WO2024117978A1

WO2024117978A1 - Methods of synthesising ionisable lipids

Info

Publication number: WO2024117978A1
Application number: PCT/SG2023/050798
Authority: WO
Inventors: Viktor BARÁT; Yee Hwee LIM
Original assignee: Agency For Science, Technology And Research (A*Star)
Priority date: 2022-11-30
Filing date: 2023-11-30
Publication date: 2024-06-06

Abstract

The present disclosure concerns methods of synthesising ionisable lipids and the ionisable lipids thereof. The methods comprises N-alkylating two acyloxy-substituted alkyl alcohols with an amino alcohol in the presence of a hydrogen borrowing catalyst, wherein the hydrogen borrowing catalyst is an iridium catalyst. The ionisable lipids comprises at least one alcohol moiety.

Description

METHODS OF SYNTHESISING IONISABLE LIPIDS

Technical Field

The present disclosure relates, in general terms, to the chemical synthesis of amphiphilic lipids. Disclosed herein are methods for synthesising ionisable lipids using hydrogen borrowing catalysis.

Background

Lipid amphiphiles are used as surfactants, emulsifiers and phase transfer agents in the manufacturing of various pharmaceuticals and food and consumer care products, lonisable lipids based on tertiary amines are a particularly important group of lipid amphiphiles for the pharmaceutical industry as these lipids are able to condense nucleic acids to form lipid nanoparticles (LNP), which efficiently deliver nucleic acids into cells. New classes of ionisable lipids are constantly being developed to improve the pharmacokinetics, biodistribution and cell uptake of nucleic acid therapeutics, especially for new generations of RNA therapeutics seeking to deliver mRNA, microRNA (miRNA) and small interfering RNA (siRNA) into cells.

Fatty aliphatic tertiary amines are specialty chemicals that have found wide-spread use in LNP formulations for nucleic acid delivery. Synthesis and purification methodologies for this class of ionisable lipids that are greener and provide higher conversion are particularly advantageous for the pharmaceutical industry.

The synthesis of tertiary amines bearing lipid chains is often tedious, as existing methodologies suffer from drawbacks. For instance, synthesis via alkylation is prone to overalkylation, and synthesis using reductive amination requires the preparation of a reactive aldehyde. Current synthesis methods can also be low-yielding while generating large quantities of toxic waste, like the chromium compounds used in reductive amination. There is thus a need for a synthesis and purification methodologies that are more concise and more environmentally sustainable without sacrificing yield.

It would be desirable to overcome at least one of the above-described problems.

Summary Disclosed herein is a method of synthesising an ionisable lipid of Formula (I), or a pharmaceutically acceptable salt, solvate or isomer thereof:

wherein each Ri is independently optionally substituted oxo, optionally substituted oxyacyl, optionally substituted acyloxyl, optionally substituted silyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene; 2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl; and n is an integer selected from 1 to 10; the method comprising:

N-a Ikylating at least two molar equivalence of alcohols of Formula (II) with 1 molar equivalence of amino alcohol of Formula (III) in the presence of a hydrogen borrowing catalyst;

R<^LI'OH _(II)

wherein the hydrogen borrowing catalyst is an iridium catalyst.

Disclosed herein is a method of synthesising an ionisable lipid of Formula (la), or a pharmaceutically acceptable salt, solvate or isomer thereof:

wherein each 3 is independently optionally substituted alkyl, optionally substituted heterocyclyl, or optionally substituted aryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene;

R2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl; and n is an integer selected from 1 to 10; the method comprising:

N-alkylating two acyloxy-substituted alkyl alcohols of Formula (Ila) with an amino alcohol of Formula (III) in the presence of a hydrogen borrowing catalyst;

wherein the hydrogen borrowing catalyst is an iridium catalyst.

In some embodiments, the hydrogen borrowing catalyst is a cyclopentadienyl iridium complex, wherein the cyclopentadienyl is optionally substituted.

In some embodiments, the hydrogen borrowing catalyst is cyclopentadienyl iridium dichloride dimer ([Cp*IrCl2]2), wherein the cyclopentadienyl is optionally substituted.

In some embodiments the hydrogen borrowing catalyst is added at a concentration of about 1 mol% to about 5 mol% relative to the hydroxyl-substituted alkyl amine.

In some embodiments, each R3 is independently C10-C24 alkyl, optionally substituted with halo.

In some embodiments, each Li is independently C1-C10 alkylene, optionally substituted with halo.

In some embodiments, n is an integer selected from 1 to 5.

In some embodiments, the method further comprises a step of protecting the hydroxyl moiety on the amino alcohol.

