

Figures 1A and IB present the cell injury assay using a lactate dehydrogenase (LDH) release and the cell viability assay obtained with curcumin.

Figure 1 A presents the cell injury assay obtained with curcumin at 5, 10 and 20 μΜ.

x-axis: LDH release (% total).

y-axis: CON: control; black histogram: 1% Triton; grey histograms from left to right: 5,

10 and 20 μΜ of curcumin. Figure IB presents the cell viability assay obtained with curcumin at 5, 10 and 20 μΜ. x-axis: Cell viability (% total).

y-axis: White histogram: CON: control; grey histograms from left to right: 5, 10 and 20 μΜ of curcumin.

Figures 1A and IB show that curcumin is toxic from 5μΜ (14% of LDH release) and a decrease of 88% of cell viability.

Increased concentrations in curcumin (10 and 20 μΜ) give a total LDH release and a complete loss of viability.

Figures 2A and 2B present the cell injury assay using a lactate dehydrogenase (LDH) release and the cell viability assay obtained with AA136 (example 1).

Figure 2A presents the cell injury assay obtained with AA136 at 5, 10 and 20 μΜ.

x-axis: LDH release (% total).

y-axis: CON: control; black histogram: 1% Triton; grey histograms from left to right: 5, 10 and 20 μΜ οί AA136.

Figure 2B presents the cell viability assay obtained with AA136 at 5, 10 and 20 μΜ. x-axis: Cell viability (% total).

y-axis: White histogram: CON: control; grey histograms from left to right: 5, 10 and 20 μΜ of AA136.

Figures 2A and 2B show that AA136 is less toxic than curcumin. AA 136 gives only an increase of LDH release at 20μΜ and a decrease of 12% of cell viability at 5 and 10 μΜ and 38% at 20μM.

Figures 3A and 3B present the cell injury assay using a lactate dehydrogenase (LDH) release and the cell viability assay obtained with AA138 (example 4).

Figure 3A presents the cell injury assay obtained with AA138 at 5, 10 and 20 μΜ.

x-axis: LDH release (% total).

y-axis: CON: control; black histogram: 1% Triton; grey histograms from left to right: 5, 10 and 20 μΜ οί AA138.

Figure 3B presents the cell viability assay obtained with AA138 at 5, 10 and 20 μΜ. x-axis: Cell viability (% total).

y-axis: White histogram: CON: control; grey histograms from left to right: 5, 10 and 20 μΜ of AA138. Figures 3 A and 3B show that AA138 is less toxic than AA 138 and much less than cur cumin. AA 138 gives only an increase of LDH release at 20 μΜ (7%) and no loss of cell viability at 5 and 10 μΜ and 20μΜ.

Figures 4A and 4B present the cell injury assay using a lactate dehydrogenase (LDH) release and the cell viability assay obtained with AA164 (example 2).

Figure 4A presents the cell injury assay obtained with AA164 at 5, 10 and 20 μΜ.

x-axis: LDH release (% total).

y-axis: CON: control; black histogram: 1% Triton; grey histograms from left to right: 5, 10 and 20 μΜ οί AA164.

Figure 4B presents the cell viability assay obtained with AA164 at 5, 10 and 20 μΜ. x-axis: Cell viability (% total).

y-axis: White histogram: CON: control; grey histograms from left to right: 5, 10 and 20 μΜ of AA164.

Figures 4A and 4B show that AA164 is less toxic than curcumin. AA 164 gives no increase of LDH release at 5, 10 and 20μΜ and only a slight decrease of cell viability of

10% at 10 μΜ and 25% at 20μΜ.

Figures 5A and 5B present the cell injury assay using a lactate dehydrogenase (LDH) release and the cell viability assay obtained with AA168 (example 4).

Figure 5 A presents the cell injury assay obtained with AA168 at 5, 10 and 20 μΜ.

x-axis: LDH release (% total).

y-axis: CON: control; black histogram: 1% Triton; grey histograms from left to right: 5, 10 and 20 μΜ οί AA168.

Figure 5B presents the cell viability assay obtained with AA168 at 5, 10 and 20 μΜ. x-axis: Cell viability (% total).

y-axis: White histogram: CON: control; grey histograms from left to right: 5, 10 and 20 μΜ of AA168.

Figures 5A and 5B show that AA168 is less toxic than AA 164 and much less than curcumin. AA 168 gives no increase of LDH release at 5, 10 and 20μΜ and no loss of cell viability at 5 and 10 μΜ and 20μΜ. Figures 6A to 6D present the heme oxygenase activity assay on endothelial cells treated with curcumin (ΙΟμΜ), AA 136 (1, 5 and ΙΟμΜ), AA 138 (1, 5 and ΙΟμΜ), AA 164 (1, 5 and ΙΟμΜ) and AA 168 (1, 5 and ΙΟμΜ).

Figure 6A: heme oxygenase activity of curcumin (ΙΟμΜ) and AA 136 (1, 5 and ΙΟμΜ). x-axis: Heme oxygenase activity (pmoles bilirubin/mg protein/h)

y-axis: white histogram: control; and from left to right histograms: curcumin (ΙΟμΜ), AA 136 (1, 5, 10 μΜ).

Figure 6B: heme oxygenase activity of curcumin (ΙΟμΜ) and AA 138 (1, 5 and ΙΟμΜ). x-axis: Heme oxygenase activity (pmoles bilirubin/mg protein/h)

y-axis: white histogram: control; and from left to right histograms: curcumin (10μΜ), AA

138 (1, 5, 10 μΜ).

Figure 6C: heme oxygenase activity of curcumin (10μΜ) and AA 164 (1, 5 and 10μΜ). x-axis: Heme oxygenase activity (pmoles bilirubin/mg protein/h)

y-axis: white histogram: control; and from left to right histograms: curcumin (10μΜ), AA 164 (1, 5, 10 μΜ).

Figure 6D: heme oxygenase activity of curcumin (10μΜ) and AA 168 (1, 5 and 10μΜ). x-axis: Heme oxygenase activity (pmoles bilirubin/mg protein/h)

y-axis: white histogram: control; and from left to right histograms: curcumin (10μΜ), AA 168 (1, 5, 10 μΜ).

Figures 6A to 6D show that the heme activity of endothelial cells is much higher with complexed curcumin derivatives than with non complexed curcumin derivatives or curcumin alone. At 10 μΜ, after 6 hours of exposure of endothelial cells, the heme oxygenase activity is 465±30 pmoles bilirubin/mg protein/h (curcumin), 1290±201 pmoles bilirubin/mg protein/h (AA136), 1768±146 pmoles bilirubin/mg protein/h (AA 138); 1043±115 pmoles bilirubin/mg protein/h (AA164) and 1592±198 pmoles bilirubin/mg protein/h (AA 168).

Thus, the presence of a metal carbonyl on a curcumin derivative increases significantly the HO-1 expression in comparison with the one of the non complexed curcumin derivative or curcumin alone.

Figure 7 presents the effect of AA168 on inflammation in vitro. Murine RAW264.7 monocyte macrophages were exposed to 1 μg/ml lipopolysaccharide (LPS) for 24 h in the presence or absence of 10 μΜ AA168. At the end of the incubation period, nitrite levels were measured as index of inflammation using the Griess reagent method. A comparison was made with control cells incubated with medium alone (CON). We can observe from Figure 7 that the presence of AA168 in the culture media significantly attenuated the increase in nitrite induced by LPS. Bars represent the mean±SEM of 4 independent experiments. * P<0.05 vs. CON; ^† P<0.05 vs. LPS.

x-axis: Nitrite (μΜ)

y-axis: from left to right: CON (Control), LPS, LPS + AA 168

Figures 8A to 8B present the heme oxygenase activity assay determined by Western Blot on macrophage cells treated with curcumin (5 and 10 μΜ), AA 164 (5 and 10μΜ), AA 168 (5 and 10μΜ), Co ₂ (CO) ₈ (5 and 10 μΜ), Fe ₂ (CO) ₉ (1, 5 and 10μΜ) and LPS (10μΜ).

