Production method of sphinganine- and phytosphingosine-based ceramides

Abstract

The present invention refers to a process for the production of a specific sphingolipid in an organic solvent in the presence of a base at 15 to 30 °C. Furthermore, the present invention refers to the compound obtained by the process as well as its use in a pharmaceutical composition.

Description

[0001] 202400175 Foreign Filing 1

[0002] Production method of sphinganine- and phytosphingosine-based ceramides

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to a method for the production of a specific sphingolipid in an organic solvent in the presence of a base at 15 to 30 °C. Furthermore, the present invention refers to the compound obtained by the process as well as its use in a pharmaceutical composition.

[0005] BACKGROUND OF THE INVENTION

[0006] Production methods for sphingolipids are known in the art. However, when looking for sphingolipids, which should be used in pharmaceutical applications, high requirements with regard to purity are given. In particular, GMP standards need to be fulfilled in order for the sphingolipids to be suitable to be used in pharmaceutical compositions. Therefore, there is a need for efficient methods which can provide GMP grade sphingolipids. In particular, the methods should provide the sphingolipids in a form that complex and time consuming purification methods are not required and the methods should be suitable for industrial scale production.

[0007] The inventors of the present invention have surprisingly found that these needs can be fulfilled by the method of the present invention.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect the present invention refers to a method for the production of a sphingolipid of formula (I):

[0010]

[0011] wherein

[0012] R1is -H or selected from linear, branched or cyclic, preferably linear or branched, hydrocarbon groups having up to 30 carbon atoms, which can contain up to 3 double bonds and in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH; and

[0013] R2is selected from linear or branched hydrocarbon groups having up to 30 carbon atoms, which can contain up to 3 double bonds and in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH;202400175 Foreign Filing 2

[0014] comprising the steps:

[0015] providing a compound of formula (II),

[0016]

[0017] wherein

[0018] R2is defined as in formula (I); and allowing the compound to react at 15 to 30 °C with an ester of formula (HI)

[0019] o

[0020] A

[0021] R3O R1(III); wherein

[0022] R1is defined as in formula (I); and

[0023] R3is selected from linear or branched hydrocarbon groups containing up to 30 carbon atoms;

[0024] in an organic solvent in the presence of a base.

[0025] In a second aspect the present invention refers to a compound of formula (I) obtained by the method according to the present invention.

[0026] In a third aspect the present invention refers to a pharmaceutical composition comprising at least one compound of formula (I) according to the present invention.

[0027] These and other aspects, embodiments, features, and advantages of the invention will become apparent to a person skilled in the art through the study of the following detailed description and claims. Any feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it will readily be understood that the examples contained herein are intended to describe and illustrate the invention but not to limit the invention and that, in particular, the invention is not limited to these examples.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0030] Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as, "comprises", "comprising", “contain”, and “containing” are to be construed in an open and inclusive sense, that is, as "including, but not limited to".202400175 Foreign Filing 3

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art. As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0032] Numerical ranges that are indicated in the format “from x to y” also include the stated values. If several preferred numerical ranges are indicated in this format, it is self-evident that all ranges that result from the combination of the various endpoints are also included.

[0033] "One or more", as used herein, relates to at least one and comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, "at least one" means one or more, i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. "At least one", as used herein in relation to any component, refers to the number of chemically different molecules, i.e. to the number of different types of the referenced species, but not to the total number of molecules. For example, "at least one compound of formula (I)" means that at least one type of molecule falling within the definition of the compound of formula (I) is used but that also two or more different types of compounds of formula (I) can be present, but does not mean that only one or more molecules of one type of compounds of formula (I) are present.

[0034] All percentages given herein in relation to the compositions relate to wt.-% relative to the total weight of the respective composition, if not explicitly stated otherwise.

[0035] In particular, the present invention refers to:

[0036] A process for the production of a compound of formula (I):

[0037]

[0038] ; wherein

[0039] R1is -H or selected from linear, branched or cyclic, preferably linear or branched, hydrocarbon groups having up to 30 carbon atoms, which can contain up to 3 double bonds and in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH; preferably saturated linear or branched hydrocarbon groups having up to 30 carbon atoms in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH; more preferably saturated linear or branched C5-C21 hydrocarbon groups in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH; and

[0040] R2is selected from linear or branched hydrocarbon groups having up to 30 carbon atoms, in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH; preferably linear or branched, most preferably linear, C12-C16 hydrocarbon groups, in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH;

[0041] comprising or consisting of the steps:202400175 Foreign Filing 4

[0042] providing a compound of formula (II), preferably (Ila)

[0043]

[0044] wherein

[0045] R2is defined as in formula (I); and allowing the compound to react at 15 to 30 °C, preferably 20 to 30 °C , more preferably 20 to 25 °C, with an ester of formula (III)

[0046]

[0047] R1is defined as in formula (I); and

[0048] R3is selected from linear or branched hydrocarbon groups containing up to 30 carbon atoms, preferably linear or branched hydrocarbon groups containing up to 6 carbon atoms, more preferably is -CH3; in an organic solvent in the presence of a base.

