HOMOCYSTEINE ANALOGUES Technical Field

The present invention relates generally to the pharmacological field and more particularly relates to a new class of compounds which are homocysteine analogues. Further, the invention relates to the use of this class of compounds to prepare a drug for treating a disorder linked to the action of homocysteine. Background Art

Homocysteine is a sulfurated amino acid whose production in the body occurs by methionine demethylation. Normally, homocysteine levels are regulated so as to stay below 13-15 μM, especially by methionine remethylation, in a reaction which requires the presence of folate and vitamin B 12, and cysteine synthesis, during which homocysteine is condensed with serine. However, plasma values of homocysteine can increase in case of metabolic dysfunctions or as a consequence of pharmacological therapies: for example, in subjects treated with L-DOPA a conspicuous increase in the plasma value of homocysteine is observed, since L-DOPA metabolism leads to the formation of this sulfurated amino acid.

It has been found that a high level of homocysteine in plasma, a condition known as hyperhomocysteinemia, is a common risk factor for vascular diseases such as ictus and myocardial infarction and also for many forms of tumor, such as glioblastoma. Moreover, a link has been suggested between hyperhomocysteinemia and diseases such as spina bifida, meningocele, autism, depression, schizophrenia, Alzheimer's disease, Parkinson's disease and Huntington's disease.

Experimental data acquired over time have demonstrated that the pathological action of homocysteine is linked to the formation of free oxygen radicals by means of a self-oxidation mechanism. Moreover, homocysteine also has a more specific action which involves precise molecular targets. It has in fact been demonstrated that homocysteine has a negative

allosteric action on D2 receptors, the receptors involved for example in the treatment of Parkinson's disease and of schizophrenia (Agnati et al., J. Proteome Res., Vol. 5, p. 3077-3083, 2006). Homocysteine binds to D2 receptors in an allosteric site which is different from the active site and is constituted by a region which is rich in arginine, with which homocysteine can establish a bond by means of electrostatic interactions. As demonstrated by Agnati et al., the interaction between D2 receptors and homocysteine leads to a reduction in signal transmission mediated by the D2 receptor pathway. This mechanism partly helps to explain the reduction in effectiveness, over time, of L-DOPA therapy in patients with Parkinson's disease: the administration of L-DOPA leads to an increase in homocysteine levels and, while L-DOPA activates D2 receptors, homocysteine tends to have a negative action on the same receptors; therefore, an ever increasing quantity of L- DOPA has to be administered in order to contrast the inhibiting effects of homocysteine.

The action of homocysteine on NMDA receptors occurs in a similar manner, by means of the binding of the sulfurated amino acid at the level of an allosteric site on the NMDA receptor (Lipton et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 94, p. 5923-5928, 1997). The effects of this interaction are characterized in the form of a positive allosteric action, with consequent overstimulation of NMDA receptors, which is followed by a high release of calcium ions, whose massive inflow into cells triggers apoptosis effects (neurotoxicity).

Further experimental evidence shows that homocysteine has a specific action in the development of Alzheimer's disease: circular dichroism studies have shown that homocysteine interacts with the amyloid beta 40 and 42 (AB ₄₀ and AB ₄₂ ) peptide, facilitating its acquisition of the so-called "B-sheet" configuration, which is the first step in the formation of amyloid aggregates and toxic compounds which have a pathogenetic significance in Alzheimer's disease.

As regards instead the development of forms of tumor, particularly glioblastoma, it has been discovered that homocysteine can act at the level of the p53 protein, whose genetic dysfunction is often the basis of forms of tumor, both by controlling its cell expression and by facilitating a conformational change therein, with cascade effects on cell replication and/or survival.

