WO2014097335A1 - A method of synthesizing creatine derivatives - Google Patents

A method of synthesizing creatine derivatives Download PDF

Info

Publication number
WO2014097335A1
WO2014097335A1 PCT/IT2013/000323 IT2013000323W WO2014097335A1 WO 2014097335 A1 WO2014097335 A1 WO 2014097335A1 IT 2013000323 W IT2013000323 W IT 2013000323W WO 2014097335 A1 WO2014097335 A1 WO 2014097335A1
Authority
WO
WIPO (PCT)
Prior art keywords
creatine
formula
boc
group
iii
Prior art date
Application number
PCT/IT2013/000323
Other languages
French (fr)
Inventor
Enrico Millo
Maurizio Balestrino
Gianluca Damonte
Patrizia GARBATI
Enrico ADRIANO
Annalisa Salis
Original Assignee
Universita' Degli Studi Di Genova
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universita' Degli Studi Di Genova filed Critical Universita' Degli Studi Di Genova
Priority to US14/650,157 priority Critical patent/US20150368192A1/en
Priority to EP13828886.5A priority patent/EP2935207A1/en
Publication of WO2014097335A1 publication Critical patent/WO2014097335A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C277/00—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C277/08—Preparation of guanidine or its derivatives, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups of substituted guanidines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/222—Amides of phosphoric acids

