Compounds and Processes for the Synthesis of Sphingomyelin

Abstract

Novel compounds useful for the synthesis of sphingomyelin, such as N-palmitoyl-D-erythro-sphingomyelin, a novel process for producing sphingomyelin using the novel compounds, preparations of sphingomyelin produced by the process, and compositions containing sphingomyelin.

Description

Technical Field

[0001] 1. Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Patent Application No. 63 / 356,178, filed on June 28, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] 2. Sequence Listing This application contains a sequence listing submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML sequence listing was created on June 20, 2023, named CRN - 047WO_SL.xml, and its size is 2,162 bytes.

Background Art

[0003] 3. Background Sphingomyelin (SM) is a major phospholipid component of biological membranes and plasma lipoprotein complexes, such as high - density lipoprotein (HDL). Sphingomyelin is also a component of some HDL - mimetic complexes, such as HDL - mimetics containing CER - 001, apolipoprotein A - I, sphingomyelin, and the negatively charged lipid DPPG.

[0004] Commercially available preparations of sphingomyelin are typically mixtures of naturally occurring sphingomyelin, for example, from chicken eggs, pig brain, or bovine milk. The actual composition can vary depending on the source and can contain various fatty acid chain lengths. N - palmitoyl sphingomyelin is a major component of natural sphingomyelin, which mainly contains the naturally occurring isomer N - palmitoyl - D - erythro - sphingomyelin.

[0005]

Chemical Formula

[0006] While a process for synthesizing sphingomyelin is described (see WO 2014 / 140787), commercially available sphingomyelin typically remains of natural origin. Thus, there is still a need for a new process for synthesizing sphingomyelin. SUMMARY OF THE INVENTION

[0007] 4. Summary The present disclosure provides novel compounds useful for the synthesis of sphingomyelin, such as N-palmitoyl-D-erythro-sphingomyelin, a novel process for making sphingomyelin using the novel compounds, preparations of sphingomyelin made by the process of the present disclosure, and compositions comprising sphingomyelin made by the process of the present disclosure.

[0008] In one aspect, the present disclosure provides a compound of formula (I):

[0009] [Chemical formula] and salts thereof, wherein: X is a halogen, such as Br, Cl, F, or I; R 1 is an acyl group having 3 to 36 carbon atoms and 0 to 6 carbon-carbon double bonds, such as palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonoyl, or eicosapentaenoyl; R 2 is a protecting group, such as benzoyl] is provided.

[0010] In a further aspect, the present disclosure provides a process for synthesizing a compound of formula (I) and salts thereof.

[0011] In some embodiments, the compound of formula (I) and salts thereof are of formula (0a):

[0012] [Chemical formula] a compound of or a salt thereof

[0013] [Chemical formula] [wherein, R 1 , R 2 , and X are as defined in the compound of formula (I)] It can be synthesized by reacting with

[0014] In other embodiments, the compound of formula (I) and a salt thereof are of formula (0c):

[0015] [Chemical formula] [wherein, R 1 , R 2 , and X are as defined in the compound of formula (I)] a compound of or a salt thereof can be synthesized by reacting with MgX2.

[0016] Further exemplary features of the compound of formula (I) and a salt thereof, and the process for synthesizing the compound of formula (I) and a salt thereof are described in Sections 6.1.2 and 6.1.5 below, and in Specific Embodiments 1 - 7 and 50 - 66.

[0017] The compound of formula (I) can be used, for example, in the synthesis of sphingomyelin and sphingomyelin precursors, such as protected sphingomyelin.

[0018] Accordingly, in a further aspect, the present disclosure provides a process for synthesizing sphingomyelin or a salt thereof. The process typically involves reacting a compound of formula (I) or a salt thereof with trimethylamine to form a compound of formula (II)

[0019] [Chemical formula] [wherein, R1 and R 2 is as defined in the compound of formula (I) step of producing a compound of or a salt thereof; and R 2 removing a protecting group from the compound of formula (II) or a salt thereof to give a compound of formula (III):

[0020]

Chemical formula

[0021] In a further aspect, the present disclosure provides a process for synthesizing a protected sphingomyelin or a salt thereof. The process typically involves reacting a compound of formula (I) or a salt thereof with trimethylamine to give a compound of formula (II)

[0022]

Chemical formula

[0023] In a further aspect, the present disclosure provides sphingomyelin and salts thereof and protected sphingomyelin and salts thereof produced by the processes of the present disclosure.

[0024] Exemplary features of the processes for making sphingomyelin, the processes for making protected sphingomyelin, and the products of such processes are described in Sections 6.1.3 and 6.1.5 below, as well as in Specific Embodiments 8 - 30 and 33 - 49.

[0025] In a further aspect, the present disclosure provides a composition comprising sphingomyelin or a salt thereof and a process for its production. For example, sphingomyelin or a salt thereof can be incorporated into a lipid-bound protein-based complex, such as CER-001. Exemplary features of such compositions and processes are described in Sections 6.1.4 below, as well as in specific embodiments 31, 32, and 66-84.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

Figure 1-1

Figure 1-2

Figure 2

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

MODE FOR CARRYING OUT THE INVENTION

[0027] 6. DETAILED DESCRIPTION 6.1.1. Terms A fatty acid is a carboxylic acid with a long aliphatic tail, which may be saturated or unsaturated. An unsaturated fatty acid has one or more carbon-carbon double bonds, and each carbon-carbon double bond can occur in a cis or trans configuration. A fatty acid residue is a fatty acid with fewer -OH groups of the carboxyl group of the fatty acid. As used herein, the terms "Ac" or "acyl" refer to a fatty acid residue.

[0028] In certain embodiments of the present disclosure, a fatty acid or a fatty acid residue has 3 to 36 carbons and 0 to 6 carbon-carbon double bonds. Such a fatty acid residue can be represented as a group of the general formula -C(O)R [wherein R is an alkyl group having 2 to 35 carbons and 0 to 6 carbon-carbon double bonds]. In certain embodiments of the present disclosure, a fatty acid or a fatty acid residue has 4 to 28 carbons and 0 to 6 carbon-carbon double bonds. In a further embodiment, a fatty acid or a fatty acid residue has 11 to 25 carbons and 0 to 6 carbon-carbon double bonds. In yet a further embodiment, a fatty acid or a fatty acid residue has 11 to 25 carbons and 1 or 2 carbon-carbon double bonds. In a further embodiment, a fatty acid or a fatty acid residue has 14 to 20 carbons and 0 to 6 carbon-carbon double bonds. In yet a further embodiment, a fatty acid or a fatty acid residue has 15 to 17 carbons and 0 to 6 carbon-carbon double bonds. In a particular embodiment, the fatty acid is palmitic acid and the fatty acid residue is palmitoyl.

[0029] Suitable fatty acids also include omega fatty acids such as omega-3 or omega-6 or omega-9 fatty acids; and include, but are not limited to, essential fatty acids such as linoleic acid (LA), alpha-linolenic acid (ALA), n-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0030] Suitable fatty acids useful in the present disclosure include, but are not limited to, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, henicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, gedanic acid, ceroplastic acid, hexatriacontylic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, α-linolenic acid, and erucic acid.

[0031] When the fatty acid is a monounsaturated fatty acid, the fatty acid can be a cis or trans monounsaturated fatty acid such as, but not limited to, oleic acid, elaidic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, and erucic acid.

[0032] The "benzoyl" or "Bz" protecting group refers to the following structure:

[0033]

Chemical formula

[0034] Benzoylating reagents include, but are not limited to, reagents such as benzoyl halides like benzoyl chloride.

[0035] The "Boc" protecting group refers to the tert-butyloxycarbonyl protecting group.

[0036] As used herein, "C 13 H 27 - " and "C15 H 31 The “-”s each represent CH3(CH2) 12 - and CH3(CH2) 14 respectively mean

[0037] As used herein, when referring to a reaction, the term “completed” means that the reaction system has reached equilibrium such that either the concentrations of the reactants and products are maintained constant or at least one of the reactants of the reaction has decreased to less than 20% (by mass) of its initial amount. In some embodiments, the remaining reactants of a reaction that has proceeded to completion are less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1%. In some embodiments of the processes described herein, one or more (e.g., all) of the reaction steps of the reaction processes described herein are capable of proceeding to completion. The reaction can be monitored, for example, by thin layer chromatography or HPLC to determine when the reaction is nearing completion or has reached completion.

