Patent Application
20250034195
The present invention relates to a novel and efficient method for the production of sphingolipids via the N-acylation of lysosphingolipids such as sphingoid bases and glycosylated sphingoid bases. The method is based on the use of 1,3,5-triazine-based acylating agents, and is especially suitable for the production of ceramides and glycosphingolipids.
Sphingolipids are an important class of polar lipids mainly found on the surface of eukaryotic cells. Sphingolipids are structurally characterized by a sphingoid base backbone and can be divided in different classes such as, ceramides, and glycosphingolipids.
Ceramides are N-acylated sphingoid bases lacking additional head groups at the 1-position of the sphingoid base backbone, and wherein the N-acyl group of ceramides typically derives from a fatty acid. Glycosphingolipids (GSLs) are glycoconjugates deriving from ceramides, wherein a glycan moiety is linked to the 1-hydroxyl group of a ceramide via a glycosidic linkage.
Sphingolipids are involved in diverse biological processes and play important structural and functional roles such as cell-cell recognition, communication, and intercellular adhesion. Particularly, GSLs such as gangliosides are found in the brain and play roles in neurological diseases (Kolter, ISRN Biochem. 2012), whereas ceramides are the main constituent of the stratum corneum lipid layer and have a major role in the water-retaining properties of the epidermis, as well as in the barrier function of the skin.
Accordingly, sphingolipids hold great potential as therapeutics, cosmetics, and as tools for the study of important biological processes. However, they are not readily available for fundamental and clinical research. In fact, sphingolipids such as ceramides and GSLs are characterized by a high structural complexity and their preparation represents a challenge.
Current processes for the preparation of sphingolipids are typically based on extraction from animal brains, or animal epidermal tissues (EP 3095451 A1, U.S. Pat. No. 5,532,141 A). However, extraction and isolation of sphingolipids from animal sources is a laborious and costly process, and typically yields the desired compounds in low amounts and with low purities. Furthermore, the obtained sphingolipids may be potentially unsafe due to the presence of hazardous biological contaminants.
Alternatively, sphingolipids such as ceramides and GSLs may be obtained via the N-acylation of lysosphingolipids.
Typically, lysosphingolipids are defined as sphingolipid breakdown products which lack the amide-linked fatty acyl group at the 2-position of the sphingoid base backbone. Accordingly, for each parental sphingolipid there is a corresponding lysosphingolipid that has an identical head group at the 1-position but lacks the amide-bound fatty acyl group at the 2-position (Hannun et al., Science 1989, 243, 500-507).
Attempts have been made for the N-acylation of lysosphingolipids via chemical or enzymatic approaches.
Current chemical N-acylation methods are typically based on the use of standard amide bond coupling reagents such as N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbodiimide (DCC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Skolova et al, Biochim Biophys Acta Biomembr. 2017, 1859, 824-834, WO0172701 A1). Disadvantages connected to these methods include the use of expensive and unstable coupling reagents, which typically require the use of water-free solvents and/or the reaction must be performed under exclusion of air, thus rendering the scale up difficult.
Current enzymatic approaches are based on the use of lipases (WO1994026919A1) which, however, are typically not specific for the amino group of a lysosphingolipid but may also act on hydroxyl groups, thus leading to the formation of mixtures of N- and O-acylation products, and thus requiring a lengthily and costly purification of the target compound.
Furthermore, current chemical and enzymatic approaches for the N-acylation of lysosphingolipids mainly rely on the use of lysosphingolipids obtained from animal sources.
Accordingly, there is a demand for the development of novel and improved methodologies which enable the efficient and large-scale N-acylation of lysosphingolipids characterized by high technological feasibilities and low costs.
In a first aspect, the present invention relates to a method for the production of a sphingolipid of formula (1):
represents a conjugated system of bonds such that either two or three double bonds are present in the ring,
In a second aspect, the present invention relates to a method for the production of a sphingolipid of formula (1), wherein the method further comprising steps of producing the triazine-based acylating agent formula (3), or the combination thereof.
In a third aspect, the present invention relates to method for the production of a sphingolipid of formula (1), wherein the triazine-based acylating agent of formula (3), or the combination thereof, is produced via the steps of:
In a fourth aspect, the present invention relates to a triazine based acylating agent of formula (3), or a combination thereof:
represents a conjugated system of bonds such that either two or three double bonds are present in the ring;
In a fifth aspect, the present invention relates to a combination comprising triazine base acylating agents of formula (10)-(13):
and wherein each of said compounds is present, in said combination, in the amount from about 1% to about 99%.
The present inventors have established for the first time an efficient and economic method for the production of sphingolipids via the chemical N-acylation of lysosphingolipids, and wherein the lysosphingolipids used as the starting materials are preferably obtained via synthetic and/or biotechnological approaches.
Surprisingly, the present inventors have found that lysosphingolipids can be N-acylated using a robust and easily accessible 1,3,5-triazine-based acylating agent of formula (3), or a combination thereof:
represents a conjugated system of bonds such that either two or three double bonds are present in the ring;
The process described in the present invention gives access to a diverse set of biologically relevant sphingolipids in high purity and yields. Furthermore, the process does not require the use of water-free solvents and/or exclusion of air, and the produced sphingolipids can be isolated directly from the reaction mixture via precipitation in high purity and yields.
Non-limiting embodiments of different aspects of the invention are described below and illustrated by non-limiting examples.