In some embodiments, the hydroxyl moiety is protected with a protecting group selected from 2-tetra hydropyranyl (THP), benzyl or dimethyl-tert-butylsilyl (TBS), tertbutyldiphenylsilyl (TBDPS), optionally substituted benzyl ether or other ethers such as methoxymethyl ether (MOM), P-methoxybenzyl (PMB).

In some embodiments, a molar ratio of the amino alcohol to the alcohol is about 1 :2 to about 1 :20.

In some embodiments, the N-alkylation step is performed in the presence of NaHCOs and toluene.

In some embodiments, the N-alkylation step is performed at a temperature of about 80°C to about 110°C.

In some embodiments, the N-alkylation step is performed for about 16 hr to about 24 hr.

In some embodiments, the method further comprises a step of purifying the ionisable lipid from the amino alcohol and/or the alcohol.

In some embodiments, the purification step is performed using column chromatography in the presence of dichloromethane, methanol and ammonia.

In some embodiments, the purification step is performed in the presence of magnesium silicate (Florisil) or silica gel.

In some embodiments, the method further comprises a step of purifying the ionisable lipid from the hydrogen borrowing catalyst.

In some embodiments, the purification of the ionisable lipid from the hydrogen borrowing catalyst is performed in the presence of a metal scavenger.

In some embodiments, the method further comprises a step of deprotecting the hydroxyl moiety on the ionisable lipid.

In some embodiments, the deprotection step is performed in the presence of hydrochloric acid and methanol. In some embodiments, the method further comprises a step of isolating a free amine form of the ionisable lipid.

In some embodiments, the isolation step is performed in the presence of ammonia, methanol and ethyl acetate or in the presence of diethyl ether, water and sodium hyd roxide.

In some embodiments, the deprotection step and the isolation step are performed sequentially in a reaction vessel.

In some embodiments, the N-alkylation step, deprotection step and isolation step method are performed sequentially in a reaction vessel.

In some embodiments, the ionisable lipid of Formula (I) is selected from:

Also disclosed herein is a method of synthesizing ALC-0315, or a pharmaceutically acceptable salt, solvate or isomer thereof:

the method comprising:

N-a Ikylating an amino alcohol of Formula (III) R₂

H^^OH n (III) wherein R2 is independently H; and n is 4; with two acyloxy-substituted alkyl alcohols of Formula (Ila)

wherein R3 is independently C15 alkyl bonded to the acyl moiety at a C7 position; and Li is independently Ce alkylene; in the presence of a cyclopentadienyl iridium dichloride dimer ([Cp*IrCl2]2) catalyst.

In some embodiments, the method further comprises a step of protecting the hydroxyl moiety on the amino alcohol with a 2-tetra hydropyranyl (THP) group.

Detailed description

"Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 25 carbon atoms or more preferably 1 to 15 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n- butyl, /so-butyl, n-hexyl, and the like.

"Alkylene" refers to divalent alkyl groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. Examples of such alkylene groups include methylene (-CH2-), ethylene (-CH2CH2-), and the propylene isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-), and the like.

"Alkenyl" refers to monovalent alkenyl groups which may be straight chained or branched and preferably have from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds. Examples include ethenyl (-CH=CH2), n-propenyl (-CH2CH = CH2), /so-propenyl (-C(CH3)=CH₂), but-2-enyl (-CH₂CH = CHCH3), and the like.

"Alkenylene" refers to divalent alkenyl groups preferably having from 2 to 8 carbon atoms and more preferably 2 to 6 carbon atoms. Examples include ethenylene (- CH = CH-), and the propenylene isomers (e.g., -CH2CH=CH- and -C(CH3)=CH-), and the like. Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

"Oxo/hydroxy" refers to groups =0, HO-.

"Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

"Oxyacyl" refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

"Acyloxy" refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and -C(O)O-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.

In this specification "optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono-and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues.

Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. "Optically-enriched," as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound of the present disclosure is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972).

Without being bound by theory, the inventors have found that hydrogen borrowing catalysis (also known as hydrogen auto-transfer) may be used for the synthesis of tertiary amine ionisable lipids, and may provide a more direct route which circumvents some of the challenges associated with current approaches of making tertiary amines, including the generation of undesired and/or toxic by-products. Amino alcohols have not been used as reagents for this type of catalysis, and tertiary amines with long aliphatic chains have not been synthesised using this catalytic approach. The inventors found that two-fold N-alkylation of a primary aliphatic amine (amino alcohol) may be achieved using hydrogen transfer catalysis to yield a tertiary amine (amino alcohol), and that N-alkylation with a long-chain fatty alcohol is possible to give ionisable amino lipids.

Accordingly, disclosed herein is a method of synthesising an ionisable lipid of Formula (I), or a pharmaceutically acceptable salt, solvate or isomer thereof:

N-a Ikylating two alcohols of Formula (II) with an amino alcohol of Formula (III) in the presence of a hydrogen borrowing catalyst;

wherein the hydrogen borrowing catalyst is an iridium catalyst.