[0038] Certain compounds of the present disclosure can be in the form of salts. In some embodiments, the salts are pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, for example, acid addition salts and base addition salts. The acids that form the acid addition salts can be organic or inorganic acids. The bases that form the base addition salts can be organic or inorganic bases. In some embodiments, the pharmaceutically acceptable salts are metal salts. In some embodiments, the pharmaceutically acceptable salts are ammonium salts.

[0039] Acid addition salts can be obtained by adding an acid to the free base form of the compound. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. Non-limiting examples of suitable acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, phosphoric acid, nicotinic acid, isonicotinic acid, lactic acid, salicylic acid, 4-aminosalicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, citric acid, oxalic acid, myricenic acid, hydroxymyricenic acid, methylmyricenic acid, glycolic acid, malic acid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, phenylacetic acid, N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid, 3-phosphoglyceric acid, glucose-6-phosphate, and amino acids.

[0040] Non-limiting examples of suitable acid addition salts include hydrochloride, hydrobromide, hydroiodide, nitrate, nitrite, sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate, bicarbonate, nicotinate, isonicotinate, lactate, salicylate, 4-aminosalicylate, tartrate, ascorbate, gentisinate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, pantothenate, acetate, propionate, butyrate, fumarate, succinate, citrate, oxalate, maleate, hydroxymaleate, methylmaleate, glycolate, malate, cinnamate, mandelate, 2-phenoxybenzoate, 2-acetoxybenzoate, embonate, phenylacetate, N-cyclohexylsulfamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, ethane-1,2-disulfonate, 4-methylbenzenesulfonate, naphthalene-2-sulfonate, naphthalene-1,5-disulfonate, 2-phosphoglycerate, 3-phosphoglycerate, glucose-6-phosphate, and amino acid salts.

[0041] Metal salts can be obtained by adding an inorganic base to a compound of the present invention having a carboxyl group. The inorganic base consists of a metal cation that pairs with a basic counterion such as, for example, a hydroxide ion, carbonate ion, bicarbonate ion, or phosphate ion. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. Non-limiting examples of suitable metals include lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, and zinc.

[0042] Non-limiting examples of suitable metal salts include lithium salts, sodium salts, potassium salts, cesium salts, cerium salts, magnesium salts, manganese salts, iron salts, calcium salts, strontium salts, cobalt salts, titanium salts, aluminum salts, copper salts, cadmium salts, and zinc salts.

[0043] The ammonium salts can be obtained by adding ammonia or an organic amine to the compound of the present invention having a carboxyl group. Non-limiting examples of suitable organic amines include triethylamine, diisopropylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole, imidazole, pyrazine, pipyrazine, ethylenediamine, N,N'-dibenzylethylenediamine, procaine, chloroprocaine, choline, dicyclohexylamine, and N-methylglucamine.

[0044] Non-limiting examples of suitable ammonium salts include triethylammonium salts, diisopropylammonium salts, ethanolammonium salts, diethanolammonium salts, triethanolammonium salts, morpholinium salts, N-methylmorpholinium salts, piperidinium salts, N-methylpiperidinium salts, N-ethylpiperidinium salts, dibenzylammonium salts, piperazinium salts, pyridinium salts, pyrazolium salts, imidazolium salts, pyrazinium salts, ethylenediammonium salts, N,N'-dibenzylethylenediammonium salts, procaine salts, chloroprocaine salts, choline salts, dicyclohexylammonium salts, and N-methylglucamine salts.

[0045] Unless otherwise required by context, for example, the disclosure regarding the compounds of formula (I), formula (II), or sphingomyelin should also be understood as a disclosure regarding the salts of the compounds.

[0046] 6.1.2. Compounds of formula (I) and processes for preparing compounds of formula (I) In some embodiments, the present disclosure provides compounds of formula (I):

[0047]

Chemical formula

[0048] In a preferred embodiment, X is Br.

[0049] In a preferred embodiment, R 1 is palmitoyl.

[0050] In some embodiments, the compound of formula (I) is a compound of formula (Ia):

[0051]

Chemical formula

[0052] Particularly preferred compounds of formula (I) are

[0053]

Chemical formula

[0054] In certain embodiments, the compound of formula (I) has an enantiomeric purity of at least 85% and contains 15% or less of its corresponding inverse enantiomer. In further embodiments, the compound of formula (I) has an enantiomeric purity of at least 90% and contains 10% or less of its corresponding inverse enantiomer. In still further embodiments, the compound of formula (I) has an enantiomeric purity of at least 95% and contains 5% or less of its corresponding inverse enantiomer. In still further embodiments, the compound of formula (I) has an enantiomeric purity of at least 98% and contains 2% or less of its corresponding inverse enantiomer. Methods for determining enantiomeric purity are known in the art and are described, for example, in WO 2014 / 140787.

[0055] The compound of formula (I) and its salts are of formula (0a):

[0056]

Chemical formula

[0057]

Chemical formula

[0058] The process can include reacting a compound of formula (0b)

[0059]

Chemical formula

[0060]

Chemical formula

[0061] The compounds of formula (I) and their salts can also be synthesized by reacting a compound of formula (0c):

[0062]

Chem.

[0063] The compound of formula (0c) can be synthesized, for example, by reacting

[0064]

Chem.

[0065] The process can include the step of reacting a compound of formula (0d):

[0066]

Chem.

[0067]

Chem.

[0068] Exemplary schemes for synthesizing the compound of formula (I) and precursors of formula (I) such as the compound of formula (0) are described in Section 6.1.5.

[0069] 6.1.3. Process for synthesizing sphingomyelin, protected sphingomyelin and products thereof The present disclosure provides a process for making sphingomyelin using the compound of formula (I) and salts thereof. The process typically involves reacting the compound of formula (I) or a salt thereof with trimethylamine to form a compound of formula (II)

[0070]

Chemical formula

[0071]

Chemical formula

[0072] In some embodiments, step (a) is of formula (Ia)

[0073]

Chemical formula

[0074]

Chemical formula

[0075] The step of reacting the compound of formula (I) or a salt thereof with trimethylamine can be carried out in a suitable solvent such as water, methanol, or a solvent mixture such as a methanol:THF mixture (e.g., in a 2:1 vol:vol ratio). Preferably, an excess of trimethylamine is used, for example, 10 to 100 equivalents, 20 to 80 equivalents, 30 to 70 equivalents, or 40 to 60 equivalents of trimethylamine per equivalent of the compound of formula (I) or a salt thereof. In some embodiments, 50 equivalents of trimethylamine are used. The reaction can be carried out at any suitable temperature, e.g., 30°C to 60°C or 40°C to 50°C. The progress of the reaction can be monitored by using TLC to monitor, for example, the disappearance of the starting material to determine when the reaction has reached completion. In some embodiments, the reaction may be allowed to proceed for 18 to 30 hours, optionally 22 to 26 hours.

[0076] The step of reacting the compound of formula (I) or a salt thereof with trimethylamine provides protected sphingomyelin. The protected sphingomyelin can be worked up from the reaction mixture and optionally purified, e.g., by chromatography (as described, for example, in Sections 6.1.5 and 7).

[0077] R 2 Sphingomyelin is produced by removing the protecting group. R 2The removal of the benzoyl group can proceed as follows: Sphingomyelin protected with benzoyl can be dissolved in a protic polar solvent, and a base is added. The reaction proceeds at about 22 °C for 8 to 24 hours. In one embodiment, the protic polar solvent is methanol, ethanol, n-propanol, isopropanol, or a mixture thereof. In another embodiment, the base is sodium methoxide, potassium carbonate, lithium hydroxide. In a particular embodiment, the base is sodium methoxide.

[0078] Sphingomyelin produced by the process described herein, such as N-acyl-D-erythro-sphingomyelin like N-palmitoyl-D-erythro-sphingomyelin, can be post-treated and / or purified by one or more means such as recrystallization, silica gel chromatography, high performance liquid chromatography or other methods known to those skilled in the art. In some embodiments, the post-treatment and / or purification includes distillation, filtration, washing, and drying, as described, for example, in section 7. In some embodiments, the sphingomyelin is washed with acetone and dried under vacuum (e.g., at 30 °C for 30 to 50 hours) and packaged in a nitrogen-purged container.

[0079] The process of the present disclosure can be carried out on a commercial scale, for example, to produce N-acyl-D-erythro-sphingomyelin, particularly N-palmitoyl-D-erythro-sphingomyelin, in commercial scale amounts. For example, the process can be carried out on a scale to produce 1 kg to 50 kg or more, such as 1 kg to 25 kg, 1 kg to 10 kg, 5 kg to 25 kg, 5 kg to 10 kg, 10 kg to 25 kg, or 25 kg to 50 kg of N-acyl-D-erythro-sphingomyelin, particularly N-palmitoyl-D-erythro-sphingomyelin.