Definitions

  • This invention relates to a method of synthesizing creatine derivatives.
  • Intracellular ATP levels are maintained constant through the reversible phosphorylation of creatine to phosphocreatine, performed by the enzyme creatine kinase.
  • Phosphocreatine is in fact capable of donating a phosphate group to ADP, restoring ATP levels. Creatine thus has a central part to play in cell energy metabolism. Its action is of great importance in all cell types, mainly in muscular tissue and in the brain.
  • creatine transfers a phosphate group to ATP using the enzyme creatine kinase according to the following reaction:
  • ADP Adenosine diphosphate
  • Creatine is synthesized in the kidneys, liver, pancreas and brain, or it is obtained from food sources such as fresh meat and fish. It is transported through the blood and enters the cells of tissues, particularly those having a high energy demand, such as in particular muscle and brain cells, using its own specific transporter (CrT). The transporter is required so that the creatine can cross the blood-brain barrier.
  • Creatine deficiency syndromes represent a group of diseases caused by mutations in the genes for arginine glycine amidinotransferase (AGAT, EC 2.1.4.1) and guanidinoacetate methyltransferase (GAMT, EC 2.1.1.2), two enzymes which are required for the synthesis of creatine, and the SLC6A8 gene which codes for the specific creatine transporter.
  • AGAT arginine glycine amidinotransferase
  • GAMT guanidinoacetate methyltransferase
  • SLC6A8 gene which codes for the specific creatine transporter.
  • Patients affected by these syndromes manifest severe neurological symptoms in early infancy, which typically include mental retardation and epileptic crises of variable severity, but there are often other symptoms such as delayed language development, movement disorders and behavioural disorders, including autism, hyperactivity and self-harming.
  • Creatine transporter deficiency is currently an incurable disease and one possible solution might lie in the administration of creatine in a form capable of crossing biological membranes without the help of the specific creatine transporter, which is absent in these patients.
  • the administration of creatine would be of great benefit, including for other diseases characterised by creatine deficiency, which also include ischaemic jaundice in addition to the abovementioned AGAT and GAMT deficiency syndromes.
  • creatine is a polar molecule which is not readily able to cross biological membranes. In order to overcome this disadvantage it is therefore necessary to have creatine derivatives which increase its lipophilic nature and therefore make it suitable for crossing biological membranes, preferably without the help of its specific transporter.
  • An alternative strategy comprises binding it to other molecules which can perform the function of carrier and therefore carry it across biological membranes by means of other transporters.
  • the bond with the molecule of interest should be a covalent bond which does not involve the guanidine group of the creatine, which must be left free to interact with the enzyme creatine kinase.
  • (Boc) 2 -creatine is synthesized in the aqueous phase by causing creatine to react with N,N-bis(t-butoxycarbonyl)anhydride.
  • This method however has the disadvantage that it offers low yields because of the instability and low solubility of the creatine.
  • the object of this invention is therefore to provide a method of synthesizing (Boc) 2 - creatine and subsequently creatine derivatives which overcomes the problems in the prior art.
  • the first step in the method according to the invention provides for the use of a sarcosine ester of formula (I) as a precursor which is converted into (Boc) 2 -creatine ester of formula (II) according to a simple procedure.
  • the ester of formula (II) is in fact obtained through using a guanylating agent protected with t-Boc on both nitrogen atoms, which allows it to be synthesized directly.
  • the sarcosine ester of formula (I) used as a precursor in the first step of the method according to the invention has the structural formula illustrated below:
  • R is a linear or branched saturated or unsaturated alkyl or aryl group having from 1 to 8 carbon atoms.
  • R is a linear alkyl group having 1 to 8 carbon atoms; even more preferably, R is ethyl and formula (I) therefore represents the ethyl ester of sarcosine.
  • the method according to the invention advantageously makes it possible to achieve high yields and optimum purity for the final (Boc) 2 -creatine product.
  • l,3-bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea (CAS 107819-90-9) or N,N-bis(t- butoxycarbonyl)l-guanyl pyrazole (CAS 152120-54-2) is used as the guanylating agent.
  • the yields obtained with these two guanylating agents are substantially similar.
  • the (Boc) 2 -creatine of formula (III) obtained by the method described above is subsequently used to synthesize a creatine derivative through conjugation using conventional procedures with a molecule comprising a functional group capable of reacting with the free carboxyl group of the creatine, thereby obtaining a (Boc) 2 -creatine derivative.
  • Non-limiting examples of molecules comprising a functional group capable of reacting with the free carboxyl group of (Boc) 2 -creatine of formula (III) are amino acids and their esters, amines, alcohols, thiols, lipids, vitamins and carbohydrates.
  • the two t-Boc groups may be easily removed from the (Boc) 2 - creatine derivative by treatment in an acid environment in order to obtain a creatine derivative which optionally may in turn be used as a precursor for the synthesis of further derivatives in which the guanidine group of the creatine is modified by bonding to any molecule comprising a functional group capable of reacting with the guanidine group of the creatine.
  • Preferred derivatives modified on the guanidine group of the creatine are illustrated by the following structural formula (IV):
