The present invention relates to novel biacyl creatine derivatives, methods of synthesis and therapeutic uses thereof.

Creatine, or methyl-guanidino-acetic acid, is an amino acid of which 95% is found in skeletal muscle, and 5% is distributed between heart, testes, brain and retina.

The creatine present in blood is transported against the concentration gradient into target cells by means of the saturable, sodium- and chlorine-dependent creatine transporter (SLC6A8, CrTl, CT1 or CrT) that crosses the plasma membrane.

The expression of the creatine transporter is present in almost all tissues, but is much higher in tissues with high energy requirements (skeletal muscle, heart, brain, retina) or with creatine uptake function.

Creatine is a polar molecule that is not capable of crossing biological barriers in the absence of its specific transporter (Ohtsuki S, Tachikawa M, Takanaga H, Shimizu H, Watanabe M, Hosoya K, Terasaki T. (2002) The blood-brain barrier creatine transporter is a major pathway for supplying creatine to the brain. J Cereb Blood Flow Metab 22:1327- 35). Even in the presence of such a transporter, it crosses slowly and only partially the blood-brain barrier and cell membranes.

Deficiency of the creatine transporter is a rare genetic disease due to the absence, or failure, of the protein that constitutes the mechanism designed for creatine transport through the blood-brain barrier and cell membranes.

Failure of the creatine transporter results in the absence of creatine in the brain with the consequence, for affected children, of severe neurological damage.

Creatine administered systemically is not capable, in the absence of the transport mechanism, of crossing biological barriers and for this reason the disease is currently incurable. Deficiency of the creatine transporter (OMIM number 300352) is the most frequent inborn error of creatine metabolism, as well as one of the most common causes of X-linked mental retardation. This is a disease that affects infants in the perinatal period, but it is also not infrequent to detect learning and/or behaviour disorders in carriers. Learning disabilities of various degrees were found in about 50% of female carriers.

A form of creatine capable of crossing cell membranes and the blood-brain barrier independently from the creatine transporter could represent a therapeutic tool. This might in fact restore the creatine content inside nerve cells. Also other diseases due to primary shortage of brain creatine, as the deficiencies of arginine glycine amidinotransferase (AGAT, EC 2.1.4.1) and guanidinoacetate methyl transferase GAMT (EC 2.1.1.2), which are the two enzymes that lead to formation of creatine from arginine, glycine and methionine, could benefit from a more bioavailable form of creatine.

Creatine esters are mostly lipophilic, but their enzymatic cleavage provides creatinine (Lunardi G, Parodi A, Perasso L, Pohvozcheva AV, Scarrone S, Adriano E, Florio T, Gandolfo C, Cupello A, Burov SV, Balestrino M. (2006) The creatine transporter mediates the uptake of creatine by brain tissue, but not the uptake of two creatine-derived compounds. Neuroscience 142(4):991-997; Adriano E, Garbati P, Damonte G, Salis A, Armirotti A, Balestrino M. (201 1) Searching for a therapy of creatine transporter deficiency: some effects of creatine ethyl ester in brain slices in vitro. Neuroscience 199:386-393).

The state of the art discloses the obtainment of a creatine derivative that bears an amide group in place of the carboxylic group. According to the teachings of H. R. Ing (Creatine and Creatinine. Part 1 1. Alleged Acyl Derivatives of Creatine. Journal of the Chemical Society Issue 0, page 2198, year 1932) diacetylation of creatine is obtained, where one of the two acetyl groups is bound to the carboxyl group and the other one is bound to one of the nitrogen atoms of the guanidine group, as shown in formula (A) that follows:

FORMULA (A)

However, this molecule is not sufficiently lipophilic.

An object of the present invention is thus to overcome the above shortcomings of the state of the art by providing a creatine derivative that is capable of crossing biological barriers by diffusion and therefore without the aid of the creatine transporter.

Another object of the present invention is to provide a creatine derivative that is effective in the treatment of diseases caused by deficiency of cerebral creatine.