[0080] In certain embodiments, N-palmitoyl-D-erythro-sphingomyelin has an enantiomeric purity of at least 85% and contains 15% or less of its corresponding inverse enantiomer. In further embodiments, N-palmitoyl-D-erythro-sphingomyelin has an enantiomeric purity of at least 90% and contains 10% or less of its corresponding inverse enantiomer. In still further embodiments, N-palmitoyl-D-erythro-sphingomyelin has an enantiomeric purity of at least 95% and contains 5% or less of its corresponding inverse enantiomer. In still further embodiments, N-palmitoyl-D-erythro-sphingomyelin has an enantiomeric purity of at least 98% and contains 2% or less of its corresponding inverse enantiomer. Methods for determining enantiomeric purity are known in the art and are described, for example, in International Publication No. WO 2014 / 140787.

[0081] 6.1.4. Compositions Containing Sphingomyelin In various aspects, the present disclosure provides sphingomyelin produced by the processes of the present disclosure and compositions containing sphingomyelin produced by the processes of the present disclosure.

[0082] Sphingomyelin can, in some embodiments, be in a crystalline form. Without being bound by theory, it is believed that the crystalline form of sphingomyelin indicates that the sphingomyelin is very pure.

[0083] The synthetic sphingomyelin produced by the processes of the present disclosure can be used in the production of lipid-bound protein-based complexes such as CER-001, Apomer, and Cargomer.

[0084] CER-001 is described in Example 4 of International Publication No. WO 2012 / 109162. International Publication No. WO 2012 / 109162 refers to CER-001 as a complex having a ratio of lipoprotein weight:total phospholipid weight of 1:2.7 and containing a weight:weight ratio of SM:DPPG of 97:3. Example 4 of International Publication No. WO 2012 / 109162 also describes a method for manufacturing CER-001. As used in the context of the present disclosure, CER-001 refers to a lipoprotein complex that may vary by up to 20% for each CER-001, such that its individual components are as described in Example 4 of International Publication No. WO 2012 / 109162. In certain embodiments, the components of the lipoprotein complex may vary by up to 10% for each CER-001, as described in Example 4 of International Publication No. WO 2012 / 109162. Preferably, the components of the lipoprotein complex are the components described in Example 4 of International Publication No. WO 2012 / 109162 (with variations within the manufacturing plus / minus tolerance). The lipoprotein, apolipoprotein A-I (ApoA-I), in CER-001 preferably has an amino acid sequence corresponding to amino acids 25 to 267 of SEQ ID NO: 1 of International Publication No. WO 2012 / 109162. SEQ ID NO: 1 of International Publication No. WO 2012 / 109162 is set forth as SEQ ID NO: 1 in the present application. ApoA-I can be purified from an animal source (particularly a human source) or can be produced recombinantly. In a preferred embodiment, the ApoA-I of CER-001 is recombinant ApoA-I. CER-001 is preferably highly homogeneous, as reflected by a single peak in gel permeation chromatography, for example having a homogeneity of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. See, for example, section 6.4 of International Publication No. WO 2012 / 109162.

[0085] In certain embodiments, the ApoA-I of CER-001 is recombinant ApoA-I produced by mammalian host cells. The host cells can be derived from any mammalian cell line. The polynucleotide encoding ApoA-I can be codon-optimized for expression in recombinant host cells. Preferred host cells are mammalian host cells including, but not limited to, Chinese hamster ovary cells (e.g., CHO-K1; ATCC number CCL 61; CHO-S (GIBCO Life Technologies Inc., Rockville, MD, catalog number 11619012)), VERO cells, BHK (ATCC number CRL 1632), BHK 570 (ATCC number CRL 10314), HeLa cells, COS-1 (ATCC number CRL 1650), COS-7 (ATCC number CRL 1651), MDCK cells, 293 cells (ATCC number CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), 3T3 cells, myeloma cells (especially mouse), PC12 cells and W138 cells. In certain embodiments, mammalian cells such as CHO-S cells (Invitrogen™, Carlsbad CA) are suitable for growth in serum-free media. Additional suitable cell lines are known in the art and are available from public depositories such as the American Type Culture Collection (Manassas, Va).

[0086] In a preferred embodiment, recombinant ApoA-I is produced by CHO cells. As will be appreciated by those skilled in the art, expression by mammalian host cells such as CHO cells can undergo post-translational processing (e.g., glycosylation, etc.). The resulting recombinant ApoA-I can have one or more structural features (e.g., glycosylation patterns) different from ApoA-I purified from human plasma.

[0087] Apomer and Cargomer are described in International Publication Nos. 2019 / 030575 and 2019 / 030574, respectively, the contents of which are incorporated herein by reference in their entirety.

[0088] Complexes based on lipid-binding proteins such as CER-001 can be formulated for the intended route of administration according to techniques known in the art, for example, as described in Allen et al., eds., 2012, Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, London, UK. CER-001 intended for administration by injection can be formulated in a phosphate buffer containing excipients of sucrose and mannitol, for example, as described in WO 2012 / 109162.

[0089] Complexes based on lipid-binding proteins such as CER-001 can be used as drug carriers. See, for example, WO 2012 / 109162. Thus, in some embodiments, the CER-001 of the present disclosure is a carrier for a drug, such as a hydrophobic, lipophilic or non-polar activator. Exemplary activators include, but are not limited to, fatty acids, drugs, nucleic acids, vitamins, and / or nutrients. Suitable hydrophobic, lipophilic or non-polar activators are not limited to pharmacotherapeutic classifications and include, for example, analgesics, anti-inflammatory agents, antihelmimthics, antiarrhythmics, antibacterial agents, antiviral agents, anticoagulants, antidepressants, antidiabetic agents, antiepileptic agents, antifungal agents, antigout agents, antihypertensive agents, antimalarial agents, antimigraine agents, antimuscarinic agents, antineoplastic agents, erectile dysfunction improvers, immunosuppressants, anti-protozoal agents, antithyroid agents, anxiolytics, sedatives, hypnotics, anxiolytics, β-blockers, inotropic agents, corticosteroids, diuretics, anti-Parkinson's disease agents, gastrointestinal agents, histamine receptor antagonists, keratolytics, lipid regulators, antianginal agents, cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants, nutrients, nucleic acids (e.g., small interfering RNA), opioid analgesics, protease inhibitors, sex hormones, stimulants, muscle relaxants, anti-osteoporosis agents, anti-obesity agents, cognitive enhancers, anti-incontinence agents, nutritional oils, anti-benign prostatic hyperplasia agents, essential fatty acids, non-essential fatty acids, and mixtures thereof.

[0090] Specific non-limiting examples of suitable hydrophobic, lipophilic, or non-polar active agents include acitretin, albendazole, albuterol, aminoglutethimide, amiodarone, amlodipine, amphetamine, amphotericin B, atorvastatin, atracurium, azithromycin, baclofen, beclomethasone, benazepril, benzonatate, betamethasone, bicalutanide, budesonide, bupropion, busulfan, butenafine, calcifediol, calcipotriene, calcitriol, camptothecin, candesartan, capsaicin, carbamezepine, carotene, celecoxib, cerivastatin, cetirizine, chlorpheniramine, cholecalciferol, cilostazol, cimetidine, cinarizine, ciprofloxacin, cisapride, clarithromycin, clemastine, clomiphene, clomipramine, clopidogrel, codeine, coenzyme Q10, cyclobenzaprine, cyclosporine, danazol, dantrolene, dextrochlorpheniramine, diclofenac, dicumarol, digoxin, dehydroepiandrosterone, dihydroergotamine, dihydrotachysterol, dirithromycin, donezepil, efavirenz, eposartan, ergocalciferol, ergotamine, essential fatty acid source, etodolac, etoposide, famotidine, fenofibrate, fentanyl, fexofenadine, finasteride, fluconazole, flurbiprofen, fluvastatin, fosphenytoin, frovatriptan, furazolidone, gabapentin, gemfibrozil, glibencamide, glipizide, glibenclamide, glimepiride, griseofulvin, halofantrine, ibuprofen, irbesartan, irinotecan, isosorbide dinitrate, isotretinoin, itraconazole, ivermectin, ketoconazole, ketorolac, lamotrigine, lansoprazole, leflunomide, lisinopril, loperamide, loratadine, lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone, mifepristone, mefloquine, megestrol acetate, methadone, methoxsalen, metronidazole, miconazole,Midazolam, Miglitol, Minoxidil, Mitoxantrone, Montelukast, Nabumetone, Nalbuphine, Naratriptan, Nelfinavir, Nifedipine, Nilsolidipine, Nilutanide, Nitrofurantoin, Nizatidine, Omeprazole, Oprevelkin, Estradiol, Oxaprozin, Paclitaxel, Paracalcitol, Paroxetine, Pentazocine, Pioglitazone, Pizofetin, Pravastatin, Prednisolone, Probucol, Progesterone, Pseudoephedrine, Pyridostigmine, Rabeprazole, Raloxifene, Rofecoxib, Repaglinide, Rifabutin, Rifapentine, Remexolone, Ritanovir, Rizatriptan, Rosiglitazone, Saquinavir, Sertraline, Sibutramine, Sildenafil Citrate, Simvastatin, Sirolimus, Spironolactone, Sumatriptan, Tacrine, Tacrolimus, Tamoxifen, Tamsulosin, Taliglucerase, Tazarotene, Telmisartan, Teniposide, Terbinafine, Terazosin, Tetrahydrocannabinol, Tiagabine, Ticlopidine, Tirofibran, Tizanidine, Topiramate, Topotecan, Toremifene, Tramadol, Tretinoin, Troglitazone, Trovafloxacin, Ubiquinone, Valsartan, Venlafaxine, Verteporfin, Vigabatrin, Vitamin A, Vitamin D, Vitamin E, Vitamin K, Zafirlukast, Ziluton, Zolmitriptan, Zolpidem, and Zopiclone. Salts, isomers and derivatives of the above-listed agents, as well as mixtures, can also be used.,