[0049] In one embodiment in R1no hydrogen atoms are replaced by -Cl, -F, -Br or -OH.

[0050] In one embodiment R1contains three double bonds.

[0051] In one embodiment R1contains two double bonds.

[0052] In one embodiment R1contains one double bond.

[0053] In one embodiment R1contains a double bond in a,p position to the carbonyl group.

[0054] In one embodiment R1does not contain double bonds.

[0055] In one embodiment R1is a linear hydrocarbon group.

[0056] In one embodiment R1is a branched hydrocarbon group.

[0057] In one embodiment R1is a saturated linear C5-C21 hydrocarbon group.

[0058] In one embodiment R1is -H.

[0059] In one embodiment in R2no hydrogen atoms are replaced by -Cl, -F, -Br or -OH.

[0060] In one embodiment in R2no hydrogen atoms are replaced by -Cl, -F, -Br or -OH and a double bond is contained in R2.202400175 Foreign Filing 5

[0061] In one embodiment in R2no hydrogen atoms are replaced by -Cl, -F, -Br or -OH and a double bond is contained in R2, which is bound to the remainder of the molecule of formula (I).

[0062] In one embodiment in R2at least one hydrogen atom is replaced by -OH.

[0063] In one embodiment in R2one hydrogen atom is replaced by -OH.

[0064] In one embodiment in R2a hydrogen atom on the carbon atom, which is bound to the remainder of the molecule of formula (I) is replaced by -OH.

[0065] In one embodiment R2contains three double bonds.

[0066] In one embodiment R2contains two double bonds.

[0067] In one embodiment R2contains one double bond.

[0068] In one embodiment R2contains a double bond bound to the remainder of the molecule of formula (I).

[0069] In one embodiment R2does not contain double bonds.

[0070] In one embodiment R2is a linear hydrocarbon group.

[0071] In one embodiment R2is a branched hydrocarbon group.

[0072] In one embodiment R2is a linear C12-C16 hydrocarbon group.

[0073] In one embodiment R1is a saturated linear C5-C21 hydrocarbon group and in R2a hydrogen atom on the carbon atom, which is bound to the remainder of the molecule of formula (I) is replaced by -OH.

[0074] In one embodiment R1is a saturated linear C5-C21 hydrocarbon group and R2contains a double bond bound to the remainder of the molecule of formula (I).

[0075] In one embodiment R1is a saturated linear C5-C21 hydrocarbon group and in R2a hydrogen atom on the carbon atom, which is bound to the remainder of the molecule of formula (I) is replaced by -OH and is a C12-C16 hydrocarbon group.

[0076] In one embodiment R1is a saturated linear C5-C21 hydrocarbon group and R2contains a double bond bound to the remainder of the molecule of formula (I) and is a C12-C16 hydrocarbon group.

[0077] In one embodiment the compound of formula (I) is selected from202400175 Foreign Filing 6

[0078]

[0079] As used herein, "organic solvent" refers to a solvent having at least one carbon atom. Non-limiting examples of organic solvents include Ci-Ca alcohols, tetrahydrofuran ("THF"), 2-methyl

[0080] tetra hydrofuran ("Me-THF"), acetone, A / ,A / -dimethylformamide ("DMF"), 1 ,2-dimethoxyethane (“DME”), cyclopropylmethyl ether ("CPME"), petroleum ether, methyl tert-butyl ether (“MTBE”), A / -methyl-2-pyrrolidone ("NMP"), A / ,A / '-dimethylpropyleneurea (“DMPU”) trifluorotoluene, chlorobenzene, nitrobenzene, benzene, toluene (“PhMe”), dimethyl sulfoxide, heptane, or cyclohexane or any mixture thereof.

[0081] In an embodiment the organic solvent is selected from Ci-Ca alcohols, tetra hydrofuran, 2-methyl tetra hydrofuran, 1 ,2-dimethoxyethane, methyl tert-butyl ether, or toluene or any mixture thereof.202400175 Foreign Filing 7

[0082] In a preferred embodiment the organic solvent is a Ci-Ce alcohol, in particular methanol.

[0083] In general any base is suitable in the method of the present invention.

[0084] In one embodiment the base is selected from inorganic bases or organic bases.