In view of the involvement of homocysteine in the diseases cited above, for example by means of the ability to modulate specific receptors, this compound is potentially useful in the field of pharmacological therapies. However, as mentioned, plasma levels of homocysteine above 13-15 μM are characterized by toxic effects, caused partly by the formation of oxygen radical during self-oxidation of the homocysteine itself and partly by neurotoxicity phenomena induced for example by overstimulation of NMDA receptors. Therefore, there is the need to provide compounds which are homocysteine analogues and can have therapeutic effects, also by modulating the activity of receptors which are naturally the target of homocysteine, but lack the characteristic contraindications of homocysteine, such as neurotoxicity and the capacity to form free radicals. Disclosure of the Invention

The aim of the present invention is to provide a homocysteine analogue compound capable of acting on the same molecular targets as homocysteine, but without exhibiting its contraindications.

Within this aim, an object of the invention is to provide a use of the homocysteine analogue compound according to the invention to prepare a drug for treating a disease selected from the group constituted by spina bifida, meningocele, autism, depression, schizophrenia, an extrapyramidal disease, a neurodegenerative disease, a form of dementia, a memory deficit, a motor deficit, Huntington's disease, a form of tumor and a cardiocerebrovascular disease.

Summary of the Invention

This aim and this and other objects which will become better apparent hereinafter are achieved by a compound selected from the groups of compounds having the formula:

( D

( H ) , or

( I I I )

where:

Rl and R2 are, independently of each other, hydrogen, -alkyl-SH, -CO-alkyl, -(CH ₂ ) _n -X _l5 -(CH ₂ ) _n -Z, -COH or -CS-alkyl;

R3 and R4 are, independently of each other, hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, -(CH^-X, or -(CH ₂ ) _n -Z; R5 is -OH, -O-alkyl, -O-haloalkyl, -SH, -S-alkyl or -N(R6)(R7);

R6 and R7 are, independently of each other, hydrogen, alkyl, alkenyl, alkynyl or haloalkyl;

X is hydrogen, alkyl, alkenyl, alkynyl, haloalkyl or -(CH ₂ ) -;

X ₂ is hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, -(CH ₂ ) - or -(CH ₂ ) _n -X ₁ ; when X or X are not hydrogen, Z and Z are, independently of each other, hydrogen, -SH, -OH, -SeH, -TeH, -0-PO ₃ H ₂ , -O-SO ₃ H or form a ring with Rl, R2, R3 or R4 in which Z or Z ₂ are -O-, -S-, -Se- or -Te-; m is 0, 1 or 2; n is 1, 2 or 3; p is 0, 1, 2 or 3.

Ways of carrying out the Invention

In the context of the present description and the claims set forth herein, the following terms have the meaning given hereinafter.

The term "alkyl" identifies a linear or branched hydrocarbon chain containing at least one carbon atom. Non-limiting examples of an alkyl are constituted by a functional group selected among methyl, ethyl, n-propyl, iso- propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylbutyl, 3-methylhexyl, 3,3-dimethylbutyl, 2,2- dimethylpentyl, 2,3-dimethylpentil-n-heptyl, n-octyl, n-nonyl and n-decyl. The term "alkenyl" identifies a linear or branched hydrocarbon chain containing at least two atoms of carbon and at least one double carbon-carbon bond formed by the removal of two hydrogen atoms. Non-limiting examples of an alkenyl are represented by a functional group selected among ethenyl, 2- propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l-heptenyl and 3-decenyl.

The term "alkynyl" identifies a linear or branched hydrocarbon chain containing at least two carbon atoms and at least one triple carbon-carbon bond. Non-limiting examples of an alkynyl are represented by a functional group selected among acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-

pentynyl and 1-butynyl.

The term "haloalkyl" identifies an alkyl group as defined above, in which at least one atom is substituted by a halogen, i.e., -Cl, -Br, -I or -F.

Non-limiting examples of an alkyl halide are represented by a functional group selected among chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl and 2-chloro-3-fluoropentyl.