  • FORMULA (IV) in which X is a residue of a molecule as defined in the appended claims and R is selected from the group comprising -OH, -PO(Ri)(R 2 ), -COR3 and -S0 2 R4, in which Ri and R 2 are independently selected from the group comprising hydrogen, hydroxyl and -OR5, and in which R 3 , R4 and R5 are independently selected from the group comprising linear or branched CI -CI 6 alkyl and heteroalkyl groups, cycloalkyl groups and C3-C8 heterocycloalkyls, which may be substituted, and aryl and heteroaryl groups which may be substituted.
  • Particularly preferred creatine derivatives included in formula (IV) are phosphocreatine derivatives in which -R is -PO(OH)(OH), which are obtained by causing the precursor to react with a phosphorylating agent.
  • t-Boc as a group protecting the guanidine group according to the method of the invention is particularly advantageous for the synthesis of creatine derivatives.
  • the present inventors have in fact tried using other protected groups described in the literature, and have experimented with different methods to protect the guanidine group, such as the insertion of the p-toluenesulfonyl group, the insertion of a trityl (triphenylmethyl) group and the insertion of the Pbf (2,2,4,6,7-pentamemyldihydrobenzofuran-5-sulfonyl) group without however achieving satisfactory results, in that the attempts resulted in degradation of the product and/or yields which were too low.
  • reaction scheme 1 Method A using l,3-bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea as the guanylating agent (Reaction scheme 1) 1.1 equivalents of HgCl 2 were added to a solution of sarcosine ethyl ester (1.2 equivalents), l,3-bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea (1 equivalent) and triethylamine (3 equivalents) in anhydrous N,N dimethylformamide. The suspension was kept stirring at ambient temperature until completion of the reaction, which was monitored using thin layer chromatography (TLC). Indicatively, depending upon the quantities used, reaction times varied from 18 to 24 hours.
  • TLC thin layer chromatography
  • reaction scheme 1 On completion the reaction mixture was taken up in ether with the formation of an abundant white precipitate. This precipitate was filtered off under vacuum. The ethereal solution obtained was washed twice with deionised water and subsequently a further 2 times with a solution of NaCl (0.1M). The ether phase was evaporated to minimum volume and subsequently lyophilised. A solution of acetonitrile and IN NaOH in a 1:1 ratio was added to the product so obtained, while stirring, in order to hydrolyse the ethyl group. This reaction was also monitored using TLC. On completion of the reaction the pH of the solution was raised to 6 using IN HC1. The compound was then centrifuged to remove any precipitate. The supernatant was lyophilised, yielding a white powder. The structure of the molecule was verified by mass spectrometric analysis, which confirmed the expected molecular weight. Reaction scheme 1 :
  • Example 3 Synthesis of (Boc ⁇ -creatine bound to an esterified amino acid.
  • One equivalent of (Boc) 2 -creatine was dissolved in anhydrous N,N-diethylformamide.
  • 1 equivalent of isobutyl chloroformate and 1 equivalent of N-methylmorpholine was added to this solution, kept with stirring at a temperature of 0°C. After 10 minutes the reaction was brought to ambient temperature and protected from the light.
  • Esterified amino acid previously prepared by stirring the amino acid ester present in the form of hydrochloride (1.5 equivalents) with triemylamine (3 equivalents) in anhydrous N,N-dimemylformamide for 30 minutes was added to this solution.
  • the mixture obtained was then centrifuged and the supernatant added to the mixture containing activated (Boc) 2 -creatine.
  • the compound was kept stirring at ambient temperature for between 24 and 48 hours, depending upon the amino acid, the progress of the reaction being monitored using TLC.
  • the synthesis mixture was centrifuged and the supernatant obtained was lyophilised. This product was taken up in ether or ethyl acetate, depending upon the polarity of the amino acid used, and washed with deionised water. The organic phase was then evaporated to minimum volume.
  • the creatine derivative so obtained was purified by reverse phase HPLC (high performance liquid chromatography).
  • reaction scheme 3 A solution of dichloromethane and trifluoroacetic acid in a 1:1 ratio was added to the final product, brought to a temperature of 0°C (Reaction scheme 3). On completion of the reaction, which was monitored by TLC, the compound was added dropwise to cold ether, yielding a white precipitate. The precipitate was separated out by centrifuging and dried to a powder by lyophilisation.
  • the following derivatives were prepared by this method: creatine-piperidine, creatine- paratoluidine, creatine-morpholine, creatine-diethylamine.
  • the structure of the derivatives prepared was confirmed by mass spectrometric analysis, confirming the expected molecular weights.
  • the creatine derivative obtained through the synthesis according to examples 3 and 4 (1 equivalent) was dissolved in anhydrous tetrahydrofuran in the presence of triethylamine (1 equivalent).
  • the reaction was cooled to 10°C and 1 equivalent of diphenylchlorophosphate dissolved in anhydrous tetrahydrofuran was added dropwise to it. After addition the reaction temperature was raised to 40°C until the product formed, monitoring using TLC (ethyl acetate: hexane, 6:4). On completion of the reaction the mixture was evaporated to minimum volume. The compound was then taken up in diethylether and washed several time with deionised water. The organic phase was men evaporated to minimum volume and finally lyophilised.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A method of synthesizing (Boc)2-creatine derivatives of formula (III) which comprises a first step in which a sarcosine ester is reacted with a guanylating agent comprising two nitrogen atoms each protected with a t-butoxycarbonyl (t-Boc) group to form a (Boc)2- creatine ester, and a second step in which the (Boc)2-creatine ester is subjected basic hydrolysis to form (Boc)2-creatine of formula (III) is described. The (Boc)2-creatine so obtained can be used in methods of synthesizing creatine and phosphocreatine derivatives in which the free carboxyl group of the creatine is conjugated with a desired molecule.