Figure 8A: Western Blot after 6h of treatment

Figure 8B: Western Blot after 24h of treatment

Molecules of the invention have a long lasting effect as usually HO-1 expression on macrophages treated with a CO releasing compound presents a peak at 6 hours and then decrease. In contrast, the molecules of the invention present an activity at 24h. EXAMPLES

CHEMICAL PART

General remarks:

All air-sensitive reactions were carried out under argon atmosphere, using standard Schlenk and vacuum-line techniques. THF was distilled from sodium/benzophenone. Dichloromethane was distilled from calcium hydride. Anhydrous piperidine was obtained by distillation from potassium hydroxide. All other chemical reagents and solvents were used as received without further purification. Preparative thin layer chromatography was performed on silica gel 60 GF254.

Column flash chromatography was performed on silica gel Merck 60 (40-63 μνα).

Chromatography solvents were used as received. Melting points were measured with a Kofler device. 1H and ¹³ C NMR spectra were recorded on a Bruker Avance300 or a Bruker Avance400 spectrometer at 20 °C at 300 MHz and 75 MHz or 400 MHz and 100 MHz, respectively. Chemical shifts (δ) are given in ppm, referenced to the residual proton resonance of the solvents (7.26 for CDC1 ₃ ; 5.32 for CD ₂ C1 ₂ ; 2.50 for (CD ₃ ) ₂ SO; 2.05 for (CD ₃ ) ₂ CO) and carbon resonance of the solvents (77.16 for CDC1 ₃ ; 53.10 for CD ₂ C1 ₂ ; 39.52 for (CD ₃ ) ₂ SO; 29.84 for (CD ₃ ) ₂ CO). Attenuated total reflection (ATR) infrared spectra were recorded on a JASCO FT/IR-4100 spectrometer. Mass spectra were obtained by the "Service de Spectrometrie de Masse" of the ENSCP, Paris. High-resolution mass spectroscopy was carried out by the "Groupe de spectroscopie de Masse" of the laboratory "Structure et Fonction de Molecules Bioactives" at the University of Pierre et Marie Curie, Paris. Elemental analyses were carried out by the "Service de Microanalyse" at the Institute de Chimie des Substances Naturelles (ICSN), Gif-sur-Yvette.

Example 1: Synthesis of AA 136 (non complexed curcumin derivative)

The scheme I below presents the scheme synthesis:

AA136

SCHEME I

Detailed synthesis:

Compounds M-Curc, (Lin, L.; Shi, Q.; Nyarko, A. K.; Bastow, K. F.; Wu, C.-C; Su, C. Y.; Shih, C. C.-Y.; Lee, K.-H. J. Med. Chem. 2006, 49, 3963-3972). AA160, (Acharya, S. P.; Brown, H. C. J. Am. Chem. Soc. 1967, 89, 1925-1932) AA134 (Prasada Rao Lingam, V. S.; Vinodkumar, R.; Mukkanti, K.; Thomas, A.; Gopalan, B. Tetrahedron Lett. 2008, 49, 4260-4264) and azido-glycine (Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2001, 3, 781-783) were synthesized as described in the literature.

(3Z,5E)-4-hydroxy-6-(3-methoxy-4-(prop-2-ynyloxy)phenyl)h exa-3,5-dien-2-one (AA159):

A suspension of 2,4-pentanedione (4.86 mL, 47 mmol) and boron oxide (0.88 g, 13 mmol) was heated for 30 minutes at 50 °C under argon atmosphere. A solution of AA134 (3.00 g, 16 mmol) in anhydrous ethyl acetate (10 mL) was added, followed by tributyl borate (4.30 mL, 16 mmol) and stirring was continued at 50 °C for further 30 minutes. w-Butylamine (1.56 mL, 16 mmol) diluted in anhydrous ethyl acetate (10 mL) was added dropwise over 15 minutes and stirring was maintained at 50 °C for 2 hours. The dark red suspension was then hydrolyzed by adding an aqueous 1N-HC1 solution and stirring for 1 hour at 50 °C. The mixture was filtered and the aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic extracts were washed until neutral with water and brine, dried over magnesium sulfate and evaporated to dryness to give a dark red oil. The crude product was purified by flash chromatography using n- hexane/ethyl acetate 2:1 as eluent, yielding AA159 as a dark yellow solid (2.39 g, 55%). Mp:

115-116 °C. ¾ NMR (300 MHz, CDC1 ₃ ) δ 7.54 (d, J = 15.8 Hz, 1H, H6), 7.1-7.0 (m, 3H, H12/H8/H9), 6.35 (d, J= 15.8 Hz, 1H, H5), 5.64 (s, 1H, H3), 4.80 (d, J= 2.4 Hz, 2H, H13), 3.95 (s, 3H, OCH ₃ ), 2.53 (t, J = 2.4 Hz, 1H, H15), 2.16 (s, 3H, HI). ¹³ C NMR (75 MHz, CDC1 ₃ ) δ 197.3 (C2), 177.4 (C4), 149.7 (CIO), 148.5 (Cl l), 139.6 (C6), 129.2 (C7), 121.8 and 121.1 (C5/C8), 113.8 (C9), 110.3 (C12), 100.9 (C3), 78.0 (C14), 76.2 (C15), 56.6 (C13), 55.9 (OC¾), 26.9 (CI). IR (ATR, cm ^"1 ) v 2124 (C≡C), 1635 (CO), 1617 (CO). MS (APCI): m/z 273.7 [M+H] ⁺ . Anal. Calcd for Ci ₆ Hi ₆ 0 ₄ : C, 70.57; H, 5.92. Found: C, 70.65; H, 5.95.

[(l£',4Z,6£)-l-(3-methoxy-4-hydroxyphenyl)-3-hydroxy-7- (3-methoxy-4-(prop-2- ynyloxy)phenyl)-hepta-l,4,6-trien-5-one (AA136):

The product already known in the literature (Shi, W.; Dolai, S.; Rizk, S.; Hussain, A.; Tariq, H.; Averick, S.; L'Amoreaux, W.; El Idrissi, A.; Banerjee, P.; Raja, K. Org. Lett. 2007, 9, 5461-5464) was synthesized by alternative routes, as follows:

Route 1 :

A suspension of M-Cur (567 mg, 2.42 mmol) and boron oxide (118 mg, 1.69 mmol) in anhydrous ethyl acetate (3 mL) was heated under argon for 30 minutes at 70 °C under argon atmosphere. A solution of AA134 (460 mg, 2.42 mmol) in dry ethyl acetate (4 mL) was added to the viscous suspension, followed by tributyl borate (1.31 mL, 4.84 mmol) and stirring was maintained for 30 minutes at 60 °C. Freshly distilled piperidine (0.24 mL, 2.42 mmol) diluted in dry ethyl acetate (2 mL) was added dropwise and stirring was continued for 2 hours at 60 °C. Hydrolysis was done by adding an aqueous 1N-HC1 solution (10 mL) and stirring for 30 minutes at 60 °C. The aqueous layer was then extracted with ethyl acetate (3 x 15 mL) and the combined organic extracts were washed until neutral with water and brine, dried over magnesium sulfate, filtered and evaporated to dryness. The crude product was purified by flash chromatography using ft-hexane/ethyl acetate 3:2 as eluent, yielding AA136 as a dark yellow solid (130 mg, 13%). Route 2:

A suspension of AA159 (810 mg, 2.98 mmol) and boron oxide (145 mg, 2.08 mmol) in anhydrous ethyl acetate (6 mL) was heated for 30 minutes at 50 °C under argon atmosphere. A solution of Vanillin (450 mg, 2.98 mmol) in dry ethyl acetate (10 mL) was added to the viscous suspension, followed by tributyl borate (1.61 mL, 5.95 mmol) and stirring was maintained for 30 minutes at 50 °C. Freshly distilled piperidine (0.29 mL, 2.98 mmol) diluted in dry ethyl acetate (5 mL) was added dropwise and stirring was continued for 2 hours at 50 °C. Hydrolysis was done with by adding an aqueous 1N-HC1 solution (12 mL) and stirring for 30 minutes at 60 °C. The aqueous layer was then extracted with ethyl acetate (3 x 15 mL) and the combined organic extracts were washed until neutral with water and brine, dried over magnesium sulfate, filtered and evaporated to dryness to give a dark red oil. The crude product was purified by flash chromatography using w-hexane/ethyl acetate 3:2 as eluent, yielding AA136 as a dark yellow solid (244 mg, 20%). The product was confirmed by NMR spectroscopy and mass analysis.