[0091] 6.1.5. Exemplary Scheme Exemplary schemes for preparing sphingomyelin, such as N-palmitoyl-D-erythro-sphingomyelin, and synthetic intermediates useful for preparing sphingomyelin are presented below. Those skilled in the art will understand that modifications to the schemes shown below (e.g., use of alternative solvents, protecting groups, etc.) can be used. In the schemes below, the intermediates can be isolated between steps, but do not necessarily have to be. In some embodiments, one or more of the intermediates shown in the bracketed schemes are not isolated. Thus, in some embodiments, many of the steps of the entire synthetic scheme can be carried out as a one-pot synthesis. The halo and bromo intermediates shown in Schemes II(a) - IV(b) are shown at the end or beginning of the scheme, but the reactions shown in the following schemes can be shown in brackets that can be part of a larger overall synthetic scheme, and the halo and bromo intermediates may not need to be isolated. For example, the entire synthetic scheme for the synthesis of sphingomyelin can include the reactions shown in Schemes I(a), II(a) and IV(a), the reactions shown in Schemes I(a), III(a) and IV(a), the reactions shown in Schemes I(b), II(b) and IV(b), or the reactions shown in Schemes I(b), III(b) and IV(b). The reactions shown in the scheme can be monitored, for example, by TLC or HPLC for the reduction or disappearance of the starting materials.

[0092]

Chemical formula

[0093] Scheme I(a) shows an exemplary synthetic scheme that can be used to prepare N-acyl-3-O-benzoyl-D-erythro-sphingosine (intermediate 10a). The acyl group is an acyl group having 3 to 36 carbon atoms and 0 to 6 carbon-carbon double bonds, such as palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonoyl, or eicosapentaenoyl. The scheme and process for preparing intermediate 10a are further described in International Publication No. WO 2014 / 140787, the content of which is incorporated herein by reference in its entirety.

[0094]

Chemical formula

[0095] Scheme I(b) shows an exemplary synthetic scheme that can be used to prepare N-palmitoyl-3-O-benzoyl-D-erythro-sphingosine (intermediate 10). Scheme I(b) corresponds to Scheme I(a), and the acyl group is palmitoyl.

[0096]

Chemical formula

[0097] Scheme II(a) shows an exemplary synthetic scheme that can be used to prepare exemplary halo intermediate compounds of formula (I) starting from intermediate 10a. In the scheme, intermediate 10a is reacted with 2-chloro-2-oxo-1,3,2-dioxaphospholane (CCP) in the presence of trimethylamine (NMe3) and THF to produce intermediate 11a. The molar ratio of intermediate 10a to CCP can be in the range of, for example, 3:1 to 1:3 or 2:1 to 1:2, such as a molar ratio of 1:1 or 1:1.5. The solution of trimethylamine and THF can be, for example, a 2M solution of trimethylamine in THF. The molar ratio of intermediate 10 to trimethylamine can be in the range of, for example, 10:1 to 1:10. In an exemplary embodiment, 5 to 10 equivalents, for example, 6 equivalents of trimethylamine are used. The reaction can be carried out at a suitable temperature, for example, in the range of -10°C to 20°C or 0 to 10°C. The reaction can be monitored by TLC.

[0098] Intermediate 11a can be reacted with magnesium halide, for example, MgBr2, to produce the halo intermediate compound of formula (I). The molar ratio of intermediate 11 to magnesium halide can be in the range of, for example, 3:1 to 1:3 or 2:1 to 1:2, such as a molar ratio of 1:1, 1:1.5 or 1:2. The reaction can be carried out at a suitable temperature, for example, in the range of 20°C to 40°C, such as 25 to 35°C. The reaction can be monitored by TLC.

[0099]

Chemical formula

[0100] Scheme II(b) shows an exemplary synthetic scheme that can be used to prepare an exemplary bromo intermediate compound of formula (I) starting from intermediate 10. In the scheme, intermediate 10 is reacted with 2-chloro-2-oxo-1,3,2-dioxaphospholane (CCP) in the presence of trimethylamine (NMe3) and THF to produce intermediate 11. The molar ratio of intermediate 10 to CCP can be in the range of, for example, 3:1 to 1:3 or 2:1 to 1:2, such as a molar ratio of 1:1 or 1:1.5. The solution of trimethylamine and THF can be, for example, a 2M solution of trimethylamine in THF. The molar ratio of intermediate 10 to trimethylamine can be in the range of, for example, 10:1 to 1:10. In an exemplary embodiment, 5 to 10 equivalents, such as 6 equivalents, of trimethylamine are used. The reaction can be carried out at a suitable temperature, for example, in the range of -10°C to 20°C or 0 to 10°C. The reaction can be monitored by TLC.

[0101] Intermediate 11 is reacted with MgBr2 to produce the bromo intermediate compound of formula (I). In some embodiments, MgBr2 is in the form of magnesium bromide ethyl etherate. The molar ratio of intermediate 11 to MgBr2 can be in the range of, for example, 3:1 to 1:3 or 2:1 to 1:2, such as a molar ratio of 1:1, 1:1.5, or 1:2. The reaction can be carried out at a suitable temperature, for example, in the range of 20°C to 40°C, such as 25 to 35°C. The reaction can be monitored by TLC.

[0102]

Chemical formula

[0103] Scheme III(a) shows an exemplary synthetic scheme that can be used to prepare an exemplary halo intermediate compound of formula (I) starting from intermediate 10a. In the scheme, intermediate 10a is reacted with 2-haloethyl phosphorodichloridate, for example, 2-bromoethyl phosphorodichloridate, to produce a halo intermediate compound of formula (I). The synthesis of the halo intermediate by this route avoids the use of CCP reagents and any ring-opening impurities due to incomplete reaction of intermediate 11a in Scheme II(a).

[0104] [Chemical formula]

[0105] Scheme III(b) shows an exemplary synthetic scheme that can be used to prepare an exemplary bromo intermediate compound of formula (I) starting from intermediate 10. In the scheme, intermediate 10 is reacted with 2-bromoethyl phosphorodichloridate to produce a bromo intermediate compound of formula (I). The synthesis of the bromo intermediate by this route avoids the use of CCP reagents and any ring-opening impurities due to incomplete reaction of intermediate 11 in Scheme II(b).

[0106] [Chemical formula]

[0107] Scheme IV(a) shows an exemplary synthetic scheme for the synthesis of N-acyl-D-erythro-sphingomyelin starting from a halo intermediate, for example, a bromo intermediate. The acyl group can be an acyl group having 3 to 36 carbon atoms and 0 to 6 carbon-carbon double bonds, such as palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonoyl, or eicosapentaenoyl.

[0108] The halo intermediate can react with trimethylamine (e.g., liquid) to produce Intermediate 12a. The reaction can be carried out in a suitable solvent, such as methanol or water, at a suitable temperature, such as a temperature in the range of 30°C to 60°C or 40°C to 50°C. Trimethylamine is preferably used in excess, for example, 10 to 100 equivalents, 30 to 80 equivalents, or 40 to 60 equivalents, such as 50 equivalents. The reaction can be monitored by TLC.