Description

A method of synthesizing creatine derivatives
This invention relates to a method of synthesizing creatine derivatives. Intracellular ATP levels are maintained constant through the reversible phosphorylation of creatine to phosphocreatine, performed by the enzyme creatine kinase. Phosphocreatine is in fact capable of donating a phosphate group to ADP, restoring ATP levels. Creatine thus has a central part to play in cell energy metabolism. Its action is of great importance in all cell types, mainly in muscular tissue and in the brain.
As is well known, creatine transfers a phosphate group to ATP using the enzyme creatine kinase according to the following reaction:
Cr + ATP «→ PCr + ADP + if
Cr = Creatine
PCr = Phosphocreatine
ATP = Adenosine triphosphate
ADP = Adenosine diphosphate
Creatine is synthesized in the kidneys, liver, pancreas and brain, or it is obtained from food sources such as fresh meat and fish. It is transported through the blood and enters the cells of tissues, particularly those having a high energy demand, such as in particular muscle and brain cells, using its own specific transporter (CrT). The transporter is required so that the creatine can cross the blood-brain barrier. Creatine deficiency syndromes represent a group of diseases caused by mutations in the genes for arginine glycine amidinotransferase (AGAT, EC 2.1.4.1) and guanidinoacetate methyltransferase (GAMT, EC 2.1.1.2), two enzymes which are required for the synthesis of creatine, and the SLC6A8 gene which codes for the specific creatine transporter. Patients affected by these syndromes manifest severe neurological symptoms in early infancy, which typically include mental retardation and epileptic crises of variable severity, but there are often other symptoms such as delayed language development, movement disorders and behavioural disorders, including autism, hyperactivity and self-harming.
Creatine transporter deficiency is currently an incurable disease and one possible solution might lie in the administration of creatine in a form capable of crossing biological membranes without the help of the specific creatine transporter, which is absent in these patients. Thus the administration of creatine would be of great benefit, including for other diseases characterised by creatine deficiency, which also include ischaemic jaundice in addition to the abovementioned AGAT and GAMT deficiency syndromes. However creatine is a polar molecule which is not readily able to cross biological membranes. In order to overcome this disadvantage it is therefore necessary to have creatine derivatives which increase its lipophilic nature and therefore make it suitable for crossing biological membranes, preferably without the help of its specific transporter. An alternative strategy comprises binding it to other molecules which can perform the function of carrier and therefore carry it across biological membranes by means of other transporters.
One technical problem associated with the synthesis of creatine derivatives, however, lies in the fact that it is not very reactive with other molecules, because of its low solubility in water and organic solvents.
US 2009/0297685 describes a method of synthesizing imino-sugars bound to creatine which in a first step provides for the synthesis of creatine protected by t-butoxycarbonyl (hereafter indicated as "(Boc)2-creatine") on the two nitrogen atoms of the guanidine group; this form is in fact more stable and more reactive than unprotected creatine. (Boc)2-creatine also has the advantage that the carboxyl group is unprotected and therefore free to react with other molecules to form the desired derivative. In creatine derivatives which are suitable for the treatment of creating deficiency syndromes it is in fact necessary that the bond with the molecule of interest should be a covalent bond which does not involve the guanidine group of the creatine, which must be left free to interact with the enzyme creatine kinase.
According to the teaching in US 2009/0297685, (Boc)2-creatine is synthesized in the aqueous phase by causing creatine to react with N,N-bis(t-butoxycarbonyl)anhydride. This method however has the disadvantage that it offers low yields because of the instability and low solubility of the creatine. The object of this invention is therefore to provide a method of synthesizing (Boc)2- creatine and subsequently creatine derivatives which overcomes the problems in the prior art.
This object is accomplished through a method of synthesizing (Boc)2-creatine as defined in the characterising part of Claim 1.
(Boc)2-creatine synthesized by the method according to the invention has the structural formula illustrated below as formula (III):
Figure imgf000005_0001
FORMULA (III)
The first step in the method according to the invention provides for the use of a sarcosine ester of formula (I) as a precursor which is converted into (Boc)2-creatine ester of formula (II) according to a simple procedure. The ester of formula (II) is in fact obtained through using a guanylating agent protected with t-Boc on both nitrogen atoms, which allows it to be synthesized directly.
The sarcosine ester of formula (I) used as a precursor in the first step of the method according to the invention has the structural formula illustrated below:
Figure imgf000006_0001
FORMULA (I) in which R is a linear or branched saturated or unsaturated alkyl or aryl group having from 1 to 8 carbon atoms. In a preferred embodiment, R is a linear alkyl group having 1 to 8 carbon atoms; even more preferably, R is ethyl and formula (I) therefore represents the ethyl ester of sarcosine.
The (Boc)2-creatine ester of formula (II) has the following structural formula:
Figure imgf000006_0002
FORMULA (Π) in which R is as defined for formula (I).
In the next step of the method, the (Boc)2-creatine ester of formula (II) is subjected to basic hydrolysis to form the (Boc)2-creatine of formula (III).
