Method for preparing creatine fatty esters, creatine fatty esters thus prepared and uses thereof

Abstract

The present invention concerns a method for preparing a creatine fatty ester or derivative thereof comprising at least one step consisting in reacting a diprotected creatinine with a molecule bearing at least one alcohol functional group and of formula ROH in which R represents a hydrocarbon radical containing at least 4 carbon atoms. The present invention also concerns particular creatine fatty esters or derivative thereof and medical uses thereof.

Claims

1. A method for preparing a creatine fatty ester or derivative thereof comprising: at least one step including reacting a diprotected creatinine with a molecule bearing at least one alcohol functional group and of formula (I):
ROH(I) in which R is a hydrocarbon radical containing at least 4 carbon atoms, wherein the diprotected creatinine includes two protecting groups each replacing a hydrogen atom substituent of a nitrogen atom of the diprotected creatinine, the two protecting groups being identical or different, wherein the creatine fatty ester derivative has a carboxylic acid group (COOH) replacing at least one hydrogen atom of the methylguanidinyl group of the creatine fatty ester, and wherein the creatine fatty ester derivative is obtained after a partial deprotection of a diprotected creatinine, the protecting groups of which are formula C(O)OR.sub.4 with R.sub.4 being a hydrocarbon group.

2. The method according to claim 1, wherein the radical R is chosen from the group consisting of an alkyl radical with 4 to 30 carbon atoms, an alkenyl radical with 4 to 30 carbon atoms, and an aryl radical with 6 to 30 carbon atoms.

3. The method according to claim 1, wherein the radical R is of the following formula (VIII):
CH.sub.2R.sub.1(VIII) in which R.sub.1 is a hydrocarbon radical containing at least 3 carbon atoms.

4. The method according to claim 1, wherein the radical R is a glucosyl radical optionally substituted.

5. The method according to claim 1, wherein said method comprises the following successive steps: a) reacting creatinine with a protective agent to obtain the diprotected creatinine; b) reacting the diprotected creatinine obtained at step (a) with a molecule bearing at least one alcohol functional group and of formula (I) to obtain a diprotected creatine fatty ester; and c) deprotecting the diprotected creatine fatty ester obtained at step (b), in order to obtain said creatine fatty ester or derivative thereof.

6. The method according to claim 5, wherein said protective agent is of formula (XII):
ClC(O)OR.sub.4(XII) in which the radical R.sub.4 is a hydrocarbon group.

7. The method according to claim 5, wherein a solvent in a solution containing the creatinine and the protective agent implemented at step (a) is dichloromethane (DCM).

8. The method according to claim 5, wherein a solution containing the creatinine and the protective agent implemented at step (a) contains the Hnig's base or N,N-diisopropyl ethylamine (DIEPA).

9. The method according to claim 5, wherein, at step (b), for one equivalent of the diprotected creatinine, the amount of molecule bearing at least one alcohol functional group and of formula (I) expressed in equivalents is between 1 and 15.

10. The method according to claim 5, wherein said step (b) is carried out during 1 to 20 h.

11. The method according to claim 5, wherein said step (b) is carried out at a temperature between 60 and 100 C.

12. A compound prepared by a method according to claim 1, said compound having the formula (III), (IV) or (V):
(NH.sub.2)C(NH)N(CH.sub.3)CH.sub.2COOR(III) in which radical R is a glucosyl radical optionally substituted, or a salt thereof.

13. A composition comprising at least one compound according to claim 12, in an acceptable vehicle, wherein said composition is a food additive or a nutritional supplement.

14. A pharmaceutical, diagnostic or imaging composition comprising at least one compound according to claim 12, in an acceptable pharmaceutical vehicle.

15. A method for treating or preventing at least one disease, disorder, or condition selected from the group consisting of hypoxia and ischemic brain disease, comprising administering to a subject in need, a therapeutic amount of a compound according to claim 12.

16. A method for treating or preventing at least one disease, disorder or condition selected from the group consisting of hypoxia and ischemic brain disease, comprising administering to a subject in need, a therapeutic amount of a compound according to claim 14.

17. The method according to claim 9, wherein the amount of molecule bearing at least one alcohol functional group and of formula (I) expressed in equivalent is between 1 and 10.

18. The method according to claim 10, wherein said step (b) is carried out during 2 to 16 h.

19. The method according to claim 18, wherein said step (b) is carried out during 2 to 10 h.

20. The method according to claim 11, wherein said step (b) is carried out at a temperature between 70 and 90 C.

21. The method according to claim 20, wherein said step (b) is carried out at a temperature at around 80 C. (i.e. 80 C.5 C.).