[0109] The post-treatment of Intermediate 12a can be carried out before further use. For example, Intermediate 12a can be concentrated (e.g., under vacuum below 50°C) and co-distilled with dichloromethane. The residue can be dissolved in a mixture of dichloromethane:methanol (e.g., 2:1, 15 vol) and washed twice with water (e.g., 5 vol). The organic layer can be concentrated to produce Intermediate 12a. If desired, Intermediate 12a can be purified by chromatography using silica gel of 230 to 400 mesh containing, for example, a mixture of dichloromethane and methanol.

[0110] Deprotect Intermediate 12a to produce N-acyl-D-erythro-sphingomyelin by reacting Intermediate 12a with sodium methoxide (NaOMe) in, for example, methanol (MeOH) at a suitable temperature, such as a temperature in the range of 10°C to 40°C or 20°C to 30°C, such as 20°C. The reaction can be monitored by HPLC. Exemplary conditions for deprotecting Intermediate 12a are further described in International Publication No. WO 2014 / 140787.

[0111] [Chemical formula] #2 Bromo Intermediate #3 Intermediate #4 N-Palmitoyl-D-erythro-sphingomyelin

[0112] Scheme IV(b) shows an exemplary synthetic scheme for the synthesis of N-palmitoyl-D-erythro-sphingomyelin starting from a bromo intermediate.

[0113] The bromo intermediate can be reacted with trimethylamine (e.g., liquid) to produce Intermediate 12. The reaction can be carried out in a suitable solvent, such as methanol or water, at a suitable temperature, such as in the range of 30 °C to 60 °C or 40 °C to 50 °C. Trimethylamine is preferably used in excess, for example, in an amount of 10 to 100 equivalents, 30 to 80 equivalents, or 40 to 60 equivalents, such as 50 equivalents. The reaction can be monitored by TLC.

[0114] The work-up of Intermediate 12 can be carried out before further use. For example, Intermediate 12 can be concentrated (e.g., under vacuum below 50 °C) and co-distilled with dichloromethane. The residue can be dissolved in a mixture of dichloromethane:methanol (e.g., 2:1, 15 vol) and washed twice with water (e.g., 5 vol). The organic layer can be concentrated to produce Intermediate 12. If desired, Intermediate 12 can be purified by chromatography using silica gel of 230 - 400 mesh containing, for example, a mixture of dichloromethane and methanol.

[0115] Deprotect Intermediate 12 to produce N-palmitoyl-D-erythro-sphingomyelin by reacting Intermediate 12 with sodium methoxide (NaOMe) in, for example, methanol (MeOH) at a suitable temperature, such as in the range of 10 °C to 40 °C or 20 °C to 30 °C, such as 20 °C. The reaction can be monitored by HPLC. Exemplary conditions for deprotecting Intermediate 12a are further described in International Publication No. WO 2014 / 140787.

Example

[0116] [Example 1] 7.1. Synthesis of Sphingomyelin via Bromo Intermediate 7.1.1 Synthesis of N - palmitoyl - D - erythro - sphingosine (Intermediate 7) Intermediate 6 (15.0 g, 0.050 mol), HBTU (20.89 g, 0.055 mol), and palmitic acid (12.85 g, 0.050 mol) in DMF (105 ml, 7 vol) and THF (360 ml, 24 vol) were charged into a transparent dry round - bottom flask (RBF). The reaction mass was cooled to 0 - 5 °C, and a solution of triethylamine (13.78 g, 0.136 mol) in THF (15.0 ml, 1.0 vol) was added. The reaction mass was stirred for 3 - 4 hours. The absence of starting materials was confirmed by HPLC. A 5% citric acid solution (240 ml, 16.0 vol) was slowly added to the reaction mass and stirred at approximately 20 °C for 2 - 3 hours. The reaction mass was filtered and washed with water. Then, the wet cake was slurried twice with water at approximately 20 °C, filtered, and washed with water and acetone. The resulting wet cake was stirred with acetone (420 ml, 28 vol) at approximately 20 °C for 1 - 2 hours, filtered, and washed with acetone (75 mL, 5 vol). The isolated solid was vacuum - dried at 30 - 40 °C for 10 - 12 hours to obtain N - palmitoyl - D - erythro - sphingosine (Intermediate 7) as a solid product (production amount: 14.4 g, yield: 53.45%).

[0117] 7.1.2 Synthesis of N - palmitoyl - 3 - O - benzoyl - D - erythro - sphingosine (Intermediate 10) Intermediate 7, ethyl acetate (8.0 vol), and triethylamine (2.0 equivalents) were charged into a transparent RBF at approximately 20 °C. A solution of trityl chloride (1.1 equivalents) in ethyl acetate (5 vol) was slowly added, and the temperature was raised to 70 - 80 °C. The mixture was stirred at 70 - 80 °C for 10 - 12 hours. A solution of trityl chloride in ethyl acetate (1 vol) was slowly added to the mixture. The mixture was held at 70 - 80 °C for 6 - 7 hours. The absence of starting materials was confirmed by HPLC. The reaction mass was filtered, and the ethyl acetate layer was washed with water. The aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated NaHCO3 solution (5 vol). The organic layer was distilled under vacuum and co - distilled with dichloromethane to form Intermediate 8 in dichloromethane.

[0118] A solution of Intermediate 8 in dichloromethane was combined with additional dichloromethane (12 vol), triethylamine (2 eq), and DMAP (0.1 eq). The reaction mass was cooled to 0 - 5 °C. Then, benzoyl chloride (1.5 eq) was added to the reaction mass under a nitrogen atmosphere. Then, the temperature of the reaction mass was raised to approximately 20 °C and stirred for 10 - 12 hours. The absence of starting material was confirmed by HPLC. Water was slowly added to the reaction and then the layers were separated. The aqueous layer was extracted with dichloromethane and combined with the organic layer. The combined organic layer was washed with aqueous sodium bicarbonate solution followed by brine solution. The organic layer was concentrated under vacuum to produce Intermediate 9.

[0119] Methanol (16 vol) and dichloromethane (16 vol) were added to the crude Intermediate 9. The mixture was cooled to 0 - 10 °C. A solution of p-toluenesulfonic acid (0.5 eq) was slowly added to the reaction mass and the temperature was raised to approximately 20 °C. The reaction mass was stirred for 6 - 10 hours. The absence of starting material was confirmed by HPLC. Triethylamine (0.25 vol) was added to the reaction mass. The reaction mass was concentrated under vacuum below 45 °C and co-distilled twice with methanol (2 vol). The crude Intermediate 10 (N-palmitoyl-3-O-benzoyl-D-erythro-sphingosine) was recrystallized using methanol and cooled to 0 - 5 °C. The solid was filtered, washed with methanol, and vacuum dried at 35 - 40 °C for 6 - 10 hours.

[0120] The results from three runs are shown in Table 1.

[0121]

Table 1

[0122] The crude intermediate 10 was purified by column chromatography using silica of 230 - 400 mesh and a mixture of ethyl acetate and hexane. The product was eluted with 15 - 25% ethyl acetate / hexane. The pure fractions were collected separately and analyzed by TLC / HPLC. The fractions with a purity of over 90% were combined and concentrated under vacuum to yield the purified intermediate 10 as a solid compound. Table 2 shows the yields and purities for three runs.

[0123]

Table 2

[0124] 7.1.3. Synthesis of N - palmitoyl - 3 - O - benzoyl - D - erythro - sphingomyelin (Intermediate 12) Intermediate 10 in THF (15.0 vol) was charged into a clear dry RBF and cooled to 0 - 10 °C. A 2 M solution of trimethylamine in THF (6.0 eq) was added. A solution of 2 - chloro - 2 - oxo - 1,3,2 - dioxaphospholane (CCP) (1.5 eq) in acetonitrile (2.5 vol) was slowly added at 0 - 10 °C and reacted to produce Intermediate 11. The absence of starting material was monitored by TLC. The reaction mass was cooled to 0 - 5 °C and magnesium ethyl etherate bromide (2.0 eq) was added. The temperature of the reaction mass was raised to approximately 20 °C and then stirred at 25 - 35 °C for 12 - 14 h under a nitrogen atmosphere to produce the bromo - intermediate. The absence of Intermediate 11 was confirmed by TLC. The reaction mass was completely concentrated under vacuum below 45 °C. Methanol (14.0 vol) and THF (7.0 vol) were charged into the residue and the mass was transferred to an autoclave. Pre - cooled (-10 to -15 °C) liquid trimethylamine (50.0 eq) was charged into the reaction mass and the temperature was raised to 40 - 50 °C. The reaction mass was stirred at 40 - 50 °C for 24 h. The absence of the bromo - intermediate was confirmed by TLC. The reaction mass was completely concentrated under vacuum below 50 °C and co - distilled with dichloromethane. The residue was dissolved in a mixture of dichloromethane:methanol (e.g., 2:1, 15 vol) and washed twice with water (5 vol). The organic layer was concentrated to produce Intermediate 12.