According the same procedure, the following curcuminoid derivatives can be synthesized:

wherein R', R" , R" ' and R" " represent, independently from each other, H or C¾.

Example 2: Synthesis of AA 164 (non complexed curcumin derivative)

The scheme II below resents the scheme synthesis:

SCHEME II

Detailed synthesis:

(lE,4Z,6E)-3-hydroxy-l,7-bis(4-hydroxy-3-methoxyphenyl)-4-(p rop-2-ynyl)hepta-l,4,6- trien-5-one (AA164) (keto nd enol product)

To a stirred solution of AA160 (1.05 g, 7.60 mmol) in anhydrous ethyl acetate (3 mL) was added boron oxide (370 mg, 5.32 mmol) and stirring was continued for 30 minutes at 40 °C under argon atmosphere. A solution of Vanillin (2.30 g, 15 mmol) in dry ethyl acetate (10 mL) was added drop wise, followed by tributyl borate (4.10 mL, 15 mmol) and stirring was maintained for 30 minutes at 40 °C. Butylamine (1.12 mL, 11 mmol) diluted in dry ethyl acetate (10 mL) was added over 15 minutes and stirring was continued over night at 40 °C. Hydrolysis was done by adding an aqueous 1N-HC1 solution (10 mL) and stirring for 30 minutes at 50 °C. The aqueous layer was extracted with ethyl acetate (3 x 20 mL) and the combined organic extracts were washed until neutral with water and brine, dried over magnesium sulfate, filtered and evaporated to dryness to yield a dark red oil. The crude product was purified by flash chromatography using w-hexane/ethyl acetate 1 :1 as eluent, yielding AA164 (keto-enol/diketo 1.4: 1) as a bright orange solid (772 mg, 25%). Mp: 88-90 °C. 1H NMR (300 MHz, CDC1 ₃ ): δ 7.76 (d, J = 15.8 Hz, 2H, H1/H7), 7.73 (d, J = 15.4 Hz, 2H, H1/H7), 7.20 (dd, J = 8.3, 1.9 Hz, 2H, H9/H19), 7.12 (dd, J = 8.3, 1.9 Hz, 2H, H9/H19), 7.07 (d, 2H, J= 1.9 Hz, H13/H15), 7.04 (d, 2H, J = 2.1 Hz, H13/H15), 6.99 (d, J = 15.8 Hz, 2H, H2/H6), 6.95 (d, J = 8.3 Hz, 1H, H10 or HI 8), 6.92 (d, J = 8.1 Hz, 3H, ¾(Η10/Η18)), 6.75 (d, J = 15.8 Hz, 2H, H2/H6), 5.94 (s, 2H, OH _dlketo ), 5.89 (s, 2H, OH _keto-enol ), 4.34 (t, J = 7.4 Hz, 1H, H4 _dlket0 ), 3.96 (s, 6H, OCH ₃ ), 3.92 (s, 6H, OCH ₃ ), 3.44 (d, J= 2.7 Hz, 2H, H20 _keto-enol ), 2.91 (dd, J = 7.5 Hz, J = 2.7 Hz, 2H, H20 _dlketo ), 2.15 (t, J = 2.6 Hz, 1H, H22 _diket0 ), 2.02 (t, J = 2.6 Hz, 1H, H22 _keto-eno i). ¹³ C NMR (75 MHz, (CD ₃ ) ₂ CO) δ 193.7 (CO), 183.8 (CO), 150.7, 150.2, 148.8 (CI 1/C17/C12/C16), 145.5 and 143.1 (C1/C7), 128.5 and 127.4 (C8/C14), 124.7, 124.0, 123.2 (C2/C6/C9/C19), 118.7 and 116.3 (C10/C18), 112.1 and 111.8 (C13/C15), 107.7 (C4 _keto-eno i), 84.2 (C21), 82.5 (C21), 71.3 (C22), 70.6 (C22), 62.2 (C4 _diket0 ), 56.4 (OCH ₃ ), 56.3 (OCH ₃ ), 18.2 (C20), 16.1 (C20). IR (ATR, cm ^"1 ) v 2121 (C≡C), 1617 (COH), 1584 (CO). MS (APCI): m/z 40Ί.Ί [M+H] ⁺ , 405.3 [M-H] ^" . Anal. Calcd for C ₂₄ H ₂₂ 06*l/2 H ₂ 0: C, 69.93; H, 5.58. Found: C, 68.99; H, 5.24.

Example 3: synthesis of [(l£',4Z,6£)-l,7-bis(3-methoxy-4-(prop-2-ynyloxy)phi hydroxy-hepta-l,4,6-trien-5-one (Di-O-Prop-Curc): The product already known in the literature (Gomes, D. d. C. F.; Alegrio, L.; Freire de Lima, M. E.; Leon, L. L.; Araujo, C. A. C. Arzneim.-Forsch. 2002, 52, 120-124) was synthesized by an alternative route, as follows:

A suspension of 2,4-pentanedione (1.08 mL, 11 mmol) and boron oxide (513 mg, 7.36 mmol) was heated for 30 minutes at 60 °C under argon atmosphere. A solution of AA134 (4.00 g, 21 mmol) in anhydrous ethyl acetate (24 mL) was added, followed by tributyl borate (5.67 mL, 21 mmol) and stirring was continued at 40 °C for further 30 minutes. w-Butylamine (1.56 mL, 16 mmol) diluted in anhydrous ethyl acetate (10 mL) was added dropwise over 15 minutes and stirring was maintained at 40 °C overnight. The dark red suspension was then hydrolyzed by adding an aqueous 1N-HC1 solution and stirring for 1 hour at 50 °C. The mixture was filtered and the aqueous layer was extracted with ethyl acetate (3 x 30 mL). The combined organic extracts were washed until neutral with water and brine, dried over magnesium sulfate and evaporated to dryness to give a dark red oil. The crude product was purified by flash chromatography using n- hexane/ethyl acetate 3:2 as eluent, yielding unreacted AA134 (1.47 g, 36 %) in the first fraction, and Di-O-Prop-Curc as a dark yellow solid (1.11 g, 38 %) in the second fraction.

The product was confirmed by NMR spectroscopy and mass analysis.

Example 4: General procedure of coordination of dicobalthexacarbonyle to an alkyne (AA138, AA168)

Dicobalthexacarbonyle (1 eq) was added in one portion to a stirred solution of the appropriate curcumin in dry THF (5 mL) and stirring was maintained for 3 hours under argon atmosphere. The solvent was evaporated in vacuum to yield a dark red solid. The crude product was purified by flash chromatography using w-hexane/ethyl acetate 3:2 as eluent.