Description

SHORT DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 presents the translocation of creatine fatty ester C18 through the BBB.

(2) FIG. 2 presents the conversion of C18 into creatine.

(3) FIG. 3 presents the translocation of creatine fatty ester C2 through the BBB.

(4) FIG. 4 presents scheme 1 depicting the preparation of creatine esters.

(5) FIG. 5 presents scheme 2 depicting the protection of creatine.

(6) FIG. 6 presents scheme 3 depicting the synthetic sequences of a creatine stearoyl ester preparation.

(7) FIG. 7 presents scheme 4 depicting the protection of creatinine.

DETAILED DISCUSSION OF PARTICULAR EMBODIMENTS

(8) All the reagents are purchased from Aldrich (Steinheim, Germany). TLC was performed on Merck F 254 plates using specified solvent system. Analytical and preparative LC/MS were performed on Waters Autopurify System (SCBM). Biological evaluation of targets was performed on yyy (SPI). .sup.1H and .sup.13C NMR spectra were recorded at 400 MHz on a Bruker spectrometer.

(9) I. Comparative Preliminary Results.

(10) I.1. Direct Synthesis of Esters from Creatine.

(11) The inventors tried to prepare creatine stearoyl ester using the proceedings disclosed in [7-8]. The results are presented in Table 1:

(12) TABLE-US-00001 TABLE 1 Substrate (1 mM) Reagents Conditions Yield Creatine, Stearyl alcohol 40 C., overnight 0% hydrate 10 eq SOCl.sub.2 3 eq Creatine, KOH in situ acyl 0% hydrate Stearyl alcohol chloride production 10 eq Potassium salt of SOCl.sub.2 3 eq creatine Reflux 1 h Creatine Stearyl alcohol Sealed tube 0.1% anhydrous 10 eq 80 C., 6 h Purified by SOCl.sub.2 3 eq LC/MS Creatine Stearyl alcohol Microwave 0% anhydrous 10 eq 160 C. 20 min Production of SOCl.sub.2 3 eq creatine acyl chloride Creatine Stearyl alcohol 130 C. 2 h 30 0% anhydrous 10 eq CH.sub.3SO.sub.3H 3 eq Creatine Ethanol, SOCl.sub.2 Reflux 60 C. 1 h 50% anhydrous RT 72 h crystallized

(13) I.2. Protection of Creatine.

(14) The inventors tried to prepare creatine fatty esters by using protected creatine. Two protective groups (PG) were developed on creatine (see Table 2) according to [12-13] (see scheme 2 in FIG. 5).

(15) TABLE-US-00002 TABLE 2 protection of creatine Protective Conditions Reagent Stoech. (RT) Base Stoech. Yield FmocCl 3 eq DCM DIEPA 3 eq 5% FmocCl 1 eq DCM TMSCl/DIEPA 4 eq/3 eq 11% Z.sub.2O 4 eq Dioxane/Water NaOH 1 eq 0% ZCl 3 eq DCM DIEPA 3 eq 33%

(16) With creatine, only the monoprotected derivative was produced. Unfortunately this compound was not reactive for the following step of the synthesis. Moreover, the inventors isolated a new compound not useful obtained by addition of the chloroacetyle on the methylene.

(17) In addition, synthesis with the FMOC group led to diprotected creatinine after self cyclization in the reaction media.

(18) I.3. Synthesis of Esters from Monoprotected Creatine.

(19) The protocols and yields are summarized in Table 3.

(20) TABLE-US-00003 TABLE 3 Substrate Reagents Yield (Z)creatine DMF/DCC/DMAP 0% (Z)creatine THF/TEA/trichlorobenzoylchloride 0% (Z)creatine Stearyl alcohol/ 1.3% 4-pyrrolidinopyridine (Z)creatine-TriCl in situ production 0% benzoyle Stearyl alcohol/NaH/THF

(21) Even with the activation of the acid function of creatine by trichlorobenzoyl chloride on protected creatine, only very poor yields were obtained.

(22) II. Synthesis of Creatine Stearoyl Ester According to the Present Invention

(23) II.1. General Procedure.

(24) This synthesis is carried out by opening the ring of di-protected creatinine, (Z2)-creatinine, by stearoyl alcohol followed by deprotection under hydrogen/Pd. Hereinafter, scheme 3 in FIG. 6 shows the synthetic sequences of creatine stearoyl ester preparation.