The terms, definitions and embodiments described throughout the specification of the invention relate to all aspects and embodiments of the invention.
The term “a” grammatically is a singular, but it may as well mean the plural of e.g., the intended compound. For example, a skilled person would understand that in the expression “a lysosphingolipid of formula (2)”, the provision of not only one single a lysosphingolipid of formula (2), but of a variety of lysosphingolipids of the same type is meant.
In formulas representing a moiety, or a group such as for example the alkyl moiety of formula (8) or (9), or the acyl groups of formula (20)-(22), or (23)-(31) the symbol means a point of attachment to another group or atom.
As used herein, the various functional groups or substituents represented will be understood to have a point of attachment at the functional group or atom having the dash (-). For example, in the case of —C(═O) it will be understood that the point of attachment is the carbon atom. If a group is listed without a dash, then the attachment point is indicated by the plain and ordinary meaning of the recited group.
As used herein the letters N, C, O and H refer to a nitrogen atom, to a carbon atom, to an oxygen atom and to a hydrogen atom, respectively.
The skilled person would understand that when speaking of position C-1, C-2, C-3, C-4, C-5 etc., reference is herein always made to the respective carbon atoms of sphingolipids of formula (1), (32), or (33), or lysosphingolipids of formula (2), (14), or (15).
The expression “one of Xa and Xb is N and the other group is NR6,” means that when Xa is N the other group Xb is NR6, or when Xb is N the other group Xa is NR6.
As used herein, the term “alkyl” refers to an acyclic straight or branched hydrocarbyl radical having 1-50 carbon atoms which may be saturated or contain one or more double and/or triple bonds (so, forming for example an alkenyl or an alkynyl), and/or which may be substituted or unsubstituted, as herein further described. Examples of “alkyl” include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neo-pentyl, n-hexyl, ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, methylpentenyl, dimethylbutenyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, pentynyl, and hexynyl, each of which may be substituted or unsubstituted. Typically, the term alkyl refers to a straight acyclic hydrocarbyl group having 1-32 carbons, which may be substituted or unsubstituted.
As used herein, the term “aryl” refers to an aromatic cyclic hydrocarbyl group having 5-14 ring carbon atoms, which may be mono- or polycyclic, which may contain fused rings, preferably 1 to 3 fused or unfused rings, and which may contain one or more heteroatoms, and/or which may be substituted or unsubstituted, as herein further described. Examples of “aryl” include, but are not limited to, phenyl, naphtyl, anthracyl, phenantryl, pyrrolyl, imidazolyl, thiophenyl, furanyl, oxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, and benzofuranyl, each of which may be substitute or unsubstituted. Typically, the term “aryl” refers to a substituted or unsubstituted phenyl.
As used herein, the term “acyl” refers to a group derived by the removal of one or more hydroxyl group from an oxoacid, preferably from a carboxylic acid. The acyl group according to the present invention is typically a saturated C130 acyl, which may be substitute or unsubstituted.
As used herein the term “alkylene” refers to a bivalent saturated or unsaturated aliphatic radical deriving from a substituted or unsubstituted alkane by removal of two hydrogen atoms from different carbon atoms, preferably from the terminal carbon atoms. The alkylene according to the present invention, typically is a C6-28 alkylene which may be saturated or contain one or more double and/or triple bonds, and/or which may be substituted or unsubstituted, as herein further described.
As used herein, the term “substituted” means that the group in question is substituted with a group which typically modifies the general chemical characteristics of the group in question. The substituents can be used to modify characteristics of the molecule, such as molecule stability, molecule solubility and the ability of the molecule to form crystals. The person skilled in the art will be aware of other suitable substituents of a similar size and charge characteristics, which could be used as alternatives in a given situation.
In connection with the terms “alkyl”, “aryl”, “acyl”, and “alkylene” the term substituted means that the group in question is substituted one or several times, preferably 1 to 3 times, with group(s) selected from hydroxy (which when bound to an unsaturated carbon atom may be present in the tautomeric keto form), oxo, C1-6-alkoxy (i.e. C1-6-alkyl-oxy), C2-6-alkenyloxy, carboxy, oxo, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy, arylamino, arylcarbonyl, heteroaryl, heteroarylamino, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)aminocarbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkylcarbonylamino, cyano, guanidino, carbamido, C1-6-alkyl-sulphonyl-amino, aryl-sulphonyl-amino, heteroaryl-sulphonyl-amino, C1-6-alkanoyloxy, C1-6-alkyl-sulphonyl, C1-6-alkyl-sulphinyl, C1-6-alkylsulphonyloxy, nitro, C1-6-alkylthio, halogen, where any alkyl, alkoxy, and the like representing substituents may be substituted with hydroxy, C1-6-alkoxy, C2-6-alkenyloxy, carboxy, C1-6-alkylcarbonylamino, halogen, C1-6-alkylthio, C1-6-alkyl-sulphonyl-amino, or guanidino.
The term “glycosyl moiety” when used herein is defined to encompass a moiety derived from a monosaccharide or from an oligosaccharide (more than one monosaccharide units), wherein the anomeric carbon of the monosaccharide or the anomeric carbon at the reducing end of the oligosaccharide is engaged in a glycosidic bond with another chemical entity, and the bond, if not further specified, may be an alpha or a beta glycosidic bond. A glycosyl moiety having more than one monosaccharide unit may represent a linear or a branched structure.