Example 4.1: synthesis [(l£',4Z,6£)-l-(3-methoxy-4-hydroxyphenyl)-3-hydroxy-7-(3- methoxy-4-(prop-2-ynyloxy)phenyl)-hepta-l,4,6-trien-5-one]-d icobalt-hexacarbonyl

(AA138):

The synthesis was carried out as described above by using dicobalthexacarbonyle (109 mg, 0.32 mmol) and AA136 (130 mg, 0.32 mmol), yielding AA138 as a dark red solid (130 mg, 60 %). Mp 67-70 °C. 1H NMR (300 MHz, CDC1 ₃ ) δ 7.61 (d, J = 15.8 Hz, 1H, HI or H7), 7.60 (d, J = 15.8 Hz, 1H, H7 or HI), 7.2-7.1 (m, 4H, H10/H18/H9/H19), 6.94 (s, 2H, H13/H15), 6.51 (d, J = 15.7 Hz, 1H, H2 or H6), 6.49 (d, J = 15.8 Hz, 1H, H6 or H2), 6.06 (s, 1H, OH), 5.86 (s, 1H, H4), 5.85 (s, 1H, H22), 5.32 (bs, 2H, H20), 3.95 (s, 3H, OCH ₃ ), 3.87 (s, 3H, OCH ₃ ). ¹³ C NMR (100 MHz, CD ₂ C1 ₂ ) δ 199.1 (Co-CO), 183.4 (CO), 182.9 (COH), 150.1 , 149.5, 147.7, 146.7 (C11/C17/C12/C16), 140.1 and 139.7 (C1/C7), 128.6 and 127.4 (C8/C14), 122.6, 121.9, 121.8, 121.4 (C2/C6/C9/C19), 114.3 and 113.7 (C10/C18), 110.4 and 109.3 (C13/C15), 101.1 (C4), 88.7 (C21), 72.1 (C22), 69.1 (C20), 55.7 (OCH ₃ ), 55.4 (OCH ₃ ). IR (ATR, cm ^"1 ) v 2096, 2053 and 2020 (CO), 1625 (COH), 1583 (CO). MS (ESI): m/z 693.6 [M+H] ⁺ , 691.6 [M-H] ^" . Anal. Calcd for C ₃ oH ₂₂ Co ₂ Oi ₂ : C, 52.04; H, 3.20. Found: C, 52.05; H, 3.32.

Example 4.2: synthesis of [(lE,4Z,6E)-3-hydroxy-l,7-bis(4-hydroxy-3-methoxyphi (prop-2-ynyl)hepta-l,4,6-trien-5-one]-dicobalt-hexacarbonyl (AA168)

The synthesis was carried out as described above by using dicobalthexacarbonyle (168 mg, 0.49 mmol) and AA164 (200 mg, 0.49 mmol), yielding AA168 as a dark red solid (120 mg, 35 %). Mp: 162-164 °C. 1H NMR (300 MHz, CDCI ₃ ) δ 7.79 (d, J = 15.4 Hz, 2H, H1/H7), 7.15 (d, J = 8.5 Hz, 2H, H9/H19), 7.10 (s, 2H, H13/H15), 6.99 (d, J= 15.6 Hz, 2H, H2/H6), 6.95 (d, J = 8.7 Hz, 2H, H10/H18), 6.05 (s, 1H, H22), 5.89 (s, 2H, OH), 4.13 (s, 2H, H20), 3.91 (s, 6H, OCH ₃ ). ¹³ C NMR (100 MHz, CD ₂ C1 ₂ ) δ 199.4 (Co-CO), 182.7 (C3/C5), 148.0 (CI 1 /CI 7), 146.7 (C12/C16), 142.2 (C1/C7), 127.5 (C8/C14), 123.3 (C9/C19), 117.2 (C2/C6), 114.3 (C10/C18), 110.6 (C4), 109.27 (C13/C15), 97.2 (C21), 73.7 (C22), 55.6 (OCH ₃ ), 30.6 (C20). IR (ATR, cm ^"1 ) v 2090, 2050 and 2009 (CO), 1618 (COH), 1590 (CO). MS (ESI): m/z 693.5 [M+H] , 691.6 [M- H] ^" . Anal. Calcd for C30H22C02O12: C, 52.04; H, 3.20. Found: C, 52.21 ; H, 3.16.

Example 5: synthesis of (i£,£)-tricarbonyl(hexa-2,4-dienal)iron (AA257)

4 3

6 5 ^^2 ¹

H ₃ C^ I ^CHO

Fe(CO) ₃

AA257 has been synthesized after the following procedure:

Nonacarbonyl diiron (3.98 g, 10.90 mmol) was suspended under argon in anhydrous diethyl ether (20 mL) and a dilution of 2,4-hexadienal (1.10 mL, 9.96 mmol) in anhydrous diethyl ether (15 mL) was added. The mixture was sonolyzed and stirred for 24 hours under inert atmosphere. The green -brownish suspension was then filtered over celite and carefully evaporated under reduced pressure. The crude product was then purified by flash column chromatography using petroleum ether/diethyl ether 2:1 as eluent, yielding AA257 as yellow oil (1.22 g, 52 %). ¾ NMR (400 MHz, CDC1 ₃ ) δ 9.25 (d, J = 4.4 Hz, HI), 5.8-5.7 (m, 1H, H3), 5.3- 5.2 (m, 1H, H4), 1.7-1.6 (m, 1H, H5), 1.51 (d, J = 6.0 Hz, 3H, H6), 1.3 -1.2 (m, 1H, H2). ¹³ C NMR (300 MHz, C ₆ D ₆ ) δ 210.0 (CO), 195.1 (CI), 89.2 (C3), 81.2 (C4), 60.0 (C5), 55.0 (C2), 18.7 (C6). IR (ATR, cm ^"1 ) v 2061 (CO), 2001 (CO), 1987 (CO). MS (EI): m/z 237.06 [M+H] ⁺ , 253.86 [M+NH ₄ ] ⁺

Example 6: General procedure for (£',£)-tricarbonyl(hexa-2,4-dienylidene)iron Knoevenagel condensates AA261, AA268

The appropriate curcumin (1 eq) was dissolved in a minimum amount of anhydrous DMF and a dilution of AA257 (1 eq) in anhydrous DMF containing piped dine (0.5 eq) was added dropwise. The solution was allowed to stir for 6-24 hours under argon atmosphere. After hydrolysis, the mixture was extracted with ethyl acetate, and the organic phase was dried on magnesium sulfate, filtered and evaporated to dryness. The Knoevenagel condensate was purified by flash chromatography using w-hexane/ethyl acetate as eluent.

Example 6.1: synthesis of (l£',6£')-l,7-bis(3,4-methoxyphenyl)-4-((£',£)-tricarbon yl(hexa- 2,4-dienylidene)iron)-hepta-l,6-diene-3,5-dione (AA261)