The method according to the invention advantageously makes it possible to achieve high yields and optimum purity for the final (Boc)2-creatine product. In a preferred embodiment l,3-bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea (CAS 107819-90-9) or N,N-bis(t- butoxycarbonyl)l-guanyl pyrazole (CAS 152120-54-2) is used as the guanylating agent. The yields obtained with these two guanylating agents are substantially similar. In a second aspect of the invention, the (Boc)2-creatine of formula (III) obtained by the method described above is subsequently used to synthesize a creatine derivative through conjugation using conventional procedures with a molecule comprising a functional group capable of reacting with the free carboxyl group of the creatine, thereby obtaining a (Boc)2-creatine derivative.
Non-limiting examples of molecules comprising a functional group capable of reacting with the free carboxyl group of (Boc)2-creatine of formula (III) are amino acids and their esters, amines, alcohols, thiols, lipids, vitamins and carbohydrates. Finally, if so desired, the two t-Boc groups may be easily removed from the (Boc)2- creatine derivative by treatment in an acid environment in order to obtain a creatine derivative which optionally may in turn be used as a precursor for the synthesis of further derivatives in which the guanidine group of the creatine is modified by bonding to any molecule comprising a functional group capable of reacting with the guanidine group of the creatine. Preferred derivatives modified on the guanidine group of the creatine are illustrated by the following structural formula (IV):
Figure imgf000007_0001
FORMULA (IV) in which X is a residue of a molecule as defined in the appended claims and R is selected from the group comprising -OH, -PO(Ri)(R2), -COR3 and -S02R4, in which Ri and R2 are independently selected from the group comprising hydrogen, hydroxyl and -OR5, and in which R3, R4 and R5 are independently selected from the group comprising linear or branched CI -CI 6 alkyl and heteroalkyl groups, cycloalkyl groups and C3-C8 heterocycloalkyls, which may be substituted, and aryl and heteroaryl groups which may be substituted.
Particularly preferred creatine derivatives included in formula (IV) are phosphocreatine derivatives in which -R is -PO(OH)(OH), which are obtained by causing the precursor to react with a phosphorylating agent.
The use of t-Boc as a group protecting the guanidine group according to the method of the invention is particularly advantageous for the synthesis of creatine derivatives. The present inventors have in fact tried using other protected groups described in the literature, and have experimented with different methods to protect the guanidine group, such as the insertion of the p-toluenesulfonyl group, the insertion of a trityl (triphenylmethyl) group and the insertion of the Pbf (2,2,4,6,7-pentamemyldihydrobenzofuran-5-sulfonyl) group without however achieving satisfactory results, in that the attempts resulted in degradation of the product and/or yields which were too low.
The examples below are provided for merely illustrative purposes and do not limit the scope of the invention as defined by the appended claims. Example 1 : Synthesis of (Boc)?-creatine
Method A using l,3-bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea as the guanylating agent (Reaction scheme 1) 1.1 equivalents of HgCl2 were added to a solution of sarcosine ethyl ester (1.2 equivalents), l,3-bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea (1 equivalent) and triethylamine (3 equivalents) in anhydrous N,N dimethylformamide. The suspension was kept stirring at ambient temperature until completion of the reaction, which was monitored using thin layer chromatography (TLC). Indicatively, depending upon the quantities used, reaction times varied from 18 to 24 hours. On completion the reaction mixture was taken up in ether with the formation of an abundant white precipitate. This precipitate was filtered off under vacuum. The ethereal solution obtained was washed twice with deionised water and subsequently a further 2 times with a solution of NaCl (0.1M). The ether phase was evaporated to minimum volume and subsequently lyophilised. A solution of acetonitrile and IN NaOH in a 1:1 ratio was added to the product so obtained, while stirring, in order to hydrolyse the ethyl group. This reaction was also monitored using TLC. On completion of the reaction the pH of the solution was raised to 6 using IN HC1. The compound was then centrifuged to remove any precipitate. The supernatant was lyophilised, yielding a white powder. The structure of the molecule was verified by mass spectrometric analysis, which confirmed the expected molecular weight. Reaction scheme 1 :
Figure imgf000009_0001
Example 2: Synthesis of (Boc -creatine Method B using N,N-bis(t-butoxycarbonyl)-l-guanyl pyrazole as guanylating agent (Reaction scheme 2)
A solution of sarcosine ethyl ester (1.2 equivalents), N,N-bis(t-butoxycarbonyl)-l-guanyl pyrazole (1 equivalent) and triethylamine (3 equivalents) in anhydrous N,N- dimethylfonnamide was maintained at ambient temperature with stirring until the reaction was complete. The reaction was monitored using TLC. Indicatively, depending upon the quantities used, reaction times varied from 18 to 24 hours. On completion the reaction mixture was taken up in ether and washed twice with an equivalent quantity of deionised water. The ether phase was evaporated to minimum volume and subsequently lyophilised. A solution of acetonitrile and IN NaOH in a 1:1 ratio was added to the product so obtained, with stirring, in order to hydrolyse the ethyl group. On completion of the reaction the pH of the solution was increased to 6 using IN HCl. The compound was then centrifuged to remove any precipitate. The supernatant was lyophilised to yield a white powder. The structure of the molecule was checked by mass spectrometric analysis, confirming the molecular weight expected. Reaction scheme 2:
Figure imgf000010_0001
Example 3: Synthesis of (Boc^-creatine bound to an esterified amino acid. One equivalent of (Boc)2-creatine was dissolved in anhydrous N,N-diethylformamide. 1 equivalent of isobutyl chloroformate and 1 equivalent of N-methylmorpholine was added to this solution, kept with stirring at a temperature of 0°C. After 10 minutes the reaction was brought to ambient temperature and protected from the light. Esterified amino acid previously prepared by stirring the amino acid ester present in the form of hydrochloride (1.5 equivalents) with triemylamine (3 equivalents) in anhydrous N,N-dimemylformamide for 30 minutes was added to this solution. The mixture obtained was then centrifuged and the supernatant added to the mixture containing activated (Boc)2-creatine. The compound was kept stirring at ambient temperature for between 24 and 48 hours, depending upon the amino acid, the progress of the reaction being monitored using TLC. On completion of the reaction the synthesis mixture was centrifuged and the supernatant obtained was lyophilised. This product was taken up in ether or ethyl acetate, depending upon the polarity of the amino acid used, and washed with deionised water. The organic phase was then evaporated to minimum volume. The creatine derivative so obtained was purified by reverse phase HPLC (high performance liquid chromatography). A solution of dichloromethane and trifluoroacetic acid in a 1:1 ratio was added to the final product, brought to a temperature of 0°C (Reaction scheme 3). On completion of the reaction, which was monitored by TLC, the compound was added dropwise to cold ether, yielding a white precipitate. The precipitate was separated out by centrifuging and dried to a powder by lyophilisation.
The structure of the derivatives obtained was confirmed by mass spectrometric analysis, confirming the expected molecular weight.
Reaction scheme 3:
Figure imgf000011_0001
Example 4: Synthesis of creatine amides
One equivalent of (Boc)2-creatine was dissolved in anhydrous N,N-dimethylformamide. 1 equivalent of isobutyl chloroformate and 1 equivalent of N-methylmorpholine were added to this solution, which had been brought to a temperature of 0°C. After 10 minutes, the reaction was brought to ambient temperature and protected from the light. 1.5 equivalents of amine were added to this solution. The mixture was kept stirring at ambient temperature for between 24 and 48 hours, depending upon the amine used, monitoring the reaction using TLC. On completion of the reaction, the suspension was centrifuged and the supernatant was lyophilised. The lyophilised compound was then taken up in ether and washed with deionised water. The organic phase was then evaporated to minimum volume and finally lyophilised. The product so obtained was purified by means of reverse phase HPLC. A solution of dichloromethane and trifluoroacetic acid in a 1:1 ratio was added to the final product, brought to a temperature of 0°C (Reaction scheme 3). On completion of the reaction, which was monitored using TLC, the product obtained was added dropwise to cold ether, yielding a precipitate. The precipitate was separated out by centrifuging and dried to a powder by lyophilisation.
The following derivatives were prepared by this method: creatine-piperidine, creatine- paratoluidine, creatine-morpholine, creatine-diethylamine. The structure of the derivatives prepared was confirmed by mass spectrometric analysis, confirming the expected molecular weights.
Of course the same procedure may be used for the synthesis of any creatine derivative which can be obtained through reaction with a molecule having at least one amine group. Example 5: Synthesis of phosphocreatine derivatives with a protected phosphate group
The creatine derivative obtained by the synthesis according to examples 3 and 4 (1 equivalent) was dissolved in anhydrous pyridine and dichloromethane (1:5). DMAP (1 equivalent) was added to the mixture, which was kept stirring at ambient temperature. A solution of diphenylchlorophosphate (1 equivalent) in anhydrous pyridine and dichloromethane (1:5) was subsequently added dropwise to the mixture.
The reaction was monitored using TLC (hexane: ethyl acetate, 1:1). On completion of the reaction the suspension was centrifuged and the supernatant was evaporated to minimum volume. The compound was then taken up in diethylether and washed several times with deionised water. The organic phase was then evaporated to minimum volume and finally lyophilised. The product so obtained was purified by reverse phase HPLC. Example 6: Synthesis of phosphocreatine derivatives with a protected phosphate group
The creatine derivative obtained through the synthesis according to examples 3 and 4 (1 equivalent) was dissolved in anhydrous tetrahydrofuran in the presence of triethylamine (1 equivalent). The reaction was cooled to 10°C and 1 equivalent of diphenylchlorophosphate dissolved in anhydrous tetrahydrofuran was added dropwise to it. After addition the reaction temperature was raised to 40°C until the product formed, monitoring using TLC (ethyl acetate: hexane, 6:4). On completion of the reaction the mixture was evaporated to minimum volume. The compound was then taken up in diethylether and washed several time with deionised water. The organic phase was men evaporated to minimum volume and finally lyophilised.
The product was subsequently purified using HPLC and lyophilised. Example 7: Synthesis of phosphocreatine derivatives with an unprotected phosphate group
The product obtained in examples 5 and 6 was dissolved in tetrahydrofuran and water (5:2) in the presence of NaOH (1 equivalent). The mixture was stirred at a temperature of 40°C and monitored using TLC (ethyl acetate and methanol, 9:1) until completion. The product was then purified using HPLC and lyophilised.

