Process for the preparation of sphingosine 1-phosphate

Abstract

A process for the preparation of sphingosine 1-phosphate (11a) or a stereoisomer thereof, comprising the steps: (a) Conversion of L-serine (1a) to methyl ester (2a) or a salt thereof: (b) protecting the amino function of methyl ester (2a) or the salt thereof to form methyl ester (3a),: where Pg stands for a protecting group, (c) protecting the primary hydroxyl group and the amino group of methyl ester (3a) to form methyl ester (4a), where R 1 and R 2 independently of one another can be hydrogen, an optionally substituted C1-C6 alkyl radical or an optionally substituted phenyl radical, or wherein R 1 and R 2 together can form an optionally substituted C4-C7 cycloalkyl radical (d) Reduction of methyl ester (4a) to form alcohol (5a): (e) Oxidation of alcohol (5a) to form aldehyde (6a): (f) Reaction of aldehyde (6a) with 1-pentadecyne and a base to form alkyne (7a): (g) partial deprotection of alkyne (7a) to form alkyne (8a): (h) Reducing the triple bond of alkyne (8a) to a trans double bond to form alkene (9a): (i) Phosphorylating the primary hydroxyl group of alkene (9a) to form phosphate (10a): and (j) Deprotection of phosphate (10a) to form sphingosine 1-phosphate (11a): wherein the method has at least one of the following features: Oxidation of alcohol (5a) to form aldehyde (6a) using oxalyl chloride, DMSO and less than 5.5 equivalents of Hünig base, Oxidation of alcohol (5a) to form aldehyde (6a) at a temperature above -70°C, Reacting aldehyde (6a) with 1-pentadecyne and a base to form alkyne (7a) using at least 1.5 equivalents of 1-pentadecyne and / or Purification of the alkyne (7a), which is obtained after reacting aldehyde (6a) with 1-pentadecyne and a base to form alkyne (7a), by distilling off unreacted 1-pentadecyne.

Description

[0001] The present invention relates to a process for the preparation of sphingososine 1-phosphate or a stereoisomer thereof. Background of the invention

[0002] Sphingosine 1-phosphate (S1P), whose (2S,3R,4E)-D-erythro isomer is shown below, is a signaling protein. Sphingolipids are a large class of biologically important cell membrane components, characterized by a particular aliphatic amino alcohol, sphingosine, shown below. When sphingolipids are present in cells, they are metabolized to form sphingosine as a metabolic intermediate. Phosphorylation of sphingosine to S1P is catalyzed by sphingosine kinase. S1P is a potent messenger. It functions both intracellularly and intercellularly and plays a major role in signal transduction in various tissues. One of the main targets of S1P is members of the lysophospholipid receptor family.

[0003] Although S1P is important throughout the human body, it appears to be particularly important for the skin. It modulates the proliferation of skin cells. This is especially true for keratinocytes, while fibroblasts are not affected in this way. Besides cell growth and differentiation, S1P is responsible for chemotaxis and angiogenesis. While S1P suppresses epidermal proliferation, similar to glucocorticoids, it differs from them in that it does not reduce the proliferation of dermal fibroblasts. In fact, S1P activates fibroblast-based extracellular matrix protein production. Due to its hyperproliferative effect against epidermal cells, S1P has been considered as a pharmaceutically active ingredient against hyperproliferative skin diseases, particularly psorias vulgaris and acne vulgaris.

[0004] F. Ruan et al. (Bioorg. & Med. Chem. Let., 2(9), 1992, pp. 973-978) report the chemical synthesis of S1P. First, the starting material, a D-erythro olefinic alcohol, was prepared from L-serine. The starting material possesses both a primary and a secondary hydroxyl group, as well as an amino group. The synthesis begins with the protection of the amino function and the secondary hydroxyl group of the starting material. The primary hydroxyl group, which remains unprotected, is then phosphorylated, and the protecting groups of the amino group and the secondary hydroxyl group are finally removed to yield S1P. The synthesis leads to only a single isomer, but is lengthy (10 steps from L-serine to S1P with approximately 98 h of synthesis time) and provides S1P in a relatively low yield (approximately 10.7% overall yield).

[0005] B. Kratzer et al. (THL; 34(11), 1993, p. 1761-1764) describe the synthesis of S1P using D-erythro 2-azido-3-O-TBDMS-sphingosine as the starting material. This starting material, which possesses a primary and a secondary hydroxyl group as well as an azide group, is obtained from D-glucose in a nine-step process in 15% yield, with the secondary hydroxyl functionality being protected with a TBDMS group. Once the starting material is prepared, the azide group is reduced to the amino group, which is then protected using a carbonate group. The unprotected primary hydroxyl group of the resulting component is then phosphorylated using bis(2-cyanoethoxy)(diisopropylamino)phosphine to form a phosphite intermediate, which is then oxidized to a phosphate triester. The 2-cyanoethyl groups and the protecting groups of the secondary hydroxyl group and the amino group are then removed to obtain S1P.On the one hand, the synthesis yields only one isomer; on the other hand, the synthesis is very long (14 steps from D-glucose to S1P with an approximate duration of 318 h) and yields S1P only in a relatively low yield (about 8.4% overall yield). In addition, the synthesis requires hazardous chemicals such as dioxane and especially sodium azide, a highly explosive compound, especially in the presence of halogenated solvents.

[0006] WO 95 / 21848 describes the chemical synthesis of S1P and its derivatives. Sphingosine serves as the starting material. First, the amino group in sphingosine is protected, for example, with a Boc group. Then, the amino-protected sphingosine is reacted with bis(2-cyanoethoxy)(diisopropylamino)phosphine, and the resulting compound is oxidized to the phosphate triester. Finally, the 2-cyanoethyl groups and the amine protecting group are removed to yield S1P. The synthesis described in patent WO 95 / 21848 has several disadvantages that make it unsuitable for industrial-scale production, including difficulties in preparing the starting material sphingosine and also in preparing reagents such as bis(cyanoethoxy)(diisopropylamino)phosphine, as well as low yields.

[0007] International patent application WO 2008 / 107365 A1 discloses the synthesis of a chiral peptide. It also demonstrates the oxidation of an alcohol to an aldehyde with oxalyl chloride at temperatures above -78°C. The reaction of the aldehyde with an alkyne occurs with more than 1.5 equivalents of alkyne per aldehyde.

[0008] In Morita, X, et al: Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphongosin-1-phosphate therapy, Nature, 2000, Vol. 6, No. 10, pp. 1109-1114, a medical use of sphingosine-1-phosphate is described.

[0009] A synthesis of gamma-aminophosphonates was described in Foss, W. et al.: Synthesis and biological evaluation of gamma-aminophosphonates as potent, subtype-selective sphingosiine-1-phosphate receptor agonists and antagonists, Bioorganic & Medical Chemistry 2007, Vol. 15, pp. 663-677. This article also describes a sequence of synthetic routes starting from serine to a 1,3-oxazolidine-4-carbaldehyde according to steps a) to e) of the procedure described below.

[0010] In Garner, P. et al.: A Stereodivergent Synthesis of D-erythro-Sphingosine and D-threo-sphingosine from L-Serine: Journal of Organic Chemistry 1988, Vol. 53, pp. 4395 - 4398, a reaction of an aldehyde with pentadecine using 1.3 equivalents of alkyne is described, among other things.

[0011] In Herold, O.: Synthesis of D-erythro- and D-threo-sphingoisoine Derivatives from L-Seribe, Helvetiva Chimica Acta 1988, Vol. 71, 354 - 362, synthetic steps are described that involve the opening of a pentadecine-substituted 3-O-xazolidine-4-carbaldehyde.

[0012] In Lim, H. et al.: Synthesis of Sphingosine-1-phosphate Stereoisomers and Analogues and Their Interaction with EDG Receptors, Bioorganic & Medical Chemistry Letters, 2003, Vol. 13, 237 - 240, synthetic steps are described that involve the opening of a tridecine-substituted 3-oxazolidine-4-carbaldehyde.

[0013] Therefore, there is a need for a process for the production of S1P and its stereoisomers and derivatives, particularly for the production of S1P, its stereoisomers and derivatives on an industrial scale, particularly by increasing the yields and thus the quantities accessible for these components in a shorter time while minimizing the use of hazardous or expensive chemicals. Summary of the invention

[0014] In the context of this application, the terms "sphingosine 1-phosphate" and "S1P" are used interchangeably and refer to the (2S,3R,4E)-D-erythro isomer 11a, as shown in the first figure. Analogously, the term "sphingosine" refers to the (2S,3R,4E)-D-erythro isomer.

[0015] For the purposes of the present description, compound 11R includes compounds 11Ri and 11Rii; compound 11Ri includes compounds 11a, 11c, 11e, and 11g; and compound 11Rii includes compounds 11b, 11d, 11f, and 11h. Compound 10R includes compounds 10Ri and Rii; compound 10Ri includes compounds 10a, 10c, 10e, and 10g; and compound 10Rii includes compounds 10b, 10d, 10f, and 10h. Compound 9R includes compounds 9Ri and 9Rii; compound 9Ri includes compounds 9a, 9c, 9e, and 9g; and compound 9Rii includes compounds 9b, 9d, 9f, and 9h. Compound 8R includes compounds 8a to 8h. Compound 7R includes compounds 7a to 7h. Compound 6 includes compounds 6a and 6e. Compound 5 includes compounds 5a and 5e.Compound 4 includes compounds 4a and 4e. Compound 3 includes compounds 3a and 3e. Compound 2 includes compounds 2a, 2e, and 2x. Compound 1 includes compounds 1a and 1e.

[0016] In the context of the present description, the term "substantially enantiomerically pure" means that the compound in question consists of more than 90% of a single stereoisomer, preferably more than 95%, very preferably more than 98%, particularly preferably more than 98.5%, more preferably more than 99%, even more preferably more than 99.3%, most preferably more than 99.5% (as measured by chiral HPLC).

[0017] The process according to the invention involves the preparation of O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11R or a stereoisomer thereof and / or a derivative thereof. This preparation involves step (e) and / or step (f) of the process according to the invention, as described below: Step (e) Oxidation of alcohol 5 to aldehyde 6:wherein step (e) is carried out using oxalyl chloride, DMSO and less than 5.5 eq. Hünig base and / or at a temperature not below -70°C. Step (f) Reaction of aldehyde 6 with a 1-alkyne HCECR3 and a base to give alkyne 7R:

[0018] Wherein step (f) is carried out using at least 1.5 eq. of 1-alkyne and / or wherein alkyne 7R is purified by distilling off unreacted 1-alkyne; where Pg is a protecting group; where R 1 and R 1independently of one another, hydrogen, an optionally substituted C1 to C6 alkyl chain, or an optionally substituted phenyl group, or the radicals R1 and R2 together form an optionally substituted C4 to C6 cycloalkyl group and wherein R3 is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl radical, each of which may be optionally substituted.

[0019] Alcohol 5 is preferably obtained by a process comprising the following steps (a) to (d): (a) Conversion of serine 1 to ester 2 or a salt thereof, wherein R' is an unsubstituted C1-6 alkyl, phenyl or benzyl radical: (b) protecting the amino group of ester 2 or a salt thereof to form ester 3, wherein Pg is a protecting group: (c) protecting the primary hydroxyl group and the amino group of ester 3 to give ester 4, wherein R1 and R2 may independently be hydrogen, optionally substituted C1-C6 alkyl radicals, or optionally substituted phenyl radicals, or R1 and R2 may together form an optionally substituted C4-C7 cycloalkyl radical. (d) Reduction of ester 4 to alcohol 5:R' can be an unsubstituted C 1-6 alkyl, phenyl, or benzyl radical. According to the invention, R' is methyl.

[0020] Preferably in step (a) Serine 1 is a serine methyl ester hydrochloride salt.

[0021] The esterification of step (a) can, for example, be carried out acid-catalyzed, or using a carbodiimide such as N,N'-dicyclohexylcarbodiimide (DCC), or by activating the carboxyl function of serine 1 by conversion to an acid chloride, or by activating the carboxyl function of serine 1 by conversion to an anhydride.

[0022] After aqueous workup, the product from step (a), ester 2 or its salt, requires no further purification. Preferably, ester 2 or its salt is obtained in a yield of 98%, 99%, 99.5%, 99.9%, or more.

[0023] Particularly preferably, a methyl ester is prepared in step (a). A preferred salt obtained in step (a) is the hydrochloride.

[0024] In a preferred embodiment, the esterification of step (a) can be carried out using thionyl chloride and methanol. Preferably, the thionyl chloride is used in a slight excess (more preferably, about 1.05 eq). Preferably, the reaction mixture is refluxed for 10-30 hours. When thionyl chloride and methanol are used in step (a), serine methyl ester hydrochloride is obtained.

[0025] When -Pg (protecting group) is -CO-OtBu (Boc), di-tert-butyl dicarbonate (Boc2O) and a base such as triethylamine in a solvent such as dichloromethane can be used in step (b).

[0026] Di-tert-butyl dicarbonate (Boc2O) is preferably used in a slight excess (particularly preferably about 1.02 eq). The base is preferably used in excess (particularly preferably 2-3 eq). The reaction is preferably carried out under a protective gas atmosphere consisting of nitrogen. After the aqueous workup, no further purification of the product ester 3 is necessary.

[0027] Preferably, ester 3 is obtained in a yield of 98%, 99%, 99.5%, 99.9% or more.

[0028] Step (c) can be carried out starting from an aldehyde or ketone, or a derivative thereof, to form the acetal or ketal 4. The reaction can be catalyzed by acid or Lewis acid. After aqueous workup, the product ester 4 can be purified by filtration through silica gel using, for example, n-hexane and / or ethyl acetate as eluent. Preferably, ester 4 is obtained in a yield of 75%, 80%, 84%, or more.

[0029] R 1 and R 2 are independently hydrogen, an optionally substituted C1-C6 alkyl radical or an optionally substituted phenyl radical, or R 1 and R 2 can together form an optionally substituted C4-C7 cycloalkyl radical. Optional substituents include -F, -Cl, -Br, -I, -CF3, -CCI3, -CBr3, -Cl3, -OH, -SH, -NH2, -CN, -NO2, -OMe, -SMe, -NMe2, -CO2H, -CHO, and -CO2Me.

[0030] In a preferred embodiment, R 1 and R 2 both methyl. Then, step (c) can be carried out using 2,2-dimethoxypropane and an acid, acetone and an acid, 2-methoxypropene and a Lewis acid, acetone and iodine or copper sulfate, or 2-chloro-2-methoxypropane and an acid. In a preferred embodiment, 2,2-dimethoxypropane and an acid, such as toluenesulfonic acid, in a solvent such as dichloromethane can be used in step (c). Preferably, the 2,2-dimethoxypropane is used in excess (more preferably 4-6 eq). Preferably, the acid is used in catalytic amounts (more preferably 0.1-0.2 eq). The reaction can be carried out at room temperature in 1-4 days.

[0031] The reduction in step (d) can be carried out with LiAlH4, DIBAL, NaBH4 and LiCl, NaBH4 and AlCl3, BH3 / THF, LiBEt3H, NaAlEt2H2, or hydrogen and a catalyst. In a preferred embodiment, the reduction in step (d) can be carried out using LiAlH4 in a solvent such as THF. Preferably, the LiAlH4 is used in excess (more preferably 1.5-2 eq). The reaction can be carried out at room temperature in 1-24 hours. After aqueous workup, the product, alcohol 5, requires no further purification. Preferably, alcohol 5 is obtained in a yield of 84%, 90%, 95%, or more.

[0032] In one embodiment, substantially enantiomerically pure L-serine 1a is used to obtain substantially enantiomerically pure alcohol 5a. Preferably, substantially enantiomerically pure alcohol 5a is converted to substantially enantiomerically pure aldehyde 6a. Preferably, substantially enantiomerically pure aldehyde 6a is converted to substantially enantiomerically pure alkyne 7a or 7c.

[0033] In an alternative embodiment, substantially enantiomerically pure L-serine 1e is used to obtain substantially enantiomerically pure alcohol 5e. Preferably, substantially enantiomerically pure alcohol 5e is converted to substantially enantiomerically pure aldehyde 6e. Preferably, substantially enantiomerically pure aldehyde 6e is converted to substantially enantiomerically pure alkyne 7e or 7g.

[0034] In one embodiment, alkyne 7R is converted into O-trans-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11Ri or a stereoisomer thereof and / or a derivative thereof using a process comprising steps (g) to (j): (g) partial deprotection of alkyne 7R to alkyne 8R: (h) Reduction of the triple bond in alkyne 8R to a trans double bond to form alkene 9Ri: (i) Phosphorylation of the primary hydroxyl group of 9Ri to phosphate 10Ri: (j) Deprotection of phosphate 10Ri to form O-trans-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11Ri or a stereoisomer thereof:

[0035] Preferably, substantially enantiomerically pure alkyne 7a, 7c, 7e or 7g is converted into substantially enantiomerically pure sphingosine 1-phosphate 11a, 11c, 11e or 11g.

[0036] In an alternative embodiment, alkyne 7R is converted into O-cis-2-amino-3-hydroxyoctadec-4-en-1-yl phosphate 11Rii or a stereoisomer thereof and / or a derivative thereof, the process comprising one or more steps (g) to (j): (g) partial deprotection of alkyne 7R to form alkyne 8R: (h) Reduction of the triple bond of alkyne 8R to a cis double bond to form alkene 9Rii: (i) Phosphorylation of the primary hydroxyl function in alkene 9Rii to form 10Rii: (j) Deprotection of phosphate 10Rii to form O-cis-2-amino-3-hydroxyoctadec-4-en-1-yl phosphate 11Rii or a stereoisomer thereof:

[0037] Preferably, substantially enantiomerically pure alkyne 7b, 7d, 7f or 7h is converted into substantially enantiomerically pure sphingosine 1-phosphate 11b, 11d, 11f or 11h:

[0038] Step (e) can be carried out by a Swern oxidation using oxalyl chloride, DMSO and a base such as Hünig's base (N,N-diisopropylethylamine) in a solvent such as dichloromethane.

[0039] Preferably, the oxalyl chloride is used in excess (particularly preferably about 1.5 eq). Preferably, the DMSO is used in excess (particularly preferably about 3 eq). Preferably, Hünig base is used in excess (more preferably 4-5.5 eq, particularly preferably 4.3-5 eq). The inventors of the present development have determined that less than 5.5 eq of Hünig base are necessary for the reaction to proceed with complete conversion. Accordingly, step (e) is preferably carried out using oxalyl chloride, DMSO, and less than 5.5 eq of Hünig base.

[0040] Severe oxidations, particularly the reaction of DMSO and oxalyl chloride, are carried out at low temperatures, usually -78°C. The present inventors have discovered that it is possible to carry out step (e) at a temperature not lower than -70°C. Thus, step (e) is preferably carried out at a temperature not lower than -70°C. Very preferably, the reaction of DMSO and oxalyl chloride (the formation of a dimethylchlorosulfonium chloride intermediate) is carried out at a temperature of -40°C to -70°C, more preferably at -40°C to -60°C, even more preferably at -40°C to -55°C, most preferably at -40°C to -50°C.

[0041] Preferably, the following addition of alcohol 5 (formation of an alkoxysulfonium ion intermediate) is carried out at a temperature of -40°C to -70°C, very preferably at -40°C to -60°C, more preferably at -40°C to -55°C, most preferably at -40°C to -50°C.

[0042] The Swern oxidation in step (e) can be carried out at a temperature not lower than -70°C for 2-10 hours. Preferably, the reaction is carried out under a protective nitrogen atmosphere. After aqueous workup, the product aldehyde 6 requires no further purification. Preferably, aldehyde 6 is obtained in a yield of 75%, 80%, 85%, 90%, 94%, or more.

[0043] The preferred 1-alkyne HClCR 3 used in step (f) is 1-pentadecine HCOC-C 13 H 27 .

[0044] Step (f) can be carried out using a 1-alkyne (preferably 1-pentadecine) and a base such as an organolithium reagent or a Grignard reagent in a solvent such as THF to obtain an erythro isomer (preferably erythro isomer 7a or 7g). Preferred organolithium reagents are butyllithium, methyllithium, and phenyllithium. Butyllithium is particularly preferred. Optionally, the reaction can be carried out in the presence of additives such as HMPA, TMEDA, or tributoxyzirconium chloride.

[0045] Alternatively, step (f) can be carried out using a 1-alkyne (preferably 1-pentadecyne) and a base, such as an organolithium reagent or Grignard reagent, in a solvent such as THF in the presence of zinc bromide or copper iodide to obtain the threo isomer (preferably threo isomer 7c or 7e). Preferred organolithium reagents are butyllithium, methyllithium, and phenyllithium. Butyllithium is particularly preferred.

[0046] The inventors of the present invention have found that a better yield can be achieved if step (f) is carried out using at least 1.5 eq of 1-alkyne. Thus, step (f) is preferably carried out using an excess of 1-alkyne, very preferably at least 1.5 eq, particularly preferably 1.8-2 eq. The base is preferably used in excess (at least 1.5 eq, preferably 1.6-2 eq, more preferably 1.6-1.8 eq). The reaction can be carried out at a temperature of -60°C to -10°C, preferably at -50°C to -15°C, more preferably at -40°C to -20°C, in 4-8 hours. The inventors of the present invention have found that alkyne 7R can be purified by distilling off unreacted 1-alkyne. Preferably, alkyne 7R is obtained in a yield of 75%, 80%, 84%, 87% or more.

[0047] Step (g) can be carried out under acid catalysis. In a preferred embodiment, step (g) can be carried out using Amberlyst ® 15 in a solvent such as methanol. The reaction can be carried out at room temperature in 10-100 hours. After aqueous workup, the product alkyne 8R can be purified by filtration through silica gel, using n-hexane and / or ethyl acetate as eluent. Alkyne 8R is preferably obtained in a yield of 45%, 48%, 50%, 60%, 70%, or more.

[0048] The reduction in step (h) is preferably carried out using a reducing agent that reduces the triple bond of alkyne 8R to a trans double bond to lead to the formation of alkene 9Ri, such as sodium in liquid ammonia or Red-Al. In a preferred embodiment, the trans reduction in step (h) is carried out using Red-Al in a solvent such as THF.

[0049] Red-Al is preferably used in excess (preferably 3-5 eq, particularly preferably 4-4.5 eq). The reaction can be carried out at a temperature between 0°C and room temperature in 15-20 hours.

[0050] Alternatively, the reduction in step (h) can be carried out using a reducing agent that reduces the triple bond of alkyne 8R to a cis double bond to form alkene 9Rii, such as hydrogen and a catalyst such as Pd on barium sulfate, hydrogen and a catalyst such as activated zinc, formic acid and a transition metal catalyst, DIBAL, isobutylmagnesium chloride and cp2 TiCl2 or a dialkylborohydride such as dicyclohexylborohydride (cy2BH) in acetic acid, bis(1,2-dimethylpropyl)borohydride (Sia2BH) in acetic acid or 9BBN in methanol. In a preferred embodiment, the cis reduction in step (h) can be carried out using hydrogen and palladium on barium sulfate in the presence of quinoline in a solvent such as diethyl ether. The reaction can be carried out at room temperature in 15-20 hours.

[0051] After aqueous workup, the product, alkene 9Ri or 9Rii, requires no further purification. Preferably, alkene 9Ri or 9Rii is obtained in a yield of 75%, 80%, 85%, 90%, 95%, or more.

[0052] Step (i) can be carried out using phosphoric acid, a phosphoric acid derivative such as an ester, chloride, bromide, fluoride, or anhydride, or trimethyl phosphite, tetrabromomethane, and a base. Preferably, step (i) is carried out using trimethyl phosphite, tetrabromomethane, and a base such as pyridine, which can also serve as a solvent. Preferably, trimethyl phosphite is used in excess (more preferably approximately 1.25-1.7 eq). Preferably, tetrabromomethane is used in excess (more preferably approximately 1.1-1.5 eq). Preferably, pyridine is used in a large excess and thus also serves as a solvent. The reaction can be carried out at a temperature of 0°C to room temperature in 5-10 hours. After aqueous workup, the product, phosphate 10Ri or 10Rii, can be purified by filtration over silica gel, using, for example, n-hexane and / or ethyl acetate as eluents.Preferably, phosphate 10Ri or 10Rii is obtained in a yield of 40%, 50%, 60%, 64% or more.

[0053] Step (j) can be carried out using trimethylsilyl bromide in a solvent such as dichloromethane. Preferably, trimethylsilyl bromide is used in excess (more preferably 3-6 eq, even more preferably about 4.5 eq). The reaction can be carried out at a temperature of 0°C to room temperature in 2-10 hours. After an aqueous workup, the product O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11Ri or 11Rii can be purified by crystallization from a solvent such as THF and / or water. Preferably, the O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11Ri or 11Rii is obtained in a yield of 65%, 68%, 75%, 85% or more.

[0054] The process according to the invention for the preparation of sphingosine 1-phosphate 11a or a stereoisomer thereof and / or a derivative thereof comprises steps (a) to (j): (a) Conversion of L-serine 1a to methyl ester 2a or a salt thereof: (b) protecting the amino function of methyl ester 2a or a salt thereof to form methyl ester 3a, where Pg is a protecting group: (c) protecting the primary hydroxyl group and the amino group of methyl ester 3a to form methyl ester 4a, wherein R 1 and R 2 independently of one another can be hydrogen, an optionally substituted C1-C6 alkyl radical or an optionally substituted phenyl radical or R 1 and R 2 together can form an optionally substituted C4-C7 cycloalkyl radical: (d) Reduction of methyl ester 4a to form alcohol 5a: (e) Oxidation of alcohol 5a to form aldehyde 6a: (f) Reaction of aldehyde 6a with 1-pentadecyne and a base to form alkyne 7a: (g) partial deprotection of alkyne 7a to form alkyne 8a: (h) Reduction of the triple bond of alkyne 8a to a trans double bond to form alkene 9a: (i) Phosphorylation of the primary hydroxyl group of alkene 9a to form phosphate 10a: (j) deprotecting phosphate 10a to form sphingosine 1-phosphate 11a: wherein the process comprises at least one of the following: - step (e) is carried out using oxalyl chloride, DMSO and less than 5.5 equivalents of Hünig's base; - step (e) is carried out at a temperature not lower than -70°C; - step (f) is carried out using at least 1.5 equivalents of 1-pentadecine; or - Purification of alkyne 7a obtained in step (f) by distilling off unreacted 1-pentadecyne.

[0055] The esterification in step (a) can be carried out, for example, by an acid-catalyzed esterification; using a carbodiimide such as N,N'-dicyclohexylcarbodiimide (DCC); by activating the carboxylic acid functionality of L-serine 1a by conversion to an acid chloride; or by activating the carboxylic acid functionality of L-serine 1a by conversion to an acid anhydride. After an aqueous workup, the product of step (a), methyl ester 2a or a salt thereof, does not require further purification. Preferably, the methyl ester 2a or a salt thereof is obtained in a yield of 98%, 99%, 99.5%, 99.9%, or more. A preferred salt obtained in step (a) is the hydrochloride 2x. In a particularly preferred embodiment, the esterification in step (a) can be carried out using thionyl chloride and methanol. Preferably, thionyl chloride is used in slight excess (preferably 1.05 equivalents).Preferably, the reaction mixture is refluxed for 10-30 hours. When thionyl chloride and methanol are used in step (a), methyl ester 2a is obtained as the hydrochloride salt 2x: .

[0056] When Pg is -CO-OtBu (Boc), step (b) can be carried out using di-tert-butyl dicarbonate (Boc2O) and a base such as triethylamine in a solvent such as dichloromethane. Preferably, the di-tert-butyl dicarbonate is used in a slight excess (more preferably about 1.02 equivalents). Preferably, the base is used in excess (more preferably 2-3 equivalents). The reaction can be carried out at room temperature in 10-20 hours. Preferably, the reaction is carried out under a protective atmosphere of nitrogen. After aqueous workup, the product methyl ester 3a requires no further purification. Preferably, the product, methyl ester 3a, is obtained in a yield of 98%, 99%, 99.5%, 99.9%, or greater.

[0057] Step (c) can be carried out using an aldehyde or ketone, or a derivative thereof, to form an acetal or ketal 4a. The reaction can be catalyzed by acid or Lewis acid. After aqueous workup, the product, methyl ester 4a, can be purified by filtration through silica gel using n-hexane and / or ethyl acetate as eluent. Preferably, methyl ester 4a is obtained in a yield of 75%, 80%, 84%, or more.

[0058] R 1 and R 2 are independently hydrogen, an optionally substituted C1-C6 alkyl radical, or an optionally substituted phenyl radical, or R1 and R2 together form an optionally substituted C4-C6 cycloalkyl radical. Optional substituents include -F, -Cl, -Br, -I, -CF3, -CCl3, -CBr3, -Cl3, -OH, -SH, -NH2, -CN, -NO2, -OMe, -SMe, -NMe2, -CO2H, -CHO, and -CO2Me.

[0059] In a preferred embodiment, R1 and R 2 both methyl. Then, step (c) can be carried out using 2,2-dimethoxypropane and an acid, acetone and an acid, 2-methoxypropene and an acid, or Lewis acid, acetone and iodine, or copper sulfate, or 2-chloro-2-methoxypropane and an acid.

[0060] In a particularly preferred embodiment, step (c) can be carried out using 2,2-dimethoxypropane and an acid, such as toluenesulfonic acid, in a solvent such as dichloromethane. Preferably, the 2,2-dimethoxypropane is used in excess (more preferably 4-6 equivalents). Preferably, the acid is used in catalytic amounts (more preferably 0.1-0.2 equivalents). The reaction can be carried out at room temperature in 1-4 days.

[0061] The reduction in step (d) can be carried out using LiAlH4, DIBAL, NaBH4 and LiCl; NaBH4 and AlCl3, BH3 / THF, AlH3, LiBEt3H, NaAlEt2H2 or hydrogen and a catalyst.

[0062] In a preferred embodiment, the reduction in step (d) can be carried out using LiAlH4 in a solvent such as THF. Preferably, the LiAlH4 is used in excess (more preferably 1.5-2 equivalents). The reaction can be carried out at room temperature in 1-24 hours. After aqueous workup, the product, alcohol 5a, requires no further purification. Preferably, alcohol 5a is obtained in a yield of 84%, 90%, 95%, or higher.

[0063] Step (e) can be carried out by means of a Swern oxidation using oxalyl chloride, DMSO and a base such as Hünig base (N,N-diisopropylamine) in a solvent such as dichloromethane. Preferably, the oxalyl chloride is used in excess (more preferably about 1.5 equivalents). Preferably, the DMSO is used in excess (more preferably about 3 equivalents). Preferably, the Hünig base is used in excess (more preferably 4-5.5 equivalents, even more preferably 4.3-5 equivalents). The inventors of the present development have discovered that less than 5.5 equivalents of Hünig base are necessary to complete the reaction. Accordingly, step (e) is preferably carried out using oxalyl chloride, DMSO and less than 5.5 equivalents of Hünig base.

[0064] Severe oxidations, in particular the reaction of DMSO and oxalyl chloride, are carried out at low temperatures, usually -78°C. The present inventors have found that it is possible to carry out step (e) at a temperature not lower than -70°C. Thus, step (e) is preferably carried out at a temperature not below -70°C. Particularly preferably, the reaction of DMSO and oxalyl chloride (formation of a dimethylchlorosulfonium chloride intermediate) is carried out at a temperature of -40°C to -70°C, more preferably at -40°C to -60°C, most preferably between -40°C and -50°C. Preferably, the following addition of alcohol 5a (formation of an alkoxysulfonium ion intermediate) is carried out at a temperature of -40°C to -70°C, particularly preferably between -40°C and -60°C, more preferably between -40°C and -55°C, most preferably between -40°C and -50°C.Preferably, the addition of the base (formation of the product, aldehyde 6a) is carried out at a temperature of -30°C to -70°C, particularly preferably between -30°C and -60°C, more preferably between -30°C and -55°C, most preferably at -30°C to -50°C.

[0065] The Swern oxidation in step (e) can be carried out at a temperature not lower than -70°C for 2-10 hours. Preferably, the reaction is carried out under a nitrogen blanket. After aqueous workup, the product, aldehyde 6a, requires no further purification. Preferably, aldehyde 6a is obtained in a yield of 75%, 80%, 85%, 90%, 94%, or more.

[0066] Step (f) can be carried out using 1-pentadecyne and a base, such as an organolithium reagent or a Grignard reagent, in a solvent such as THF to form the D-erythro isomer 7a. Particularly preferred organolithium reagents are butyllithium, methyllithium, and phenyllithium, even more preferably butyllithium. Optionally, the reaction can be carried out in the presence of additives such as HMPA, TMEDA, or tributylzirconium chloride. The present inventors discovered that a better yield can be achieved when step (f) is carried out using at least 1.5 equivalents of 1-pentadecyne. Thus, step (f) is preferably carried out using 1-pentadecyne in excess (particularly preferably at least 1.5 equivalents, more preferably 1.6-2 equivalents, most preferably 1.6-1.8 equivalents).The reaction can be carried out at a temperature of -60°C to -10°C, preferably at -50°C to -15°C, most preferably at -40°C to -20°C, for 4-8 hours. The present inventors have found that alkyne 7a can be purified by distillative removal of 1-pentadecyne. Accordingly, after an aqueous workup, product 7a is preferably purified by distillative removal of unreacted 1-pentadecyne. Preferably, alkyne 7a is obtained in a yield of 75%, 80%, 84%, 87%, or more.

[0067] Alternatively, step (f) can be carried out using 1-pentadecyne and a base, such as an organolithium reagent or a Grignard reagent, in a solvent such as THF in the presence of zinc bromide or copper iodide to form the L-threo isomer 7c. Particularly preferred organolithium reagents are butyllithium, methyllithium, and phenyllithium, most preferably butyllithium.

[0068] Step (g) can be carried out by acid catalysis. In a preferred embodiment, step (g) is carried out using Amberlyst ® 15 in a solvent such as methanol. The reaction can be carried out at room temperature in 10–100 hours. After aqueous workup, the product, alkyne 8a, can be purified by filtration through silica gel using n-hexane and / or ethyl acetate as eluents. Preferably, alkyne 8a is obtained in a yield of 45%, 48%, 50%, 60%, 70%, or more.

[0069] The reduction in step (h) is preferably carried out using a reducing agent that converts the triple bond of alkyne 8a to a trans double bond to form alkene 9a, such as sodium in liquid ammonia or Red-Al. In a preferred embodiment, the trans reduction in step (h) is carried out using Red-Al in a solvent such as THF. More preferably, Red-Al is used in an excess (even more preferably 3-5 equivalents, most preferably 4-4.5 equivalents). The reaction can be carried out at a temperature of 0°C in 15-50 hours.

[0070] Alternatively, the reduction in step (h) can be carried out using a reducing agent that reduces the triple bond of alkyne 8a to a cis double bond to form alkene 9b, such as hydrogen and a catalyst such as Pd on barium sulfate, hydrogen and a catalyst such as activated zinc, formic acid and a transition metal catalyst, DIBAL, isobutylmagnesium chloride and cp2TiCl2, or a dialkylborohydride such as dicyclohexylborohydride (cy2BH) in acetic acid, bis(1,2-dimethylpropyl)borohydride (Sia2BH) in acetic acid, or 9-BBN in methanol:

[0071] In a preferred embodiment, the cis reduction in step (h) is carried out using hydrogen and palladium on barium sulfate in the presence of quinoline in a solvent such as diethyl ether. The reaction can be carried out at room temperature for 15-20 hours. Then, in step (i), the primary hydroxyl group of alkene 9b can be phosphorylated to form phosphate 10b: and in step (j), phosphate 10b can be formed to form stereoisomer 11b of sphingosine-1-phosphate 11a:

[0072] After aqueous workup, the product, alkene 9a or 9b, requires no further purification. Preferably, alkene 9a or 9b is obtained in a yield of 75%, 80%, 85%, 90%, 95%, or more.

[0073] Step (i) can be carried out using phosphoric acid, a phosphoric acid derivative such as an ester, a chloride, a bromide, a fluoride, or anhydride, or trimethyl phosphite, tetrabromomethane, and a base. Preferably, step (i) is carried out using trimethyl phosphite, tetrabromomethane, and a base such as pyridine, which can also be used as a solvent. Preferably, trimethyl phosphite is used in excess (more preferably about 1.1 to 1.7 equivalents). Preferably, tetrabromomethane is used in excess (more preferably 1.1 to 1.5%). Preferably, pyridine is used in a large excess and also serves as a solvent. The reaction can be carried out at a temperature of 0°C to room temperature for 5-10 hours.After aqueous workup, the product phosphate 10a or 10b can be purified by filtration through silica gel, using, for example, n-hexane and / or ethyl acetate as eluents. Phosphate 10a or 10b is preferably obtained in yields of 40%, 50%, 60%, 64%, or more.

[0074] Step (j) can be carried out using trimethylsilyl bromide in a solvent such as dichloromethane. Preferably, the trimethylsilyl bromide is used in excess (more preferably 3-6 equivalents, most preferably about 4.5 eq). The reaction can be carried out at a temperature of 0°C to room temperature in 2-10 hours. After an aqueous workup, sphingosine 1-phosphate 11a or stereoisomer 11b can be purified by crystallization from a solvent such as THF and / or water. Preferably, sphingosine 1-phosphate 11a or stereoisomer 11b is obtained in a yield of 65%, 68%, 75%, 85%, or more.

[0075] The invention encompasses a process for the preparation of O-2-amino-3-hydroxyoctadec-4-en-1-yl phosphate 11R: or a stereoisomer and / or a derivative thereof. Derivatives of O-2-amino-3-hydroxyoctadec-4-en-1-yl phosphate 11R encompassed by the present invention include: wherein R a , R b , R c , R d and R e may independently be hydrogen, or an alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl, or alkynylaryl radical, each of which may be optionally substituted. Optional substituents include -F, -Cl, -Br, -I, -CF3, -CBr3, -Cl3, -OH, -SH, -NH2, -CN, -NO2, -OMe, -SMe, -NMe2, -CO2H, -CHO, and -CO2Me.

[0076] In all embodiments of the process according to the invention, the protecting group Pg can be any suitable amino protecting group. Suitable protecting groups are state of the art, as described, for example, in Chapter 7 of "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts (Wiley-Interscience, 3rd edition, 1999), where they are fully discussed by references.

[0077] Preferably, -Pg is -CO-OR or -CO-R, where R is independently alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl, or alkynylaryl, each of which may be optionally substituted. Optional substituents include -F, -Cl, -Br, -I, -CF3, -CCl3, -CBr3, -Cl3, -OH, -SH, -NH2, -CN, -NO2, -OMe, -SMe, -NMe2, -CO2H, -CHO, and -CO2Me.

[0078] An alkyl radical may be straight-chain or branched or may include cyclic radicals. Preferably, an alkyl radical is a C1-C 20Alkyl radical, more preferably a C1-C 15 An alkenyl radical may be straight-chain or branched, or may include cyclic radicals. Preferably, an alkenyl radical is a C2-C 20 Alkenyl radical, more preferably a C2-C 15 Alkyl radical. An alkynyl radical can be straight-chain or branched or include cyclic radicals. Preferably, an alkynyl radical is a C2-C 20 Alkyl radical, more preferably a C2-C 15 Alkynyl radical. Preferably, an aryl radical is a C4-C 30 Aryl residue.

[0079] Preferably -Pg is -CO-O t Bu (Boc), -CO-OCH2Ph ​​(Cbz or Z), -CO-O-CH2-C6H4-pOMe (Moz), -CO-O-CH2-C6H4-pNO2, -CO-O-CH2-C6H4-pBr, -CO-O-CH2-C6H4pCl, -CO-O-CH2-C6H3-2,4-CL2, -CO-OCH2-C 13 H9 (Fmoc), -CO-O-CHPh2, -CO-OMe, -CO-OEt, -CO-OCH2CCl3 (Troc), -CO-OCH2CH2SiMe3 (Teoc), -CO-O-CH2CH2SMe or -CO-OCH2CH2SO2Me. -Pg-CO-OtBu (Boc) is particularly preferred.

[0080] Preferably, the process according to the invention is carried out on an industrial scale, preferably to produce an intermediate or O-2-amino-3-hydroxyoctade-4-en-1-yl phosphate 11Ri or 11Rii or a stereoisomer thereof (particularly preferably sphingosine 1-phosphate 11a) or a derivative thereof in batches of 100 g, 500 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg or more.

[0081] To make the process according to the invention suitable for industrial scale, the process is preferably carried out without the use of chromatography. However, filtration through a small amount of silica gel is also acceptable on an industrial scale.

[0082] In order to make the process according to the invention suitable for industrial scale, the process is preferably carried out at temperatures not lower than -70°C.

[0083] In order to make processes according to the invention suitable for production on an industrial scale, the process preferably produces O-2-amino-3-hydroxyoctadec-4-en-1-yl phosphate 11Ri or 11Rii or a stereoisomer thereof (preferably sphingosine 1-phosphate 11a) or a derivative thereof starting from serine 1 in a total yield of 7% or more and / or starting from aldehyde 6 in a total yield of 11% or more.

[0084] A second aspect of the present invention provides the intermediate phosphate 10a, having the formula: wherein Pg is a protecting group selected from -CO-OCH2Ph ​​(Cbz or Z), -CO-O-CH2-C6H4-pOMe (Moz), -CO-O-CH2-C6H4-pNO2, -CO-O-CH2-C6H4-pBr, -CO-O-CH2-C6H4pCl, -CO-O-CH2-C6H3-2,4-Cl2, -CO-OCH2-C 13 H9 (Fmoc), -CO-O-CHPh2, -CO-OMe, -CO-OEt, -CO-OCH2CCl3 (Troc), -CO-OCH2CH2SiMe3 (Teoc), -CO-O-CH2CH2SMe or -CO-OCH2CH2SO2Me.

[0085] A further aspect of the present invention is that O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11a or a stereoisomer or derivative thereof obtained by carrying out the process becomes a part of the present invention. This further aspect of the present invention also provides that O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11R (preferably sphingosine 1-phosphate 11a) or a derivative thereof is obtained substantially enantiomerically pure. This further aspect of the present invention also provides that O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11R (preferably sphingosine 1-phosphate 11a) or a derivative thereof is obtained substantially free of impurities.This further aspect of the present invention also provides that O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11R or a stereoisomer thereof (preferably sphingosone 1-phosphate 11a) and derivatives thereof are suitable for use in medicine, for example for the treatment or prevention of inflammatory dermatoses, hyperproliferative dermatoses, psorias, psorias vulgaris, acne vulgaris, dermatoheliosis, actinic keratosis or solar keratosis.

[0086] For the purpose of the present invention, the term "substantially enantiomerically pure" means that the component in question consists of more than 90% of a single stereoisomer, preferably more than 95%, more preferably more than 98%, more preferably more than 99%, even more preferably more than 99.2% and most preferably more than 99.5% (by HPLC measurement).

[0087] Yet another aspect of the present invention provides a method for treating or preventing in need patients an inflammatory dermatosis, hyperproliferative dermatosis, psorias, psorias vulgaris, acne vulgaris, dermatoheliosis, actinic keratosis, or solar keratosis, comprising providing a therapeutically or prophylactically effective amount of O-2-amino-3-hydroxy-octadec-4-en-1-yl phosphate 11R or a stereoisomer thereof (preferably sphingosine 1-phosphate 11a) or a derivative thereof. Preferably, the patient is a mammal, more preferably a human. Brief description of the schemes

[0088] The present invention will now be described by way of examples with reference to the accompanying schemes in which: Scheme 1 shows sphingosine 1-phosphate (S1P) 11a and its stereoisomers. Scheme 2 shows a schematic representation of the reaction sequence included in the present invention. Schemes 3 and 4 show schematic representations of the reaction sequence, which differs in its stereochemistry from that shown in Scheme 2. Detailed description of the invention

[0089] The present invention will now be described using synthetic examples. Examples 1-10 follow the reaction sequence shown in Scheme 2. Examples 11-13 follow the reaction sequence shown in Scheme 3. Examples 14-18 follow the reaction sequence shown in Scheme 4. Synthetic examplesExample 1: L.Serine methyl ester hydrochloride 2x (a) L-Serine 1a (107 g; 1.018 mol) was suspended in methanol (800 mL). Thionyl chloride (78 mL; 1.075 mol; 1.05 eq) was added dropwise over 35 minutes. The resulting yellow solution was refluxed for 15 hours and concentrated in vacuo to isolate 2x as a white solid (159.2 g; quantitative). 1 H NMR (DMSO-d6): δ 8.64 (s, 2H, NH2); 5.62 (s(br), 1H, OH); 4.07 (t, J=3.5Hz; 1H; CHNH2); 3.83 (d, J=3.5Hz; 2H; CH2OH); 3.75 (s; 3H; OCH3). (b) L-Serine 1a (107 g; 1.018 mL) was suspended in methanol (800 mL). Thionyl chloride (78 mL; 1.075 mol; 1.05 eq.) was added dropwise over 26 minutes. The resulting yellow solution was refluxed for 21 hours and then purified in vacuo to afford the product 2x as a white solid (158.8 g, quant.). 1 H-NMR (DMSO-d6): see above. (c) The reaction was carried out on a large scale as in Example 1a. 2 kg of L-serine was used to obtain 2.93 kg (98.9%) of L-serine methyl ester hydrochloride 2x. 1H NMR (DMSO-d6): see above. Example 2: N-(tert-Butoxycarbonyl)-L-serine methyl ester 3a (with Boc as Pg)(a) 158.6 g (1.02 mol) of L-serine methyl ester hydrochloride 2x were suspended in 2 L of dichloromethane under N2, and 223 g (2.2 mol) of triethylamine were added. The suspension was cooled to 6°C, and a solution of 227.6 g (1.03 mol) of di-tert-butyl dicarbonate in 500 ml of dichloromethane was added dropwise over a period of 1 h 15 min (Ti=6°C). The mixture was then stirred at RT overnight. 1 L of saturated NaHCO3 solution was added to the yellow solution, and stirring was continued for one hour. The mixture was transferred to a separatory funnel, and after adding a further 750 ml of NaHCO3 solution, the phases were separated (partially solids at the interface). The aqueous phase was extracted three times with 250 ml of dichloromethane each time, the combined organic phases were dried over 770 g of Na2SO4, the desiccant was removed by suction, and the residue was washed with 500 ml of dichloromethane. The combined organic phases were concentrated under vacuum.

[0090] 220.17 g (98.5% of theory) of the product 3a were obtained as a yellow viscous oil. 1 H-NMR (CDCl3); material still contains traces of NEt3.

[0091] 1 H (CDCl3): 5.47 (s(br); 1H); 4.37 (s(br); 1H); 3.95 (dd; 1H; J=4 / 11 Hz); 3.89 (dd;1H; J= 3-4 / 11 Hz); 3.77 (s; 3H); 2.94 (s(br); 0.3H); s(br) 2.54 (0.7H); s 1.45 (9H)

[0092] [α] D : -16.2° (c=4; MeOH)

[0093] b) 157.7 g (1.01 mol) of L-serine methyl ester hydrochloride 2x were suspended in 2 L dichloromethane under N2, and 225.8 g (2.23 mol) of triethylamine were added. The suspension was cooled to 5°C, and a solution of 227.7 g (1.04 mol) of di-tert-butyl dicarbonate in 500 ml of dichloromethane was added dropwise over 1 h 15 min. The mixture was then stirred at RT overnight. 1 L of saturated NaHCO3 solution was added to the yellow reaction mixture, and the mixture was stirred for 1.5 h. The mixture was transferred to a separatory funnel, and the phases were separated. The aqueous phase was extracted four times with 250 ml of dichloromethane each time. The combined organic phases were dried over 636 g of Na2SO4, the drying agent was filtered off with suction, and the mixture was washed with 2 x 500 ml of dichloromethane. The combined organic phases were completely concentrated. The material was then dried in a vacuum oven. 220.46 g (99%) of product 3a was obtained as a yellow viscous oil.

[0094] 1H-NMR (CDCl3): see above.

[0095] The large-scale reaction was carried out completely analogously to Example 2b. The reaction was carried out in two portions. 1.74 kg of L-serine methyl ester hydrochloride 2x yielded 2.43 kg of N-tert-butoxycarbonyl-L-serine methyl ester 3a (99.1%), and 1.34 kg yielded 1.92 kg (quantitative). Example 3: Methyl N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidine-4(S)-carboxylate 4a (with Boc as Pg and methyl as R1 and R2). N-(tert-butoxycarbonyl)-L-serine methyl ester 3a from Example 2a (210.67 g; 0.96 mol) was dissolved in dichloromethane (1 L). 2,2-Dimethoxypropane (208.2 g; 2.0 mol) and p-toluenesulfonic acid (3.34 g; 17.6 mmol) were added, and the reaction mixture was stirred overnight at room temperature under exclusion of moisture. Since the reaction was not yet complete, additional p-toluenesulfonic acid (to a total of 30.36 g; 160 mmol) and 2,2-dimethoxypropane (to a total of 452.8 g; 4.35 mol) were added, and the mixture was stirred at room temperature for another 2.5 days. K2CO3 (22.4 g) was then added, filtered, and the filtrate was washed with saturated NaHCO3 solution (0.5 l). The phases of the 2-phase mixture were separated. The organic phase was dried over Na2SO4 and concentrated under vacuum to afford crude product 4a as a yellow oil (203.2 g; 81.6%).

[0096] 1 H NMR(C6D6): 4.45 (dd; 0.3H; J: 2.5 / 7 Hz); 4.17 (dd; 0.6H; J=3 / 7 Hz); 3.78 (dd; J= 2.5 / 9 Hz); 3.77 (dd; J: 3 / 9 Hz; both together 3.78 and 3.77 1H); 3.65 (dd; J=7 / 9 Hz); 3.61 (dd; J= 7 / 9 Hz; both together 3.65 and 3.61 1H; signals strongly superimposed); s 3.27; s 3.25 (both together 3H); 1.89 (s; 1.8H); 1.77 (s; 1.04H); s 1.55 (1.82H); further signals superimposed by the following signal; 1.39; 1.37 (s; both together 9H); NMR shows that a mixture of two rotamers is present. [α] D = -45.5° (C=1, CHCl3).

[0097] To remove polar impurities, a portion of crude product 4a (156 g) was purified by dissolving it in a 2:1 mixture of n-hexane and ethyl acetate and filtering it through 424 g of silica gel. The filtrate was concentrated under vacuum, and 4a was isolated as a pale yellow oil (140.9 g).

[0098] 1 H-NMR(C6D6): as above, shows rotamers.

[0099] Some crude product 4a (13 g) was purified by chromatography. Clean product 4a was isolated as a pale yellow oil. [α] D = -52.0° (C=1, CHCl3).

[0100] (b) N-(tert-Butoxycarbonyl)-L-serine methyl ester 3a from Example 2b (220.1 g, 1 mol) was dissolved in dichloromethane (1 L). 2,2-Dimethoxypropane (210.9 g; 2.02 mol) and p-toluenesulfonic acid (18.18 g, 96 mmol) were added, and the reaction mixture was stirred for 23 hours at room temperature under exclusion of moisture. Since the reaction was still incomplete, further p-toluenesulfonic acid (to a total of 34.2 g; 180 mmol) and further 2,2-dimethoxypropane (to a total of 545.1 g; 5.23 mol) were added, and the mixture was stirred for a further 2.5 days at room temperature. K2CO3 (10.6 g) was then added, and the mixture was stirred for one hour, filtered, and the filtrate was washed with sat. NaHCO3 (0.8 l). The phases of the biphasic mixture were separated. The aqueous phase was extracted with dichloromethane (3 x 250 ml). The combined organic phases were dried over Na2SO4 and concentrated under vacuum to isolate crude product 4a (221.2 g).

[0101] 1H-NMR(C6D6): as above, shows rotamers.

[0102] To remove polar impurities, the product was dissolved in a 1:1 mixture of n-hexane and ethyl acetate (150 mL) and filtered through a layer of silica gel (4 cm thick; 265 g), then washed with additional eluent (2 l). The filtrate was concentrated to afford filtered product 4a as a pale yellow oil (215.4 g; 82.7%).

[0103] 1 H-NMR(C6D6): as above, shows rotamers.

[0104] A portion of the filtered product 4a (1.3 g) was purified by distillation to give clean product 4a as a colorless oil (0.81 g).

[0105] 1 H-NMR(C6D6): as above, shows rotamers.

[0106] (c) The reaction was carried out on a large scale as in Example 3b. From 4.35 kg of N-(tert-butoxycarbonyl)-L-serine methyl ester 3a, 4.36 kg (84.7%) of methyl N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxyzolidine 4-(S)-carboxylate 4a were obtained.

[0107] 1 H-NMR(C6D6): as above, shows rotamers. Example 4: N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(R)-yl methanol 5a (with Boc as Pg and methyl as R1 and R2)(a) LiAlH4 (2.52 g; 66 mmol) was suspended in THF (100 mL). Then, a solution of the crude product 4a from Example 3a (10.1 g; 39.0 mmol) in THF (50 mL) was added dropwise over 30 minutes, and the reaction mixture was stirred at room temperature for 2.5 hours. A 10% KOH solution (25 mL) was added dropwise under ice-cooling, and the reaction mixture was stirred overnight at room temperature. The resulting LiAlH4 slag and byproducts had a slimy consistency and could not be filtered. Therefore, the reaction mixture was decanted. The remaining salts were dissolved in water, stirred with methyl tert-butyl ether (3 x 50 ml), and filtered. The two-phase filtrates were separated, and the aqueous phase was exhaustively extracted with methyl tert-butyl ether. The combined organic phases were washed with sat.The residue was washed with brine (250 ml), dried over Na2SO4, and concentrated in vacuo. Product 5a was isolated as a viscous pale yellow oil (8.2 g; 91.0%).

[0108] 1 H NMR (C6D6): 3.75-3.95 (m; 1H); 3.5-3.7 (m; 3H); 3.50-3.70 (m; 3H); 3.35-3.70 (m; 1H); 1.55 (s; 3H); 1.41 (s; 3H); 1.36 (s; 9H)

[0109] (b) LiAlH4 (2.98 g; 18.5 mmol) was added to THF (100 mL), and a solution of the filtered product 4a from Example 3a (9.96 g; 38.4 mmol) in 50 mL of THF was added dropwise over 30 minutes, and then the reaction mixture was stirred at room temperature for 1 hour. Then, water (2.5 mL), a 10% NaOH solution (3.5 mL), and finally more water (8 mL) were added dropwise under ice-bath cooling, and stirring was continued for 40 minutes. Methyl tert-butyl ether (20 mL) was added, and the reaction mixture was dried over MgSO4 and concentrated in vacuo to isolate product 5a as a pale yellow viscous oil (7.75 g; 87.2%).

[0110] 1 H-NMR(C6D6): as above.

[0111] (c) LiAlH4 (32.26 g; 0.85 mol) was initially charged in THF, and a solution of the filtered product 4a in THF (650 mL) was added dropwise over 50 minutes. The reaction mixture was stirred at room temperature, and then water (24 mL), a 10% NaOH solution (33 mL), and more water (60 mL) were added dropwise while cooling in an ice bath. The reaction mixture was stirred overnight. Methyl tert-butyl ether (200 mL) was added, and the reaction mixture was dried over MgSO4 and concentrated in vacuo to afford product 5a as a pale yellow oil (93.9 g; 80.4%).

[0112] 1 H-NMR(C6D6): as above.

[0113] (d) The reaction was carried out on a large scale as in Example 4b. From 2.29 kg of methyl N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidine-4(S)-carboxylate 4a, 1.73 kg (84.7%) of N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-o-xazolidin-4(R)-yl methanol 5a were obtained.

[0114] 1 H-NMR(C6D6): as above. Example 5: N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-yl methanal 6a (with Boc as Pg and methyl as R1 and R2)(a) N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(R)-yl methanol 5a (7.24 g; 31.3 mmol) was dissolved in dichloromethane (60 ml) and then Dess-Martin periodinane (DMP) (20.4 g; 48.2 mmol) was added (exothermic). The reaction mixture was cooled, moist dichloromethane (125 ml) was added and stirred for 30 minutes. The reaction mixture was filtered through a small amount of silica gel, which was washed with further dichloromethane. The combined filtrates were washed with a 1:1 mixture of sat. NaHCO3 solution and 1M Na2S2O7 solution (160 ml), dried over Na2SO4 and concentrated under vacuum to obtain product 6a as a yellow oil (3.75 g; 52.5%).

[0115] 1H NMR: 9,33 (s(br); 1H); 3,7-4,2 (m; 1 H); 3,55-7,70 (m; 1H); 3,45-3,55 (m; 1H); 1,5-1,7 (m; 3H); 1,4-1,5 (m; 3H); 1,33 (s; 9H)

[0116] (b) Oxalyl chloride (4.5 mL; 6.62 g; 1.5 eq.) was dissolved in dichloromethane (80 mL) and cooled to -78°C. DMSO (7.4 mL; 8.1 g; 104 mmol; 3 eq.) was dissolved in dichloromethane (10 mL) and added dropwise to the reaction mixture over 25 minutes, during which the internal temperature rose to -70°C. The reaction mixture warmed to -60°C within 20 minutes. N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxyzolidin-4(R)-yl methanol 5a (8 g; 35 mmol) was dissolved in dichloromethane (60 mL) and added dropwise to the reaction mixture over 50 minutes, during which the internal temperature rose to -55°C. The reaction mixture was then stirred for 30 minutes until the internal temperature rose to -45°C. A mixture of Hünig's base (36 ml; 6.2 eq) and dichloromethane (5 ml) was added dropwise over 5 minutes, during which the internal temperature reached -32°C. The color of the reaction mixture changed from pale pink to clear yellow.The reaction mixture was allowed to warm to 12°C over one hour and then cooled again to -8°C. The reaction mixture was poured into ice-cold 1M HCl solution (130 ml) and stirred until two phases formed, which were separated. The aqueous phase was extracted with dichloromethane (3 x 30 ml). The combined organic phases were washed with phosphate buffer (pH 7) (4 x 80 ml), dried over Na2SO4, and concentrated in vacuo. Product 6a was obtained as a yellow oil (7.29 g; 91.9%).

[0117] 1 H-NMR(C6D6): as above. [α] D = -63°

[0118] (c) Oxalyl chloride (4.5 mL; 6.62 g; 52 mmol; 1.5 eq) was dissolved in dichloromethane (80 mL) and cooled to -78°C. DMSO (7.4 mL; 8.1 g; 104 mmol; 3 eq) was dissolved in dichloromethane (10 mL) and added dropwise to the oxalyl chloride solution, whereby the internal temperature rose to -70°C. The reaction mixture was stirred for 45 minutes, during which it warmed to -60°C. N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(R)-ylmethanol 5a (8 g; 35 mmol) was dissolved in dichloromethane (60 mL) and added dropwise to the reaction mixture over a period of 50 minutes. The reaction mixture was then stirred for 45 minutes, during which it warmed to -45°C. A mixture of Hünig's base (36 ml; 6.2 eq) and dichloromethane (5 ml) was added dropwise over 20 minutes. The reaction mixture was then stirred for 45 minutes, warming to 0°C. The reaction mixture was poured into ice-cold 1M HCl solution (130 ml) and stirred until two phases formed, which were separated.The aqueous phase was extracted with dichloromethane (3 x 30 ml). The combined organic phases were dried over Na2SO4 and concentrated in vacuo. Product 6a was isolated as a yellow oil (7.36 g; 92.8%).

[0119] 1 H-NMR(C6D6): as above.

[0120] (d) The following reaction was carried out under nitrogen: Oxalyl chloride (2.83 mL; 33 mL; 1.5 eq) was dissolved in dry dichloromethane (80 mL) and cooled to -50°C. DMSO (4.69 mL; 66 mmol; 3 eq) was dissolved in dry dichloromethane (10 mL) and added dropwise over 45 minutes, during which the internal temperature rose to -46°C. Gas evolution occurred at the beginning of the addition. The reaction mixture was then stirred at -50°C for 20 minutes. N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolin-3(R)-ylmethanol 5a (5 g; 22 mmol) was dissolved in dry dichloromethane (60 mL) and added dropwise over 1.25 hours. The reaction mixture was then stirred at -50°C for 30 minutes. A mixture of Hünig's base (22.5 ml; 6.2 eq) and dry dichloromethane (5 ml) was added dropwise over 5 minutes, with the internal temperature not exceeding -35°C.The reaction mixture was then stirred until the internal temperature rose to 0°C and then poured into an ice-cold 1M HCl solution (130 ml). The phases of the resulting two-phase mixture were separated, and the aqueous phase was extracted with dichloromethane (3 x 30 ml). The combined organic phases were washed with phosphate buffer (Na2HPO4 / NaH2PO4, 0.5M with respect to phosphate) (4 x 80 ml), dried over Na2SO4, and concentrated under vacuum. Product 6a was obtained as a yellowish-brown oil (3.78 g; 76.3%). 1 H-NMR(C6D6): as above. [α] D = -73.7° (c=1.3).

[0121] (e) The following reaction was carried out under nitrogen: Dichloromethane (700 mL) was cooled to -50°C, oxyyl chloride (47 mL; 542 mmol; 1.5 eq) was added dropwise, and the dropping funnel was rinsed with dichloromethane (140 mL). DMSO (77 mL; 1.09 mL; 3 eq) was dissolved in dichloromethane (105 mL) and added dropwise to the oxalyl chloride solution. The internal temperature reached -43°C, and gas evolution was observed at the beginning of the addition. The reaction mixture was stirred for 20 minutes. N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(R)-yl methanol 5a (83.46 g; 361 mmol) was dissolved in dichloromethane (650 mL) and added dropwise over 3 hours at an internal temperature of -43°C. The dropping funnel was rinsed with dichloromethane (2 x 50 mL), and the reaction mixture was stirred at -50°C for 40 minutes. A mixture of Hünig's base (367 mL; 6.2 eq) and dichloromethane (60 mL) was added dropwise over 20 minutes, during which the internal temperature increased.The reaction mixture was stirred until 5°C was reached. Then, an ice-cold 1M HCl solution (1.4 L) was added dropwise. The phases of the biphasic mixture were separated. The aqueous phase was extracted with dichloromethane (3 x 300 mL). The combined organic phases were washed with phosphate buffer (151 g NaH2PO4 dihydrate and 117 g NaH2PO4 monohydrate in 3.41 L water) (4 x 850 mL), dried over Na2SO4, and concentrated under vacuum. Product 6a was obtained as a yellow oil (63.55 g; 76.8%).

[0122] 1 H-NMR(C6D6): as above. [α] D = -75.2° (c=1.34; CHCl3).

[0123] (f) The following reaction was carried out under nitrogen: Oxalyl chloride (2.83 mL; 33 mmol; 1.5 eq) was dissolved in dichloromethane (13 mL) and cooled to -50°C. DMSO (4.69 mL; 66 mmol; 3 eq) was dissolved in dichloromethane (13 mL) and added dropwise to the oxalyl chloride solution over 30 minutes, during which the internal temperature reached -45°C and gas evolution was observed upon addition. The reaction mixture was then stirred at -50°C for 20 minutes. N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(R)-ylmethanol 5a (5 g; 22 mmol) was dissolved in dichloromethane (31 mL) and added dropwise to the reaction mixture over 1 hour. The reaction mixture was then stirred at -50°C for 30 minutes. A mixture of Hünig's base (15.7 ml; 4.3 eq) and dichloromethane (4 ml) was added dropwise over 5 minutes, during which the internal temperature reached -35°C. The reaction mixture was then allowed to warm to 0°C.Ice-cold 1M HCl solution (51 ml) was then added dropwise to the reaction mixture. The resulting two-phase mixture was separated. The aqueous phase was extracted with dichloromethane (30 ml). The combined organic phases were washed with phosphate buffer (14.29 g Na2HPO4 dihydrate and 11.13 g NaH2PO4 monohydrate in 320 ml water) (4 x 80 ml), dried over Na2SO4, and concentrated under vacuum to isolate product 6a as a yellow oil (4.5 g; 90.8%).

[0124] 1 H-NMR(C6D6): as above. [α] D = -76.6° (c=1.34; CHCl3).

[0125] (g) The reaction was carried out on a large scale as in Example 5f. 1.62 kg of N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxyzolidin-4(R)-yl methanol 5a, 1.5 eq of oxalyl chloride, 3 eq of DMSO, and 4.3 eq of Hünig's base in more concentrated solution were used to afford 1.51 kg (94.0%) of N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-o-xazolidin-4(S)-yl methanal 6a.

[0126] 1H-NMR(C6D6): as above.

[0127] The experiments in Example 5 demonstrate that alcohol 5a can be oxidized to aldehyde 6a by various methods, such as Dess-Martin oxidation (Example 5a) and Swern oxidation (Examples 5b-g). They also demonstrate that the Swern oxidation can be successfully performed at -78°C (Examples 5b-c), as described in the literature, but also at -40°C to -50°C (Examples 5d-g). Furthermore, they demonstrate that the reaction can be carried out at acceptably high concentrations (Examples 5f-9 compared to 5d-g), which makes it possible to conduct it on an industrial scale. Example 6: 1(R)-[N-(tert-Butoxycarbonyl)-2',2'-dimethyl-1',3'-oxazolidin-4'(S)-yl]-hexadec-2-yn-1-ol 7a (with Boc as Pg and methyl as R1 and R2)(a) 1-Pentadecyne (2.31 mL; 8.8 mmol; 2.0 eq) was dissolved in dry THF (50 mL) and cooled to below -20°C. BuLi (3.2 mL; 2.5 M; 7.8 mmol, 1.8 eq) was added while maintaining the internal temperature below -20°C. A milky white reaction mixture was obtained, which was stirred for 1.5 h at a temperature below -20°C. N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-ylmethanal 6a (1 g; 4.4 mmol) was dissolved in dry THF (30 mL), cooled to -20°C, and added to the reaction mixture. The reaction mixture was kept below -20°C for 4 hours, during which time the turbidity slowly dissipated. The reaction was quenched by adding NH4Cl solution (50 mL). Precipitated salts were dissolved by adding water (30 mL). The phases of the biphasic mixture were separated.The aqueous phase was extracted with dichloromethane (4 x 25 ml). The combined organic phases were washed with brine (2 x 25 ml), dried over Na2SO4, and concentrated in vacuo to obtain crude product 7a as an orange-brown oil (2.78 g). To remove unreacted 1-pentadecyne, crude product 7a was purified by chromatography (54 g silica gel; eluent first n-hexane, then 6:1 n-hexane:ethyl acetate), yielding the chromatographed product 7a as a yellow oil (1.37 g; 3.2 mmol; 71.8%).

[0128] 1 H NMR (C6D6, 400 MHz): 0.89 (t; J=6.5 Hz: 3H); 1.1-1.5 (m; 37H); 2.1 (German; 1.5 / 7 Hz,;2H); 3.70-3.85 (m; 1H); 3.85-3.95 (m; 1H); 3.95-4.05 (m; 1H); 4.4-4.5 (m; 1H); 5.41 (d; 1H; J= 6 Hz). Diastereoselectivity 1:7.

[0129] (b) 1-Pentadecine (2.1 mL; 7.9 mmol; 1.8 eq) was dissolved in dry THF (50 mL) and cooled to -30°C. BuLi (2.82 mL; 2.5 M; 7.0 mmol; 1.6 eq) was added, whereby the internal temperature rose to -25°C. A milky white solution was obtained, which was stirred for a further 1.5 hours at a temperature below -20°C. N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-ylmethanal 6a (1 g; 4.4 mmol) was dissolved in dry THF (30 mL), cooled to -20°C, and then added to the reaction mixture. The reaction mixture was stirred for 4 hours at a temperature below -20°C, during which the turbidity slowly dissolved. The reaction was quenched by the addition of NH4Cl solution (40 mL). The phases of the two-phase system were separated. The aqueous phase was extracted with dichloromethane (4 x 25 mL). The combined organic phases were washed with brine (2 x 25 mL), dried over Na2SO4, and concentrated under vacuum. The crude product 7a was isolated as an orange-brown oil (2.77 g).To separate unreacted 1-pentadecine, crude product 7a was purified by chromatography (51 g silica gel; eluent first n-hexane, then 6:1 n-hexane:ethyl acetate), whereby the chromatographed product 7a was obtained as a yellow oil (1.38 g; 3.2 mmol; 72.3%).

[0130] 1 H NMR (C6D6): as above. Diastereoselectivity: 1:8.

[0131] (c) 1-Pentadecine (2.1 mL; 7.9 mmol; 1.8 eq) was dissolved in THF (50 mL) and cooled to -20°C. BuLi (2.82 mL; 2.5 M; 7 mmol; 1.6 eq) was added, and the reaction mixture was stirred below -20°C for 1.5 h and then cooled to -40°C. N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)ylmethanal 6a (1 g; 4.4 mmol) was dissolved in THF (2.5 mL) and added to the reaction mixture. The reaction mixture was allowed to warm to -20°C and stirred at this temperature for 4.5 h. The reaction was quenched by addition of NH4Cl solution (50 mL). Precipitated salts were dissolved in additional NH4Cl solution (130 ml) and water (25 ml). The phases of the two-phase system were separated, and the aqueous phase was extracted with dichloromethane (4 x 25 ml). The combined organic phases were dried over Na2SO4 and concentrated under vacuum. Crude product 7a was isolated as a yellow oil (2.77 g).To separate unreacted 1-pnetadecine, crude product 7a was purified by chromatography (53 g silica gel; eluent first n-hexane, then 6:1 n-hexane: ethyl acetate), yielding chromatographed product 7a as a yellow oil (0.71 g; 37.2%).

[0132] 1 H-NMR(C6D6): as above. Diastereoselectivity 1:8 to 1:12.

[0133] (b) THF (2.4 L) was cooled to -20°C, and 1-pentadecine (124 mL; 471 mmol; 1.8 eq) was added. BuLi (167 mL; 2.5 M; 419 mmol; 1.6 eq) was added dropwise over 25–30 minutes, maintaining the internal temperature at approximately -20°C. The reaction mixture was stirred at -20°C for 1.5 hours, during which a milky white suspension formed, and then cooled to -40°C. N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-yl methanal 6a (60 g; 262 mmol) was dissolved in THF (200 mL) and added dropwise to the reaction mixture over 35 minutes while maintaining the internal temperature at approximately -40°C. The reaction mixture was allowed to warm to -20°C and then held at this temperature for 4 hours. The reaction was quenched by slow addition of NH4Cl solution (730 mL) at an internal temperature of -15°C. Then, further NH4Cl solution (1700 mL) was added. Precipitated salts were dissolved by addition of water (1 L).The phases of the two-phase mixture were separated. The aqueous phase was extracted with dichloromethane (1.6 l; 1.1 l; 1 l; 1 l). The combined organic phases were washed with NaCl solution (2 x 1 l), dried over Na2SO4, and concentrated under vacuum, yielding crude product 7a as an orange-brown oil (157.27 g).

[0134] Some crude product 7a (5.08 g) was purified by distilling off unreacted 1-pentadecine. The unreacted 1-pentadecine was obtained as a colorless distillate (1.64 g), and the product 7a purified by distillation was obtained as a brown oil (3.11 g).

[0135] 1 H-NMR(C6D6): as above.

[0136] The 1H-NMR shows that distillation neither decomposes product 7a nor affects the diastereoisomer ratio. To determine the diastereoisomer ratio in the product, distilled product 7a was further purified by chromatography (56 g of silica gel; eluent first n-hexane, then 6:1 n-hexane:ethyl acetate), yielding chromatographed product 7a as a yellow oil (1.17 g; 31.6% calculated overall yield).

[0137] 1 H NMR (C6D6): as above. Diastereoselectivity: 1:5.

[0138] The bulk of crude product 7a (152 g) was purified by distilling off unreacted 1-pentadecine. Unreacted 1-pentadecine was obtained as a pale yellow distillate (74.11 g), and the distillatively purified product 7a was obtained as a brown oil (81.75 g; 73.9% overall yield). A portion of the distillatively purified product 7a (10.04 g) was further purified by filtration over silica gel (42 g; eluent first n-hexane, then 6:1 n-hexane:ethyl acetate), yielding filtered product 7a as an orange oil (6.06 g; 44.6% overall yield).

[0139] 1 H-NMR(C6D6): as above.

[0140] (e) 1-Pentadecine (74 mL; 279 mmol; 1.8 eq) was dissolved in dry THF (1.4 L) and cooled to -20°C. BuLi (100 mL; 2.5 M; 248 mmol; 1.6 eq) was added dropwise over 25 minutes while maintaining the internal temperature at approximately -20°C. The reaction mixture was stirred for 1.5 hours at a temperature below -20°C to form a milky suspension, which was then cooled to -40°C. N-(tert-butoxycarbonal)-2,2-dimethyl-1,3-oxazolidin-4(S)-ylmethanal 6a (35.45 g; 155 mmol) was dissolved in THF (120 mL) and added dropwise to the reaction mixture over 45 minutes at an internal temperature of approximately -40°C. The reaction mixture was allowed to warm to -20°C and then stirred at this temperature for a further 4 hours. The reaction was quenched by the dropwise addition of NH4Cl solution (430 ml). Precipitated salts were dissolved by the addition of further NH4Cl solution (11) and water (400 ml).The phases of the two-phase mixture were separated. The aqueous phase was extracted with dichloromethane (4 x 590 ml), and the combined organic phases were washed with brine (2 x 190 ml), dried over Na2SO4, and concentrated in vacuo. The crude product 7a was isolated as an orange-brown oil (99.77 g).

[0141] 1 H-NMR (C6D6): as above. The crude product 7a was purified by distilling off the unreacted 1-pentadecine. The unreacted 1-pentadecine was obtained as a colorless distillate (56.23 g), and the distillatively purified product 7a was obtained as a brown oil (36.21 g; 53.5%).

[0142] 1 H-NMR(C6D6): as above. Diastereoselectivity 1:4.

[0143] (f) 1-Pentadecine (11.6 mL; 44.2 mmol, 2.3 eq) was dissolved in THF (144 mL) and cooled to -20°C. BuLi (12 mL; 2.5 M; 31 mmol; 1.6 eq) was added dropwise while maintaining the internal temperature below -20°C. The reaction mixture was stirred for 1.5 h at an internal temperature below -20°C. N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-ylmethanal 6a (4.39 g; 19.1 mmol) was dissolved in THF (11 mL), cooled to -20°C, and added dropwise to the reaction mixture at -20°C. The reaction mixture was stirred for 4 h at an internal temperature below -20°C. The reaction was quenched by adding NH4Cl solution (50 ml). Precipitated salts were dissolved by adding further NH4Cl solution (130 ml) and water (50 ml). The phases of the two-phase system were separated. The aqueous phase was extracted with dichloromethane (4 x 100 ml).The combined organic phases were dried over Na2SO4 and concentrated in vacuo, yielding crude product 7a as a yellow oil (13.18 g). A portion of crude product 7a (3 g) was purified by chromatography (58 g silica gel; eluent first n-hexane, then 6:1 n-hexane:ethyl acetate). The chromatographed product 7a was obtained as a yellow oil (1 g; 52.4%).

[0144] 1 H-NMR(C6D6): as above.

[0145] (g) The reaction was carried out on a large scale as in Example 6f in 2 runs: 610 g each of N-(tert-butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-yl methanal 6a were used to obtain 994 g (85.4%) and 1019 g (87.5%) of 1(R)-[N-(tert-butoxycarbonyl)-2',2'-dimethyl-1',3'-oxazolidin-4'(S)-yl]-hexadec-2-yn-1-ol 7a, which was purified by distilling off unreacted 1-pentadecyne but not by filtration over silica gel.

[0146] 1H-NMR(C6D6): as above.

[0147] The experiments of Example 6 demonstrate that 1.8 eq of 1-pentadecine is sufficient to achieve complete reaction (Examples 6a-b). They also demonstrate that it is possible to perform it at a sufficiently high concentration (Example 6c-g compared to 6a-b), making it suitable for industrial scale production. Furthermore, they demonstrate that it is possible to purify product 7a by distillation and / or filtration over silica gel for large-scale production. Likewise, the experiments of Example 7 below demonstrate that it is not necessary to purify product 7a by filtration over silica gel and that the crude product or distillatively purified product 7a can be directly used in the next step, the deprotection of the primary hydroxyl and amino groups. Example 7: 2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-yne-1,3(R)-diol 8a (with Boc as Pg)(a) Distillatively purified product 7a (3.1 g) was dissolved in methanol (70 ml) and Amberlyst ® 15 (3.67 g) was added. The reaction mixture was stirred at room temperature for 81 hours, dried over Celite ® filtered and concentrated under vacuum to obtain product 8a as a yellow oil (2.04 g; 72.4%).

[0148] 1 H NMR (DMSO-d6, 400MHz): δ 0.85 (t; J=6.5 Hz; 3H); 1.1-1.5 (m; 31H); 2.14 (dt; J= 1.5 / 7 Hz; 2H); 3.4-3.6 (m; 3H); 4.2-4.3 (m; 1H); 4.48 (t; J=5Hz; 1H); 5.29 (d; J=6Hz; 1H); 6.13 (d; J: 7.5Hz; 1H).

[0149] (b) Distillatively purified product 7a (10.02 g) was dissolved in methanol (230 ml) and Amberlyst ® 15 (11.71 g) was added. The reaction mixture was stirred at room temperature for 39 hours, dried over Celite ®filtered and concentrated under vacuum to obtain product 8a as an orange-brown oil (7.66 g; 84.2%).

[0150] 1 H NMR (DMSO-d6): as above.

[0151] (c) Distillatively purified product 7a (33.53 g) was dissolved in methanol (220 ml) and Amberlyst ® 15 (39.43 g) was added. The reaction mixture was stirred at room temperature for 16 hours, dried over Celite ®filtered and reduced under vacuum to a methanolic solution of approximately 500 mL, which was extracted with n-hexane (3 x 250 mL). The methanolic solution was concentrated under vacuum, and product 8a was isolated as a brown oil (8.76 g). The n-hexane extracts were concentrated under vacuum to obtain impure product 8a, which was filtered through silica gel (55 g; eluent first n-hexane, then 2:1 n-hexane: ethyl acetate) to obtain filtered product 8a (2.91 g). The extractively purified product 8a and the filtered product 8a were combined to give a total yield of 11.67 g (38.3%).

[0152] 1 H NMR (DMSO-d6) as above.

[0153] (d) Distillatively purified product 7a (36.21 g) was dissolved in methanol (240 ml) and Amberlyst ® 15 (42.58 g) was added. The reaction mixture was stirred at room temperature for 41 hours, dried over Celite ®filtered and reduced under vacuum to 500 mL of a methanolic solution, which was extracted with n-hexane (700 mL, then 5 x 250 mL). The methanolic phase was concentrated under vacuum to isolate product 8a as a brown oil (10.08 g). The n-hexane phases were concentrated under vacuum to isolate impure product 8a as a brown oil (12.23 g), which was filtered through silica gel (190 g; eluent first n-hexane, then 4:1 n-hexane:ethyl acetate) to isolate filtered product 8a (3.72 g). The extractively purified product 8a and the filtered product 8a were combined to give a total of 13.80 g (42.0%).

[0154] 1 H NMR (DMSO-d6) as above.

[0155] (e) The reaction was carried out on a large scale as in Example 7b. Starting from 2.01 kg of 1(R)-[N-tert-butoxycarbonyl)-2',2'-dimethyl-1',3'-oxazolidin-4'(S)-yl]-hexadec-2-yn-1-ol 7a, 1.46 kg (80%) of crude 2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-yn-1,3(R)-diol 8a were obtained, which was filtered through silica gel to isolate 0.88 kg (48.2%) of filtered product 8a.

[0156] 1 H NMR (DMSO-d6) as above. Example 8: trans-2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-ene-1,3(R)-diol (N-Boc-sphingosine) 9a, (with Boc as Pg)(a) 2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-yne-1,3(R)-diol 8a (0.99 g; 2.5 mmol) was dissolved in dry THF (25 mL) and cooled to 0°C. Red-Al (3 mL; 70%; 10.8 mmol; 4.3 eq) was dissolved in THF (23 mL) and pre-cooled to 0°C. The solution of alkyne 8a was added to the solution of Red-Al at 0°C, with gas evolution. The reaction mixture was then allowed to warm to room temperature and stirred for a further 16.5 h. The reaction mixture was cooled again to 0°C and quenched with methanol (2 ml), which again resulted in gas evolution. A potassium sodium tartrate solution (26 g / 100 ml) (100 ml) was added to the reaction mixture, and the mixture was stirred at room temperature for 3.5 hours. The phases of the two-phase mixture were separated.The organic phase was washed with potassium sodium tartrate solution (50 ml) and NaCl solution (50 ml), dried over Na2SO4 and concentrated under vacuum to give product 9a as a colorless oil (0.93 g; 93.5%).

[0157] 1 H (DMSO-d6, 400 MHz): δ 0.85 (t; J=7Hz; 3H); 1.1-1.4 (m; 31H); 1.9-2.0 (m; 2H); 2.9-3.0 (m; 1H); 3.4-3.5 (m; 2H); 3.85 (dt; J= 5.5 / 6.5Hz; 1H); 4.38 (t; J= 5.5Hz; 1H); 4.74 (d; 5 Hz; 1H); 5.40 (dd, J=6.5 / 15.5Hz; 1H); 5.53 (dt; J=6.5 / 15; 1H); 6.16 (d; J=9Hz; 1H).

[0158] (b) 2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-yne-1,3(R)-diol 8a (13.8 g; 35 mmol) was dissolved in THF (50 mL) and cooled to 0°C. Red-Al (41 mL; 70%; 147 mmol, 4.2 eq) was dissolved in THF (50 mL) and cooled to 0°C. The solution of alkyne 8a was added to the Red-Al solution at 0°C. The reaction mixture was allowed to warm to room temperature and stirred at RT for 41 h. The reaction mixture was cooled to 0°C and slowly quenched with methanol, with gas evolution. A saturated sodium potassium tartrate solution (99.34 g / 150 mL) (150 mL) was added, and the mixture was stirred at room temperature for a further 5 h. The phases of the two-phase mixture were separated. The aqueous phase was extracted with methyl tert-butyl ether (3 x 150 mL). The organic phase was washed with sodium potassium tartrate solution (92.23 g / 150 mL) (150 mL) and NaCl solution (150 mL), dried over Na2SO4, and concentrated under vacuum to isolate product 9a (10.43 g; 75.5%).

[0159] 1 H NMR (DMSO-d6) as above.

[0160] (c) The reaction was carried out on a large scale as in Example 8b, using 883 g of 2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-yne-1,3(R)-diol 8a to obtain 844 g (95.1%) of trans-2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-ene-1,3(R)-diol 9a. 1 H NMR (DMSO-d6) as above. Example 9: O,O'-Dimethyl-O''-[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate (N-Boc-sphinqosin 1-phosphate) 10a (with Boc as Pg)(a) trans-2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-en-1,3(R)-diol 9a (9.95 g; 25 mmol) was dissolved in pyridine (60 mL) and cooled to 0°C. Tetrabromomethane (12.44 g; 37.5 mmol; 1.5 eq) was added. Trimethyl phosphite (5 mL, 42.5 mmol; 1.7 eq) was added dropwise over 1 hour at an internal temperature below 10°C. The reaction mixture was then allowed to warm to room temperature and stirred at RT for a further 5 hours. Methyl tert-butyl ether (200 ml) was added to the reaction mixture, and it was washed with 2M HCl solution (195 ml). To achieve phase separation, a further 50 ml of methyl tert-butyl ether was added. The reaction mixture was washed again with 2M HCl solution (195 ml). To achieve phase separation, methyl tert-butyl ether was added.-butyl ether (140 ml), NaCl solution (80 ml), 1M NaOH solution (100 ml), and NaHCO3 solution (200 ml) were added. The organic phase was washed with NaHCO3 solution (100 ml). After addition of solid NaCl, the mixture separated into three phases: an organic phase, a brown intermediate layer, and an aqueous phase. The organic phase was washed with NaCl solution (100 ml), dried over Na2SO4, and concentrated under vacuum to isolate crude product 10a as a brown oil (13.37 g). Crude product 10a was filtered through silica gel (95 g; eluent: first 1:1 ethyl acetate:n-hexane, then 2:1) to obtain filtered product 10a as a yellow oil (8.18 g; 64.7%).

[0161] 1 H NMR (DMSO-d6): δ 0.85 (t; J=7Hz; 3H); 1.2-1.3 (m; 22H); 1.36 (s; 9H); 1.9-2.1 (m; 2H); 3.4-3.6 (m; 1H); 3.65 (d; J= 11Hz; 6H); 3.75-3.85 (m; 1H); 3.85-4.0 (m; 1H); 4.1 (ddd; J= 3-4 / 6 / 10 Hz; 1H); 5.0 (d; J= 5 Hz; 1H); 5.39 (dd(br); J= 7 / 15 Hz; 1H); 5.58 (dt; J= 7 / 15 Hz; 1H); 6.68 (d; J= 9Hz; 1H).

[0162] (b) The reaction was carried out on a large scale as in Example 9a. From 832 g of trans-2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-en-1,3(R)-diol 9a, 446 g (42.2%) of O,O'-dimethyl-O''-[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate 10a were obtained.

[0163] 1 H NMR (DMSO-d6) as above. Example 10: O-trans-2(S)-amino-3(R)-hydroxy-octadec-4-en-yl phosphate (Sphingosine 1-phosphate) 11a(a) O,O'-Dimethyl-O''-[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate 10a (500 mg; 0.99 mmol) was dissolved in dichloromethane (12.5 mL) and cooled to approximately 2°C. Trimethylsilyl bromide (0.6 mL; 4.5 mmol, 4.5 eq) was added dropwise over 5 minutes. The reaction mixture was allowed to warm to room temperature and stirred at RT for 2 hours. The reaction mixture was concentrated and methanol (12.5 mL) was added to the residue. The reaction mixture was stirred at room temperature for 2 hours and then concentrated again under vacuum to yield a pink foam (0.5 g), which was crystallized from a 2:1 mixture of THF and water (7 mL). The solid was washed with ice-cold n-hexane and dried under vacuum. Sphingosine 1-phosphate 11a was isolated as a white solid (320 mg; 85.6%).

[0164] 1H (DOAc, 70°C): 6,3-6,5 (m; 1H); 5,9-6,1 (m; 1H); 4,9-5,1 (m; 1H); 4,7-4,9 (m; 2H); 4,1-4,3 (m; 1H); 2,5-2,6 (m; 2H); 1,7-2,0 (m; 22H); 1,34 (t; 3H; J: 6,5 Hz).

[0165] (b) O,O'-Dimethyl-O''-[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate 10a (5.16 g; 10.2 mmol) was dissolved in dichloromethane (125 mL) and cooled to approximately 2°C. Trimethylsilyl bromide (6.1 mL; 46 mmol, 4.5 eq) was added dropwise over 5 minutes. The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 1.25 hours. The reaction mixture was concentrated under vacuum, and the residue was taken up in methanol (125 mL). The reaction mixture was stirred at room temperature for 2 hours and then concentrated again under vacuum to give a pink foam, which was crystallized from a 2:1 mixture of THF and water (75 mL). The solid was washed with ice-cold n-hexane and dried under fine vacuum at 30°C. Sphingosine 1-phosphate 11a was isolated as a white solid (3.0 g; 77.8%).

[0166] 1 H-NMR (DOAc): as above.

[0167] (c) The reaction was carried out on a large scale as in Example 10b. From 281 g and 165 g of O,O'-dimethyl-O''-[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate 10a, 144 g (68.6%) and 69 g (55.9%) of sphingosine 1-phosphate 11a were obtained, respectively.

[0168] 1 H-NMR (DOAc): as above.

[0169] The sphingosine 1-phosphate 11a obtained by the method of the present invention was analyzed by HPLC. It was found that the purity was 99.2% and the enantiomeric purity was 99.3%, as determined by chiral HPLC. Example 11: cis-2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-ene-1,3(R)-diol 9b (with Boc as Pg)(a) 2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-yne-1,3(R)-diol 8a (250 mg: 0.63 mmol) was dissolved in diethyl ether (35 mL). Quinoline (0.05 mL) and Pd on BaSO4 (10%) (645 mg) were added. The reaction mixture was purged three times with nitrogen and then stirred under a H2 atmosphere for 16 h at room temperature. The reaction mixture was dried over Celite. ® filtered and concentrated in vacuo to obtain crude product 9b as a yellow oil (340 mg). The crude product 9b was purified by chromatography (37 g silica gel; eluent first 6:1 n-hexane:ethyl acetate, then 2:1) to obtain product 9b (150 mg; 59.7%).

[0170] 1 H (DMSO-d6): δ 6.13 (d; J:=9Hz; 1H); 5.2-5.5 (m; 2H); 4.69 (d; J=5 Hz; 1H); 4.39 (t; J=6Hz; 1H); 4.15-4.30 (m; 1H); 3.45-3.55 (m; 2H); 3.3-3.5 (m; 1H); 2.0-2.1 (m; 2H); 1.36 (s; 9H); 1.2-1.3 (m 22H); 0.85 (t; J=6.5 Hz; 3H)

[0171] (b) 2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-yne-1,3(R)-diol 8a (1.57 g; 3.95 mmol) was dissolved in diethyl ether (220 mL). Quinoline (0.31 mL) and Pd on BaSO4 (10%) (645 mg) were added, and the reaction mixture was purged three times with nitrogen. The reaction mixture was stirred under a hydrogen atmosphere at room temperature for 17.5 h. The reaction mixture was then dried over Celite. ® filtered and concentrated in vacuo to obtain crude product 9b as a yellow oil (1.97 g). Crude product 9b was purified by column chromatography (40 g of silica gel; eluent: first 6.1 n-hexane:ethyl acetate, then 2:1) to obtain product 9b as a yellow oil (1.05 g; 66.5%), which was obtained as a crystalline solid.

[0172] 1 H (DMSO-d6): as above. Example 12: O,O'-Dimethyl-O''-[cis-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate 10b (with Boc as Pg)cis-2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-en-1,3(R)-diol 9b (700 mg; 1.75 mmol) was dissolved in pyridine (6 mL). Tetrabromomethane (871 mg, 2.63 mmol; 1.5 eq) was added and the reaction mixture was cooled to 0°C. Trimethyl phosphite (0.35 mL; 2.98 mmol; 1.75 eq) was added dropwise over 10 minutes. The reaction mixture was then allowed to warm to room temperature and stirred for a further 4.5 hours. The reaction mixture was diluted with diethyl ether (45 ml) and washed with 1M HCl solution (45 ml). The phases of the biphasic mixture were separated. The aqueous phase was extracted with diethyl ether (3 x 45 ml). The combined organic phases were washed with NaHCO3 solution (45 ml) and NaCl solution (45 ml), dried over Na2SO4, and concentrated under vacuum to afford crude product 10b as a red oil (1.1 g).Crude product 10b was purified by column chromatography (100 g silica gel; eluent 2:1 ethyl acetate / n-hexane) to obtain product 10b as a yellow oil (570 mg; 64.1%).

[0173] 1 H (DMSO-d6): δ 6.68 (d; J=9 Hz; 1H); 5.3-5.5 (m; 1H); 5.2-5.3 (m; 1H); 4.8-5.1 (s(br); 1H); 4.0-4.2 (m; 1H); 3.9-4.0 (m; 1H); 3.65 (d; J=11 Hz; 6H); 3.5-3.6 (m; 1H); 3.3-3.4 (m; 1H); 1.95-2.10 (m; 2H); 1.35 (s; 9H); 1.2-1.3 (m; 22H); 0.85 (t; 6.5 Hz; 3H). Example 13: O-cis-2(S)-amino-3(R)-hydroxy-octadec-4-en-1-yl phosphate (cis-Sphinqosin 1-phosphate) 11b O,O'-Dimethyl-O''-[cis-2(S)-N-(tert-butoxycarbonyl)amino-3(R)-hydroxy-octadec-4-en-1-yl] phosphate 10b (0.58 g; 1.18 mmol) was dissolved in dichloromethane (14.5 mL) and cooled to 0°C. Trimethylsilyl bromide (0.7 mL; 5.3 mmol; 4.5 eq) was added dropwise over 5 minutes. The reaction mixture was allowed to warm to room temperature and stirred for a further 2.5 hours. The reaction mixture was concentrated under vacuum and the residue was taken up in methanol (15 mL). The reaction mixture was stirred at room temperature for 2 hours and again concentrated under vacuum to obtain a brown foam (0.54 g), which was crystallized from a 2:1 mixture of THF and water and dried under vacuum.

[0174] Cis sphingosine 1-phosphate 11b was obtained as a white solid (0.25 g; 55.8%).

[0175] 1H-NMR (DOAc): δ 5,99 (dt, J=7-8 / 11 Hz; 1H); 5,76 (dd; J=9 / 11 Hz; 1H); 5,17 (dd; J= 5 / 9 Hz; 1H; 4,45-4,65 (m; 2H); 3,95 (dt; J= 4-5 / 5-6 Hz; 1H); 2,35-2,50 (m; 2H); 1,50-1,75 (m; 22H); 1,14 (t; J: 6-7 Hz; 3H). Beispiel 14: 1(S)-[N-(tert-Butoxycarbonyl)-2',2'-dimethyl-1',3'-oxazolidin-4'(S)-yl]-hexadec-2-in-1-ol 7c (mit Boc als Pg und Methyl als R 1and R2)1-pentadecine (2.5 mL; 9.7 mmol; 2.2 eq) was dissolved in diethyl ether (30 mL) and cooled to -30°C. BuLi (3.5 mL; 8.8 mmol; 2 eq) was added while maintaining the temperature below -15°C. The reaction mixture was then stirred for 1 h at -25°C. Anhydrous ZnBr2 (2.36 g) was dried by melting under vacuum and then dissolved in diethyl ether (20 mL) by heating to form a cloudy, yellow solution, which was added to the reaction mixture. The reaction mixture was then stirred for 1 h at 0°C. A white, viscous solution formed, which was diluted by addition of diethyl ether (20 mL) and then cooled to -70°C. N-(tert-Butoxycarbonyl)-2,2-dimethyl-1,3-oxazolidin-4(S)-ylmethanal 6a (1 g; 4.4 mmol) was dissolved in diethyl ether (20 mL), cooled to -70°C, and added dropwise to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for a further 19 hours.The reaction mixture was then cooled to -20°C and quenched with NH4Cl solution (50 mL). Precipitated salts were dissolved by addition of water (30 mL). The phases of the biphasic mixture were separated. The aqueous phase was extracted with diethyl ether (2 x 50 mL). The combined organic phases were washed with NaCl solution (50 mL), dried over Na2SO4, and concentrated under vacuum to obtain crude product 7c (2.84 g). The crude product 7c was purified by filtration through silica gel (56 g; eluent; first n-hexane, then 6:1 n-hexane:ethyl acetate) to obtain the filtered product 7c (1.61 g; 84.3%).

[0176] 1 H NMR (DMSO-d6): δ 4.75-4.85 (m; 1H); 4.25 (s(br); 1H); 4.17 (dd; J= 2 / 9 Hz; 1H); 3.81 (dd; J= 6 / 9 Hz; 1H); 2.16 (dt; J= 2 / 7 Hz; 2H); 1.34 (s); 1.50-1.20 (m; both together 1.34 and 1.50-1.20 37-38 H); 0.89 (t; J: 6.5 Hz; 3H). Example 15: 2(S)-N-(tert-Butoxycarbonyl)amino-octadec-4-yn-1,3(S)-diol 8c (with Boc as Pg)1(S)-[tert-Butoxycarbonyl)-2',2'-dimethyl-1',3'-oxazolidin-4'(S)-yl]-hexadec-2-yn-1-ol 7c (1.55 g; 3.5 mmol) was dissolved in methanol (37 ml) and Amberlyst ® 15 (1.95 g) was added. The reaction mixture was stirred at room temperature for 93 hours, dried over Celite ® filtered and concentrated under vacuum to isolate product 8c (0.97 g; 68.9%).

[0177] 1 H NMR (DMSO-d6, 400MHz): 0.86 (t; J: 6.5 Hz; 3H); 1.2-1.6 (m; 31H); 2.15 (t(br); J=7 Hz; 2H); 3.2-3.6 (m; 3H); 4.2-4.3 (m; 1H); 4.58 (t; J=5Hz; 1H); 5.17 (d; J=6.5Hz; 1H); 6.12 (d; J=7.5Hz; 1H) Example 16: trans-2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-ene-1,3(S)-diol 9c (with Boc as Pg)2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-yne-1,3(S)-diol 8c (970 mg; 2.44 mmol) was dissolved in diethyl ether (15 mL) and cooled to 0°C. Red-Al (3.7 mL; 65%; 12.2 mmol; 5 eq) was dissolved in diethyl ether and cooled to 0°C. The solution of alkyne 8c was added to the solution of Red-Al at 0°C. The reaction solution was allowed to warm to room temperature and stirred for a further 43 h. The reaction mixture was then cooled to 0°C and quenched by dropwise addition of methanol, which resulted in gas evolution. Diethyl ether (25 ml) and potassium sodium tartrate solution (25 ml) were added, and the reaction mixture was stirred for 21 hours. The phases of the biphasic system were separated. The aqueous phase was extracted with diethyl ether (2 x 25 ml).The combined organic phases were washed with potassium sodium tartrate solution (25 mL) and NaCl solution (25 mL), dried over Na2SO4, and concentrated under vacuum to isolate crude product 9c (870 mg). Crude product 9c was purified by column chromatography (73 g silica gel; eluent: first 2:1 n-hexane:ethyl acetate, then 3:2) to obtain chromatographed product 9c (480 mg; 49.2%).

[0178] 1 H (DMSO-d6, 400 MHz): δ 0.86 (d; J = 6.5 Hz; 3H); 1.2-1.4 (m; 31H); 1.8-2.0 (m; 2H); 3.2-3.5 (m; 5H); 4.0-4.2 (m; 1H); 5.40 (dd; J=4.5 / 16Hz; 1H); 5.57 (ddt; J=1 / 6.5 / 16Hz; 1H); 5.92 (d; J= 8Hz; 1H). Example 17: O,O'-Dimethyl-O''[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(S)-hydroxy-octadec-4-en-1-yl] phosphate 10c (with Boc as Pg)trans-2(S)-N-(tert-butoxycarbonyl)amino-octadec-4-en-1,3(S)-diol 9c (0.44 g; 1.15 mmol) was dissolved in pyridine (3 mL) and cooled to 0°C. Tetrabromomethane (572 mg; 1.73 mmol; 1.5 eq) was added. Trimethyl phosphite (0.23 mL; 1.95 mmol; 1.7 eq) was added dropwise at an internal temperature below 10°C within 5 minutes. The reaction mixture was then allowed to warm to room temperature and stirred for a further 4.5 hours. Ethyl acetate (40 ml) was added to the reaction mixture, and it was washed with 1 M HCl solution (2 × 30 ml). The phases of the biphasic mixture were separated, and the aqueous phase was extracted with ethyl acetate (2 × 50 ml).The combined organic phases were washed with NaHCO3 solution and NaCl solution, dried over Na2SO4, and concentrated under vacuum to obtain product 10c as a brown-orange oil (0.61 g). Crude product 10c was filtered through silica gel (60 g; eluent: 2:1 ethyl acetate:n-hexane) to isolate the filtered product 10c (0.42 g; 74.5%).

[0179] 1 H NMR (CDCl3): δ 5.77 (ddt; J= 1 / 6-7 / 15 Hz; 1H); 5.46 (dd; J= 7 / 15 Hz; 1H); 4.29-4.37 (m; 1H); 4.09 (t; J=7-8Hz; 1H); 3.78 (d; J=11 Hz; 3H); 3.77 (d; J=11Hz; 3H); 3.55-3.70 (m; 1H); 3.03 (s(br); 1H); 1.95-2.10 (m; 2H); 1.42 (s; 9H); 1.20-1.40 (m; 22H); 0.86 (t; J=7Hz; 3H) Example 18: O-trans-2(S)-amino-3(S)-hydroxy-octadec-4-en-1-yl phosphate (D-threo-sphingosin 1-phosphate) 11c O,O'-Dimethyl-O''-[trans-2(S)-N-(tert-butoxycarbonyl)amino-3(S)-hydroxy-octadec-4-en-1-yl] phosphate 10c (0.42 g, 0.85 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. Trimethylsilyl bromide (0.5 mL; 3.8 mmol; 4.5 eq) was added dropwise over 10 minutes. The reaction mixture was allowed to warm to room temperature and stirred for a further 2 hours. The reaction mixture was concentrated under vacuum and the residue taken up in methanol (10 mL). The reaction mixture was stirred at room temperature for 12 hours and then concentrated under vacuum to isolate crude product 11c, which was crystallized from a 2:1 mixture of THF and water (2 ml), dried under vacuum and obtained as a white solid (170 mg; 52.4%).

[0180] 1H NMR (DOAc): 6.37 (dt; J=7 / 15Hz; 1H); 5.94 (dd; J=7 / 15 Hz; 1H); 4.83 (t; J=7 Hz; 1H); 4.64-4.76 (m; 1H); 4.50-4.64 (m; 1H); 3.85-4.00 (m; 1H); 2.48-2.60 (m; 2H); 1.68-1.90 (m; 22H); 1.32 (t; J= 6-7 Hz; 3H).

[0181] The present invention has been described only by way of example. These examples are not intended to limit the scope of the invention. Numerous modifications and embodiments are possible without departing from the scope and spirit of the invention, which are defined only by the following claims. LIST OF REFERENCE SYMBOLS

[0182] no reference symbols

Claims

[1] A process for the preparation of sphingosine 1-phosphate (11a) or a stereoisomer thereof, comprising the steps: (a) Conversion of L-serine (1a) to methyl ester (2a) or a salt thereof: (b) protecting the amino function of methyl ester (2a) or the salt thereof to form methyl ester (3a),: where Pg stands for a protecting group, (c) protecting the primary hydroxyl group and the amino group of methyl ester (3a) to form methyl ester (4a), where R 1 and R 2 independently of one another can be hydrogen, an optionally substituted C1-C6 alkyl radical or an optionally substituted phenyl radical, or wherein R 1 and R 2 together can form an optionally substituted C4-C7 cycloalkyl radical (d) Reduction of methyl ester (4a) to form alcohol (5a): (e) Oxidation of alcohol (5a) to form aldehyde (6a): (f) Reaction of aldehyde (6a) with 1-pentadecyne and a base to form alkyne (7a): (g) partial deprotection of alkyne (7a) to form alkyne (8a): (h) Reducing the triple bond of alkyne (8a) to a trans double bond to form alkene (9a): (i) Phosphorylating the primary hydroxyl group of alkene (9a) to form phosphate (10a): and (j) Deprotection of phosphate (10a) to form sphingosine 1-phosphate (11a): wherein the method has at least one of the following features: Oxidation of alcohol (5a) to form aldehyde (6a) using oxalyl chloride, DMSO and less than 5.5 equivalents of Hünig base, Oxidation of alcohol (5a) to form aldehyde (6a) at a temperature above -70°C, Reacting aldehyde (6a) with 1-pentadecyne and a base to form alkyne (7a) using at least 1.5 equivalents of 1-pentadecyne and / or Purification of the alkyne (7a), which is obtained after reacting aldehyde (6a) with 1-pentadecyne and a base to form alkyne (7a), by distilling off unreacted 1-pentadecyne. [2] Method according to claim 1 characterized by Replacing the reduction of the triple bond of alkyne (8a) to a trans double bond to form alkene (9a) in step (h) by reducing the triple bond of alkyne (8a) to a cis double bond to form alkene (9b): [3] Method according to one of claims 1 or 2, characterized by Pg is -CO-OR, -CO-R or -CO-OtBu (Boc). [4] Method according to one of claims 1 to 3, characterized by the intermediate (10a) with the structure Pg gleich -CO-OCH2Ph ​​(Cbz oder Z), -CO-O-CH2-C6H4-pOMe (Moz), -CO-O-CH2-C6H4-pNO2, -CO-O-CH2-C6H4-pBr, -CO-O-CH2-C6H4pCl, -CO-O-CH2-C6H3-2,4-CL2, -CO-OCH2-C 13 H9 (Fmoc), -CO-O-CHPh2, -CO-OMe, -CO-OEt, -CO-OCH2CCl3 (Troc), -CO-OCH2CH2SiMe3 (Teoc), -CO-O-CH2CH2SMe or -CO-OCH2CH2SO2Me.