The synthesis was carried out as described above by using DMC (350 mg, 0.88 mmol), piperidine (44 μΐ _^ , 0.44 mmol) and AA257 (288 mg, 1.22 mmol). After column flash chromatography (w-hexane/ethyl acetate 1 :1), AA261 was obtained as a golden-yellow solid (183 mg, 34 %). Mp: 86-88 °C. 1H NMR (300 MHz, CDC1 ₃ ) δ 7.65 (d, J = 15.6 Hz, 1H, HI or H7), 7.49 (d, J = 15.8 Hz, 1H, H7 or HI), 7.19 (dd, J = 8.3 Hz, J = 1.7 Hz, 2H, H9/H19), 7.08 (d, J = 1.6 Hz, 1H, H13 or H15), 7.03 (d, J = 1.6 Hz, 1H, H15 or H13), 6.9-6.8 (m, 5H, H2/H6/H10/H18/H20), 5.5-5.4 (m, 1H, H22), 5.2-5.1 (m, 1H, H23), 3.92 (s, 3H, OCH ₃ ), 3.91 (s, 3H, OCH ₃ ), 3.89 (s, 3H, OCH ₃ ), 3.87 (s, 3H, OCH ₃ ), 2.1-2.0 (m, 1H, H24), 1.8-1.7 (m, 1H, H21), 1.45 (d, J = 6.2 Hz, 3H, H25). ¹³ C NMR (75 MHz, CDCI ₃ ) δ 210.7 (CO), 194.6 (CO), 187.5 (CO), 151.7 and 151.5 (C11/C17), 149.3 and 149.2 (C12/C16), 148.8 (C20), 146.2 and 144.6 (C1/C7), 138.7 (C4), 127.6 and 127.4 (C8/C14), 124.9, 123.8, 123.4 and 120.9 (C9/C19/C2/C6), 111.1 and 111.0 (C10/C18), 110.0 and 109.9 (C13/C15), 88.2 (C22), 84.5 (C23), 59.7 (C24), 56.0 and 55.9 (OCH ₃ ), 53.6 (C21), 19.1 (C25). IR (ATR, cm ^"1 ) v 2042 (CO), 1977 (CO), 1967 (CO), 1635 (CO), 1592 (CO). MS (APCI): m/z 615.3 [M+H] ⁺ , 649.5 [M+Cl] ^" . Anal. Calcd for C ₃₂ H ₃₀ FeO ₉ : C, 62.55; H, 4.92. Found: C, 62.95; H, 5.21. HRMS (ESI) calcd. for C ₃₂ H ₃₀ FeO ₉ Na ⁺ : 637.1132, found: 637.1146.

Example 6.2: synthesis of (l£',6£)-l,7-bis(4-(prop-2-ynyloxy)-3-methoxyphenyl)-4-((� �',£)- tricarbonyl(hexa-2 -dienylidene)iron)-hepta-l,6-diene-3,5-dione (AA268)

The synthesis was carried out as described above by using Di-O-Prop-Curc (580 mg, 1.31 mmol), piperidine (65 \iL, 0.65 mmol) and AA257 (431 mg, 1.83 mmol). After column flash chromatography (w-hexane/ethyl acetate 2:1), AA268 was obtained as a golden -yellow solid (312 mg, 36 %). Mp: 69-71 °C. 1H NMR (300 MHz, CDC1 ₃ ) δ 7.64 (d, J = 15.5 Hz, 1H, HI or H7), 7.48 (d, J = 16.0 Hz, 1H, H7 or HI), 7.13 (dd, J = 8.3 Hz, J = 2.0 Hz, 2H, H9/H19), 7.08 (d, J = 1.9 Hz, 1H, H13 or H15), 7.1-7.0 (m, 3H, H10/H18/H15 or H13), 6.87 (d, J= 12.4 Hz, 1H, H20), 6.86 (d, J = 15.3 Hz, 1H, H2 or H6), 6.83 (d, J = 16.0 Hz, 1H, H6 or H2), 5.5-5.4 (m, 1H, H22), 5.2-5.1 (m, 1H, H23), 4.79 (d, J = 2.5 Hz, 1H, H29 or H26), 4.78 (d, J = 2.5 Hz, 1H, H26 or H29), 3.86 (s, 3H, OC¾), 3.88 (s, 3H, OC¾), 2.53 (t, J = 2.3 Hz, 1H, H31 or H28), 2.52 (t, J = 2.3 Hz, 1H, H28 or H31), 2.0-1.9 (m, 1H, H24), 1.8-1.7 (m, 1H, H21), 1.43 (d, J = 6.2 Hz, 3H, H25). ¹³ C NMR (100 MHz, CDCI3) δ 210.9 (CO), 194.6 (CO), 187.7 (CO), 149.9 (C11/C17), 149.5 (C12/C16), 149.3 (C20), 146.0 and 144.5 (C1/C7), 138.7 (C4), 128.9 and 128.7 (C8/C14), 125.5, 123.4, 123.0 and 121.6 (C9/C19/C2/C6), 113.8 and 113.7 (C10/C18), 110.8 (C13/C15), 88.5 (C22), 84.7 (C23), 78.1 and 78.0 (C27/C30), 76.6 and 76.5 (C28/C31), 60.0 (C24), 56.7 (C26/C29), 56.2 and 56.1 (OCH ₃ ), 53.7 (C21), 19.3 (C25). IR (ATR, cm ^"1 ) v 2120 (C≡C), 2043 (CO), 1978 (CO), 1966 (CO), 1636 (CO), 1592 (CO). MS (APCI): m/z 663.4 [M+H] ⁺ , 697.6 [M+Cl] ^" . Anal. Calcd for C36H3oFe0 ₉ *l/8CH ₂ Cl ₂ : C, 64.46; H, 4.53. Found: C, 64.22; H, 4.72. HRMS (ESI) calcd. for C ₃ 6H3oFe0 ₉ Na ⁺ : 685.1132, found: 685.1155.

Example 7: synthesis of (l£',6£)-l-(3-methoxy-4-{methoxy-lH-l,2,3-triazol-l-yl)ace tic acid}phenyl)-4-((£',£)-tricarbonyl(hexa-2,4-dienylidene)ir on)-7-(4-(prop-2-ynyloxy)-3- methoxyphenyl)-hepta-l,6-diene-3,5-dione and (l£',6£)-l-(4-(prop-2-ynyloxy)-3- methoxyphenyl)-4-((£',£)-tricarbonyl(hexa-2,4-dienylidene) iron)-7-(3-methoxy-4-{methoxy- lH-l,2,3-triazol-l-yl)acetic acid}phenyl)-hepta-l,6-diene-3,5-dione (AA270)

AA268 (100 mg, 0.15 mmol) was suspended in a i-butanol/water l :l-mixture (1-2 niL) and three drops of chloroform were added. Azido-glycine (15 mg, 0.15 mmol) diluted in the same solvent mixture (1 mL) was added, followed by freshly prepared aqueous copper sulfate pentahydrate solution (1 mol%, 1.5 μΐ. of a ImM solution, 1.5 μηιοΐ) and sodium ascorbate solution (1 mol%, 15 μΐ. of a ImM solution, 15 μηιοΐ) and the reaction mixture was stirred vigorously over night. After evaporation of the solvent, the residue was filtered and washed again with distilled water to remove any traces of copper sulfate or sodium ascorbate. The remaining solid was dissolved in acetone, dried on magnesium sulfate, filtered and evaporated to dryness, yielding pure AA270 as an orange-red solid (105 mg, 91 %). Mp: 102-104 °C. ^X H NMR (300 MHz, (CD ₃ ) ₂ SO): 8.2-8.1 (m, 2H, H28/H28'), 7.53 (d, J = 16.0 Hz, 1H, H _viny i), 7.52 (d, J = 15.1 Hz, 1H, Hvinyi), 7.3-7.2 (m, 16H, 4*H _viny i/10*H _ar /H20/H20'), 7.07 (d, J = 8.3 Hz, 1H, H _ar ), 7.03 (d, J = 8.5 Hz, 1H, ¾), 6.88 (d, J= 16.0 Hz, 1H, H _viny i), 6.87 (d, J= 16.0 Hz, 1H, H^i), 5.9-5.8 (m, 2H, H22/H22'), 5.57-5.5-5.4 (m, 2H, H23/H23'), 5.2-5.1 (m, 4H, H26/H29/H26'/H29'), 4.83 (d, J= 2.5 Hz, 4H, H31/H31 '), 3.80 (s, 3H, OC¾), 3.79 (s, 3H, OC¾), 3.77 (s, 3H, OC¾), 3.76 (s, 3H, OC¾), 3.58 (bs, 2H, H33/H33'), 1.9-1.8 (m, 2H, H24/H24'), 1.8-1.7 (m, 2H, H21/H21 '), 1.36 (d, J = 6.2 Hz, 6H, H25/H25'). ¹³ C NMR (100 MHz, (CD ₃ ) ₂ SO): δ 211.4 (CO), 195.4 and 187.3 (C3/C5/C37C5'), 168.9 (C30/C30'), 150.3, 149.5, 149.3, 149.2 and 149.0 (C11/C17/C12/C16/C117C177C127C16'), 148.2 (C20/C20'), 145.5, 145.3, 143.4 and 143.2 (C1/C7/C17C7'), 142.2 (C27/C27'), 139.0 (C4/C4'), 128.5, 128.1, 127.9 and 127.6 (C8/C14/C87C14'), 126.4, 125.7 and 125.5 (C9/C19/C97C19'), 123.6, 123.3, 122.9 and 122.5 (C2/C6/C27C6'), 120.4 and 120.1 (C28/C28'), 113.9, 113.6, 113.4 and 113.1 (C10/C18/C107C18'), 111.9, 111.7, 111.2 and 111.0 (C13/C15/C137C15'), 88.7 (C22/C22'), 85.2 (C23/C23'), 79.1 and 78.8 (C32/C33/C327C33'), 61.7 (C26/C26'), 60.5 (C24/C24'), 56.2 (C31/C31 '), 56.1, 55.9 and 55.8 (OC¾), 54.4 (C21/C21 '), 51.6 (C29/C29'), 19.0 (C25/C25'). IR (ATR, cm ^"1 ) v 2120 (C≡C), 2043 (CO), 1979 (CO), 1964 (CO), 1734 (CO), 1636 (CO), 1591 (CO), 1579 (N=N). MS (ESI): m/z 762.9 [M+Cl] ^" . HRMS (ESI) calcd. for C ₃ 8H33FeOiiN ₃ Na ⁺ : 786.1357, found: 786.1378.