Claims

1. A method of synthesizing (Boc)2-creatine of formula (III), characterised comprises the steps of:
(i) reacting a sarcosine ester of formula (I)
Figure imgf000014_0001
FORMULA (I) wherein R is a linear or branched, saturated or unsaturated alkyl or aryl group having 1 to 8 carbon atoms with a guanylating agent comprising two nitrogen atoms each protected with a t-butoxycarbonyl group (t-Boc), to form a (Boc)2-creatine ester of formula (II)
Figure imgf000014_0002
FORMULA (Π) wherein R is a linear or branched, saturated or unsaturated alkyl or aryl group having 1 to 8 carbon atoms;
(ii) subjecting the (Boc)2-creatine ester of formula (II) to basic hydrolysis, to form (Boc)2- creatine of formula III)
Figure imgf000014_0003
FORMULA (III)
2. The method according to Claim 1 , in which R is a linear alkyl group.
3. The method according to Claim 2, in which R is a linear saturated alkyl group.
4. The method according to Claim 3, in which R is ethyl.
5. The method according to any of Claims 1 to 4, in which the guanylating is l,3-bis(t- butoxycarbonyl)-2-methyl-2-thiopseudourea or N,N-bis(t-butoxycarbonyl)- 1-guanyl- pyrazole.
6. A method of synthesizing a creatine derivative, characterised in that it comprises synthesizing (Boc)2-creatine of formula (III)
Figure imgf000015_0001
FORMULA (III) by a method according to any of Claims 1 to 5, and conjugating the (Boc)2-creatine of formula (III) with a molecule comprising a functional group capable of reacting with the free carboxyl group of (Boc)2-creatine of formula (III), thereby obtaining a derivative of (Boc)2-creatine.
7. The method according to Claim 6, in which the molecule comprising a functional group capable of reacting with the free carboxyl group of the (Boc)2-creatine of formula (III) is selected from the group comprising amino acids and their esters, amines, alcohols, thiols, lipids, vitamins and carbohydrates.
8. The method according to Claim 5 or 6, comprising the further step of removing the t- butoxycarbonyl groups from the (Boc)2-creatine derivative through treatment in an acid environment, thereby obtaining a creatine derivative.
9. The method according to Claim 8, comprising the further step of reacting the creatine derivative with a molecule comprising one or more functional groups capable of reacting with the guanidine group of the creatine derivative, thus obtaining a creatine derivative which is modified on the guanidine group.
10. The method according to Claim 9, in which the creatine derivative modified on the guanidine group is represented by structural formula (IV):
Figure imgf000016_0001
FORMULA (IV) in which X is a residue of a molecule as defined in Claims 6 or 7 and R is selected from the group comprising -OH, -PO(Ri)(R2), -COR3 and -S02R4, in which Ri and R2 are independently selected from the group comprising hydrogen, hydroxyl and -OR5, and in which R3, R4 and R5 are independently selected from the group comprising linear or branched CI -CI 6 alkyl and heteroalkyl groups, cycloalkyl groups and C3-C8 heterocycloalkyl groups, which may be substituted, and aryl and heteroaryl groups which may be substituted.
11. The method according to Claim 11, in which R is -PO(Ri)(R2) and Ri and R2 are both hydroxyl.
PCT/IT2013/000323 2012-12-18 2013-11-18 A method of synthesizing creatine derivatives WO2014097335A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US14/650,157 US20150368192A1 (en) 2012-12-18 2013-11-18 A method of synthesizing creatine derivatives
EP13828886.5A EP2935207A1 (en) 2012-12-18 2013-11-18 A method of synthesizing creatine derivatives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT001098A ITTO20121098A1 (en) 2012-12-18 2012-12-18 PROCEDURE FOR SYNTHESIZING CREATINE DERIVATIVES
ITTO2012A001098 2012-12-18

Publications (1)

Publication Number Publication Date
WO2014097335A1 true WO2014097335A1 (en) 2014-06-26

Family

ID=47633439

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IT2013/000323 WO2014097335A1 (en) 2012-12-18 2013-11-18 A method of synthesizing creatine derivatives

Country Status (4)

Country Link
US (1) US20150368192A1 (en)
EP (1) EP2935207A1 (en)
IT (1) ITTO20121098A1 (en)
WO (1) WO2014097335A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017106687A1 (en) 2015-12-18 2017-06-22 Lonza Inc. Method and composition for increasing muscle protein synthesis and/or functional strength in mammals as well as method of producing a composition
JP2018502911A (en) * 2014-12-22 2018-02-01 ファーミントン ファーマ ディベロップメントＦａｒｍｉｎｇｔｏｎ Ｐｈａｒｍａ Ｄｅｖｅｌｏｐｍｅｎｔ Creatine prodrug, composition thereof, and method of use thereof
CN111269149A (en) * 2020-04-08 2020-06-12 南京优氟医药科技有限公司 Production process of 5- (3,3-dimethylguanidino) -2-oxopentanoic acid
WO2021076920A1 (en) 2019-10-16 2021-04-22 Capsugel Belgium Nv Method and composition for increasing muscle protein synthesis
US11021501B2 (en) 2015-03-30 2021-06-01 Farmington Pharma Development Creatine phosphate analog prodrugs, compositions and methods of use thereof
US11332438B2 (en) 2017-12-01 2022-05-17 Ultragenyx Pharmaceutical Inc. Creatine prodrugs, compositions and methods of use thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITTO20131070A1 (en) 2013-12-24 2015-06-25 Univ Degli Studi Genova THERAPEUTIC APPLICATION OF A CREATINE DERIVATIVE

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6759553B1 (en) * 1998-09-10 2004-07-06 Basf Aktiengesellschaft Process for the preparation of creatine or creatine monohydrate
US20090163739A1 (en) * 2006-04-06 2009-06-25 Franz Thalhammer Process for Preparing Creatine, Creatine Monohydrate or Guanidinoacetic Acid
US20090297685A1 (en) * 2008-05-30 2009-12-03 Heuer Marvin A Preparations containing creatine and imino sugars

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007146085A2 (en) * 2006-06-06 2007-12-21 Xenoport, Inc. Creatine phosphate prodrugs, compositions and uses thereof
WO2013043580A2 (en) * 2011-09-19 2013-03-28 Gencia Corporation Modified creatine compounds

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6759553B1 (en) * 1998-09-10 2004-07-06 Basf Aktiengesellschaft Process for the preparation of creatine or creatine monohydrate
US20090163739A1 (en) * 2006-04-06 2009-06-25 Franz Thalhammer Process for Preparing Creatine, Creatine Monohydrate or Guanidinoacetic Acid
US20090297685A1 (en) * 2008-05-30 2009-12-03 Heuer Marvin A Preparations containing creatine and imino sugars