(25) A. (Z.sub.2)-Creatinine.

(26) Benzoylchloroformate (4.2 ml-3 eq) were added to a solution of diisopropyl ethylamine (5.2 ml-3 eq) with creatinine (1.124 g-1 eq) in 100 ml of anhydrous dichloromethane under nitrogen. The benzylchloroformate is added dropwise in an ice bath. The mixture is allowed to react with stirring 30 min in the ice bath and overnight at room temperature.

(27) The reaction is controlled by CCM (Silica, Heptane/Ethyl Acetate) and LC/MS. The reaction medium is extracted by addition of dichloromethane and water. The dichloromethane phase is washed 3 times with water and dried by magnesium sulphate.

(28) The dichloromethane solution is concentrated by evaporation under vacuum and allowed to crystallize. 3.25 g of crude (Z.sub.2)-creatinine are obtained (87%). The (Z.sub.2)-creatinine can be purified on silica gel (Heptane/Ethyl Acetate gradient) for the determination of structure. The crude product is used to perform the esterification step.

(29) B. (Z.sub.2)Creatine Stearoyl Ester.

(30) Stearoyl alcool (0.5 g-2 eq) is allowed to react with 1 eq of crude (Z.sub.2)-creatinine in a tube heated to 80 C. during 5 h. The reaction is monitored by CCM (Silica, Heptane/Ethyl Acetate) and LC/MS.

(31) The crude (Z.sub.2)-creatine stearoyl ester is purified on silica gel (Heptane/Ethyl Acetate gradient) to yield 286 mg of pure (Z.sub.2)-creatine stearoyl ester with a 50% yield.

(32) C. Creatine Stearoyl Ester.

(33) Pure (Z.sub.2)-creatine stearoyl ester (140 mg) is dissolved in anhydrous dichloromethane/methanol solution (6 ml/12 ml) under nitrogen. Pd/Al.sub.2O.sub.3 5% (20 mg) is added. The reaction mixture is degassed under vacuum, frozen to be purged and the vacuum is broken by hydrogen. The purge with hydrogen is done three times. Then the medium is allowed to reach room temperature and react under vigorous stirring.

(34) The reaction is monitored by CCM (Silica, Heptane/Ethyl Acetate) and LC/MS. When the reaction is complete, generally after 3 h, filtration on 0.5 m filter gives creatine stearoyl ester solution.

(35) The creatine stearoyl ester solution is evaporated under vacuum to yield 75 mg (quantitative yield).

(36) II.2. Variants of the Method According to the Present Invention.

(37) A. Protection of Creatinine.

(38) TABLE-US-00004 TABLE 4 Protective Reagent Stoech. Conditions Base Stoech. Yield Boc.sub.2O 5 eq RT, NaOH 1 eq 0.6% Dioxane/Water Boc.sub.2O 6 eq RT, DCM DIEPA 6 eq 43% FmocCl 1.5 eq RT, DCM DIEPA 1.5 eq 28% ZCl 3 eq RT, DCM DIEPA 3 eq 87%

(39) Different protective reagents were tested in order to prepare protected creatinine (see scheme 4 in FIG. 7). Results are presented in Table 4.

(40) Reaction of creatinine with Boc.sub.2O presented very poor yield under aqueous conditions and reaction with Boc.sub.2O under anhydrous conditions led to a new and unuseful compound with 3 Boc functions by addition of the Boc group on the methylene with a 43% isolated yield.

(41) Fortunately, the reaction with the benzoyl chloroformate (ZCI) yielded 87% of diprotected creatinine used as a crude product after crystallisation in hexane.

(42) B. Nucleophilic Addition of Alcohols on Diprotected Creatinine.

(43) Different molecules bearing a alcohol group were tested for nucleophilic addition on diprotected creatinine. Results are presented in Table 5.

(44) TABLE-US-00005 TABLE 5 R Stoech. Conditions Yield C1 as solvent RT 2 h 92% C2 as solvent 80 C. 4 h 72% iPrOH 4 eq 80 C. 4 h 5.5% C4 10 eq 60 C. 4 h 60% C8 4 eq 80 C. 4 h 40% Octanol-2 4 eq 80 C. 4 h 5% C9 8 eq 80 C. 5 h 25% C12 8 eq 80 C. 7 h 47% C16 6 eq 80 C. 7 h 55% C18 1.5 eq 80 C. 16 h 25% C18 rad 10 eq 80 C. 5 h 28% (HPLC) C18 ins 4 eq 80 C. 5 h 20% Glucose 1 eq 80 C. 3 h 30 0.5% Z3,OMe Glu 3.25 eq 80 C. 5 h 21% C6OH

(45) In Table 5, C18 rad means .sup.14C labelled compound, C18 ins a C18 aliphatic chain presenting insaturation(s) and Z3,OMe Glu C6-OH means a glucose in which 3 of the 4 alcohol groups borne by the carbon atoms 1, 2, 3 and 4 are protected, while the alcohol group borne by the carbon atom 6 is free and reacts during the ring opening step in the method according to the present invention.