The term "-CO-alkyl" identifies an alkyl group as defined above, in which a carbon atom of the chain is bonded to a carbonyl group (-CO-). Non- limiting examples of a -CO-alkyl are represented by a functional group selected among -CO-methyl, -CO-ethyl and -CO-propyl.

The term "-CS-alkyl" identifies an alkyl group as defined above, in which a carbon atom of the chain is bonded to a -CS- group, where the sulfur atom and the carbon atom are joined by a double carbon-sulfur bond. Non- limiting examples of a -CS-alkyl are represented by a functional group selected among -CS-methyl, -CS-ethyl and -CS-propyl.

The term "-O-alkyl" identifies an alkyl group as defined above, in which a carbon atom of the chain is bonded to an oxygen atom. Non-limiting examples of an -O-alkyl are represented by a functional group selected among -O-methyl, -O-ethyl and -O-propyl. The term "-O-haloalkyl" identifies an alkyl group halide as defined above, in which a carbon atom of the chain is bonded to an oxygen atom. Non-limiting examples of an -O-alkyl halide are represented by a functional group selected among O-chloromethyl, -0-2-fluoroethyl, -O-trifluoromethyl, - O-pentafluoroethyl and -O-2-chloro-3-fluoropentyl. The term "-S-alkyl" identifies an alkyl group as defined above, in which a carbon atom of the chain is bonded to a sulfur atom. Non-limiting examples of an -S-alkyl are represented by a functional group selected among -S- methyl, -S-ethyl and -S-propyl.

The term "alkyl-SH" identifies an alkyl group as defined above, in

which a carbon atom of the chain is bonded to a thiol group (-SH). Non- limiting examples of an alkyl-SH are represented by a functional group selected among methyl-thiol, ethyl-thiol and propyl-thiol.

For example, the homocysteine analogue compound may be selected from the group of compounds consisting of:

or

In another aspect, the present invention consists of the use of the homocysteine analogue compound of formula (I), (II) or (III) described herein to prepare a drug for treating a disease selected from the group consisting of spina bifida, meningocele, autism, depression, schizophrenia, an extrapyramidal disease, a neurodegenerative disease, a form of dementia, a memory deficit, a motor deficit, Huntington's disease, a form of tumor and a cardiocerebrovascular disease.

The compound according to the invention may be used to treat a disease

in which a role for homocysteine has been determined, be it due to the induction of oxygen radicals, modulation of the activity of a receptor, such as for example D2 dopaminergic receptors or NMDA glutamatergic receptors, or as a consequence of direct interaction of homocysteine with specific protein complexes, such as protein p53 or an amyloid beta peptide.

In particular, the compound according to the invention may be used to prepare a drug for treating a disease falling within the group consisting of spina bifida, meningocele, autism, depression, schizophrenia, an extrapyramidal disease, a neurodegenerative disease, a form of dementia, a memory deficit, a motor deficit, Huntington's disease, a form of tumor and a cardiocerebrovascular disease.

In a preferred embodiment, the treated cardiocerebrovascular disease may be for example ictus or myocardial infarction, while the form of tumor may be for example glioblastoma. In another preferred embodiment, the treated disease is an extrapyramidal disease; in particular, it may be Parkinson's disease. In another preferred embodiment, the treated disease is a neurodegenerative disease, which may be for example Alzheimer's disease.

Typically, the disease may be treated by administering the compound according to the invention, either alone or in combination with another active ingredient, for example as part of a therapeutic regimen.

In a preferred embodiment, the invention consists of the use of the compound of formula (I), (II) or (III) to prepare a drug for treatment of a neurodegenerative disease, particularly Alzheimer's disease, by combined administration with a drug which acts on NMDA receptors. This drug may be for example memantine. As regards the doses of these drugs to be used during combined therapy, one can proceed with "patient titration", establishing the threshold minimum dose, i.e., the minimum dose that is effective to significantly reduce the symptoms in the opinion of the physician and of the

patient, and then use a dose of a drug that acts on NMDA receptors just below this threshold.