Example 8: General procedure for the synthesis of ruthenium carbonyl curcumin derivative. Example with the synthesis of (l£',6£)-l,7-bis(3,4-methoxyphenyl)-4-((£',£)- tricarbonyl(hexa-2,4-dienylidene)ruthenium)-hepta-l,6-diene- 3,5-dione

Under argon atmosphere, the curcumin derivative (leq) is dissolved in a minimum amount of anhydrous DMF and a solution of rutheniumtricarbonyl hexa^^-dienal ¹ (1.1 eq) in anhydrous DMF containing piped dine (0.5 eq.) is added dropwise. The reaction was followed by TLC. After hydrolysis, the mixture is extracted with ethyl acetate, and the organic phase is dried on magnesium sulfate, filtered and evaporated to dryness. The Knoevenagel condensate is purified by flash chromatography with hexane/ethyl acetate as eluant.

1 S. L. Ingham, S. W. Magennis, J. Organometallic Chem., 1999, 574, 302-310.

Example 9: General procedure for the synthesis of formylcyclopentadienyl metal carbonyl curcumin derivatives

Under argon atmosphere, an anhydrous DMF diluted solution of (formylcyclopentadienyl) metalcarbonyl (1 eq) containing piperidine (0.5 eq.) is added dropwise to the appropriate curcuminoid (1 eq.) dissolved in a minimum amount of anhydrous DMF. The reaction can be monitored by TLC. After hydrolysis with water, the mixture is extracted with ethyl acetate, and the organic phase is dried on magnesium sulfate, filtered and evaporated to dryness. The Knoevenagel condensate is purified by flash chromatography with hexane/ethyl acetate as eluant.

With (formylcyclopentadienyl)tricarbonylmanganese :

With (formylcyclopentadienyl)tricarbonylrhenium ² :

With (formylcyclopentadienyl)tetracarbonylvanadium ² :

S. S. Jones, Stephen, M. D. Rausch, T. E Bitterwolf, J. Organometallic Chem., 1990 , 396, 279 288.

Example 10-1: General procedure for the synthesis of mono- or di-molybdenum tricarbonyl curcumin derivative ³

Under argon atmosphere, [Mo(?7 ⁶ -C ₆ H ₆ )(CO)3] (1 eq) is added to a degassed solution of dimethylcurcumin in anhydrous THF. After stirring for 1 h and removal of volatiles, THF is added again and stirring is pursued for 1 h. After evaporation of the solvent, the residue is dissolved in dichloromethane and filtered through a pad of silica gel.

E. P. Kiindig, C.-H. Fabritius, G. Grossheimann, P. Romanens, Organometallics, 2004, 23, 3741-3744.

Example 10-2: General procedure for the synthesis of PEG-curcumin derivatives

The compounds have been synthesized according to Safavy, A ; Raisch, K. P.; Mantena, S.; Sanford, L. L.; Sham, S. W.; Krishna, N. R; Bonner, J. A. J. Med. Chem. 2007, 50, 6284- 6288. or

The PEG curcumin (R' = H) can further be transformed in PEG-curcumin derivative by reaction with K ₂ CO ₃ and propargyl bromide to give PEG-curcumin derivative (R'=CI¾CCH) and then the PEG-curcumin derivative can further be coordinated with dicobalthexacarbonyle.

BIOLOGICAL PART

Example 11: Cell culture

Bovine aortic endothelial cells (BAECs) were from Coriell Cell Repositories (Camden, NJ, U.S.A.). Hemin was from Porphyrin Products (Logan, UT, U.S.A.) and all other chemicals and reagents were purchased from Sigma. Endothelial cells were grown in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cells were cultured and maintained until confluent for the experiments as previously described (Foresti, R. et al. Biochem. J. 2003, 372, 381-390).

Example 12: Cell Injury Assay

Cells were exposed to increasing concentrations of the testing compounds for 24 h and cell injury assessed at the end of incubation. Cell injury was determined using a lactate dehydrogenase (LDH) assay kit and carried out according to the manufacturer's instructions (Roche Diagnostics, United Kingdom) as previously reported by us (Sawle, P. et al. Chem. Res. Toxicol. 2008, 21, 1484-1494). LDH is a stable cytoplasmic enzyme present in all cells. It is rapidly released into the cell culture supernatant upon damage of the plasma membrane. Samples were read on a plate reader (Molecular Devices VERSAmax tunable microplate reader) at 490 nm with a reference wavelength of 690 nm and blanked against cell culture medium. Samples were run in triplicate, and treatment of some cells with 1% triton was used as a positive control (100% LDH release). Example 13: Cell Viability Assay

Cells were exposed to increasing concentrations of the testing compounds for 24 h and viability assessed at the end of incubation. Cell viability was determined using an Alamar Blue assay kit and carried out according to the manufacturer's instructions (Serotec, Oxford, UK) as previously reported by us (Sawle, P. et al. J. Pharmacol. Exp. Therap. 2006, 318, 403-410). The assay is based on the detection of metabolic activity of living cells using a redox indicator that changes from an oxidized (blue) form to a reduced (red) form. The intensity of the red color is proportional to the metabolism of the cells, which is calculated as the difference in absorbance between 570 and 600 nm and expressed as a percentage of control. Example 14: Heme Oxygenase Activity Assay with endothelial cells

The heme oxygenase activity was determined in bovine aortic endothelial cells after treatment with the testing compounds as previously described by our group (Foresti, R. et al. Biochem. J. 2003, 372, 381-390). Briefly, harvested cells were subjected to three cycles of freeze thawing before addition to a reaction mixture consisting of phosphate buffer (1 mL final volume, pH 7.4) containing magnesium chloride (2 mM), NADPH (0.8 mM), glucose-6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 units), rat liver cytosol as a source of biliverdin reductase, and the substrate hemin (20 μΜ). The reaction was conducted at 37 °C in the dark for 1 h and terminated by the addition of 1 ml of chloroform; the extracted bilirubin was calculated by the difference in absorbance between 464 and 530 nm (ε=40 mM ^"1 x cm ^"1 ).