(46) Nucleophilic addition of alcohols occurs spontaneously in methanol at room temperature using the alcohol as solvent. The crude diprotected showed a better reactivity probably because acidic impurity catalyses the addition.

(47) Increasing the length of the aliphatic chain needed to react with heating up to 80 C. The reaction time was about 3 h. Increasing the reaction time might degrade the desired compound to provide by-compounds as compounds obtained by deprotection of one protective group and transesterification by excess alcohol.

(48) Finally, the inventors obtained 45% yields for addition of aliphatic alcohols with chain length comprised between 8 and 18 carbons.

(49) C. Deprotection of (Z.sub.2) Creatine Fatty Esters.

(50) The deprotection of the Z groups leads to the recovery of the guanidine function, well-known for its very polar character.

(51) Palladium over alumina was preferred to palladium over charcoal because the recovery of the desired product needs steps of washing the catalyst by less amounts of methanol.

(52) Creatinine was systematically obtained as by product because of the propensity for cyclization of our derivatives. Creatinine is easily eliminated on reversed phase column. The deprotection of short aliphatic chain was unsuccessful because the formation of creatinine by self-cyclization was complete and no creatine ester could be obtained.

(53) Results on the deprotection are presented in Table 6.

(54) TABLE-US-00006 TABLE 6 R Reactants Conditions Yield C1 TMSI, CH.sub.3CN 50 C., 30 min Quantitative, HPLC 40 mg/ml yield C1 MeOH PA, 1 h RT 0% (creatinine) Pd/C 5% 40 mg/ml C1 DCE PA, 4 h RT 0% (creatinine) Pd/C 5% 40 mg/ml C4 DCM PA, 4 h RT 7% (isolated by Pd/C 5% 20 mg/ml LC/MS) C8 CH.sub.3CN PA, 4 h RT 0% (mono) Pd/Al.sub.2O.sub.3 5% 1 mg/ml C8 DCM/MeOH (1/1) PA, 3 h RT 100% (+44) Pd/Al.sub.2O.sub.3 5% 10 mg/ml C8 ACN/MeOH (1/1) PA, 4 h RT 13% Pd/Al.sub.2O.sub.3 5% 10 mg/ml C9 DCE PA, 3 h RT 50% Pd/C 5% 10 mg/ml C12 CH3CN PA, 4 h RT 0% (mono) Pd/Al.sub.2O.sub.3 5% 1 mg/ml C12 DCM/MeOH (1/4) PA, 4 h RT 100% (+44) Pd/Al.sub.2O.sub.3 5% 10 mg/ml C12 DCM/MeOH (1/2) PA, 4 h RT 100% (+44) Pd/Al.sub.2O.sub.3 5% 10 mg/ml C16 CH3CN PA, 3 h RT 0% (mono) Pd/Al.sub.2O.sub.3 5% 4 mg/ml C16 DCM/MeOH (1/1) PA, RT 100% Pd/Al.sub.2O.sub.3 5% 10 mg/ml C18 DCM/MeOH (1/2) PA, 3 h RT 90% Pd/Al.sub.2O.sub.3 5% 8 mg/ml C18 rad DCM/MeOH (1/1) PA, 3 h RT 65% Pd/Al.sub.2O.sub.3 5% 2 mg/ml C18 ins DCE PA, 2 h RT 0% (saturated Pd/C 5% 20 mg/ml analogue) Glucose DCM/MeOH (1/1) PA, 3 h RT Molecular pic Pd/Al.sub.2O.sub.3 5% 3 mg/ml availalble

(55) Depending on the solvent used and the ratio of DCM/methanol or acetonitrile, the inventors produced the carbonated form of the creatine fatty ester (+44) isolated by LC/MS preparative chromatography.

(56) The deprotection of unsaturated compounds (C18 ins) gave the saturated analogues. The method with TMSI/CH.sub.3CN must be used for these compounds.