[0125] The crude intermediate 12 was purified by chromatography using silica gel of 230 - 400 mesh containing a mixture of dichloromethane and methanol. Fractions having a purity of more than 90% were combined and concentrated under vacuum. Table 3 shows the yields and purities for exemplary runs.

[0126]

Table 3

[0127] To control the impurity of 0.06 RRT, the intermediate 12 was dissolved in dichloromethane:methanol (2:1, 10 vol) and washed with water (5 vol). Table 4 shows the results for exemplary runs.

[0128]

Table 4

[0129] In another exemplary run (not washed with methanol water), purification was carried out on a C18 silica bed using a solvent of acetonitrile - methanol, resulting in a purity higher than 97% (Table 5).

[0130]

Table 5

[0131] 7.1.1. Synthesis of N - palmitoyl - D - erythro - sphingomyelin Intermediate 12 was charged in methanol (5.0 vol) in a transparent dry RBF. The reaction mass was cooled to 0 - 5 °C under a nitrogen atmosphere. A solution of 30% NaOMe in methanol (0.2 eq) was added under a nitrogen atmosphere and the temperature was raised to 25 - 35 °C. The reaction mass was stirred at approximately 20 °C for 10 - 12 hours. The absence of starting material was confirmed by HPLC. Dichloromethane (10.0 vol) and water (5.0 vol) were charged to the reaction mass and the pH was adjusted to 6 - 7 using 1 M HCl solution. The layers were separated and the aqueous layer was extracted with dichloromethane (5.0 vol). The organic layer was distilled under vacuum below 35 °C and co-distilled with methanol (2.4 vol) and dichloromethane (2.4 vol). The residue was dissolved in dichloromethane (2.4 vol) and methanol (2.4 vol), filtered through a micron filter and washed with (1:1) methanol:dichloromethane. The filtrate was distilled completely under vacuum below 35 °C. 1:1 methanol:dichloromethane (12.5 ml, 1 vol) was added and subsequently acetone (20 vol) was slowly added to the reaction mass. The reaction mass was cooled to 0 - 5 °C for 5 - 6 hours. The resulting solid was filtered and washed with cold acetone. The sphingomyelin product was dried under vacuum at 30 °C for 30 - 50 hours. The results for an exemplary run are shown in Table 6.

[0132]

Table 6

[0133] The impurity at approximately 0.06 RRT was obtained from Intermediate 12 and an impurity of 1.05% at 0.07 RRT was observed. Thus, the purity of the final sphingomyelin product can be increased by controlling the impurity at 0.06 RRT.

[0134] [Example 2] 7.2. Optimization of Intermediate 7 Synthesis Intermediate 7 is considered an important intermediate in the synthesis of N-palmitoyl-D-erythro-sphingomyelin. The synthesis of the intermediate involves the coupling of palmitic acid with Intermediate 6 using the N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) reagent in the presence of triethylamine as a base. The molar equivalents of trimethylamine and HBTU were optimized.

[0135] To evaluate the effect of different molar equivalents of triethylamine on the reaction of Intermediate 7, the reaction was carried out using different molar equivalents of triethylamine. The results are shown in Table 7.

[0136] [Table 7]

[0137] No significant effect of lower and higher equivalents of triethylamine on the formation of Intermediate 7 was observed.

[0138] HBTU activates palmitic acid in the reaction of Intermediate 7. To evaluate the effect of different molar equivalents of HBTU on the reaction of Intermediate 7, the reaction was carried out using different molar equivalents of HBTU. The results are shown in Table 8.

[0139] [Table 8]

[0140] No significant effect of lower and higher equivalents of HBTU on the formation of Intermediate 7 was observed.

[0141] [Example 3] 7.3. Characterization of Synthetic Sphingomyelin The synthetic sphingomyelin (sSph) generated according to the process described in Example 1 was characterized and compared with sphingomyelin derived from eggs (eggSph) (NOF Corporation).

[0142] of eggSph 1 The 1H-NMR spectrum is shown in Figure 1A, and that of sSph 1 The 1H-NMR spectrum is shown in Figure 1B. The spectrum indicates that eggSph has some additional impurities not observed in sSph.

[0143] Data on high performance thin layer chromatography (HPTLC) for eggSph and sSph are shown in Table 9 and Figure 2.

[0144] [Table 9]

[0145] [Example 4] 7.4. Comparison of CER-001 prepared with egg sphingomyelin and CER-001 prepared with synthetic sphingomyelin 7.4.1. Cholesterol mobilization in rabbits Pharmacological tests were performed by comparing CER-001 prepared with eggSph with CER-001 prepared with sSph generated according to the process of Example 1. The preparations were evaluated in an in vivo cholesterol mobilization test performed in rabbits.

[0146] In this model, an increase in plasma or HDL cholesterol levels indicates cholesterol mobilization from tissues and transfer to HDL. CER-001 prepared with eggSph and CER-001 prepared with sSph were injected into rabbits fasted at a dose of 5 mg / kg or 20 mg / kg and compared with the vehicle group. There were 4 animals per group. Plasma phospholipids, plasma human ApoA-I levels, plasma HDL, and plasma total cholesterol were measured at various time points after administration. The increases in all parameters other than ApoA-I were determined by subtracting the baseline values. At 30 - 34 hours after administration, these parameters returned to baseline.

[0147] When different CER-001 complexes were injected into rabbits at doses of 5 and 20 mg / kg, substantially identical dose-dependent increases were seen in plasma phospholipids and human ApoA-I at the tested doses (Figures 3A and 3B). This reflects the injection of human ApoA-I and phospholipids containing the CER-001 complex and suggests that these CER-001 preparations were stable in circulation and caused cholesterol mobilization. A similar dose-dependent increase was also observed in cholesterol mobilization as an increase in both the levels of plasma total cholesterol and HDL total cholesterol (Figures 3C and 3D).

[0148] 7.4.2. Cholesterol Efflux Assay The biological potency of the CER-001 batches was measured using a cholesterol efflux assay based on Fu5AH rat hepatocarcinoma cells. Fu5AH rat hepatocarcinoma cells have high expression of scavenger receptor class B type I (SRB1) and promote bidirectional flow of cholesterol between the cells and mature HDL. The method was selected based on the specificity of HDL-mediated cholesterol efflux activity in this experimental model.

[0149] Fu5AH cells were labeled with 3H-cholesterol for 24 hours. Receptor media for efflux were prepared for each CER-001 sample (diluted in MEM buffered with 25 mM HEPES at 30, 20 and 10 μg / ml), as well as for controls containing ApoA-I purified from human plasma (20 μg / mL); HDL3, 2% human serum and media alone. Receptor media containing the samples and controls were added to the cells for 4 hours and the efflux media and cell monolayers were evaluated to determine the percentage of cholesterol released from the Fu5AH cells. The biological activity of each test substance was expressed as the percentage of cholesterol efflux relative to a CER-001 reference standard and used as an experimental control at the same concentration as the test substance.

[0150] Table 10 shows the data on the biological activity of CER-001 prepared using eggSph and CER-001 prepared using a test using sSph synthesized in Example 1. The quality of the final product of the CER-001 data for a representative process test using sSph is summarized in Table 5 and compared with a representative production batch of CER-001 using egg-derived Sph. These data indicate the comparability of the substances.

[0151]

Table 10-1

[0152]

Table 10-2

[0153] 7.4.3. Identity, Size, Purity and Particle Size Distribution of the CER-001 Complex CER-001 prepared with eggSph or sSph was evaluated by gel permeation chromatography (GPC). This method allows the CER-001 complex to be disassembled from the free protein (ApoA-I) as well as the phospholipids (Sph and DPPG). Tosoh TSK-GEL G3000SW XL was used. Samples containing the CER-001 reference standard were prepared by diluting with the mobile phase.

[0154] The comparison of the GPC profiles of CER-001 from the eggSph reference batch and the GPC profiles of CER-001 from the sSph batch is shown in Figures 4A - 4B. The profiles indicate that the complex prepared with sSph is approximately the same size and of equivalent quality as the complex prepared with eggSph.