The monosaccharide unit can be any 5-9 carbon atom sugar, comprising aldoses (e.g. D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g. D-fructose, D-sorbose, D-tagatose, etc.), deoxysugars (e.g. L-rhamnose, L-fucose, etc.), deoxy-aminosugars (e.g. N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acids, ketoaldonic acids (e.g. sialic acid). The monosaccharide unit can form different cyclic structures such as pyranose (six-membered) cyclic structures or furanose (five-membered) cyclic structures.
The glycosyl moieties according to the present invention may be illustrated in the following style: Galβ1-4Glc1-, wherein the dash (-) represents the point of attachment of the glycosyl moiety and wherein the glycosyl moiety may be linked via an alpha or a beta glycosidic bond, preferably a beta glycosidic bond.
The term, “oligosaccharide portion of a ganglioside” as used herein is defined to encompass glycosyl moieties deriving from gangliosides, wherein the anomeric carbon at the reducing end of the oligosaccharide portion of the ganglioside is engaged in a glycosidic bond with another chemical entity, the glycosidic bond may be an alpha or a beta glycosidic bond, preferably a beta glycosidic bond. In the context of the present invention the terms oligosaccharide portion and glycosyl moiety may be used interchangeably.
In the context of the present invention, the terms “about”, “around”, or “approximate” are applied interchangeably to a particular value (e.g. “a temperature of about 25° C.”, “a temperature of around 25° C.”, or “a temperature of approximate 25° C.”), or to a range (e.g. “an amount from about 1% to about 99%”, “an amount from around 1% to around 99%”, or “an amount from approximate 1% to approximate 99%”), to indicate a deviation from 0.1% to 10% of that particular value.
The term, “triazine-based acylating agent” as used herein refers to an activated derivate of a carboxylic acid, wherein the acidic hydroxyl group of the carboxylic acid is converted into a good leaving group via the replacement of the hydrogen atom with a 1,3,5-triazynyl group. Suitable triazine-based acylating agents for use in the context of the present invention are for example those represented by formulas (3)-(7) and (10)-(13), wherein the hydrogen atom of the acidic hydroxyl group of a carboxylic acid is replaced by a 4,6-dialkoxy-1,3,5-triazin-2-yl group, or by a 4,6-diaryloxy-1,3,5-triazin-2-yl group.
Accordingly, suitable triazine-based acylating agents for use in the context of the present invention carry one acyl group.
Triazine-based acylating agents carrying one acyl group can form several isomeric structures resulting from the migration of substituents on different positions of the 1,3,5-triazine ring. Depending on conditions such as temperature, solvent, and/or the use of a certain reagent such as a certain base, these isomeric structures can be isolated in a pure form or obtained as an isomeric mixture.
Accordingly, in the context of the present invention, the term “combination of triazine-based acylating agents” refers to a mixture comprising triazine isomeric structures which differ only in the position of the substituents on the triazine ring and can all serve as acylating agents.
In some embodiments, the triazine-based acylating agent of formula (3) is a triazine-based acylating agent is of formula (4).
In some embodiments, the triazine-based acylating agent of formula (3) is a triazine-based acylating agent of formula (5).
In some embodiments, the triazine-based acylating agent of formula (3) is a triazine based acylating agent of formulas (6), or (7):
In some embodiments, the triazine-based acylating agent is a triazine-based acylating of formula (18) or (19):
In some embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (4), (5), (6) and (7), and wherein each of said triazine-based acylating agent is present, in said combination, in the amount from about 1% to about 99%.
In some embodiments, the combination of triazine-based acylating agents of formulas (4), (5), (6) and (7), further comprises triazine based acylating agents of formulas (18), and (19), and wherein each of said triazine-based acylating agent is present, in said combination, in the amount from about 1% to about 99%.
Triazine-based acylating agents for use in the context of the present invention carry one acyl group preferably deriving from a carboxylic acid, and wherein the carboxylic acid is selected from formic acid, benzoic acid, acetic acid, or a fatty acid.
The acyl group carried by the triazine-based acylating for use in the context of the present invention may be illustrated in the following stile: —C(═O)R4, or may be represented by the acyl group of formula (20):
In some preferred embodiments, R4 of the acyl group —C(═O)R4, or R4 of the acyl group of formula (20) is a substituted or unsubstituted C9-31 alkyl selected from the group consisting of substituted or unsubstituted C9-31 alkyl of formula (8) and (9):
Accordingly in some preferred embodiments, the acyl group carried by the triazine-based acylating agents for use in the context of the present invention may be represented by an acyl group of formula (21) or (22):
In some embodiments, the substituted or unsubstituted C9-31 alkyl is a substituted or unsubstituted C9-31 alkyl of formula (8), wherein Q is —H, R7 is hydrogen, and L is a straight-chain saturated C12-28 alkylene. Accordingly, in some embodiments, the acyl group carried by the triazine-based acylating agent for use in the context of the present invention is an acyl group of formula (21), wherein the acyl group of formula (21) is an acyl group deriving from a non-hydroxy fatty acids (N).
In some preferred embodiments, the acyl group carried by the triazine-based acylating agent for use in the context of the present invention, is an acyl group deriving from stearic acid [N (18:0)].