These and other objects are achieved by a creatine derivative of formula (I) which, in the following, will be referred to as "carboxylic biacyl creatine derivative", and pharmaceutically acceptable salts thereof. Formula I i shown below:

FORMULA (I)

wherein R and Ri are independently selected from the group consisting of substituted or unsubstituted, saturated or unsaturated aliphatic chains having from 1 to 18 carbon atoms, saturated or unsaturated aliphatic cycles, saturated or unsaturated heterocycles, aromatic and heteroaromatic rings, and wherein R is selected from the group consisting of substituted or unsubstituted, saturated or unsaturated aliphatic chains having from 1 to 18 carbon atoms, saturated or unsaturated aliphatic cycles, saturated or unsaturated heterocycles, aromatic and heteroaromatic rings, biomolecules, such as for example amino acids, vitamins, carbohydrates and lipids.

The compounds of formula (I) of the present invention and the pharmaceutically acceptable salts thereof solve the above mentioned technical problem as these are molecules that are more lipophilic compared to creatine and also because the two acyl groups on the guanidine group protect the molecule from enzymatic cleavage into creatinine. In fact, the ester bond is the first one to be hydrolysed because of its reduced stability, while the amide bond is more stable and therefore the guanidine group will be made available later for phosphorylation by the enzyme creatine kinase.

Furthermore, the compound of formula (A) of the state of the art is a less lipophilic derivative compared to the compounds forming the subject of the present invention in so far as: (i) the anhydride on the carboxyl group is less stable - in aqueous media in comparison with the esters present in the compounds that are the subject of the present invention, (ii) the unsubstituted nitrogen atom represents a polar moiety capable of increasing the hydrophilicity of the molecule, (iii) the partition coefficient (log Pow) calculated for the compound of formula (A) of the state of the art is -0.6862, while the molecule of formula (IA) shown here below, which is one of the less lipophilic molecules among those falling within formula (I) of the present invention, exhibits a partition coefficient of -0.0972.

FORMULA (IA)

Pow is indicated on a base-10 logarithmic scale, i.e. as log Pow:

The values obtained by the above equation for the molecules of formula (A) and (IA) mentioned above indicate that the molecule of formula (IA) is more lipophilic.

According to a preferred embodiment, R and Ri in the formula (I) are independently selected from methyl, ethyl and propyl.

According to another preferred embodiment, R ₂ is selected from methyl, ethyl and propyl.

According to a further preferred embodiment, a pharmaceutically acceptable salt is a salt of the compounds according to the present invention that has no toxicity or an acceptable degree of toxicity, obtained by addition of inorganic or organic acids or bases. The following examples are to be understood as illustrative and by no means limitative of the salts that can be prepared from the carboxylic biacyl creatine derivative of formula (I) on the basis of the specific structure accomplished: ammonium salts, hydrochlorides, hydrobromides, sulfates, hydrogen sulfates, dihydrogen phosphates, citrates, malates, fumarates, tosylates, mesylates, phosphates, salicylates, tartrates, lactates, citrates, benzoates, succinates, acetates, trifluoroacetates, 2-naphthalenesulfonates, p- toluenesulfonates, potassium salts, sodium salts, lithium salts, calcium salts, magnesium salts, zinc salts, and aluminum salts.

The carboxylic biacyl creatine derivative of formula (I) forming the subject of the present invention is synthesized through an extremely simple process, which contemplates the use as a precursor of a commercially available (e.g. from Sigma Aldrich) carboxylic creatine derivative of formula (II), wherein R ₂ has the meanings indicated above in relation to formula (I), which is converted into the carboxylic biacyl creatine derivative of formula (I). The conversion of the carboxylic creatine derivative of formula (II) into the carboxylic biacyl creatine derivative of formula (I) is obtained by the use of an acylating agent on both of the nitrogen atoms of the guanidine group, which allows for the direct synthesis thereof.

FORMULA (II) In the method of the invention, the carboxylic creatine derivative of formula (II) is preferably creatine ethyl ester.

The method of the invention advantageously allows for the obtainment of high yields and of an excellent purity of the final product carboxylic biacyl creatine derivative of formula

(I).

In a preferred embodiment of the method of the invention, acetic anhydride (CAS 108-24- 7) or butyric anhydride (CAS 106-31-0) is used as the acylating agent. The yields obtained with these two acylating agents are similar.

The reaction of the obtained compound with an anionic or cationic partner selected from the pharmacologically acceptable ones results in a salt of the carboxylic biacyl creatine derivative of formula (I). For the obtaining of the salt with basic compounds, it is possible to choose to deprive the molecule in question of the substituent R ₂ to leave the oxygen of the carboxylic group free to form the selected salt.