Example 15: Heme Oxygenase Activity Assay with macrophage cells

The heme oxygenase activity was determined in RAW264.7 cells after various treatments as previously described by (Abuarqoub, H., Foresti, R., Green, C. I, and Motterlini, R. (2006), Heme oxygenase- 1 mediates the anti-inflammatory actions of 2'-hydroxychalcone in RAW 264.7 murine macrophages. Am. J. Physiol. Cell Physiol. 290, C1092-C1099 ; Sawle, P., Foresti, R., Mann, B. E., Johnson, T. R., Green, C. J., and Motterlini, R. (2005) Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages. Br. J. Pharmacol. 145, 800-810 ; NO-mediated activation of heme oxygenase: Endogenous cytoprotection against oxidative stress to endothelium. Am. J. Physiol. Heart Circ. Physiol. 270, H107-H1 14.

Briefly, harvested cells were subjected to three cycles of freeze thawing before addition to a reaction mixture consisting of phosphate buffer (1 mL final volume, pH 7.4) containing magnesium chloride (2 mM), NADPH (0.8 mM), glucose-6- phosphate (2 mM), glucose-6- phosphate dehydrogenase (0.2 units), rat liver cytosol as a source of biliverdin reductase, and the substrate hemin (20 μΜ). The reaction was conducted at 37 °C in the dark for 1 h and terminated by the addition of 1 mL of chloroform; the extracted bilirubin was calculated by the difference in absorbance between 464 and 530 nm (ε = 40 mM ^"1 cm ^"1 ).

Example 16 : Detection of CO Release

Example 16.1: Detection of CO release using a myoglobin assay.

The release of CO from newly synthesized molecules was assessed spectrophotometrically by measuring the conversion of deoxymyoglobin (deoxy-Mb) to carbonmonoxy myoglobin (MbCO). A small aliquot of concentrated solution of the new molecules were added to 1 ml deoxy-Mb solution in phosphate buffer (final concentrations: CO- RM (metal carbonyl complex) = 50 μΜ; deoxy-Mb= 55 μΜ), and changes in the Mb spectra were recorded over time. The amount of MbCO formed were quantified by measuring the absorbance at 540 nm. Before the addition of the new testing molecule, 500 μΐ of mineral oil (Sigma Aldrich) were added on top of the aqueous solution containing Mb to prevent CO escaping and the myoglobin becoming oxygenated.

Myoglobin solutions (66 μηιοΙ/Ε final concentration) were prepared fresh by dissolving the protein in 0.04 mol/L phosphate buffer (pH 6.8). Sodium dithionite (0.1%) was added to convert myoglobin to deoxy-Mb prior to each reading.

CO released from complexed curcumin derivative was quantified by adding aliquots of stock solutions (10 iL) of the carbonyl complex in DMSO directly to the myoglobin solution. All the spectra were measured using a Helios a spectrophotometer. When CO loss from complexed curcumin derivative occurs only by photodissociation, the release of CO was induced by exposing these metal carbonyl complexes to a cold light source and allowing the gas to diffuse through a membrane before reacting with myoglobin.

Example 16.2: Detection of CO release from CO-RMs (metal carbonyl complex) using an amperometric CO sensor.

The spontaneous release of CO from the testing compounds was measured using a CO- sensitive electrode. This CO electrode (World Precision Instrument, UK) is a membrane-covered amperometric sensor which has been designed on a basic operating principle similar to the nitric oxide (NO) sensor. The CO sensor can be connected to the WPI ISO-NO Mark II meter for detection of the current signals providing that the poise potential is set to a different value (900 mV for CO as opposed to 860 mV for NO). Briefly, CO diffuses through the gas permeable membrane and is then oxidized to C0 ₂ on the working electrode. This oxidation creates a current whose magnitude can be related directly to the concentration of CO in solution. The electrode were immersed into distilled water containing 0.1 M phosphate buffer (pH=7.4) and equilibrated for 30 min prior to addition of the testing compounds. The experiments were conducted at 37 °C and the solutions were maintained at the desired temperature using a Grant W6 thermostat.

Example 17 : Protocol for assessing inflammation in vitro and in vivo

Example 17.1 : In vitro protocol:

Murine RAW264.7 monocyte macrophages were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cultures were maintained at 37 C in a 5% C0 ₂ humidified atmosphere and experiments were conducted on cells at approximately 80-90% confluence. Macrophages were exposed to 1 μg/ml lipopolysaccharide (LPS) for 24 h and nitrite levels, inducible nitric oxide synthase (iNOS) protein expression and production of tumor necrosis factor-a (TNF-a) were determined as markers of inflammation.

Example 17.2 : In vivo protocol:

Sprague Dawley rats received a single intraperitoneal dose of 10 mg/kg lipopolysaccharide (LPS) from Escherichia coli (serotype 055: B5). At different times after LPS injection (4, 12 and 24 h), blood samples were collected and the serum analyzed for markers of inflammation and oxidative stress. These include tumor necrosis factor-a (TNF-a), total nitrite/nitrate (NOx) levels, creatine kinase (CK) and lactate dehydrogenase (LDH). After 24 h, the animals were sacrificed and tissues from heart, liver and skeletal muscle were immediately collected and homogenized in ice-cold phosphate buffer (pH=7.4). The resultant homogenate (10% w/v) were used for the determination of inflammatory and anti-inflammatory markers. These include: heme oxygenase-1 (HO-1), malondialdehyde (MDA), gluathione, total nitrite/nitrate (NOx) and myeloperoxidase (MPO) activity.

Example 17.3 : Assay for the determination of nitrite levels.

Nitrite levels were determined using the Griess method. The measurement of this parameter is widely accepted as indicative of NO production and thus inflammation. Briefly, the medium from treated cells cultured in 24-well plates were removed and placed into a 96-well plate (50 μΐ per well). The Griess reagent was added to each well to begin the reaction, the plates were shaken for 10 min and the absorbance measured at 550 nm using a plate reader. The nitrite level in each sample was calculated from a standard curve generated with sodium nitrite (0-300 μΜ in cell culture medium).

Example 17.4 : Determination of cellular glutathione content.

The 5,5'dithiobis-(2-nitrobenzoic acid) colorimetric assay was used for the measurement of glutathione. Briefly, at the end of the incubation period cells were washed with PBS and 600 μΐ of a 2% (w/v) solution of 5-sulfosalicylic acid were added for cell lysis and deproteinization. The samples were centrifuged and 500 μΐ aliquots were reacted reacted with 500 μΐ of 5,5 dithiobis-(2-nitrobenzoic acid) solution (0.3 M sodium phosphate buffer, 10 mM EDTA and 0.2 mM 5,5' dithiobis-(2-nitrobenzoic acid), freshly prepared) and after 5 min the absorbance were read at 412nm (extinction coefficient = 14.3mM ^_1 cm ^"1 ). Positive and negative controls were obtained by incubating macrophages with 1 mM NAC, a precursor of glutathione, or 1 mM DL- buthionine-[S,R]sulfoximine (BSO), an inhibitor of glutathione biosynthesis.

Example 17.5 : Determination of tumor necrosis factor-a (TNF-a).