(57) In Table 6, 0% (mono) means that monoprotected creatine fatty ester was obtained and that additional deprotection step was necessary to prepare the deprotected form.

(58) Finally the prepared compounds were not or poorly soluble in water or biological media and not or poorly stable in organic solution. They had to be conserved on a solid form and proceeded to the biological protocol just before their use.

(59) That is the reason why the inventors decided to develop more hydrophilic esters by reaction with protected glucose. In such compounds, the glucose moiety acts as a ligand to favour the transport of the creatine derivatives in particular through the BBB.

(60) III. Translocation of Creatine Fatty Esters Across the In-Vitro Cell-Based BBB Model.

(61) The in-vitro cell-based BBB model which is used to assess the permeability of fatty esters consists in a coculture of glial cells and brain endothelial cells.

(62) A transport buffer (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl.sub.2, 0.2 mM MgCl.sub.2, 6 mM NaHCO.sub.3, 2.8 mM glucose and 5 mM Hepes) is added: 1500 l to the basolateral chamber (which represents the cerebral parenchyma) and 500 l to the apical chamber (which represents the blood).

(63) The fatty esters are introduced in the apical compartment. After 60 min, aliquots are removed from the apical and basolateral chambers for drug-concentration determination. The percentage of drug from the initial dosage that crossed BBB is calculated as follows: P (%)=[(Bf1500)/(A0500)]100 where Bf is the amount of tested compounds in the basolateral compartment at the end point. A0 is the initial amount in the apical compartment at time point 0.

(64) After 60 min of incubation with creatine fatty esters, the inventors noticed that fatty esters C18 but not C2 presented a good permeability through the blood brain barrier.

(65) Indeed, in the case of creatine fatty ester C18, intracellular level of creatine stearoyl ester increased within brain endothelial cells after incubation of cells with this compound (FIG. 1). Notably, this tremendous increase in creatine stearoyl ester strikingly coincided with the appearance of creatine content (about 700 nM) within brain endothelial cells as well as in the basal compartment (about 31 nM) (FIG. 2), suggesting the potential interaction of yielded creatine with cells (e.g. neuronal cells) of brain parenchyma.

(66) In the case of creatine fatty ester C2, the inventors found no translocation of this compound within brain endothelial cells or in the basal compartment of the in vitro cell-based blood-brain barrier model. The inventors found no evidence of the appearance of creatine content within cells or in the brain parenchyma compartment (FIG. 3).

(67) Taken together, the present findings report for the first time differential translocation throughout the BBB between creatine fatty esters. To sum up, the inventors bring the first evidence of the usefulness of the design molecular strategy to get creatine within brain endothelial cells and brain parenchyma compartment after administration of creatine fatty esters. Creatine fatty esters can thus represent the promising candidates for the development of new drugs useful in the treatment of creatine deficiency transporter.

REFERENCES

(68) [1] International application WO 02/22135 in the name of Board of Regents of the University of Nebraska and published on Mar. 21, 2002. [2] Patent application US 2002/0049253 in the name of Kaddurah-Daouk and published on Apr. 25, 2002. [3] Patent application US 2003/0212130 in the name of Miller et al. and published on Nov. 13, 2003. [4] U.S. Pat. No. 6,413,552 in the name of Stoll and published on Jul. 2, 2002. [5] Article of Edgar and Shiver, 1925, The equilibrium between creatine and creatinine in aqueous solution. The effects of hydrogen ion, J. Amer. Chem. Soc., vol. 47, pages 1179-1188. [6] Patent application CN 1616420 in the name of XinMao Dacron Chemical General and published on May 18, 2005. [7] Patent application US 2005/0049428 in the name of Vennerstrom and published on Mar. 3, 2005. [8] Patent application CN 1900056 in the name of Tiangcheng Pharmaceutical Co. Lt. and published on Jan. 24, 2007. [9] International application WO 2008/101309 in the name of Multi Formulations Ltd. and published on Aug. 28, 2008. [10] Patent application US 2011/0269986 in the name of Burov et al. and published on Nov. 3, 2011. [11] Patent application US 2008/0200705 in the name of Chaudhuri et al. and published on Aug. 21, 2008. [12] Article of Gers et al., 2004, Reagents for efficient conversion of amines to protected guanidines, Synthesis, vol. 2004, pages 37-42. [13] Article of Robles et al., 1999, Towards nucleopeptides containing any trifunctional amino acid, Tetrahedron, vol. 55, pages 13251-13261.