WO2006066806A1

WO2006066806A1 - Manufacture of vitamin b6

Info

Publication number: WO2006066806A1
Application number: PCT/EP2005/013547
Authority: WO
Inventors: Andrew George Gum
Original assignee: Dsm Ip Assets B.V.
Priority date: 2004-12-20
Filing date: 2005-12-16
Publication date: 2006-06-29
Also published as: CN101084192A; CN101084192B

Abstract

The present invention relates to a process for the manufacture of pyridoxamine, pyridoxine and further clodely related 3-hydroxy-pyridine derivatives and of acid salts thereof. Pyridoxamine and pyridoxine belong to the vitamin B6 group. This new multistep process is represented schematically in the following Reaction Scheme (formula (I)).

Description

Case 22258

Manufacture of Vitamin B^

The present invention concerns a process for the manufacture of pyridoxamine, pyridoxine and further closely related 3-hydroxy-pyridine derivatives and of acid salts thereof. Pyridoxamine and pyridoxine belong to the vitamin B₆ group. As well as playing an essential role in numerous biochemical processes, pyridoxamine is a useful precursor for pyridoxine (often itself, particularly as its hydrochloride salt, designated as "vitamin B₆").

Nearly all industrial syntheses of Vitamin B₆ start from inexpensive, readily available alanine, which is transformed in several chemical steps to 5-ethoxy-4-methyl-oxazle (EMO), the most expensive intermediate in this chemical production process. EMO is used as a diene in a Diels- Alder reaction with a protected 2-butene-l,4-diol. Subsequent acid catalyzed rearrangement of the Diels- Alder adducts and hydrolysis in aqueous hydrochloric acid affords pyridoxine hydrochloride, the commercial form of Vitamin B₆. This is represented schematically in the following Reaction Scheme 1, in which Et signifies ethyl and R a component of the protecting group for 2-butene-l,4-diol:

Pyridoxins hydrochloride (vitamin B₆) Reaction Scheme 1: Diels-Alder reaction and acidic rearrangement - the key production steps in the known industrial syntheses of vitamin B₆

Publications on this route from EMO and a protected 2-butene-l,4-diol to a vitamin

B₆ endproduct include the U.S. Patent Specifications 3,250,778, 3,296,275 and 3,822,274 and the Indian Patent Specification 175,617. The Diels-Alder reaction has been shown in practice to disadvantageously yield mixed products.

Because the synthesis of the starting oxazole EMO requires several chemical steps and produces large amounts of waste stream products, a synthetic route that avoids such disadvantages as well as the above indicated inefficient Diels-Alder reaction has been the object of much chemical research. It has now been found that pyridoxamine (hydrochloride) or other closely related 3-hydroxy-pyridine derivatives can surprisingly be produced by a new multistep process, also starting from the inexpensive readily available amino acid alanine or from further amino acids, as appropriate, but which avoids the involvement both of EMO or a related 4-substituted 5-alkoxy-oxazole and of the Diels- Alder reaction. Moreover, the new multistep process embraces less chemical steps from alanine or an alternative amino acid to pyridoxamine, pyridoxine or a salt thereof or related 3-hydroxy-pyridine derivative than the previous routes discussed above.

This new multistep process is represented schematically in the following Reaction Scheme 2 for the simplest case of starting from alanine (Y) itself and involving as the acyl group of the Michael acceptor 5_, i.e. the acylated γ-hydroxycrotononitrile NC-CH=CHCH₂OAC, the simplest such group, acetyl (Ac):

Diazotization and hydrolysis

8 Cl^"

- A -

Reaction Scheme 2: Example of the inventive route for ultimatively producing a pyridoxine salt or a closely related derivative thereof

In the Reaction Scheme 2 the 4-methyl-5(4H)-oxazolone 4 is given in parenthesis to signify that this compound need not be isolated after its generation from N-formyl-alanine 3: instead, the post-reaction mixture with added Michael acceptor 5 can be reacted via the next two parenthesized compounds 6 and 7, which on their part are hypothetically formed but do not need to be isolated (if indeed formed and isolable), to the first of the three 3- hydroxypyridine derivatives formed in the route, compound 8.

The process of the present invention resides essentially in the reaction of a A- substituted 5(4H)-oxazolone (exemplified in the Reaction Scheme 2 by 4-methyl-5(4H)- oxazolone, 4) with an acylated γ-hydroxy-crotononitrile (exemplified in the Reaction Scheme 2 by γ-acetyloxy-crotononitrile, 5) to the appropriate 2-substituted 3-hydroxy-4- cyano-5-acyloxymethyl-pyridine salt (exemplified in the Reaction Scheme 2 with the pyridine derivative 8 bearing as the 2-substituent methyl and as the 5-substituent acetyloxymethyl). It may optionally include the previous step of converting a N-formyl- substituted α-amino acid (exemplified by N-formyl-alanine, 3, in the Reaction Scheme 2) into the 4-substituted 5(4H)-oxazolone, especially if the oxazolone is not isolated from the post-reaction mixture before reaction with the acylated γ-hydroxy-crotononitrile. Moreover, the process of the present invention may optionally (also) include the subsequent reduction and hydrolysis step required to convert the aforementioned 2-substituted 3-hydroxy-4- cyano-5-acyloxymethyl-pyridine salt into the corresponding 2-substituted 3-hydroxy-4- aminomethyl-5-hydroxymethyl-pyridine salt (salt of pyridoxamine or a closely related derivative thereof; exemplified in the Reaction Scheme 2 by the 2-methyl compound, i.e. pyridoxamine itself, as its dihydrochloride salt, 9), and furthermore optionally the final step of converting the last-mentioned compound to the corresponding 2-substituted 3-hydroxy- 4,5-di(hydroxymethyl)-pyridine salt (salt of pyridoxine or a closely related derivative thereof; exemplified in the Reaction Scheme 2 by the 2-methyl compound, i.e. pyridoxine itself, as its hydrochloride salt, JO), by diazotization and hydrolysis.

Accordingly, the present invention provides as the main aspect a process for manufacturing a 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl-pyridine of the general formula

wherein R¹ signifies methyl, isopropyl, isobutyl, 2-butyl or benzyl, or in each case, as appropriate, hydroxyl- or mercapto-protected hydroxymethyl, 1- hydroxyethyl, mercaptomethyl or p-hydroxybenzyl, or methylthioethyl, and R² signifies an acyl group,

characterized by reacting a 4-substituted 5(4H)-oxazolone of the general formula

wherein R¹ has the above-mentioned significance,

with an acylated γ-hydroxy-crotononitrile of the general formula

wherein R² signifies an acyl group.

As a further aspect of the process of the present invention there is provided a process for manufacturing a 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl-pyridine of the general formula I as above whereby the 4-substituted 5(4H)-oxazolone of the general formula II is manufactured by dehydrating and cyclizing an N-formyl-α-amino acid of the general formula

wherein R¹ has the above-mentioned significance,

with a dehydration-activating agent, optionally in the presence of a base. The process of the present invention still further embraces the further steps leading firstly to the manufacture of a 2-substituted 3-hydroxy-4-aminomethyl-5-hydroxymethyl- pyridine salt of the general formula

wherein R¹ has the above-mentioned significance, and HX is the salt-forming acid,

whereby the 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl-pyridine of the general formula I, manufactured according to the above-defined manufacturing process therefor, is catalytically hydrogenated and hydrolysed with an aqueous mineral acid, and, leading finally to the manufacture of a 2-substituted 3-hydroxy-4,5-di(hydroxymethyl)-pyridine salt of the general formula

wherein R has the above-mentioned significance and HX is the salt-forming acid,

whereby the 2-substituted 3-hydroxy-4-aminornethyl-5-hydroxymethyl-pyridine salt of the general formula V, manufactured according to the above defined manufacturing process therefor, is diazotized and hydrolysed. In the above definition of the substituent R¹ present in the N-formyl-α-amino acid of the formula FV and in each of the compounds of the formulae π, V and VI this is the alpha substituent of those oc-amino acids which come into question, namely alanine (R¹ = methyl), valine (R¹ = isopropyl), leucine (R¹ = isobutyl), isoleucine (R¹ = 2-butyl), phenylalanine (R¹ = benzyl), serine (R¹ = hydroxymethyl), threonine (R¹ = 1-hydroxyethyl), cysteine (R¹ = mercaptomethyl), tyrosine (R¹ = p-hydroxybenzyl) or methionine (R = methylthioethyl), whereby in the case of serine, threonine, cysteine and tyrosine the hydroxyl or mercapto group of the respective alpha substituent is protected. As suitable protecting groups there come into question lower alkyl groups, suitably Ci_-6 alkyl groups, preferably methyl or ethyl; or the benzyl group. Those alkyl groups featuring 3 or more carbon atoms can be straight or branched chain. Thus the pertinent protected groups R¹ derived from serine, threonine, cysteine and tyrosine are, respectively, alkoxymethly or benzyl ox ymethyl; 1-alkoxyethyl or 1-benzyloxyethyl; alkylthiomethyl or benzylthiomethyl; and p-alkoxybenzyl or p-benzyloxybenzyl. Preferably R¹ is methyl.

The acyl group signified by R² in the acylated γ-hydroxy-crotononitrile of the formula

HI and present also in the 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl-pyridine of the formula I formed in the "core" process of the present invention is suitably straight or branched chain C_2-9 alkanoyl (the carbonyl C atom being counted as one of the 2-9 carbon atoms so that the alkyl moiety is C_1-8); aryl-C₂.g-alkanoyl, wherein the Ci_-8 alkylene moiety linking carbonyl to aryl is from C₂ straight or branched chain; or aroyl. The aryl group of aryl-C_2-9-alkanoyl and of aroyl may in particular be unsubstituted or substituted phenyl, any substituents being suitably selected from one or more lower alkyl, especially Ci_-6 alkyl, groups, halogen atoms, especially fluorine, chlorine and bromine, nitro groups and other conventional substituents for phenyl and other aromatic groups. Preferably the acyl group is acetyl, benzoyl or phenylacetyl, most preferably acetyl.

The anion X^" in the general formulae V and VI and derived from the salt-forming acid or mineral acid HX employed in the catalytic hydrogenation and hydrolysis step I -> V is suitably halide (fluoride, chloride, bromide or iodide), hydrosulphate (HSO₄ ^'), hydrophosphate (H₂PO₃ ^') or hydrophosphite (H₂PO₃ ^"), preferably chloride.

The process of the present invention involving the Michael addition of the compounds of formulae II and IH (the "core" process step) is effected conveniently in the presence of an essentially non-aqueous, nonpolar or polar aprotic organic solvent and a base, and conducted at relatively low temperatures. As the organic solvent of this type comes into question preferably a lower halogenated aliphatic hydrocarbon, e.g. methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane or 1,1,2,2-tetrachloroethane; a lower aliphatic or cyclic ether, e.g. diethyl ether, diisopropyl ether, tert. butyl methyl ether or diethyleneglycol dimethyl ether (diglyme), or appropriately, tetrahydrofuran or dioxan; a lower aliphatic ester, e.g. ethyl acetate, propyl acetate or butyl acetate; a lower aliphatic hydrocarbon, e.g. butane, pentane, hexane, heptane, octane or ligroin; an alicyclic hydrocarbon, e.g. cyclobutane, cyclopentane, cyclohexane or cyclooctane; an aromatic hydrocarbon, e.g. benzene, toluene or an xylene; or a mixture of one or more of such types of and specific solvents. As the base there may be used in general a tertiary amine-type base, such as a trialkylamine, e.g. triethylamine; pyridine or a 4-dialkylaminopyridine, e.g. 4-dimethylaminopyridine; or a diazabicycloalkene, e.g. l,5-diazabicyclo[4.3.0]non-5-ene or l,8-diazabicyclo[5.4.0]undec-7-ene.

The relatively low temperatures at which the process (Michael addition) is conveniently effected are from about -100⁰C to about +60°C, preferably from about -80°C to about +20⁰C, and most preferably from about -40⁰C to about 0⁰C.

Furthermore, the molar ratio of the acylated γ-hydroxycrotononitrile of the formula in to the 4-substituted 5(4H)-oxazolone of the formula II in the pertinent reaction mixture is conveniently from about 5 : 1 to about 1 : 1, preferably from about 2 : 1 to about 1 : 1.

The relative amount of base present in the reaction mixture is suitably from approximately stoichiometic to about double excess with respect to the 4-substituted 5(4H)-oxazolone; said oxazolone becomes activated (deprotonated) to a nucleophile for enabling its participation in the Michael addition with the acylated γ-hydroxycrotononitrile. This range is thus suitably from about 1 to about 2 equivalents of the base per equivalent of the oxazolone, but preferably from about 1 to about 1.1 equivalents of the base per equivalent of the oxazolone.

The reaction to form the desired 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl- pyridine of the formula I is generally complete within about 30 to 60 minutes.

To isolate the product such conventional methods as column chromatography, preferably using silica or an ion exchange resin as the stationary phase, or recrystallization, preferably from ethanol, may be used. As indicated above, the present invention embraces as a further aspect the process for manufacturing the 4-substituted 5(4H)-oxazolone of the general formula II prior to the involvement of this starting material in the Michael addition reaction with the acylated γ- hydroxy-crotononitrile of the formula El (the "core" process step). It involves the dehydration and cyclization of a N-formyl-α-amino acid of the general formula IV, as given and defined above, with a dehydration-activating agent, optionally in the presence of a base.

The dehydration and cyclization reaction can be carried out according to procedures known per se, e.g. as published in Roczniki Chemii 3_5, 979-984 (1961), Macromolecules 19, 1547-1551 (1986) and, solely by way of an example in its Experimental Section, Tetrahedron 52(13), 4719-4734 (1996). In such known procedures N,N'- dicyclohexylcarbodiimide (DCC) is used as the dehydration-activating agent, but the generated N,N'-dicyclohexylurea resulting from the dehydration and cyclization reaction has proved to pose certain difficulties due to its difficult separation from the post-reaction mixture. Alternative dehydration-activating agents are phosgene, thionyl chloride; formic acid derivatives, e.g. ethyl chloroformate; acid anhydrides, e.g. acetic anhydride; and further carbodiimides.

Depending on which kind of dehydration activating agent is used, a base many also be required in the reaction mixture for producing the 4-substituted 5(4H)-oxazolone. This is the case when phosgene or thionyl chloride is used, and in such cases the base is suitably an aliphatic tertiary amine, particularly a trialkylamine, e.g. triethyl amine; pyridine; dimethylaminopyridine; or piperidine.

If a base is employed, this is suitably used in up to about a 5 : 1 molar ratio with respect to the amount of N-formyl α-amino acid of the formula 1. This ratio is preferably about 2 : 1.

Suitable solvents for the cyclization and dehydration of the N-formyl-α-amino acid to the 4-substituted 5 (4H)-oxazolone are polar or nonpolar aprotic organic solvents, preferably the polar ones, and include lower halogenated aliphatic hydrocarbons, e.g. methylene chloride, chloroform and carbon tetrachloride; aromatic hydrocarbons, e.g. toluene; aliphatic ethers, e.g. diethyl ether and diglyme; and cyclic ethers, e.g. tetrahydrofuran, or mixtures of two or more such solvents, e.g. a mixture of a lower halogenated aliphatic hydrocarbon with an aliphatic ether. The cyclization and dehydration reaction is generally effected at relatively low temperatures, suitably from about -78⁰C to about +25°C, preferably from about -20°C to about O⁰C.

For the avoidance of atmospheric moisture in the reaction system, which would tend to react with the dehydration-activating agent or promote other undesired side-reactions, the reaction is suitably effected under an atmosphere of an inert gas, such as nitrogen or argon.

One particular procedure of producing the 4-substituted 5(4H)-oxazole of the formula π involving the use of phosgene as the dehydration-activating agent is effected by suspending the N-formyl-α-amino acid of the formula IV in methylene chloride at a temperature from about 0⁰C to about 25°C, treating the suspension with about 2 to about 3.2 equivalents of the preferred base triethylamine and, after cooling the mixture to about - 15⁰C, adding a solution of phosgene in toluene. Then the precipitated hydrochloride salts are removed by filtration and the filtrate is concentrated under reduced pressure to afford the desired product in crude form. The so-obtained 4-substituted 5(4H)-oxazole of the formula II can be used directly, i.e. without intermediate purification, in the reaction with the acylated γ-hydroxy-crotononitrile of the formula HI. In a similar manner, thionyl chloride can be used as the dehydration-activating agent, and in this case, too, the crude product used directly in the next reaction.

The starting N-formyl-α-amino acids of the formula IV can be produced by N- formylating and, where appropriate (in the cases of serine, threonine, tyrosine and cysteine) protecting, the appropriate α-amino acids by methods known per se and well documented in the literature: see for example J. M. Aizpurua and C. Palomo, Synth. Comm. I3_(9), 745- 752 (1983) in respect of N-formulation, and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3^rd Edn. 1999, John Wiley & Sons, Inc. in respect of protecting the aforementioned specific α-amino acids with suitable hydroxyl- and mercapto-protecting groups.

The manufacture of the 2-substituted 3-hydroxy-4-aminomethyl-5-hydroxymethyl- pyridine salt of the formula V, i.e. the next process step after the manufacture of the 2- substituted 3-hydroxy-4-cyano-5-acyloxymethyl-pyridine of the formula I, is accomplished by catalytic hydrogenation with simultaneous hydrolysis using an aqueous mineral acid, and can be based on the pertinent literature teachings, e.g. as described by S. Shimada and M. Oki in Chem. Pharm. Bull. 32(1), 39-43 (1984) drawing upon the earlier reference, T. Matsukaura and K. Shirakawa, Takugaku Zasshi 7JL, 1498 (1951). U.K. Patent Specification (UKP) 1,062,843 provides appropriate details on the catalytic hydrogenation conditions for converting the closely related 2-methyl-3-hydroxy-4-cyano-5- hydroxymethyl-pyridine to pyridoxamine or a salt thereof. It has been found that the hydrogenation is conveniently effected using a supported palladium catalyst, e.g. 5% palladium on active charcoal, in an acidified aqueous alcoholic, e.g. methanolic, medium, e.g. an approximately 0.5% vol./vol. to approximately 20% vol./vol. of 10-30% hydrochloric acid in alcohol, preferably approximately 1% vol./vol. to approximately 5% vol./vol. of about 30% hydrochloric acid in methanol. The pressure is not critical, and the catalytic hydrogenation can be efficiently performed under atmospheric pressure. Neither is elevated temperature necessarily required; effecting the process step at room temperature produces good results.

The product of formula V can be isolated and purified by methods known in the literature, e.g. involving recrystallization from an alcohol such as methanol or ethanol.

After completion of the above-described process step of catalytic hydrogenation with simultaneous hydrolysis to the 2-substituted 3-hydroxy-4-aminomethyl-5-hydroxymethyl- pyridine salt of the formula V, this may be subjected to diazotization and hydrolysis in the further optional process step, leading to the appropriate 2-substituted 3-hydroxy-4,5- di(hydroxylmethyl)-pyridine salt of the formula VI (pyridoxine hydrochloride being a prominent example of this endproduct). This process step, too, can be carried out in accordances with procedures known from the prior art, e.g. as described in the aforementioned S. Shimada et al. reference by way of two examples concerning the convertion of 4-aminomethyl-pyridine derivatives similar but not identical to the pyridine salt of formula V, or in the also aforementioned UKP 1,062,843, in particular in its Example 3.

The acylated γ-hydroxy-crotononitriles of the formula m used in the "core" process step hereinabove is a known compound in the case where the acyl group R² is acetyl. Thus, this acetylated compound, γ-acetyl-crotononitrile, and its production from known starting materials are disclosed in A. Nudelman and E. Keinan, Synthesis (Communications) August 1982, 687-689, and in the aforementioned S. Shimada et al. reference, each exemplifying a different production process.

The further γ-acylated γ-hydroxy-crotononitriles of the formula HQ, i.e. featuring R² as an acyl group other than acetyl, can be produced analogously from appropriate known starting materials.

The process of the present invention is illustrated by the following Examples. Example 1

Production of N-formylalanine

To a suspension of 179.3 g (2.01 mol) of D,L-alanine in 900 ml of dimethylformamide were carefully added 155 ml of formic acid, and the mixture was heated at reflux temperature until all the alanine had dissolved (5 hours). The solvent was then evaporated off and the residue dried under high vacuum to afford 271. Ig of crude N- formyl-alanine as an off-white solid consisting mainly of the desired N-formylalanine [85 area % by gas chromatography (GC)]. Impurities included alanine, alanine-alanine anhydride and dimethylformanide.

Example 2

Production of 4-methyl-5(4H)-oxazolone from N-formylalanine

To a flask containing a suspension of 90 g (76.9 mmol) of N-formylalanine in 150 ml of methylene chloride pre-cooled to 0⁰C and under an argon atmosphere were added in portions 15.9 g (76.9 mmol) of N^'-dicyclohexylcarbodiimide. After stirring for one hour at O⁰C the generated N,N'-dicyclohexylurea was removed by filtration through a sintered funnel, and the methylene chloride was subsequently removed by rotary evaporation at 20°C and 200 mbar (20 kPa). The crude product was carefully distilled bulb-to-bulb under a high vacuum (0.3 mbar/30 Pa) at room temperature. The purified 4-methyl-5(4H)- oxazolone was collected in a receiving flask at -78⁰C. Thereafter the receiving flask was purged with argon, and 10 ml of dry methylene chloride were added. The concentration of the oxazolone was determined to be 0.87 M (isolated yield 11%). A portion of this solution was used in the following step, exemplified in Example 3.

Example 3

Production of 2-methyl-3-hvdroxy-4-cyano-5-acetyloxymethyl-pyridine

In a flask containing 408 mg (3.26 mmol; 1.01 equivalents) of γ-acetyloxy- crotononitrile (produced as exemplified in Example 4 below) in 15 ml of methylene chloride under argon there were introduced 454 μl (3.26 mmol, 1.01 equivalents) of triethylamine followed dropwise by a 3.7 ml portion of the methylene chloride solution containing 4-methyl-5(4H)-oxazolone, as obtained in the procedure described in Example 2 above, at room temperature. An exothermic reaction resulted. After stirring the reaction mixture for one hour, it was concentrated under reduced pressure. Using column chromatography with silica gel as the stationary phase and a 1 : 9 v/v mixture of methanol and methylene chloride as the eluent 353 mg of the desired 2-methyl-3-hydroxy-4-cyano-5- acetyloxymethyl-pyridine were isolated, representing a 53% yield based on the starting oxazolone. The product could be readily crystallized from ethanol to give a yellow-orange solid of melting point 205-207⁰C (with decomposition).

Example 4

Production of γ-acetyloxy-crotononitrile

To a reaction flask containing 375 ml of absolute tetrahydrofuran at room temperature which had been degassed for 30 minutes with argon were added with stirring 3.94 g (15.0 mmol) of triphenylphosphine followed by 842 mg (3.75 mmol) of palladium (II) acetate. The resulting clear orange solution was stirred for an additional 30 minutes after the completion of addition. Then 18.8 g (18.6 ml, 150 mmol) of 2-acetyloxy-3- butenenitrile were added in one portion to the mixture in the flask, and the reaction mixture was stirred at room temperature until the starting material could be detected no longer using thin layer chromatography and GC; this occurred after about 4 hours from the addition of the nitrile reactant. The resulting dark brown solution was concentrated under reduced pressure and the remaining concentrate distilled, affording 13.69 g (109.4 mmol; 73% yield) of the desired γ-acetyloxy-crotononitrile as a pale yellow liquid. The product, consisting of a mixture of E- and Z- isomers, was used as the Michael acceptor in the procedure described in Example 3 above. Analytical date of the produced E/Z-isomeric mixture of γ-acetyloxy-crotononitrile

(C₆H₇NO₂; molecular weight 125.13):

Boiling point: 100-102°C/12 mbar (1.2 kPa);

R_f (silica gel, hexane: ethyl acetate 2 : 1 v/v): 0.3 (KMnO₄ stain); ¹H-NMR (300 MHz, CDCI₃): δ = 2.10 (s, 3H), 2.11 (s, 3H), 4.71 (m, 2H), 4.86 (m, 2H),

5.52 (d, J = 11.4 Hz, IH), 5.59 (d, J = 16.4 Hz, IH), 6.51 (dt, J = 5.7, 5.7 Hz, IH), 6.72 (d,

J = 16.4 Hz, IH);

¹³C-NMR (75 MHz, CDCI₃): δ = 20.48, 20.51, 61.9, 62.3, 101.1, 101.6, 114.7, 116.4,

147.3, 147.7, 169.9, 107.3; Mass spectroscopy: 125 [M]⁺, 110, 105, 83, 66, 55, 43, 39 m/z

Example 5

Production of 2-methyl-3-hydroxy-4-cvano-5-acetyloxymethyl-pyridine from N- formylalanine via 4-methyl-5-(4HVoxazoIone without isolating the oxazolone

The following exemplified procedure avoids the difficulties associated with the distillative isolation of the intermediate 4-methyl-5(4H)-oxazolone and embraces the two reaction steps.

To a suspension of 3.2 g (27.3 mmol) of N-formyl alanine in 100 ml of chloroform at room temperature (about 25°C) under an atmosphere of argon were added 8.8 g (12.2 ml, 87.45 mmol, 3.2 equivalents) of triethylamine, and the resulting opaque solution was cooled to -15⁰C. 15 ml of a 20% v/v solution of phosgene in toluene (1.05 equivalents of COCl₂) were added dropwise to the N-formylalanine suspension at such a rate that the internal temperature did not exceed -10°C, and the conversion of the N-formylalanine was monitored by GC. Complete conversion had occurred after about 45 minutes from the start of the dropwise addition.

To the resulting orange-brown solution at 0°C were added dropwise from a dropping funnel 3.42 g (27.33 mmol, 1 equivalent) of γ-acetyloxy-crotononitrile, and the resulting mixture was allowed to warm to room temperature over a period of 16 hours. After addition of 50 ml of toluene to said mixture, the resulting precipitate was filtered off and rinsed with two 25 ml portions of toluene. Then the solvent was slowly evaporated off at 60°C/100 mbar (10 kPa) and the residue distilled at room temperature under a reduced pressure of 0.01 mbar (1 Pa) using a dry ice trap at -78°C to recover 783 mg (6.26 mmol, 0.23 equivalents) of unconverted γ-acetyloxy-crotononitrile for recycling if desired. The remaining solid residue was dissolved in ethanol at 50°C and recrystallized at 4⁰C to yield 780 mg of 2-methyl-3-hydroxy-4-cyano-5-acetyloxymethy]-pyridine as the major product: 3.78 mmol, representing a 14% yield based on the starting N-formylalanine. However, further such product remained in the mother liquor and was isolable.

Instead of the aforementioned orange-brown solution resulting from the N- formylalanine dehydration and cyclization being processed as above and the 2-methyl-3- hydroxy^-cyano-S-acetyloxymethyl-pyridine being ultimately isolated by crystallization, the product could be isolated by column chromatography.

In this alternative procedure, 3.42 g (27.3 mmol, 1 equivalent) of γ-acetyloxy- crotononitrile were added dropwise from a dropping funnel to the orange-brown solution at 0⁰C, and the resulting mixture was allowed to slowly warm to 6°C over 2 hours. Then the solvent was slowly evaporated off at 60°C/100 mbar (10 kPa). The crude product (residue) was purified by column chromatography through silica gel to afford 1.43 g (6.9 mmol) of 2-methyl-3-hydroxy-4-cyano-5-acetyloxymethyl-pyridine; this amount of product represented a 25% yield based on the starting N-formylalanine. The purity of the product was 63% (area % by GC); unconverted γ-acetyloxy-crotononitrile was also recovered to the extent of 2.29 g (18.35 mmol, for recycling if desired).

Analytical data of the produced 2-methyl-3-hydroxy-4-cyano-5-acetyloxymethyl-pyridine

(C₁₀Hi_ON₂O₃, molecular weight 206.2):

Melting point: 205-207°C (with decomposition);

Rf (silica gel, methylene chloride: methanol 9 : 1 v/v): 0.2 (ultraviolet fluorescence at 330 nm);

¹H-NMR (300 MHz, DMSO-D₆): δ = 2.07 (s, 3H), 2.45 (s, 3H), 5.12 (s, 2H), 8.09 (s, IH);

¹³C-NMR (75 MHz, DMSO-D₆): δ = 19.6, 20.3, 61.4, 106.5, 113.7, 130.9, 138.2, 149.4,

153.6, 169.9;

IR (KBr): v = 2933, 2579, 2217, 1751, 1533, 1467, 1376, 1234, 1046 cm^"1; Mass spectroscopy (elec. spray, negative mode): 205.0 [M-H]^";

Mass spectroscopy: retention time (RT)=12.96 min., 206 [M]⁺, 164, 147, 136, 119, 10, 43 m/z; derivitized with trimethylsilane: RT = 14.37 min., 278 [M]⁺, 236, 221, 205, 118, 73 m/z\ liquid chromatography/MS: RT=10.52 min. [M+H]⁺;

UV spectroscopy: 230 (max), 255, 360 nm;

Elemental analysis: calc. C 58.25, H 4.89, N 13.59, O 23.38%; found C 58.10, H 4.97, N

13.56 O 23.47%.

Example 6

Production of 2-methyl-3-hydroxy-4-aminomethyl-5-hydroxymethyl-pyridine (pyridoxamine) dihydrochloride

214 mg (1.04 mmol) of 2-methyl-3-hydroxy-4-cyano-5-acetyloxymethyl-pyridine were dissolved in 12 mol of methanol containing 300 μl of 30% hydrochloric acid at room temperature. 40 mg of 5 w/w% palladium on charcoal was added to the solution, and the mixture was hydrogenated under atmospheric pressure and at room temperature. The catalyst was then removed by filtration and the solvent of the filtrate removed under reduced pressure. The residue was recystallized from ethanol to afford 208 mg (0.86 mmol) of pyridoxamine dihydrochloride as an off-white solid; the amount obtained represented a 83% yield. The analytical data (¹H and ¹³C NMR, LC, LC/MS and UV) obtained for the product confirmed through comparison of such data with those of a sample of commercially available pyridoxamine dihydrochloride that the product was the desired one.

Example 7

Production of 2-methyl-3-hydroxy-4,5-di(hydroxymethyl)-pyridine (pyridoxine) hydrochloride

To a solution of 208 mg (0.863 mmol) of pyridoxamine dihydrochloride in 8.6 ml of water were added 125 μl (2.3 mmol) of 16 M concentrated sulphuric acid at room temperature. The mixture was headed to 90°C, and a solution of 119 mg (1.726 mmol, 2 equivalents) of sodium nitrite in 3.9 ml of water was added dropwise with stirring. The reaction mixture was stirred for a further 2 hours. Then a solution of 56.2 mg (2.3 mmol) of barium chloride dihydrate in 3.9 ml of water was added to the hot solution, and the post- reaction mixture was cooled to room temperature. The solid was removed by filtration and washed with two 5 ml portions of water. The combined aqueous solution was evaporated under reduced pressure and the residue triturated with boiling ethanol to yield 44 mg (86 w/w% by HPLC) of pyridoxine hydrochloride, representing a 25% yield.

Claims

1. A process for manufacturing a 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl- pyridine of the general formula

characterized by reacting a 4-substitited 5(4H)-oxazolone of the general formula

wherein R¹ has the above-mentioned significance,

with an acylated γ-hydroxy-crotononitrile of the general formula

wherein R² signifies an acyl group.

2. A process according to claim 1, wherein R¹ in the formulae I and II is methyl.

3. A process according to claim 1 or claim 2, wherein R² in the formula III is acetyl, benzoyl or phenylacetyl, preferably acetyl.

4. A process according to any one of claims 1 to 3, wherein the reaction is effected in the presence of an essentially non-aqueous nonpolar or polar organic solvent and a base.

5. A process according to claim 4, wherein the solvent is a lower halogenated aliphatic hydrocarbon, a lower aliphatic or cyclic ether, a lower aliphatic ester, a lower aliphatic hydrocarbon, an alicychic hydrocarbon, an aromatic hydrocarbon, or a mixture of one or more of such types of solvents.

6. A process according to claim 4 or 5, wherein the base is a tertiary amine-type base, pyridine or a 4-dialkylaminopyridine, or a diazabicycloalkene.

7. A process according to any one of claims 1 to 6, wherein the reaction is effected at temperature from about -100°C to about +60°C, preferably from about -80°C to about

+20°C, most preferably from about -4O⁰C to about 0°C.

8. A process according to any one of claims 1 to 7, wherein the molar ratio of the γ- hydroxycrotonoritrile of the formula III to the 4-substituted 5(4H)-oxazolone of the formula II in the reaction mixture is from about 5 : 1 to about 1 : 1, preferably from about 2 : 1 to about 1 : 1.

9. A process according to any one of claims 1 to 8, wherein the range of the equivalents of the base to the 4-substituted 5(4H)-oxazolone of the formula π is from about 1 to 2 : 1 to about 1 to 1.1 : 1.

10. A process for manufacturing a 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl- pyridine of the general formula I according to any one of claims 1 to 9, whereby the 4- substituted 5(4H)-oxazolone of the general formula II is manufactured by dehydrating and cyclizing an N-formyl-α-amino acid of the general formula

wherein R¹ has the significance given in claim 1,

with a dehydration-activating agent, optionally in the presence of a base.

11. A process for manufacturing a 2-substituted 3-hydroxy-4-aminomethyl-5- hydroxymethyl-pyridine salt of the general formula

HX

wherein R¹ has the significance given in claim 1, and HX is the salt-forming acid,

characterized by catalytically hydrogenating and hydrolysing with an aqueous mineral acid the 2-substituted 3-hydroxy-4-cyano-5-acyloxymethyl-pyridine of the general formula I which has been manufactured in accordance with the process of any one of claims 1 to 10.

12. A process for manufacturing a 2-substituted 3-hydroxy-4,5-di(hydroxymethyl)- pyridine salt of the general formula

wherein R¹ has the has the significance given in claim 1, and HX is the salt-forming acid,

characterized by diazotizing and hydrolysing the 2-substituted 3-hydroxy-4-aminomethyl- 5-hydroxymethyl-pyridine salt of the general formula V which has been manufactured in accordance with the process of claim 11.

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