The level of TNF-a present in each sample was determined using a commercially available kit from R&D Systems (Abingdon, U.K.). The assay was performed according to the manufacturers' instructions. Briefly, cell culture supematants were collected immediately after the treatment and spun at 13,000g for 2 min to remove any particulates. The medium was added to a 96-well plate pre-coated with affinity-purified polyclonal antibodies specific for the mouse TNF-a. An enzyme-linked polyclonal antibody specific for the mouse TNF-a were added to the wells and left to react for 2 h followed by a final wash to remove any unbound antibody-enzyme reagent. The intensity of the color detected at 450nm (correction wavelength 570 nm) were measured after addition of a substrate solution and were proportional to the amount of TNF-a produced. Example 17.6 : Western blot analysis for HO-1 and iNOS protein expression. Samples of

RAW 264.7 cells were analyzed by Western immunoblot technique. Briefly, an equal amount of proteins (30 μg) for each sample were separated by SDS-PAGE, transferred overnight to nitrocellulose membranes, and the nonspecific binding of antibodies were blocked with 3% nonfat dried milk in PBS. Membranes were then probed with a polyclonal rabbit anti-HO-1 antibody (Bioquote, York, UK) (1 :1 ,000, dilution in Tns-buffered saline, pH 7.4) or iNOS (1 :1,000 dilution) antibodies. After three washes with PBS containing 0.05% (vol/vol) Tween 20, blots were visualized with the use of an amplified alkaline phosphatase kit from Sigma (Extra- 3A). For equal loading verification, the samples were probed with a-actin polyclonal antibodies. Example 18: Protocol for diabetes

Example 18.1 : In vitro protocol: Macrophages and endothelial cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cultures were maintained at 37 C in a 5% C02 humidified atmosphere and experiments were conducted on cells at approximately 80-90% confluence. After reaching 90% confluence, cells were exposed to either low (5.5 mM), medium (11 mM) or high (25 mM) glucose concentration (hyperglicemia) for 24 h in the presence or absence of the different concentration of the testing compounds. Cell viability, HO-1 protein expression and activity as well as inflammatory mediators (TNF-a and nitrite levels) were measured at the end of the incubation period. Example 18.2 : In vivo protocol: The Goto-Kakizaki (GK) rat is a non-obese Wistar substrain which develops Type 2 diabetes mellitus early in life. Thus, this is a well-established model of animal diabetes. GK rats were treated with different doses of the testing compounds (5, 10 and 15 mg/kg) given intraperitoneally daily for 4 weeks, a period necessary for hyperglicemia to be normalized. During the entire 4 wk of treatment, fasting glucose was monitored weekly with a glucose-meter. One day before the end of the treatment, the animals were fasted in metabolic cages for 24 h, urine samples collected, and after anesthesia (pentobarbital sodium, 50 mg/kg ip) the animals were sacrificed and blood/plasma/tissues harvested. Markers of inflammation (urinary 8-isoprostane) and oxidative stress (plasma superoxide dismutase and plasma total antioxidant capacity) as well as a direct marker of diabetes (glycated hemglobin) will be measured both in treated and untreated animals.

Example 19: in vitro and in vivo model for cardiovascular diseases

Example 19.1 : In vitro protocol:

Langendorff heart preparations were performed using male Lewis rats (300-350 g).

Hearts were excised, the aorta was cannulated, and retrograde perfusion was established at a constant flow of 15 ml/min using Krebs-Henseleit buffer (in mM: 119 NaCl, 4.7 KC1, 2.5 CaCl ₂ , 1.66 MgS0 ₄ , 24.9 NaHC0 ₃ , 1.18 KH ₂ P0 ₄ , 5.55 glucose, 2.00 sodium pyruvate, and 0.5 EGTA) bubbled with 95% 02-5% C0 ₂ at 37°C (pH 7.4). Coronary perfusion pressure (CPP) was measured by a pressure transducer connected to the aortic cannula. A latex balloon filled with saline was inserted into the left ventricle through the atrium and connected by a catheter to a second pressure transducer. The balloon was inflated to provide an initial end-diastolic pressure (EDP) of 10 mmHg. Both transducers were connected to a computer, and data were acquired with BioPac instrumentation and analyzed with the data acquisition software Acknowledge. Left ventricular developed pressure (LVDP), heart rate (HR), CPP, and EDP were continuously recorded throughout the period of perfusion. Cardiac performance was calculated at each time point from the following formula: (HR x LVDP)/1,000. Isolated hearts were allowed to equilibrate at constant flow for 20 min and were then made globally ischemic by interrupting the buffer perfusion. Hearts were kept at 37°C in the water-jacketed chamber for 30 min and then reperfused. The protocol consisted of a control group (ischemia-reperfusion without treatment) and groups of hearts where the testing compounds (5, 10 and 50 μΜ) were added to the perfusion buffer at the onset of reperfusion. After 60 min of reperfusion, hearts were removed from the aortic cannula and either dissected and fixed for electron microscopy analysis or stained for tissue viability. Example 19.2 : In vivo protocol:

Myocardial infarction was produced in mice by a 30-min coronary occlusion followed by 24 h reperfusion. Briefly, ligation of the left anterior descending coronary artery close to its origin was applied to induce ischemia-reperfusion injury. Measurement of area at risk (AR) and infarct size (IS) were assessed by staining with Evans blue dye (0.5 ml of a 1 % w/v solution in water) injected into the carotid artery catheter to delineate the area at risk (AR). The heart was removed and the left ventricle excised and weighed. After this procedure, the heart was sectioned transversely into five sections with one section being made at the site of ligature, and each section weighed. Sections of the ventricle above the site of the ligature were uniformly completely blue. Sections of the ventricle from the level of the ligature to the apex that were not blue (defining the AR) were then incubated in 1.5% w/v triphenyltetrazolium chloride-in PBS staining, viable myocardium stains brick red and the infarct appears pale white. The area of infarction for each slice was determined by computerized planimetry using an image analysis software program. The protocol consisted of a control group (myocardial infarction without treatment) and groups of animals where the testing compounds (5, 10 and 15 mg/kg) were given intraperitoneally 30 min before myocardial infarction.

Example 20 : A protocol for assessing vasodilatation in vitro and in vivo

Example 20.1 : In vitro protocol: Transverse ring sections of aorta were isolated from male Lewis rats (350 ± 450 g) and suspended under 2 g tension in an organ bath containing 9 ml of oxygenated (95% 0 ₂ /5% C0 ₂ )

Krebs Henseleit buffer (mM): (NaCl 118, KC1 4.7, KH ₂ P0 ₄ 1.2, MgS0 ₄ x 7H ₂ 0 1.2, NaHC0 ₃ 22, glucose 11, K ⁺ EDTA 0.03, CaCl ₂ 2.5). Indomethacin (10 μΜ) was present in the buffer to exclude prostaglandins as potential modulators of vascular tone. Before each protocol was carried out, rings were contracted with a standard dose of KC1 (100 mM) in order to provide an internal reference and to control for variability in contractile responsiveness between tissues. All results were subsequently expressed as a percentage of the KCl-induced contraction. Relaxation responses to acetylcholine (1 ±

1000 nM) in tissues pre-contracted with phenylephrine (3 mM) were used to confirm the integrity of the endothelium in this model. The compounds to be tested (1 -100 μΜ) were added three times at 6-10 min intervals and changes in tension recorded.

Example 20.2 : In vivo protocol: Male Sprague-Dawley rats (300 to 350 g) were housed at constant temperature (23°C) in rooms that provide automatic lighting with a 12-hour on-off cycle. Rats were acclimatized for 2 to 3 days in plastic cages and were given free access to water and food. On the day of surgery, animals were anesthetized with an intramuscular injection of the following mixture: 0.5% ketamine/0.4% acepromazine/1% xylazine (0.2 mL/100 g body wt). Specially designed femoral artery and venous catheters were then surgically implanted and then rats returned to the plastic cages and allowed to recover from the surgical procedure for at least five days. On the day of the experiment, previously instrumented and fully conscious rats were placed in a restrainer and the arterial line was connected to a pressure transducer for continuous mean arterial pressure monitoring and data were recorded using a an acquisition software (Powerlab). Animals were then treated with an intravenous injection of a vasoconstrictor agent (L-nitroarginine methyl ester or L-NAME, 10 mg/kg) followed by administration of our selected testing molecules.