In some embodiments, the substituted or unsubstituted C9-31 alkyl is a substituted or unsubstituted C9-31 alkyl of formula (8), wherein Q is —OH, R7 is hydrogen, and L is a straight-chain saturated C12-28 alkylene. Accordingly, in some embodiments, the acyl group carried by the triazine-based acylating agent for use in the context of the present invention is an acyl group of formula (21), wherein the acyl group of formula (21) is an acyl group deriving from an α-hydroxy fatty acids (A).
In some embodiments, the acyl group carried by the triazine-based acylating agent for use in the context of the present invention is an acyl group deriving from α-hydroxystearic acid [A(18:0)].
In some embodiments, the substituted or unsubstituted C9-31 alkyl is a substituted or unsubstituted C9-31 alkyl of formula (8), wherein Q is —H, L is a straight-chain saturated C12-28 alkylene, and R7 is —OR8, wherein R8 is a linoleoyl group. Accordingly, in some embodiments, the acyl group carried by the triazine-based acylating agent for use in the context of the present invention is an acyl group of formula (21), wherein the acyl group of formula (21) is an acyl group deriving from a w-linoleoyloxy-fatty acid [E(18:2)O(12-28)].
In some embodiments, the acyl group carried by the triazine-based acylating agent for use in the context of the present invention is an acyl group of formula (21), wherein the acyl group pf formula (21) is selected from the group consisting of acyl groups of formula (23)-(31):
In some preferred embodiments, the triazine-based acylating agent of formula (3), is a triazin-based acylating agent of formulas (4), (5), (6) or (7), and wherein the triazine based acylating agents of formulas (4), (5), (6) and (7), are triazine based acylating agents of formulas (10), (11), (12), and (13), respectively:
In some embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (10), (11), (12) and (13), and wherein each of said triazines is present, in said combination, in the amount from about 1% to about 99%.
In some embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (10) and (12), and wherein each of said triazines is present, in said combination, in the amount from about 1% to about 99%.
In some embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (10) and (12), and wherein the triazine-based acylating agents of formulas (10) is present, in said combination, in the amount from about 5% to about 75%.
In some embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (10) and (12), and wherein the triazine-based acylating agents of formulas (12) is present, in said combination, in the amount from about 5% to about 75%.
In some embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (11) and (13), and wherein each of said triazines is present, in said combination, in the amount from about 1% to about 99%.
In some preferred embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (11) and (13), and wherein the triazine-based acylating agents of formulas (11) is present, in said combination, in the amount from about 5% to about 50%.
In some preferred embodiments, the triazine-based acylating agent of formula (3) is a combination comprising triazine-based acylating agents of formulas (11) and (13), and wherein the triazine-based acylating agents of formulas (13) is present, in said combination, in the amount from about 5% to about 50%.
For those embodiments, describing a combination of triazine-based acylating agents, the amount of the triazine-based acylating agents comprised in the combination is typically determined via 1H NMR spectroscopy.
In a typical procedure:
The person skilled in the art will understand that the amount of the triazine-based acylating agents of the combination may represent a molar ratio or mol %, or a weight ratio or wt %. The person skilled in the art will also understand that the molar ratio or mol %, or the weight ratio or wt % of the triazine-based acylating agents of the combination, may vary over time due interconversion between the different isomeric structures of the triazine-based acylating agent.
In some preferred embodiments, R6 of the triazine-based acylating agents of formulas (3)-(7), (10)-(13), (18), or (19) is methyl.
Triazine-based acylating agents according to the present invention, or their combination thereof, may be produced by standard methods known to the skilled person. A method for the synthesis of triazine-based acylating agent is e.g. described by Z. J. Kamiński, J. prakt. Chem. 1990, 4, 579-583.
Alternatively, triazine-based acylating agents or combinations thereof may be synthesized according to the method of the present invention.
The triazine-based acylating agents according to the present invention may be utilized or produced in different polymorphic forms. Polymorphic forms as referred to herein can include crystalline and amorphous forms as well as solvate and hydrate forms, which can be further characterized as follows:
In some embodiments, the triazine-based acylating agents are present as solvates.
In some embodiments, the triazine-based acylating agents are present as hydrates, such as in the form of monohydrates, dihydrates or trihydrates.
In some embodiments, the triazine-based acylating agents are present in a crystalline form.
In some embodiments, the triazine-based acylating agents are present in an amorphous form.
The present invention describes a method for the production of a sphingolipid of formula (1), wherein the triazine-based acylating agent according to the present invention or a combination of triazine-based acylating agents according to the present invention, is reacted with a lysosphingolipid of formula (2).
In some preferred embodiments, a triazine-based acylating agent of formula (10), is reacted with a lysosphingolipid of formula (2).
In some preferred embodiments, a triazine-based acylating agent of formula (11), is reacted with a lysosphingolipid of formula (2).
In some embodiments, a combination comprising triazine-based acylating agents of formulas (10), (11), (12) and (13), is reacted with a lysosphingolipid of formula (2), and wherein each of said triazine-based acylating agent is present, in said combination, in the amount from about 1% to about 99%.
In some embodiments, a combination comprising triazine-based acylating agents of formulas (10) and (12), is reacted with a lysosphingolipid of formula (2), and wherein each of said triazine-based acylating agent is present, in said combination, in the amount from about 1% to about 99%.
In some embodiments, a combination comprising triazine-based acylating agents of formulas (10) and (12), is reacted with a lysosphingolipid of formula (2), and wherein the triazine-based acylating agents of formulas (10) is present, in said combination, in the amount from about 5% to about 75%.