Disclosed herein is a method of synthesising an ionisable lipid of Formula (I), or a pharmaceutically acceptable salt, solvate or isomer thereof:

N-alkylating at least two molar equivalence of alcohols of Formula (II) with 1 molar equivalence of amino alcohol of Formula (III) in the presence of a hydrogen borrowing catalyst;

wherein the hydrogen borrowing catalyst is an iridium catalyst.

each R3 is independently optionally substituted alkyl, optionally substituted heterocyclyl, or optionally substituted aryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene;

N-alkylating at least two molar equivalence of acyloxy-substituted alkyl alcohols of Formula (Ila) with 1 molar equivalence of amino alcohol of Formula (III) in the presence of a hydrogen borrowing catalyst;

R₂

H₂N _n0H

(HI) wherein the hydrogen borrowing catalyst is an iridium catalyst.

Compounds of the present invention comprises at least one alcohol moiety.

Hydrogen borrowing catalysis, also called hydrogen autotransfer or dehydrogenative activation, is a method to activate, for example, alcohols. In contrast, carbonyl compounds are much better electrophiles and can be used in a variety of reactions. Hydrogen borrowing catalysis uses this "chemical detour" as method of activation. In essence, the catalyst first oxidizes an alcohol by removing or "borrowing" hydrogen to form a reactive carbonyl compound. This intermediate can undergo a diverse range of subsequent transformations before the catalyst returns the "borrowed" hydrogen to liberate the product and regenerate the catalyst. In this way, alcohols may be used as alkylating agents whereby the sole byproduct of this one-pot reaction is water. The overall process allows alcohols to be converted into amines, to form C-C bonds, or to be functionalized at the p-position. The catalysts may be transition metal complexes, e.g., Ru, Ir, or Rh compounds. In addition to alcohols, borrowing hydrogen catalysis may also be applied to amines and alkanes.

In some embodiments, the hydrogen borrowing catalyst is a homogenous catalyst. In some embodiments, the hydrogen borrowing catalyst is a transition metal catalyst. In some embodiments, the hydrogen borrowing catalyst is an iridium catalyst. For example, the hydrogen borrowing catalyst may be [Ir(COD)CI]2 with Py2-NPiPr2 ligand, IrCh with 2,2' -bis(diphenylphosphino)-l,l' -binaphthyl (BINAP), or N-heterocyclic carbine (NHC) ligands. In some embodiments, the iridium catalyst is selected from:

In some embodiments, the hydrogen borrowing catalyst is cyclopentadienyl iridium complex, wherein the cyclopentadienyl is optionally substituted. In preferred embodiments, the hydrogen borrowing catalyst is cyclopentadienyl iridium dichloride dimer ([Cp*IrCl2]2), wherein the cyclopentadienyl is optionally substituted.

In some embodiments the hydrogen borrowing catalyst is added or present at a concentration of about 1 mol% to about 5 mol% relative to the hydroxyl-substituted alkyl amine. In some embodiments, the hydrogen borrowing catalyst is loaded at a concentration of about 5 mol% relative to the hydroxyl-substituted alkyl amine.

The two alcohol of Formula (II) may be the same compound, or may be different compounds. When different alcohols are N-alkylated to the amino alcohol of Formula (III), they may be N-alkylated sequentially.

In some embodiments, each Ri is independently optionally substituted oxo, optionally substituted oxyacyl, optionally substituted acyloxyl, optionally substituted silyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, each Ri is independently optionally substituted oxo, optionally substituted oxyacyl, optionally substituted acyloxyl, optionally substituted silyl, optionally substituted alkyl, optionally substituted heterocyclyl, or optionally substituted aryl. In some embodiments, the optional substituent is selected from halo, oxo, oxyacyl, acyloxyl, silyl, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, the optional substituent is selected from halo. In some embodiments, each Ri is independently optionally substituted C1-C24 alkyl, optionally substituted Ci- C24 oxo, optionally substituted C1-C24 oxyacyl, optionally substituted C1-C24 acyloxyl, optionally substituted C1-C24 silyl, optionally substituted C5-C10 heterocyclyl, or optionally substituted aryl.

In some embodiments, each R3 is independently optionally substituted alkyl. In some embodiments, the optional substituent is selected from halo, oxo, oxyacyl, acyloxyl, silyl, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, the optional substituent is selected from halo, oxo, oxyacyl, acyloxyl, silyl, alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, each R3 is independently C10-C24 alkyl, optionally substituted with halo. In preferred embodiments, Ri is C15 alkyl, optionally substituted with halo.

In some embodiments, each Li is independently optionally substituted alkylene. In some embodiments, the optional substituent is selected from halo, oxo, oxyacyl, acyloxyl, silyl, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, the optional substituent is selected from halo, oxo, oxyacyl, acyloxyl, silyl, alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, each Li is independently Ci-Cio alkylene, optionally substituted with halo. In some embodiments, each Li is independently Ci-Ce alkylene, optionally substituted with halo. In preferred embodiments, Li is Ce alkylene, optionally substituted with halo.

In some embodiments, R2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl. In some embodiments, R2 is independently H, halo, oxo, optionally substituted alkyl.

In some embodiments, n is an integer selected from 1 to 5. In preferred embodiments, n is 2-4.

In some embodiments, the alcohol of Formula (II) is:

R<^LI'OH _(II) wherein Ri is independently optionally substituted oxo, optionally substituted oxyacyl, optionally substituted acyloxyl, optionally substituted silyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene.

In some embodiments, the acyloxy-substituted alkyl alcohol is a compound of Formula (Ha):

O

R₃ r^Ll'OH _(IIa) wherein R3 is independently optionally substituted alkyl, optionally substituted heterocyclyl, or optionally substituted aryl; and

In preferred embodiments, the compound of Formula (II) is:

In some embodiments, the compound of Formula (II) comprises only 1 alcohol moiety. In other embodiments, if the compound of Formula (II) comprises more than 1 alcohol moieties, all except 1 alcohol moiety are protected.

In some embodiments, the amino alcohol is a compound of Formula (III):

wherein R2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl; and n is an integer selected from 1 to 10.

In preferred embodiments, the amino alcohol is aminobutanol.

In some embodiments, the alcohol moiety in the amino alcohol protected before the N- alkylation step. In some embodiments, the method further comprises a step of protecting the hydroxyl moiety on the amino alcohol. In some instances, it was found that amino alcohols may undergo cyclisation under hydrogen borrowing conditions. Accordingly, protecting the hydroxyl moiety on the amino alcohol may be useful for the N-alkylation reaction.

In some embodiments, the hydroxyl moiety is protected with a protecting group selected from 2-tetra hydropyranyl (THP), benzyl or dimethyl-tert-butylsilyl (TBS), tertbutyldiphenylsilyl (TBDPS), optionally substituted benzyl ether or other ethers such as methoxymethyl ether (MOM), P-methoxybenzyl (PMB). In preferred embodiments, the protecting group is THP.

It was found that the combination of an iridium catalyst and a THP protecting group provides the most advantageous conditions for forming ionisable lipids. It is believed that this is due to a combination of factors, such as solvent compatibility, temperature compatibility, catalyst stability and the ease of deprotection. Of all the protecting groups, the THP protecting group is able to balance between the tolerance to reaction conditions (heat, nucleophiles, acids) and ease of liberation at the end of the catalytic step. It was found that THP is stable enough to survive the Ir catalysis step allowing for high conversion and supresses side reactions. In addition, using THP allows the deprotection to be traceless, as it can be liberated under acidic conditions (inorganic acids) and provides the target lipid as a hydrochloride salt directly.

In some embodiments, a molar ratio of the amino alcohol to the alcohol is about 1 :2 to about 1 :20, about 1:2 to about 1 : 15, about 1 :2 to about 1 : 10, or about 1:2 to about 1 :5. In other embodiments, the molar ratio is about 1 :3 to about 1:5, or about 1:4 to about 1 :5. In some embodiments, the molar ratio is 1 :2.

It was found that a stoichiometric equivalence of 2 alcohol to 1 amino alcohol is sufficient to substantially complete the reaction.

The N-alkylation step can work (with at least 80% conversion) without a base as the amino alcohol can act as a base. Alternatively, in some embodiments, the N-alkylation step is performed in the presence of a base. The base may be an inorganic base. The base may be NaHCOs, K2CO3, KHCO3, or NH4HCO3. In some embodiments, the N- alkylation step is performed in the presence of non-polar solvent. In some embodiments, the N-alkylation step is performed in the presence of NaHCCH and toluene. In some embodiments, the N-alkylation step is performed in a sealed vessel under an inert gas. In some embodiments, the N-alkylation step is performed at a temperature of about 80°C to about 110°C, preferably about 110°C. In some embodiments, the temperature is about 100°C to about 150°C.

In some embodiments, the N-alkylation step is performed for about 16 hr to about 24 hr, preferably about 16 hr to about 32 h. In some embodiments, the N-alkylation step is performed for at least about 16 hr.

In some embodiments, when the two alcohols are different, a molar ratio of the amino alcohol to the first alcohol is about 1 :1. A molar ratio of the amino alcohol to the second alcohol is about 1 : 1 to about 1 :20. The second alcohol may be reacted sequentially after the N-alkylation of the first alcohol is substantially completed as a one pot reaction. Alternatively, after the first alcohol is N-alkylated, this intermediate may be purified before the second N-alkylation.