[0155] The average particle size and particle size distribution of the CER-001 complex were monitored by dynamic light scattering (DLS). DLS measures the velocity of CER-001 particles, which correlates with the time-dependence of the intensity fluctuations of laser light scattered from the particles diffusing in solution. DLS analysis was performed using a Malvern Zetasizer Nano ZS. The particle size distributions of the CER-001 batches tested were equivalent (see Table 10).

[0156] 8. Specific Embodiments The present disclosure is illustrated by the following specific embodiments. 1. Formula (I):

[0157] [Chemical Formula] a compound of or a salt thereof [wherein, X is halogen; R 1 is an acyl group having 3 to 36 carbon atoms and 0 to 6 carbon-carbon double bonds; R 2 is a protecting group]. 2. Formula (Ia):

[0158] [Chemical Formula] a compound of or a salt thereof as described in Embodiment 1. 3. The compound according to Embodiment 1 or Embodiment 2, wherein X is Br. 4. The compound or a salt thereof according to any one of Embodiments 1 to 3, wherein R 1 is palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonyl, or eicosapentaenoyl. 5. The compound or a salt thereof according to Embodiment 4, wherein R 1 is palmitoyl. 6. The compound or a salt thereof according to any one of Embodiments 1 to 5, wherein R 2 is a benzoyl group. 7.

[0159]

Chem.

[0160]

Chem.

[0161]

Chem.

[0162]

Chem.

[0163]

Chem.

[0164] [Chemical formula] The process according to embodiment 8, comprising the step of producing a compound of or a salt thereof. 10. The process according to embodiment 8 or embodiment 9, wherein X is Br. 11. R 1 The process according to any one of embodiments 8 to 10, wherein R is palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonyl, or eicosapentaenoyl. 12. R 1 The process according to embodiment 11, wherein R is palmitoyl. 13. R 2 The process according to any one of embodiments 8 to 12, wherein R is a benzoyl group. 14. The process according to any one of embodiments 8 to 13, wherein trimethylamine is in liquid form. 15. The process according to any one of embodiments 8 to 14, wherein step (a) comprises reacting the compound of formula (I) or a salt thereof with trimethylamine in methanol. 16. The process according to any one of embodiments 8 to 14, wherein step (a) comprises reacting the compound of formula (I) or a salt thereof with trimethylamine in a mixture of methanol and tetrahydrofuran (THF), optionally wherein the mixture is a 2:1 mixture of methanol and THF. 17. The process according to any one of embodiments 8 to 14, wherein step (a) comprises reacting the compound of formula (I) or a salt thereof with trimethylamine in water. 18. In step (a), 20 to 100 moles of trimethylamine are used per mole of the compound of formula (I) or a salt thereof, optionally 30 to 80 moles of trimethylamine are used per mole of the compound of formula (I) or a salt thereof, optionally 40 to 60 moles of trimethylamine are used per mole of the compound of formula (I) or a salt thereof, and optionally 50 moles of trimethylamine are used per mole of the compound of formula (I) or a salt thereof. The process according to any one of embodiments 8 to 17. 19. The process according to any one of embodiments 8 to 18, wherein step (a) is carried out at a temperature of 30°C to 60°C, optionally 40°C to 50°C. 20. The process according to any one of embodiments 8 to 19, wherein the reaction in step (a) is monitored by thin layer chromatography (TLC). 21. The process according to any one of embodiments 8 to 20, which allows the reaction in step (a) to proceed to completion. 22. The process according to any one of embodiments 8 to 21, which allows the reaction in step (a) to proceed for 18 to 30 hours, optionally 22 to 26 hours. 23. The process according to any one of embodiments 8 to 22, wherein step (b) comprises reacting the compound of formula (II) or a salt thereof with a base. 24. The process according to embodiment 23, wherein the base is sodium methoxide, potassium carbonate, or lithium hydroxide. 25. The process according to embodiment 24, wherein the base is sodium methoxide. 26. The process according to any one of embodiments 23 to 25, wherein step (b) is carried out in a protic polar solvent which is optionally methanol, ethanol, n-propanol, isopropanol, or a mixture of any of the foregoing. 27. The process according to embodiment 26, wherein the protic polar solvent is methanol. 28. The compound of formula (I) or a salt thereof is

[0165]

Chemical formula

[0166]

Chemical formula

[0167]

Chem.

[0168]

Chem.

[0169]

Chem.

[0170]

Chem.

[0171] [Chemical formula] The process according to embodiment 33, comprising the step of producing a compound of or a salt thereof. 35. The process according to embodiment 33 or embodiment 34, wherein X is Br. 36. R 1 The process according to any one of embodiments 33 to 35, wherein R is palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonyl, or eicosapentaenoyl. 37. R 1 The process according to embodiment 36, wherein R is palmitoyl. 38. R 2 The process according to any one of embodiments 33 to 37, wherein R is a benzoyl group. 39. The process according to any one of embodiments 33 to 38, wherein trimethylamine is in a liquid state. 40. The process according to any one of embodiments 33 to 39, comprising the step of reacting a compound of formula (I) or a salt thereof with trimethylamine in methanol. 41. The process according to any one of embodiments 33 to 39, comprising the step of reacting a compound of formula (I) or a salt thereof with trimethylamine in a mixture of methanol and tetrahydrofuran (THF). 42. The process according to any one of embodiments 33 to 39, comprising the step of reacting a compound of formula (I) or a salt thereof with trimethylamine in water. 43. The process according to any one of embodiments 33 to 42, using 20 to 100 moles of trimethylamine per mole of the compound of formula (I) or a salt thereof, optionally using 30 to 80 moles of trimethylamine per mole of the compound of formula (I) or a salt thereof, optionally using 40 to 60 moles of trimethylamine per mole of the compound of formula (I) or a salt thereof, and optionally using 50 moles of trimethylamine per mole of the compound of formula (I) or a salt thereof. 44. The process according to any one of embodiments 33 to 43, wherein the reaction is carried out at a temperature of 30°C to 60°C, optionally 40°C to 50°C. 45. The process according to any one of embodiments 33 to 44, wherein the reaction is monitored by thin layer chromatography (TLC). 46. The process according to any one of embodiments 33 to 45, which allows the reaction to proceed to completion. 47. The process according to any one of embodiments 33 to 46, which allows the reaction to proceed for 18 to 30 hours, optionally 22 to 26 hours. 48. A compound of formula (I) or a salt thereof is a product of a process comprising reacting with a compound of formula (0a)

[0172]

Chem.

[0173]

Chem.

[0174]

Chem.

[0175]

Chem.

[0176]

Chem.

[0177]

Chem.

[0178]

Chem.

[0179]

Chem.

[0180]

Chem.

[0181]

Chem.

[0182]

Chem.

[0183]

Chem.

[0184] [Chemical formula] The process according to embodiment 64, wherein the molar ratio of to CCP is from 3:1 to 1:3, optionally from 2:1 to 1:2, optionally 1:1 or 1:1.5. 66. The process according to embodiment 64 or embodiment 65, wherein the reaction to form the compound of formula (0c) is carried out at 10°C to 20°C or 0 to 10°C. 67. A complex based on a lipid-binding protein, comprising sphingomyelin or a salt thereof according to embodiment 31 or embodiment 32. 68. The complex based on a lipid-binding protein according to embodiment 67, which is CER-001. 69. The complex based on a lipid-binding protein according to embodiment 67 or embodiment 68, which is a carrier for drugs. 70. A pharmaceutical composition comprising the complex based on a lipid-binding protein according to any one of embodiments 65 to 69 and a pharmaceutically acceptable excipient. 71. A process for preparing a complex based on a lipid-binding protein, comprising the step of combining sphingomyelin or a salt thereof according to embodiment 31 or embodiment 32 with a lipid-binding protein. 72. The process according to embodiment 71, comprising the step of combining sphingomyelin or a salt thereof and a lipid-binding protein to form a mixture, and then thermocycling the mixture. 73. The process according to embodiment 71 or 72, wherein the lipid-binding protein is apolipoprotein A-I (ApoA-I). 74. The process according to embodiment 73, wherein ApoA-I is recombinant. 75. The process according to embodiment 73 or embodiment 74, wherein the amino acid sequence of ApoA-I comprises amino acids 25 to 267 of SEQ ID NO: 1. 76. The process according to any one of embodiments 73 to 75, wherein ApoA-I is pre-complexed with DPPG before being combined with sphingomyelin or a salt thereof. 77. The process according to embodiment 76, wherein the weight ratio of ApoA-I to lipid is 1:2.7. 78. The process according to embodiment 76 or embodiment 77, wherein the weight ratio of sphingomyelin to DPPG is 97:3. 79. The process according to any one of embodiments 76 to 78, comprising the steps of combining ApoA-I pre-complexed with DPPG with sphingomyelin or a salt thereof to form an ApoA-I / DPPG / sphingomyelin mixture, and thermocycling the ApoA-I / DPPG / sphingomyelin mixture at 57°C ± 10°C to 37°C ± 10°C. 80. The process according to any one of embodiments 76 to 78, comprising the steps of combining ApoA-I pre-complexed with DPPG with sphingomyelin or a salt thereof to form an ApoA-I / DPPG / sphingomyelin mixture, and thermocycling the ApoA-I / DPPG / sphingomyelin mixture at 57°C ± 5°C to 37°C ± 5°C. 81. The process according to any one of embodiments 76 to 78, comprising the steps of combining ApoA-I pre-complexed with DPPG with sphingomyelin or a salt thereof to form an ApoA-I / DPPG / sphingomyelin mixture, and thermocycling the ApoA-I / DPPG / sphingomyelin mixture at 57°C ± 2°C to 37°C ± 2°C. 82. The process according to any one of embodiments 76 to 78, comprising the steps of combining ApoA-I pre-complexed with DPPG with sphingomyelin or a salt thereof to form an ApoA-I / DPPG / sphingomyelin mixture, and thermocycling the ApoA-I / DPPG / sphingomyelin mixture at 57°C to 37°C. 83. A step of thermocycling an ApoA-I / DPPG / sphingomyelin mixture until a complex with at least 98% or at least 99% homogeneity is formed, as reflected by a single peak in gel permeation chromatography, the process according to any one of embodiments 79 to 82. 84. A lipid-binding protein-based complex obtained by or obtainable by the process according to any one of embodiments 71 to 83. 85. The process according to embodiment 74, wherein ApoA-I is produced by a mammalian host cell. 86. The process according to embodiment 85, wherein the mammalian host cell is a CHO cell. 87. A lipid-binding protein-based complex according to embodiment 68, wherein ApoA-I is recombinant and produced by a mammalian host cell. 88. A lipid-binding protein-based complex according to embodiment 87, wherein ApoA-I comprises amino acids 25 to 267 of SEQ ID NO: 1. 89. A lipid-binding protein-based complex according to embodiment 87 or 88, wherein the mammalian host cell is a CHO cell. 90. A lipid-binding protein-based complex according to any one of embodiments 87 to 89, wherein ApoA-I has undergone post-translational processing (e.g., glycosylation) such that ApoA-I has one or more structural features (e.g., glycosylation pattern) different from human ApoA-I purified from human plasma. 91. An apolipoprotein A-I ("ApoA-I") formulation comprising ApoA-I and sphingomyelin or a salt thereof according to embodiment 31 or 32. 92. The ApoA-I formulation according to embodiment 91, further comprising DPPG and optionally having a ratio of Apo-AI weight:total lipid weight of 1:2.7 and a weight:weight ratio of SM:DPPG of 97:3. 93. The ApoA-I formulation according to embodiment 91 or 92, wherein ApoA-I is produced by a mammalian host cell. 94. The ApoA-I formulation according to embodiment 93, wherein the mammalian host cell is a CHO cell. 95. An ApoA-I preparation according to any one of embodiments 91 to 94, wherein the ApoA-I contains amino acids 25 to 267 of SEQ ID NO: 1.

[0185] 9. Incorporation by reference All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document had been individually indicated to be incorporated by reference for all purposes. If there is a conflict between one or more teachings of the documents incorporated by reference and the present disclosure, the teachings of this specification are intended.

Claims

1. Equation (I): 【Chemistry 24】 Compounds or salts thereof [in the formula, X is a halogen; R １ This is an acyl group having 3 to 36 carbon atoms and 0 to 6 carbon-carbon double bonds; R ２ [is a protecting group] A process for producing, Formula (0c) 【Chemistry 25】 The compound or a salt thereof is MgX ２ A process that includes a step to trigger a reaction.

2. Formula (0d) 【Chemistry 26】 The compound or a salt thereof is MgX ２ The process according to claim 1, comprising the step of reacting with

3. X is Br; R １ is palmitoyl, myristoyl, stearoyl, oleoyl, linoleoyl, linolenoyl, arachidonoyl, or eicosapentaenoyl, preferably palmitoyl; and / or R ２ The process according to claim 1, wherein is a benzoyl group.

4. The MgX of the compound of formula (0c) ２ The process according to claim 2, wherein the molar ratio to is 1:1 to 1:3 or 2:1 to 1:

2.

5. The process according to claim 2, wherein the reaction is carried out at a temperature of 20°C to 40°C.

6. The compound of formula (0c) is 【Chemistry 27】 The process according to claim 1, comprising the step of reacting with 2-chloro-2-oxo-1,3,2-dioxaphosphorane (CCP).

7. The process according to claim 6, wherein the reaction to form the compound of formula (0c) is carried out at 10°C to 20°C or 0 to 10°C.

8. A process for synthesizing sphingomyelin or a salt thereof, a) A step of producing a compound of formula (I) by the process described in claim 1; b) Reacting the compound of formula (I) or a salt thereof with trimethylamine to obtain formula (II) 【Transformation 5】 The steps of producing a compound or a salt thereof, c) Remove the R2 protecting group from the compound of formula (II) or its salt to obtain formula (III): 【Transformation 6】 Steps to produce sphingomyelin or a salt thereof. A process that includes this.

9. Step (b) is formula (Ia) 【Transformation 7】 The compound or a salt thereof is reacted with trimethylamine, which may be in liquid form, to obtain formula (IIa). 【Transformation 8】 The process according to claim 8, comprising the step of producing a compound or a salt thereof.

10. The process according to claim 9, wherein the trimethylamine is a liquid.

11. The process according to claim 8, wherein step (b) comprises reacting a compound of formula (I) or a salt thereof with trimethylamine in (i) methanol; or (ii) a mixture of methanol and tetrahydrofuran (THF).

12. The process according to claim 8, wherein in step (b), 20 to 100 moles of trimethylamine are used per mole of the compound of formula (I) or a salt thereof.

13. The process according to claim 8, wherein step (b) is carried out at a temperature of 30°C to 60°C.

14. The process according to claim 8, wherein the reaction of step (b) is carried out to completion and / or the reaction is carried out for 18 to 30 hours.

15. The process according to claim 8, wherein step (c) comprises reacting the compound of formula (II) or a salt thereof with a base.

16. The process according to claim 15, wherein step (c) is carried out in a protic polar solvent.

17. A process for synthesizing protected sphingomyelin or a salt thereof, a) the step of preparing a compound of formula (I) or a salt thereof according to the process described in claim 1; and b) A step of reacting the compound of formula (I) or a salt thereof with trimethylamine. A process that includes this.

18. A process for synthesizing protected sphingomyelin or a salt thereof, a) A step of producing a compound of formula (I) by the process described in claim 1; b) React the compound of formula (I) or a salt thereof with liquid trimethylamine to obtain formula (II). 【Chemistry 13】 A process comprising the step of producing a compound or a salt thereof.

19. Formula (Ia) 【Chemistry 14】 The compound or a salt thereof is reacted with trimethylamine to obtain formula (IIa). 【Chemistry 15】 The process according to claim 18, comprising the step of producing a compound or a salt thereof.

20. The process according to claim 19, wherein the trimethylamine is a liquid.

21. A process for producing a lipid-binding protein-based complex, comprising the steps of synthesizing sphingomyelin or a salt thereof according to any one of claims 8 to 20, and combining the sphingomyelin or a salt thereof with a lipid-binding protein.

22. The process according to claim 21, comprising the steps of combining sphingomyelin or a salt thereof and the lipid-binding protein to form a mixture, and then thermally cycling the mixture.

23. The process according to claim 21, wherein the lipid-binding protein is apolipoprotein A-I (ApoA-I).

24. The process according to claim 23, wherein the amino acid sequence of ApoA-I includes amino acids 25 to 267 of SEQ ID NO:

1.

25. The process according to claim 23, wherein ApoA-I is pre-complexed with DPPG before being combined with sphingomyelin or a salt thereof.

26. The process according to claim 24, wherein the complex based on the lipid-binding protein comprises ApoA-I, sphingomyelin, and DPPG, with a weight ratio of ApoA-I to lipids of 1:2.7 and a weight ratio of sphingomyelin to DPPG of 97:

3.

27. ​​A complex based on a lipid-binding protein, which can be obtained or obtained by the process described in Claim 21.

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