In another preferred embodiment, the invention consists of the use of the compound of formula (I), (II) or (III) to prepare a drug for treating Parkinson's disease, by combined administration with a drug for treating Parkinson's disease. This drug may be selected from the group consisting of L-DOPA, an L-DOPA analogue, a dopaminergic agonist, amantadine, selegiline and an acetylcholine antagonist. Preferably, the dopaminergic agonist is bromocriptine. The acetylcholine antagonist may be for example selected from the group consisting of benztropine, biperiden and trihexyphenidyl. As regards the doses of these drugs to be used during combined therapy, one can proceed with "patient titration", establishing the threshold minimum dose, i.e., the minimum dose that is effective to significantly reduce the symptoms in the opinion of the physician and of the patient, and then use a dose of anti-Parkinson's drug that is just below this threshold.

In another preferred embodiment, the invention consists of the use of the compound of formula (I), (II) or (III) to prepare a drug for treating schizophrenia, by combined administration with a neuroleptic. Preferably, said neuroleptic may be selected from the group consisting of a phenothiazine, a butyrophenone and a benzamide. For example, the phenothiazine may be flupentixol. Typically, a butyrophenone used to treat schizophrenia may be haloperidol. Finally, an example of benzamide that is useful for combined treatment is sulpiride. As regards the doses of the neuroleptics to be used during combined therapy, one can proceed with "patient titration", establishing the threshold minimum dose, i.e., the minimum dose that is effective to significantly reduce the symptoms in the opinion of the physician and of the patient, and then use a dose of neuroleptic that is just below this threshold. In another preferred embodiment, the invention consists of the use of

the compound of formula (I), (II) or (III) to prepare a drug for treating a memory deficit by combined administration with a cholinergic drug. Typically, the cholinergic drug may be selected from the group consisting of an angiotensin conversion enzyme inhibitor, an alternative drug, an atypical neuroleptic and an acetylcholinesterase inhibitor. An alternative drug, such as a homeopathic product, is for example ginseng or a derivative of vitis vinifera. Typically, the atypical neuroleptic may be selected from the group consisting of amisulpride and clozapine. Examples of an acetylcholinesterase inhibitor useful in combined therapy are constituted by donezepil, rivastigmine or galantamine.

Without intending to be constrained to any theory in particular, it is believed that the therapeutic action of the compound according to the invention is due to its characteristics of homocysteine analogue, thanks to which it can interact with the molecular targets that are characteristic of the sulfurated amino acid, without however having the contraindications of homocysteine.

For example, since the activity of D2 dopaminergic receptors is linked to diseases such as Parkinson's disease and schizophrenia, the compound according to the invention may be used to treat these disorders by way of its ability to act as a D2 dopaminergic receptor modulator.

The activity of NMDA glutamatergic receptors is linked to various physiological processes, which also include learning and memory mechanisms; therefore, the use of the compound according to the invention can aid in treating diseases that depend on dysfunction in signal transduction mediated by NMDA receptors, such as for example memory deficits, without however stimulating the neurotoxic effects of homocysteine.

In the field of the treatment of Parkinson's disease, a treatment with a drug prepared starting from the compound according to the invention may be accompanied at a later time by the administration of a drug for treating Parkinson's disease, with the further advantage of being able to use reduced

doses of these compounds. It is thus possible to avoid the side effects caused for example by repeated administration of L-DOPA or of dopaminergic agonists.

Likewise, in the treatment of schizophrenia, a treatment with a drug prepared starting from the compound according to the invention may be accompanied at a later time by the administration of neuroleptic, which can be used in reduced therapeutic doses in order to avoid the induction of side effects in patients.

Also in the treatment of memory deficit, a treatment with a drug prepared starting from the compound according to the invention may be assisted by the administration of another drug, typically a cholinergic drug.

The disclosures in Italian Patent Application No. PD2007A000209 from which this application claims priority are incoiporated herein by reference.