When the two alcohols are different, the N-alkylation of the two alcohols occur in a stepwise manner. In the first N-alkylation, a first catalyst is loaded to the first alcohol and amino alcohol, and the reaction performed under suitable conditions as mentioned herein. In the second N-alkylation, a second catalyst may be loaded with a second alcohol to the intermediate of the first N-alkylation, and the reaction performed under suitable conditions as mentioned herein. The first catalyst and second catalyst may be the same catalyst and at the same concentration. Alternatively, the second catalyst may not be added. The reaction conditions in the first N-alkylation and second N-alkylation may be the same.

In some embodiments, when the two alcohols are different, the method comprises N- alkylating one molar equivalence of a first alcohol of Formula (II) with 1 molar equivalence of amino alcohol of Formula (III) in the presence of a hydrogen borrowing catalyst to form an intermediate; and

N-a Ikylating at least one molar equivalence of a second alcohol of Formula (II) with the intermediate in the presence of the hydrogen borrowing catalyst.

In some embodiments, when the two alcohols are different, the method comprises N- alkylating one molar equivalence of a first alcohol of Formula (II) with 1 molar equivalence of amino alcohol of Formula (III) in the presence of a first hydrogen borrowing catalyst to form an intermediate; and N-a Ikylating at least one molar equivalence of a second alcohol of Formula (II) with the intermediate in the presence of a second hydrogen borrowing catalyst.

In some embodiments, the second hydrogen borrowing catalyst is the same as the first hydrogen borrowing catalyst. In this regard, additional catalyst is loaded.

In some embodiments, when the two alcohols are different, the method comprises N- alkylating one molar equivalence of a first alcohol of Formula (II) with 1 molar equivalence of amino alcohol of Formula (III) in the presence of a cyclopentadienyl iridium dichloride dimer ([Cp*IrCl2]2), wherein the cyclopentadienyl is optionally substituted, to form an intermediate; and

N-a Ikylating at least one molar equivalence of a second alcohol of Formula (II) with the intermediate in the presence of [Cp*IrCl2]2.

In some embodiments, the method further comprises loading additional catalyst (or [Cp*IrCl2]2) during the second N-alkylation. The catalyst may be loaded at 1 mol% to about 5 mol% relative to the hydroxyl-substituted alkyl amine.

In some embodiments, the method further comprises a step of purifying the ionisable lipid from the amino alcohol and/or the alcohol. In some embodiments, the purification step is performed using column chromatography in the presence of dichloromethane (DCM), methanol (MeOH) and ammonia (NH3). In some embodiments, the purification step is performed in the presence of magnesium silicate (Florisil) or silica gel. In preferred embodiments, the ionisable lipid is purified on Florisil using a gradient of DCM: MeOH (with NH3). For example, the column chromatography may be performed on silica gel using a gradient over 14 min from 100% dichloromethane to 20% methanol/80% dichloromethane with ammonia.

In some embodiments, the method further comprises a step of purifying the ionisable lipid from the hydrogen borrowing catalyst. In some embodiments, the purification of the ionisable lipid from the hydrogen borrowing catalyst is performed in the presence of a metal scavenger. In preferred embodiments, the metal scavenger is SiliaMetS Imidazole in chloroform or diethyl ether.

In some embodiments, the method further comprises a step of deprotecting the hydroxyl moiety on the ionisable lipid. In some embodiments, the deprotection step is performed in the presence of hydrochloric acid and methanol. Lipids such as ALC-0315 are notoriously difficult substrates to purify as they bind strongly to silica gel and a significant product loss occurs during column chromatography (which also uses large volumes of solvent). The inventors found that the choice of the THP protecting group can ameliorate this difficulty, since THP deprotection may be performed using common acids and generates products and by-products that are either volatile or water soluble, and may be removed by simple reaction work-up and evaporation. This approach avoids the need to use column chromatography for purification of the final product.

In some embodiments, the method further comprises a step of isolating a free amine form of the ionisable lipid. In some embodiments, the isolation step is performed in the presence of ammonia, methanol and ethyl acetate or diethyl ether and sodium carbonate and water.