[0085] In one embodiment the base is selected from the group of IJ2CO3, Na2CO3, K2CO3, CS2CO3, NaOMe (sodium methoxide), KOMe (potassium methoxide), NaOEt (sodium ethoxide), KOEt (potassium ethoxide), NaOAc (sodium acetate) or a mixture thereof.

[0086] In one embodiment the base is selected from IJ2CO3, Na2CO3, K2CO3, and CS2CO3 or a mixture thereof.

[0087] In one embodiment the base is present in 0.1 to 3.0 eq. based on 1 eq. of compound (II).

[0088] In one embodiment compound (III) is present in 1.0 to 3.0 eq. based on 1 eq. of compound (II).

[0089] In one embodiment the method further comprises a purification step in a second vessel, preferably a precipitation step: the reaction is poured into a second vessel comprising acetonitrile and stirred at 23°C for 2 hours before stored at 4°C. The suspension formed is separated by filtration to obtain the desired product. The product is a white solid in high yield and purity.

[0090] The present invention further refers to a compound of formula (I) obtained by the method according to the present invention.

[0091] Moreover, the present invention refers to a pharmaceutical composition comprising the compound of formula (I) according to the present invention, in particular a parenteral pharmaceutical composition, preferably a lipid nanoparticle, most preferably a vaccine.202400175 Foreign Filing 8

[0092] Examples

[0093] General procedure for the synthesis of Sphingolipids:

[0094] The corresponding sphingosine base (compound of formula (II)) was fully dissolved in an organic solvent (approximately 20 mL / g). To the solution were added an inorganic or organic base (0.1 - 2.0 eq.) as well as a carboxylic acid ester (compound of formula (III); 1.0 - 2.0 eq.). All components were stirred at an adequate temperature to complete the reaction within a maximum of 48 hours. Upon completion of the reaction the mixture was optionally transferred to a second vessel and the solvent was removed by distillation and the residual material was dissolved in methanol. The methanol solution was slowly mixed with acetonitrile to precipitate a solid. The resulting suspension was filtered off and washed with additional solvent to yield the desired sphingolipid in high purity.02400175 Foreign Filing 9

[0095]

[0096] 02400175 Foreign Filing 10

[0097]

[0098] t was surprisingly found that the reactions performed at temperatures of at most 30 °C, in particular room temperature, showed less impurities and equal or bette ield compared to reactions performed at 45 °C or higher. It was particularly surprising that the method according to the present invention was also more economica ince despite lower reaction temperatures, no longer reaction time was required and thus the energy consumption was reduced compared to reactions carried out a igher temperatures.

Claims

202400175 Foreign Filing 11Claims1. A process for the production of a compound of formula (I):; whereinR1is -H or selected from linear, branched or cyclic hydrocarbon groups having up to 30 carbon atoms, which can contain up to 3 double bonds and in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH; andR2is selected from linear or branched hydrocarbon groups having up to 30 carbon atoms, in which up to 10 hydrogen atoms can be replaced by -Cl, -F, -Br or -OH;comprising or consisting of the steps:providing a compound of formula (II),whereinR2is defined as in formula (I); and allowing the compound to react at 15 to 30 °C with an ester of formula (III)whereinR1is defined as in formulaR3is selected from linear or branched hydrocarbon groups containing up to 30 carbon atoms; in an organic solvent in the presence of a base.

2. The method according to claim 1 , wherein the organic solvent is selected from Ci-Ca alcohols, tetrahydrofuran, 2-methyl tetra hydrofuran, acetone, A / ,A / -dimethylformamide, 1 ,2- dimethoxyethane, cyclopropylmethyl ether, petroleum ether, methyl tert-butyl ether, A / -methyl- 2-pyrrolidone, A / ,A / '-dimethylpropyleneurea, trifluorotoluene, chlorobenzene, nitrobenzene, benzene, toluene, dimethyl sulfoxide, heptane, or cyclohexane or any mixture thereof.

3. The method according to any of the preceding claims, wherein the basei) is selected from inorganic bases or organic bases; and / orii) is present in 0.1 to 3.0 eq. based on 1 eq. of compound (II).202400175 Foreign Filing 124. The method according to any of the preceding claims, wherein compound (III) is present in 1.0 to 3.0 eq. based on 1 eq. of compound (II).

5. The method according to any of the preceding claims, wherein the compound of formula (I) is selected from6. The method according to any of the preceding claims, further comprising a purification step in a second vessel.

7. A compound of formula (I) obtained by the method according to any of claims 1 to 6.202400175 Foreign Filing 138. A pharmaceutical composition comprising at least one compound of formula (I) according to claim 7, in particular a parenteral pharmaceutical composition, preferably a lipid nanoparticle, most preferably a vaccine.

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