Alternatively to the synthesis method described above, a compound of formula (I) according to the present invention can be obtained according to the following reaction scheme, in which, in a first step, an alkyl thiourea of formula (III), wherein R4 is a short- chain alkyl (preferably a C 1 -C4 alkyl, such as for example methyl), is acylated by reaction with an acylating agent of formula (IV), wherein R and R _\ are as defined above, to give an intermediate compound of formula (V), which, in a second step, is esterified by reaction with a sarcosine ester of formula (VI), wherein R ₂ is as defined above, to obtain the compound of formula (I) of the invention: F

FORMULA V

R1 +

. ₌ 0 trieth lamine

o O FORMULA VI

R

FORMULA I

The reaction of the obtained compound with an anionic or cationic partner selected from the pharmacologically acceptable ones results in a salt of the carboxylic biacyl creatine derivative of formula (I). For the obtaining of the salt with basic compounds, it is possible to choose to deprive the molecule in question of the substituent R ₂ to leave the oxygen of the carboxylic group free to form the selected salt.

The acylating agent of formula (IV) used in the synthesis method described above is also employed in the synthesis method previously described in which the carboxylic creatine derivative of formula (II) is used as the precursor.

As indicated previously, the carboxylic biacyl creatine derivative of formula (I) of the present invention is capable of crossing biological barriers and the blood-brain barrier independently from the creatine transporter and is also protected from enzymatic cleavage into creatinine. It is therefore effective in the therapeutic treatment of pathologies due to deficiency of cerebral creatine, such as creatine transporter deficiency, AGAT deficiency, and GAMT deficiency. A further aspect of the present invention is therefore the carboxylic biacyl creatine derivative of formula (I),, as defined above, for use in the therapeutic treatment of pathologies due to deficiency of cerebral creatine, such as creatine transporter deficiency, AGAT enzyme deficiency and GAMT enzyme deficiency.

Therefore, a pharmaceutical composition comprising as the active ingredient the carboxylic biacyl creatine derivative of formula (I), as defined above, is also included within the scope of the present invention. Such a pharmaceutical composition also contains additional ingredients such as pharmaceutically acceptable carriers, excipients and/or diluents known per se to those of ordinary skill in the art. The pharmaceutical composition is formulated into any suitable form, such as tablets, solutions, suspensions, aerosols, or any other appropriate form for administration of the active ingredient. The administration may for example take place by intravenous, subcutaneous or intramuscular injection, or orally, ophthalmologically, nasally, buccally, rectally, even if the parenteral administration is preferred.

When used for treating the pathologies mentioned above, the amount of the active principle will be chosen by the attending physician considering various factors such as, for example, the specific disease to be treated, the severity of the disease, the age, weight and state of health of the patient. For instance, but without limitation, there is administered an amount of active principle which is suitable to provide a blood concentration of the active ingredient in the range between 0.1 and 10 mM (millimolar). Also the frequency and the duration of the treatment will be chosen taking into account various factors related to the specific disease to be treated, the patient's status, the toxicity and the effectiveness of the active ingredient. The person of average skill in the art is able to select the correct amount of active ingredient to be administered and the correct administration regimen.

The examples that follow are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims.

The structure of the synthesized molecules was checked by mass spectrometry analysis. Example 1 : Synthesis of biacetyl creatine ethyl ester. (Ac CEE

(Reaction Scheme 1 )

A solution of DMF, acetic anhydride and triethylamine in a 9:0.5:0.5 ratio is added to a solution of creatine ethyl ester (1 equivalent) in anhydrous N,N-dimethylformamide (DMF). The suspension is kept under stirring at room temperature until the reaction is completed, as monitored by thin layer chromatography (TLC). Indicatively, depending on the quantities used, the reaction times range from 3 to 24 hours. Upon completion, the reaction mixture is evaporated to minimum volume and subsequently freeze-dried in order to remove the solvents. The oil thus obtained is taken up in ether and subsequently crystallized to obtain the final product.

Reaction Scheme 1 :

Example 2: Synthesis of dibutyryl creatine ethyl ester, (butyrylV CEE

(Reaction Scheme 2)

A solution of DMF, butyric anhydride and triethylamine in a 9:0.5:0.5 ratio is added to a solution of creatine ethyl ester (1 equivalent) in anhydrous N,N-dimethylformamide (DMF). The suspension is kept under stirring at room temperature until the reaction is completed, as monitored by TLC. Indicatively, depending on the quantities used, the reaction times range from 3 to 24 hours. Upon completion, the reaction mixture is evaporated to minimum volume and subsequently freeze-dried in order to remove the solvents. The oil thus obtained is taken up in ether and subsequently crystallized to obtain the final product.

Reaction Scheme 2:

Example 3 : Synthesis of biacetyl creatine ester

(Reaction Scheme 3)

A 5M NaOH solution is added to a solution of methylisothiourea (1 equivalent) in cold deionized water and maintained in an ice bath.

Then, the following is added: acetic anhydride (3 equivalents) and a 1M aqueous sodium carbonate solution. The pH is then maintained at 6 with 1 N NaOH. The suspension is kept under stirring in an ice bath until the reaction is completed, as monitored by TLC.

Once the reaction has occurred, the resulting suspension is filtered and washed twice with cold water.

The aqueous filtrate is then evaporated, taken up in ethyl acetate and washed with water. The organic layer thus obtained is then evaporated and subsequently freeze-dried.

The freeze-dried organic phase is dissolved in anhydrous Ν,Ν-dimethylformamide (DMF). The following is added: 1 equivalent of sarcosine ethyl ester, 3 equivalents of triethylamine and 1.1 equivalents of HgCl ₂ . The reaction is kept under stirring at room temperature until completion, as monitored by TLC. Upon completion, the reaction mixture is taken up in an organic solvent (ether or ethyl acetate according to the type of sarcosine ester used) with the formation of an abundant white precipitate. This precipitate is filtered under vacuum. The filtrate is washed with deionized water and the organic layer is evaporated to minimum volume and subsequently freeze-dried to obtain the final product.

Reaction Scheme 3:

Example 4: Biological Effects

In preliminary in vitro experiments, the effects of one of the subject compounds of the present patent, biacetyl creatine ethyl ester, were tested. These effects were assessed by using slices of mouse hippocampus in which the creatine transporter was pharmacologically blocked by the use of guanidinopropionic acid (GPA). The result of this block is an in vitro mimicry of the human genetic disease, i.e. creatine transporter deficiency.

Exposure of the slices to GPA is known to be detrimental, and this entails the survival of about 40/50% of them. This damage is due to impairment of normal absorption and of the normal exchange of creatine between glial cells and neurons (Braissant O, Beard E, Torrent C, Henry H. (2010) Dissociation of AGAT, GAMT and SLC6A8 in CNS: relevance to creatine deficiency syndromes. Neurobiol Dis 37(2): 423-33; Braissant O, Henry H, Loup M, Eilers B, Bachmann C. (2001) Endogenous synthesis and transport of creatine in the rat brain: an in situ hybridization study. Brain Res Mol Brain Res 31 ; 86(1 - 2): 193-201).

The present inventors have observed that biacetyl creatine ethyl ester is able to abolish the negative effects of GPA, which instead creatine is not able to do, thereby protecting the nervous tissue from damage. The effect of biacetyl creatine ethyl ester is statistically significant, as shown in figure 1 (the P-value is indicated for the analysis of variance, ANOVA).

Figure 2 shows that this compound is able to increase the content of phosphocreatine in mouse hippocampus slices in the presence of a blockage of the creatine transporter induced in this case by using a chlorine-free incubation medium. The creatine transporter works with a sodium-chlorine symport mechanism, therefore it does not work in the absence of chlorine (Dai W, Vinnakota S, Qian X, Kunze DL, Sarkar H . (1999) Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes. Arch Biochem Biophys 361 :75-84). This demonstrates that such a molecule is capable of entering the nervous tissue cells even when the creatine transporter is not working, as is the case in creatine transporter deficiency, and that it is capable of releasing creatine in cells, which is then phosphorylated into phosphocreatine.

Figure 3 shows the effects of biacetyl creatine ethyl ester in hippocampus slices subjected to electrophysiology experiments carried out in the presence of GPA to mimic the creatine transporter deficiency disease.

In these experiments the slices were subjected to anoxia after 3 hours of incubation with biacetyl creatine ethyl ester in the presence of GPA.

These experiments demonstrated that the slices treated with biacetyl creatine ethyl ester have population spikes that last longer during anoxia both compared to the slices treated with creatine and the untreated controls.