In some embodiments, a combination comprising triazine-based acylating agents of formulas (10) and (12), is reacted with a lysosphingolipid of formula (2), and wherein the triazine-based acylating agents of formulas (12) is present, in said combination, in the percentage from about 5% to about 75%.
In some embodiments, a combination comprising triazine-based acylating agents of formulas (11) and (13), is reacted with a lysosphingolipid of formula (2), and wherein each of said triazine-based acylating agent is present, in said combination, in the amount from about 1% to about 99%.
In some preferred embodiments, a combination comprising triazine-based acylating agents of formulas (11) and (13), is reacted with a lysosphingolipid of formula (2), and wherein the triazine-based acylating agents of formulas (11) is present, in said combination, in the amount from about 5% to about 50%.
In some embodiments, a combination comprising triazine-based acylating agents of formulas (11) and (13), is reacted with the lysosphingolipid of formula (2) and wherein the triazine-based acylating agents of formulas (13) is present, in said combination, in the amount from about 5% to about 50%.
The term “lysosphingolipid” when used herein refers to a sphingolipid breakdown product which lack the amide-linked fatty acyl group at the 2-position of the sphingoid base backbone. Suitable lysosphingolipids, for use in the context of the present invention, are sphingoid bases or glycosylated sphingoid bases and are represented by a lysosphingolipid of formula (2).
Lysosphingolipids for use in the context of the present invention are preferably obtained via synthetic and/or biotechnological approaches such as those described in WO 2021170624 A2, or in WO2019238970 A1, WO2022158993 A1, or by Sarmientos et al., Eur. J. Biochem. 1986, 160,527-535.
In some embodiments, the lysosphingolipid of formula (2) is a lysosphingolipid of formula (14), or a salt thereof:
In some embodiments, the stereochemical configuration of the C-2, C-3, and C-4 carbon atoms of the lysosphingolipid of formula (14) is (2S,3R,4E).
In some preferred embodiments, the lysosphingolipid of formula (14) is
In some embodiments, the lysosphingolipid of formula (14) is 6-hydroxy-
In some embodiments, the lysosphingolipid of formula (2) is a lysosphingolipid of formula (15), or a salt thereof:
In some embodiments the stereochemical configuration of the C-2, C-3, and C-4 carbon atoms of the lysosphingolipid of formula (15) is (2S,3S,4R).
In some preferred embodiments, the lysosphingolipid of formula (15) is
In some preferred embodiments, the lysosphingolipid of formula (15) is
In some embodiments, lysosphingolipids of formula (2), (14), and (15) may be produced or utilized in the form of salts, preferably in the form of pharmaceutical acceptable salts.
In some embodiments, the salts of compounds of formula (2), (14), and (15), may be formed from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, polyphosphoric acid, acetic acid, camphor sulfonic acid, p-toluene sulfonic acid, methane sulfonic acid, trifluoromethanesulfonic acid, perchloric acid.
Typically, the lysosphingolipid, and the triazine-based acylating agent, or a composition thereof are reacted in a polar solvent such as methanol, ethanol, propanol, isopropanol, butanol, or isobutanol.
In some preferred embodiment, the reaction is performed in methanol. In some embodiment the reaction is performed in a mixture of one or more polar solvents, such as a mixture of methanol and ethanol, methanol and propanol, methanol and isopropanol, methanol and butanol, methanol and isobutanol, or methanol and water.
In some embodiments, the lysosphingolipid and the triazine-based acylating agent, or the composition thereof are reacted in the presence of a base such as NaOH, KOH, LiOH, Ca(OH)2, triethylamine, N,N-diisopropylethylamine, and pyridine. In some preferred embodiments, the based is selected from NaOH, or KOH.
The purity of the triazine-based acylating agent according to the present invention, or the combination thereof may vary from a purity of about 99% to a purity of about 70%.
The reaction between the lysosphingolipid and the triazine-based acylating agent, or the composition thereof is typically performed at temperature from about 25° C. to about 65° C. Accordingly, in some embodiments, the reaction is performed at a temperature of about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., or 65° C. Preferably, the reaction between the lysosphingolipid and the triazine-based acylating agent, or a composition thereof is performed at temperature from about 45° C. to about 55° C. Accordingly, in some preferred embodiments the reaction is performed at a temperature of 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C.
The components of the reactions of the invention may be combined in any order, and it will be appreciated that the order of combining the reactants may be adjusted as needed. For example, the lysosphingolipid may be added to a solution of the triazine-based acylating agent, or the composition thereof. As another example the triazine-based acylating agent, or the composition thereof may be added to a solution of the lysosphingolipid. As yet another example, a solvent may be added to a flask containing the lysosphingolipid and the triazine-based acylating agent, or the composition thereof.
The lysosphingolipid and the triazine-based acylating agent, or the composition thereof, as well as any other reagent used during the reaction may be added to the reaction either as a solid or dissolved in a solvent, and in any quantities and manner effective for the intended result of the reaction.
In some embodiments, the present invention discloses a method for the production of a sphingolipid of formula (1), wherein the method further comprising steps of producing a triazine-based acylating agents of formula (3), or a combination thereof, and wherein the triazine-based acylating agents of formula (3), or the combination thereof is produced via the steps of:
In some embodiments, the present invention discloses a method for the production of a sphingolipid of formula (1), wherein the method comprising steps of:
represents a conjugated system of bonds such that either two or three double bonds are present in the ring;
Typically, the step of reacting the carboxylic acid of formula (16) with the compound of formula (17) is performed in the presence of an organic base. In some embodiments, the organic base is selected from 4-methylmorpholine, 1,4-diazabicyclo[2.2.2]octane, preferably 4-methylmorpholine.
Typically, the steps of the method for the synthesis of the triazine-based acylating agents of formula (3), or the combination thereof are performed in the same non-halogenated solvent. The non-halogenated solvent is preferably selected from a ketone, an alcohol, or an aliphatic hydrocarbon. In some embodiments, the non-halogenated solvent is a ketone selected from acetone, diethyl ketone, methyl isobutyl ketone, or butan-2-one, preferably acetone. In some embodiments, when the solvent is a ketone, water may be added to the reaction mixture. In some embodiments, the non-halogenated solvent is an alcohol selected from methanol, ethanol, propanol, isopropanol, butanol, isobutanol, preferably methanol, or a mixture thereof. In some embodiments, the non-halogenated solvent is an aliphatic hydrocarbon selected from petroleum ether, hexane, or an isomeric mixture thereof, n-heptane, or an isomeric mixture thereof, octane, or an isomeric mixture thereof. In some embodiments, the non-halogenated solvent is a mixture of two alcohols, and wherein the mixture of two alcohols is preferably selected from a mixture of methanol and ethanol, methanol and propanol, methanol and isopropanol, methanol and butanol, or methanol and isobutanol, preferably a mixture of methanol and ethanol.
In some embodiments, the step of reacting the carboxylic acid of formula (16) with the compound of formula (17), is performed at a temperature between about 30° C. and about 100° C., preferably between about 30° C. and about 55° C.
In some embodiments, step isolating the triazine-based acylating agents of formula (3), or the combination thereof is performed at a temperature between about −20° C. and about 25° C., preferably at a temperature between about −10° C. and about 25° C., even more preferably at a temperature between about 5° C. and about 25° C.
The purity of the triazine-based acylating agent according to the present invention, or the combination thereof may vary from a purity of about 99% to a purity of about 70%.
In some embodiments, stearic anhydride (from about 8 mol % to about 30 mol %) may be present in combination with the triazine-based acylating agent according to the present invention.
The reaction between the triazine-based acylating agents according to the present invention and the lysosphingolipid of formula (2), (14), or (15), or the reaction between a composition of triazine-based acylating agents according to the present invention, and the lysosphingolipid of formula (2), (14), or (15), results in the selective N-acylation of the amino group at the C-2 carbon atom of the lysosphingolipid of (2), (14), or (15), thereby producing a sphingolipid of formula (1).
In some embodiments, the sphingolipid of formula (1) is a sphingolipid of formula (32), or (33):
Sphingolipids of formulas (1), (32), or (33) carry an acyl group, preferably deriving from a carboxylic acid, and wherein the carboxylic acid is selected from formic acid, benzoic acid, acetic acid, or a fatty acid.
The acyl group carried by the sphingolipids of formulas (1), (32), or (33) may be represented by the acyl group of formula (20):
In some preferred embodiments, R4 of the acyl group of formula (20) is a substituted or unsubstituted C9-31 alkyl selected from the group consisting of substituted or unsubstituted C9-31 alkyl of formula (8) and (9):
Accordingly in some preferred embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33) may be represented by an acyl group of formula (21) or (22):
In some embodiments, the substituted or unsubstituted C9-31 alkyl is a substituted or unsubstituted C9-31 alkyl of formula (8), wherein Q is —H, R7 is hydrogen, and L is a straight-chain saturated C12-28 alkylene. Accordingly, in some embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33) is an acyl group of formula (21), wherein the acyl group of formula (21) is an acyl group deriving from a non-hydroxy fatty acid (N).
In some preferred embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33), is an acyl group deriving from stearic acid [N (18:0)].
In some embodiments, the substituted or unsubstituted C9-31 alkyl is a substituted or unsubstituted C9-31 alkyl of formula (8), wherein Q is —OH, R7 is hydrogen, and L is a straight-chain saturated C12-28 alkylene. Accordingly, in some embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33) is an acyl group of formula (21), wherein the acyl group of formula (21) is an acyl group deriving from an α-hydroxy fatty acids (A).
In some embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33) is an acyl group deriving from α-hydroxystearic acid [A(18:0)].
In some embodiments, the substituted or unsubstituted C9-31 alkyl is a substituted or unsubstituted C9-31 alkyl of formula (8), wherein Q is —H, L is a straight-chain saturated C12-28 alkylene, and R7 is —OR8, wherein R8 is a linoleoyl group. Accordingly, in some embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33) is an acyl group of formula (21), wherein the acyl group of formula (21) is an acyl group deriving from a w-linoleoyloxy-fatty acid [E(18:2)O(12-28)].
In some embodiments, the acyl group carried by the sphingolipids of formulas (1), (32), or (33) is an acyl group of formula (21), wherein the acyl group pf formula (21) is selected from the group consisting of acyl groups of formula (23)-(31):
In some embodiments for the sphingolipid of formula (1), (32), or (33), W is hydrogen, R1 is a substituted or unsubstituted C13 alkyl, R2 is hydrogen or —OH, and R3 is hydrogen. Accordingly in some embodiments, the sphingolipid of formula (1), (32), or (33) is a ceramide. Ceramides denote, in the context of the present invention, naturally occurring ceramides, analogues thereof or derivatives thereof. Preferred ceramides are those naturally occurring in humans. Naturally occurring human ceramides (CERs) include, but are not limited to, CER[NS], CER[AS], CER[EOS], CER[NH], CER[AH], or CER[EOH], CER[NP], CER[AP], or CER[EOP], CER[NdS], CER[AdS], or CER[EOdS], wherein letters in brackets refer to the shorthand nomenclature developed by Motta et al., Biochim Biophys Acta., 1993, 1182:147-151 and expanded by Rabionet et al., Biochim Biophys Acta, 2014, 1841:422-434, and by Masukawa et al., Journal of Lipid Research, 2008, 49, 1466-1476. Particularly, the letters N, A, and EO represent non-hydroxy fatty acids (N), alpha-hydroxy fatty acids (A), and omega-linoleoyloxy fatty acids (EO), respectively, wherein the number of fatty acid carbons and unsaturations may be expressed in parentheses following the letters of N, A, E, and O. The letters, S, H, P, and dS represent
In some embodiment, the sphingolipid of formula (1) is a sphingolipid of formula (32), wherein the sphingolipid of formula (32) is a ceramide selected from CER[N (16:0) S (18)], CER[A (16:0) S (18)], CER[N (18:0) S (18)], CER[A (18:0) S (18)], CER[N (20:0) S (18)], CER[A (20:0) S (18)],
In some embodiment, the sphingolipid of formula (1) is a sphingolipid of formula (32), wherein the sphingolipid of formula (32) is a ceramide selected from CER[N (16:0) H (18)], CER[A (16:0) H (18)], CER[N (18:0) H (18)], CER[A (18:0) H (18)], CER[N (20:0) H (18)], CER[A (20:0) H (18)].
In some embodiment, the sphingolipid of formula (1) is a sphingolipid of formula (33), wherein the sphingolipid of formula (33) is a ceramide selected from CER[N (16:0) P (18)], CER[A (16:0) P (18)], CER[N (18:0) P (18)], CER[A (18:0) P (18)], CER[N (20:0) P (18)], CER[A (20:0) P (18)].
In some embodiment, the sphingolipid of formula (1) is a sphingolipid of formula (33), wherein the sphingolipid of formula (33) is a ceramide selected from CER[N (16:0) dS (18)], CER[A (16:0) dS (18)], CER[N (18:0) dS (18)], CER[A (18:0) dS (18)], CER[N (20:0) dS (18)], CER[A (20:0) dS (18)].
In some preferred embodiments, W of the lysosphingolipid of formula (2), (14), or (15), and W of the sphingolipid of formula (1), (32), or (33) is a glycosyl moiety, and wherein the glycosyl moiety is selected from Glc1-, Gal1-, Galβ1-4Glc1-.
In some embodiments, W of the lysosphingolipid of formula (2), (14), or (15), and W of the sphingolipid of formula (1), (32), or (33) is a glycosyl moiety, and wherein the glycosyl moiety is the oligosaccharide portion of a ganglioside selected from GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, GM4, preferably GM4, GM3, or GD3, more preferably GM3.
In the context of the present invention the oligosaccharide portion of from GM1a, GM1b, GD1a, GD1b, GD3, GT1b, GT3, GQ1b, GM3, GM4 may be represented by the following formulas:
respectively.
In some embodiments, W of the lysosphingolipid of formula (2), (14), or (15), and W of the sphingolipid of formula (1), (32), or (33) is a glycosyl moiety, wherein the glycosyl moiety is that of a human milk oligosaccharide, and wherein the human milk oligosaccharide is preferably selected from LNT, LNnT, LNH, LNnH, 2′FL, 3FL, DFL, LNFP-1, LNFP-II, LNFP-III, LNFP-V, LNDFH-1, 3SL, 6′SL, FSL, LSTa, LSTb, LSTc, and DSLNT.
In the context of the present invention the oligosaccharide portion of LNT, LNnT, LNH, LNnH, 2′FL, 3FL, DFL, LNFP-1, LNFP-II, LNFP-Ill, LNFP-V, LNDFH-1, 3SL, 6′SL, FSL, LSTa, LSTb, LSTc, and DSLNT may be represented by the following formulas:
respectively.
In some embodiments, W of the lysosphingolipid of formula (2), (14), or (15), and W of the sphingolipid of formula (1), (32), or (33) is a glycosyl moiety, and wherein the glycosyl moiety is the oligosaccharide portion of a glycosphingolipid selected from the gala series [SP0509], the neogala series, the globo series [SP0502], the isoglobo series [SP0506], the lacto series [SP0504], the neolacto series [SP0505], the arthro series [SP0508], the muco series, the schisto series, the spirometo series, or protected analogs thereof.
Wherein the bracketed alphanumeric string indicates a link contained on the website https://www.lipidmaps.org/, wherein detailed chemical formulas and structural information for the corresponding compounds can be found.
In the context of the present invention lysosphingolipid of formula (2), (14), or (15), wherein W is a glycosyl moiety may also be referred to as glycosylated sphingoid bases.
In the context of the present invention, sphingolipids of formula (1), (32), or (33) wherein W is a glycosyl moiety may also be referred to as glycosphingolipids or glycosylated ceramides.
In the context of the present invention, glycosyl moiety may be linked via an alpha or a beta glycosidic bond, preferably a beta glycosidic bond.
In some preferred embodiments, the sphingolipid of formula (1) is a sphingolipid of formula (32), and wherein the sphingolipid of formula (32) is selected from the group consisting of sphingolipids of formulas (34)-(38):
The skilled person will understand that in formulas showing a specific compound, unless the chemical formula expressly describes a carbon atom having a particular stereochemical configuration, the formula is intended to cover compounds where such a stereocenter has an R or an S configuration, or wherein a double bond has an E or a Z configuration.
1H NMR and 13C NMR was recorded with a Bruker WM-300S (300/75.1 MHz) spectrometer. 1H and 13C chemical shifts are given in ppm (δ) relative to CDCl3 (δ=7.26, or δ=77.2) as internal standard. LCMS analysis was performed with a Shimadzu ECO 2020 LC system coupled with a Shimadzu LCMS-2020 system. TLC-analysis was performed with silica gel TLC-plates (Merck, Silica gel, F254) with detection by carring (±140° C.) with ammonium molybdate (25 g/L) and cerium ammonium sulfate (10 g/L) in 10% H2SO4. Lysosphingolipids such as for example
A fatty acid (1 eq.) was suspended in n-heptane, acetone, or a mixture of ethanol/methanol. 2-Chloro-4,6-dimethoxy-1,3,5-triazine (1 eq.), and 4-methylmorpholine (1 eq.) were added to the suspension/solution. The reaction mixture was stirred at a temperature between 20° C. to 50° C. until a TLC-analysis showed complete consumption of the starting material.
The reaction mixture was cooled down to a temperature between 5° C. to 30° C. When acetone was used as solvent, water was added to the reaction mixture prior cooling. A solid precipitated, which was filtered and dried in vacuum to obtain the final product.
Compound (10) was synthesised from stearic acid and 2-chloro-4,6-dimethoxy-1,3,5-triazine following the general procedure described in Example 1.
1H NMR (500 MHz, CDCl3) δ 4.06 (s, 6H), 2.60 (t, J=7.5 Hz, 2H), 1.73 (m, 2H), 1.77-1.68 (m, 28H), 0.88 (t, J=6.9 Hz, 3H).
Compound (11) was synthesised from stearic acid and 2-chloro-4,6-dimethoxy-1,3,5-triazine following the general procedure described in Example 1.
1H NMR (400 MHz, CDCl3) δ 4.10 (s, 3H), 3.35 (s, 3H), 2.84 (t, J=7.4 Hz, 2H), 1.78-1.67 (m, 2H), 1.43-1.19 (m, 28H), 0.87 (t, J=6.7 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 174.66, 160.13, 152.21, 148.83, 57.51, 40.30, 31.93, 29.70, 29.67, 29.66, 29.64, 29.58, 29.41, 29.36, 29.25, 28.81, 28.63, 23.61, 22.69, 14.11.
The combination of compounds (11) and (13) was synthesised from stearic acid and 2-chloro-4,6-dimethoxy-1,3,5-triazine following the general procedure described in Example 1. The amounts of compounds (11) and (13) may vary over time due to interconversion between the two isomeric structures.
1H NMR (400 MHz, CDCl3): δ 4.10 (s), 4.05 (s), 3.35 (s), 3.34 (s), 2.85 (m), 1.78-1.67 (m), 1.43-1.19 (m), 0.88 (m).
The combination of compounds (10) and (12) was synthesised from stearic acid and 2-chloro-4,6-dimethoxy-1,3,5-triazine following the general procedure described in Example 1. The amounts of compounds (10) and (12) may vary over time due to interconversion between the two isomeric structures.
1H NMR (500 MHz, CDCl3) δ 4.06 (s), 4.08 (s), 4.02 (s), 2.88 (t), 2.60 (t), 1.77-1.68 (m), 1.73 (m), 1.43-1.20 (m), 0.88 (t).
A lysosphingolipid was dissolved in methanol, the triazine-based acylating agent of formula (10) (1 eq), or the triazine-based acylating agent of formula (11) (1 eq), or the combination of triazine-based acylating agents of formulas (11) and (13) (1 eq), or the combination of triazine-based acylating agents of formula (10) and (12) (1 eq), was added. The resulting suspension was stirred at about 50° C. until a TLC-analysis showed complete consumption of the starting material. Water was added, and the suspension was subsequently heated to reflux, and then cooled down the to a temperature between about 5° C. to about 10° C. A solid precipitated, which was filtered, washed with a methanol/water (5/1) mixture, and dried in vacuum to obtain the final product.
Compound (37) was synthesised from α-N-acetylneuraminosyl-(2→3)-O-β-
MS spectrum: [M+H]+ 1181, [M+Na]+ 1203
Compound (36) was synthesised from β-
MS spectrum: [M+H]+ 890, [M+Na]+ 913
Compound (34) was synthesised from β-
MS spectrum: [M+H]+ 728, [M+Na]+ 750
| Number | Date | Country | Kind |
|---|---|---|---|
| 117615 | Dec 2021 | PT | national |
| 117616 | Dec 2021 | PT | national |
| 117639 | Dec 2021 | PT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/083675 | 11/29/2022 | WO |