In some embodiments, the ionisable lipid of Formula (I) is selected from:

In this regard, the inventors have developed a streamlined method to access, for example, ALC-0315 (the cationic lipid used in the Pfizer COVID-19 vaccine) in high yields based on hydrogen borrowing catalysis. Compared to current state of the art approach via reductive amination, the method disclosed herein may produce ALC-0315 in 44% overall yields from a common intermediate IM2 compared to 11% using the patented route in WO/2016176330 (incorporated by reference herein), which is a 400% improvement in yield. Further, some of the reagents used in the prior method are toxic and unsafe, and it would be desirable to avoid their use. Towards this end, a hydrogen borrowing catalyst is used to catalyse N-alkylation of aminobutanol with IM2 to access ALC-0315 without overalkylation. The reaction byproduct is water, which is non-toxic. Accordingly, the present disclosure provides a method of synthesizing ALC-0315, or a pharmaceutically acceptable salt, solvate or isomer thereof:

the method comprising:

N-a Ikylating an amino alcohol of Formula (III)

wherein R2 is independently H; and n is 4; with two acyloxy-substituted alkyl alcohols of Formula (Ila)

The present disclosure also provides an ionisable lipid of Formula (I), (la), ALC-0315 or a pharmaceutically acceptable salt, solvate or isomer synthesised by the method as disclosed herein.

The present disclosure also provides an ionisable lipid of Formula (I), or a pharmaceutically acceptable salt, solvate or isomer thereof:

wherein each Ri is independently optionally substituted oxo, optionally substituted oxyacyl, optionally substituted acyloxyl, optionally substituted silyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene;

R2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl; and n is an integer selected from 1 to 10.

The present disclosure also provides an ionisable lipid of Formula (la) or a pharmaceutically acceptable salt, solvate or isomer:

wherein each R3 is independently optionally substituted alkyl, optionally substituted heterocyclyl, or optionally substituted aryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene;

The compound of the disclosure can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present disclosure includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (e.g., methyl, ethyl) of the phosphate group.

Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

The compound of the disclosure may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present disclosure. Methods of solvation are generally known within the art.

The salt form may be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of a composition and not injurious to the patient.

Examples

Example 1: Synthesis of ALC-0315

The synthesis of ALC-0315 from IM2 involves 3 steps.

Scheme 1. Synthesis of ALC-0315 from IM2 using the method of this disclosure.

10% (silica)

Scheme 2. Ir-catalyzed hydrogen borrowing catalysis for the reaction between IM2 and aminobutanol with different protecting groups.

General methodology

Step 1:

Screening of optimal catalyst for the hydrogen borrowing catalysis step found that [Cp*IrCl2]2 is an appropriate catalyst for this step (Table 1). The initial screening was performed using the TBS (dimethyl-tert-butylsilyl) protected aminobutanol which had a moderate stability under the optimal reaction conditions. The condition was found to be transferrable to THP (tetra hydropyranyl) protected aminobutanol (Scheme 2).

Further conditions screening involving catalyst loading, reaction time, method of heating (conventional and microwave), and excess of IM2 alcohol identified the optimal reaction conditions to be 5mol%, [Cp*IrCl2]2 (2.5 mol% iridium complex) and sodium bicarbonate base at 110°C for 16 hrs. It has been observed that towards the end of the reaction (or in instances where the amount of IM2 alcohol is low) catalyst deactivation may happen, and higher reaction temperatures may accelerate this catalyst deactivation process. Conversely, using a larger excess of IM2 may not allow faster reaction/higher conversion, which may indicate a simultaneous effect of reaction time and IM2 concentration on the catalyst half-life.

Table 1. Optimisation of the hydrogen borrowing catalysis step (Step 1).

ALC-0315: conditions screening (Pg ~ TBS snbstrete)

SM refers to unreacted alcohol of Formula (II) in the final mixture.

Ruthenium catalyst did not work. For [Ir(cod)Cl2]2, conversion to the monosubstituted product is possible. It is possible that this catalyst was not stable or active enough to proceed to the second alkylation. This may be overcome by stabilising or activating the catalyst with other ligands.

Step 2:

Deprotection of the THP group on the THP-protected ALC-0315 may be performed by reacting THP-protected ALC-0315 with hydrochloric acid in methanol and/or dioxane. The reaction proceeds at ambient temperature over the course of a few hours. At the end of the reaction, a simple evaporation under reduced pressure yield the target compounds as its hydrochloride salt in 94% yield.

Step 3:

Although the hydrochloride salt is a pharmaceutically equivalent compound to the free amine base, the positive charge on this compound may present issues when used for formulation. To furnish the free amine base (ALC-0315), the ALC-0315 hydrochloride salt product may be treated with ammonia in methanol with some amounts of ethyl acetate. A simple filtration-evaporation workup at the end yields the target compound in up to 77% yields. Alternatively, the freebasing may be performed using aqueous sodium hydroxide or sodium carbonate solutions and extracted with diethyl ether.

Theoretically all steps presented here can be done in one-pot, given the proper process optimisation is done, yielding a fully telescoped process. In this case, the column chromatography and iridium scavenging may be performed on the final product in order to have an acceptably low Ir metal content in the final product.

Synthesis of 4-((tetrahydro-2H-pyran-2-yl)oxy)butan-l-amine

4-aminobutanol (2 mmol, 178 pL) was dissolved in 2 mL DCM, cooled in an ice bath and HCI (4M in dioxane, 2.2 mmol, 0.56 mL) was added. The mixture was stirred for 15 minutes and dihydropyran (2.2 mmol, 200 pL) was added. Cooling was removed and the mixture stirred for 1.5 hours. The reaction was treated with 2M NaOH to liberate the free amine and extracted twice with DCM. The combined organic layers were dried on sodium sulfate, filtered, and concentrated. Purified on silica gel with DCM:MeOH(NH3) 20% followed by 30%. A colourless oil was obtained: 282 mg (1.63 mmol), 82% yield.

Synthesis of O-THP-protected ALC-0315

A mixture of OTHP-protected aminobutanol (43 mg, 0.25 mmol), IM2 alcohol (178 mg, 0.5 mmol), [cp*IrCl2]2 (5 mg, 0.0063 mmol), NaHCOs (1.1 mg, 0.0125 mmol) and toluene (0.25 mL) was heated in a sealed vial under argon for 16 hours at 110 degrees, then concentrated. The crude mixture was purified on silica gel eluting with a DCM: MeOH(NH3) gradient 0 to 10%. A yellow oil was obtained (130 mg, 0.153 mmol), 61% yield.

Synthesis of ALC-0315 hydrochloride

A mixture of OTHP-protected ALC-0315 (480 mg), methanol (2 mL) and hydrochloric acid (4M in dioxane, 0.3 mL) was stirred at ambient temperature for 2-4 hours then concentrated. A yellow oil was obtained: 424 mg, 94%

Synthesis of ALC-0315

A mixture of ALC-0315 hydrochloride (465 mg), ethyl acetate (2 mL) and ammonia (7M in methanol, 0.5 mL) was stirred for 30 minutes. The mixture was filtered and concentrated yielding a faint yellow oil: 343 mg, 77%.

Example 2

Sequential addition of two different alcohols, resulting in lipids with a more diversified structure. This one-pot process allows the introduction of two different LiRi groups.

mixture of OTHP-protected aminobutanol (42 mg, 0.25 mmol), IM2 alcohol (89 mg, 0.25 mmol), [cp*IrCl2]2 (5 mg, 0.0063 mmol), NaHCOs (1.1 mg, 0.0125 mmol) and toluene (0.25 mL) was heated in a sealed vial under argon for 16 hours at 110 °C, then cooled to ambient temperature. The vessel was opened under argon, and additional [cp*IrCl2]2 (5 mg, 0.0063 mmol), NaHCOs (1.1 mg, 0.0125 mmol) and ethanol (0.29 mL, 5 mmol) was added. The vessel was sealed and heated under argon for 16 hours at 110 °C. The crude mixture was concentrated, and the residue was purified on silica gel eluting with a DCM: MeOH(NH3) gradient 0 to 20%. A yellow oil was obtained: 34 mg (26% yield). ^TH NMR (400 MHz, CDCI3) 6 4.59 - 4.55 (m, 1H), 4.05 (t, J = 6.6 Hz, 2H), 3.90 - 3.82 (m, 1H), 3.77 - 3.70 (m, 1H), 3.52 - 3.45 (m, 1H), 3.42 - 3.35 (m, 1H), 2.52 (q, J = 7.1 Hz, 2H), 2.48 - 2.37 (m, 4H), 2.33 - 2.24 (m, 1H), 1.93 - 1.16 (m, 42H), 1.01 (t, J = 7.1 Hz, 3H), 0.92 - 0.79 (m, 6H).

The obtained oil was stirred at ambient temperature with 1 mL MeOH and 0.1 mL HCI (4M in dioxane) for 3 hours. The mixture was concentrated. The residue was stirred for 30 minutes at ambient temperature with 1 mL EtOAc and 1 mL NH3 (0.1 M in MeOH), then filtered and the filtrate was concentrated. 23 mg yellow oil was obtained (80% yield). ^TH NMR (400 MHz, CDCI3) 6 4.03 (t, J = 6.7 Hz, 2H), 3.68 (t, J = 5.5 Hz, 1H), 3.25 - 2.83 (m, 6H), 2.28 (tt, J = 8.8, 5.3 Hz, 1H), 2.01 - 1.08 (m, 40H), 0.85 (t, J = 6.8 Hz, 3H). The otherwise clean product can be further purified as described in Example 1.

It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the disclosure. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the disclosure but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. A method of synthesising an ionisable lipid of Formula (I), or a pharmaceutically acceptable salt, solvate or isomer thereof:

wherein each Ri is independently optionally substituted oxo, optionally substituted oxyacyl, optionally substituted acyloxyl, optionally substituted silyl, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene; 2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl; and n is an integer selected from 1 to 10; the method comprising :

R<^LI'OH _{( II)}

wherein the hydrogen borrowing catalyst is an iridium catalyst.

2. A method of synthesising an ionisable lipid of Formula (la), or a pharmaceutically acceptable salt, solvate or isomer thereof:

wherein each R3 is independently optionally substituted alkyl, optionally substituted heterocyclyl, or optionally substituted aryl; each Li is independently optionally substituted alkylene, optionally substituted heterocyclylene, or optionally substituted arylene; 2 is independently H, halo, oxo, optionally substituted alkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted oxy, optionally substituted oxyacyl, optionally substituted acyloxyl, or optionally substituted silyl; and n is an integer selected from 1 to 10; the method comprising :

R₂ H₂N ?OH n (III) wherein the hydrogen borrowing catalyst is an iridium catalyst. The method of claim 1 or 2, wherein the hydrogen borrowing catalyst is a cyclopentadienyl iridium complex, wherein the cyclopentadienyl is optionally substituted. The method of any one of claims 1 to 3, wherein the hydrogen borrowing catalyst is cyclopentadienyl iridium dichloride dimer ([Cp*IrCl2]2), wherein the cyclopentadienyl is optionally substituted. The method of any one of claims 1 to 4, wherein the hydrogen borrowing catalyst is added at a concentration of about 1 mol% to about 5 mol% relative to the hydroxyl-substituted alkyl amine. The method of any one of claims 1 to 5, wherein each Ri is independently C10-C24 alkyl, optionally substituted with halo. The method of any one of claims 1 to 6, wherein each Li is independently C1-C10 alkylene, optionally substituted with halo. The method of any one of claims 1 to 7, wherein n is an integer selected from 1 to 5. The method of any one of claims 1 to 8, further comprising a step of protecting the hydroxyl moiety on the amino alcohol. The method of claim 9, wherein the hydroxyl moiety is protected with a protecting group selected from 2-tetra hydropyranyl (THP), benzyl or dimethyl-tert-butylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), optionally substituted benzyl ether or other ethers such as methoxymethyl ether (MOM), P-methoxybenzyl (PMB). The method of any one of claims 1 to 10, wherein the molar ratio of the amino alcohol to the alcohol is about 1:2 to about 1 :20. The method of any one of claims 1 to 11, wherein the N-alkylation step is performed in the presence of NaHCOs and toluene. The method of any one of claims 1 to 12, wherein the N-alkylation step is performed at a temperature of about 80°C to about 110°C. The method of any one of claims 1 to 13, wherein the N-alkylation step is performed for about 16 hr to about 24 hr. The method of any one of claims 1 to 14, further comprising a step of purifying the ionisable lipid from the amino alcohol and/or the alcohol. The method of claim 15, wherein the purification step is performed using column chromatography in the presence of dichloromethane, methanol and ammonia. The method of claim 15 or 16, wherein the purification step is performed in the presence of magnesium silicate (Florisil) or silica gel. The method of any one of claims 1 to 17, further comprising a step of purifying the ionisable lipid from the hydrogen borrowing catalyst. The method of claim 18, wherein the step of further purification is performed in the presence of a metal scavenger. The method of any one of claims 1 to 19, further comprising a step of deprotecting the hydroxyl moiety on the ionisable lipid. The method of claim 20, wherein the deprotection step is performed in the presence of hydrochloric acid and methanol. The method of any one of claims 1 to 21, further comprising a step of isolating a free amine form of the ionisable lipid. The method of claim 22, wherein the isolation step is performed in the presence of ammonia, methanol and ethyl acetate, or in the presence of diethyl ether, water, and sodium carbonate. The method of any one of claims 20 to 23, wherein the deprotection step and the isolation step are performed sequentially in a reaction vessel. The method of any one of claims 1 to 24, wherein the N-alkylation step, deprotection step and isolation step are performed sequentially in a reaction vessel. The method of any one of claims 1 to 25, wherein the ionisable lipid of Formula (I) is selected from:

A method of synthesising ALC-0315, or a pharmaceutically acceptable salt, solvate

the method comprising :

N-a Ikylati ng 1 molar equivalence of amino alcohol of Formula (III)

wherein R2 is independently H; and n is 4; with at least two molar equivalence of acyloxy-substituted alkyl alcohols of Formula (Ha)

wherein R3 is independently C15 alkyl bonded to the acyl moiety at a C7 position; and

Li is independently Ce alkylene; in the presence of a cyclopentadienyl iridium dichloride dimer ([Cp*IrCl2]2) catalyst. The method of claim 27 , wherein the hydroxyl moiety on the amino alcohol is protected with a 2 -tetra hydropyranyl (THP) group.