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2935207A1 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018502911A (en) * 2014-12-22 2018-02-01 ファーミントン ファーマ ディベロップメントＦａｒｍｉｎｇｔｏｎ Ｐｈａｒｍａ Ｄｅｖｅｌｏｐｍｅｎｔ Creatine prodrug, composition thereof, and method of use thereof
JP2020128379A (en) * 2014-12-22 2020-08-27 ファーミントン ファーマ ディベロップメントＦａｒｍｉｎｇｔｏｎ Ｐｈａｒｍａ Ｄｅｖｅｌｏｐｍｅｎｔ Creatine prodrugs, compositions and methods of use thereof
JP7037597B2 (en) 2014-12-22 2022-03-16 ファーミントン ファーマ ディベロップメント Creatine prodrug, its composition, and how to use it
US11407722B2 (en) 2014-12-22 2022-08-09 Farmington Pharma Development Creatine prodrugs, compositions and methods of use thereof
US11021501B2 (en) 2015-03-30 2021-06-01 Farmington Pharma Development Creatine phosphate analog prodrugs, compositions and methods of use thereof
WO2017106687A1 (en) 2015-12-18 2017-06-22 Lonza Inc. Method and composition for increasing muscle protein synthesis and/or functional strength in mammals as well as method of producing a composition
US11332438B2 (en) 2017-12-01 2022-05-17 Ultragenyx Pharmaceutical Inc. Creatine prodrugs, compositions and methods of use thereof
US11753369B2 (en) 2017-12-01 2023-09-12 Ultragenyx Pharmaceutical Inc. Creatine prodrugs, compositions and methods of use thereof
WO2021076920A1 (en) 2019-10-16 2021-04-22 Capsugel Belgium Nv Method and composition for increasing muscle protein synthesis
CN111269149A (en) * 2020-04-08 2020-06-12 南京优氟医药科技有限公司 Production process of 5- (3,3-dimethylguanidino) -2-oxopentanoic acid
CN111269149B (en) * 2020-04-08 2022-04-15 南京优氟医药科技有限公司 Production process of 5- (3,3-dimethylguanidino) -2-oxopentanoic acid

Also Published As

Publication number Publication date
ITTO20121098A1 (en) 2014-06-19
US20150368192A1 (en) 2015-12-24
EP2935207A1 (en) 2015-10-28

Similar Documents

Publication Publication Date Title
EP2935207A1 (en) A method of synthesizing creatine derivatives
JP6403671B2 (en) Method for preparing creatine fatty ester, creatine fatty ester so prepared and use thereof
CA1117965A (en) Structural analogs of natural phospholipids
CA2372068A1 (en) Sulphostin analogue and process for producing sulphostin and its analogue
EP3106465B1 (en) Method for preparing racemic or optically active glycerophosphoryl choline
CN105764909A (en) Stable pantetheine derivatives for the treatment of pantothenate kinase associated neurodegeneration (PKAN) and methods for the synthesis of such compounds
JPH0149276B2 (en)
FI91966C (en) Method for the preparation of pharmacologically valuable alkylphosphonoserines
CA2528383A1 (en) Inactivators and bivalent inhibitors of glyoxalase i and methods of inhibiting tumor growth
ES2905104T3 (en) Efficient method for the preparation of Cangrelor
Morana et al. Synthesis and characterisation of a new class of stable S-adenosyl-l-methionine salts
Arkel et al. A novel method to synthesize phosphocreatine and phosphocreatine prodrugs
EP3470403B1 (en) Taxoid compound and preparation method and use thereof
ITMI20001696A1 (en) CARNOSINE DERIVATIVES, PROCEDURE FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM.
ES2927901T3 (en) Peptide derivatives and therapeutic activity thereof
WO2011056091A1 (en) Process for preparing creatine amides
CN107998403B (en) PEG-modified water-soluble prodrugs of triacontanol
CZ303164B6 (en) Process for preparing phosphonic acid substituted diamides
JPS6188886A (en) Preparation of phospholipid long-chain alcohol derivative by enzymatic process
US20200010500A1 (en) Synthesis of polyphosphorylated molecules from polyphosphates
CN102344452B (en) The new method of synthesis pemetrexed disodium
CN117919199A (en) Application of blank lipid nanoparticles in preparation of in-vivo delivery products
CN116234812A (en) Preparation method of cannabinoid compounds
CN117942406A (en) Use of preformed vectors for the preparation of products for in vitro gene delivery of immune cells and stem cells
KR20130010102A (en) Process for preparing sodium cyclic phosphatidic acid and composition comprising the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13828886

Country of ref document: EP

Kind code of ref document: A1

REEP Request for entry into the european phase

Ref document number: 2013828886

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2013828886

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 14650157

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE