Methods for the preparation of chiral piperazine-2-carboxylic acids

Abstract

The present invention relates to a compound of formula (I) TIFF2023523961000036.tif2643 relates to a novel process for the preparation of chiral piperazine-2-carboxylic acid or its salts. Chiral piperazine-2-carboxylic acid derivatives of formula (I) are key intermediates for the preparation of fused heteroaryldihydropyrimidines useful for the treatment and prevention of hepatitis B virus infection.

Description

[Technical Field]

[0001] The present invention relates to a compound of formula I [ka] This invention relates to a novel method for the preparation of chiral piperazine-2-carboxylic acids. [Background technology]

[0002] Chiral piperazine-2-carboxylic acid derivatives of formula I are key intermediates for the preparation of fused heteroaryldihydropyrimidines useful in the treatment and prevention of hepatitis B virus infection (PCT Publication No. WO 2015 / 132276).

[0003] A method for the preparation of chiral piperazine-2-carboxylic acids is described in Eichhorn et al., Tetrahedron Asymmetry, Vol. 8, No. 15, pp. 2533-2536, 1997. Racemic piperazine-2-carboxamides have been kinetically isolated in bacterial cells from Klebsiella terrigena and Burkholderia species. However, for technical-scale synthesis, it would be desirable to use isolated and characterized enzymes and perform the process at higher enzyme and substrate concentrations. Therefore, the objective of the present invention was to create a process that could be carried out on a technical scale. Summary of the Invention

[0004] The objective can be achieved in the manner outlined below, which comprises: a) Formula II [ka] by catalytic hydrogenation of pyrazine-2-carboxamide of formula III [ka] forming piperazine-2-carboxamide and b) enzymatic conversion of piperazine-2-carboxamide of formula III with a hydrolase to form chiral piperazine-2-carboxylic acid of formula I or a salt thereof. Includes: DETAILED DESCRIPTION OF THE INVENTION

[0005] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

[0006] The term "amino protecting group" refers to an acid- or Lewis acid-sensitive substituent conventionally used to prevent the reactivity of amino groups. Suitable acid- or Lewis acid-sensitive amino protecting groups are described in Green T., "Protective Groups in Organic Synthesis", 4 th Ed. by Wiley Interscience, 2007, Chapter 7, 696 ff. Thus, the amino-protecting group suitable for PG can be selected from Boc (tert-butoxycarbonyl), benzyl, 4-methoxybenzyl, benzhydryl, Fmoc (fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), Moz (p-methoxybenzylcarbonyl), Troc (2,2,2-trichloroethoxycarbonyl), Teoc (2-(trimethylsilyl)ethoxycarbonyl), Adoc (adamantoxycarbonyl), formyl, acetyl, or cyclobutoxycarbonyl. The preferred amino-protecting group is Boc.

[0007] helical bond [ka] teeth [ka] and thus indicates the chirality of the molecule.

[0008] Whenever a chiral carbon is present in a chemical structure, all stereoisomers associated with that chiral carbon are intended to be encompassed by the structure, both as pure stereoisomers and as mixtures thereof.

[0009] Process a) Step a) is a compound of formula II [ka] by catalytic hydrogenation of pyrazine-2-carboxamide of formula III [ka] This requires a step of forming piperazine-2-carboxamide of formula (I).

[0010] Pyrazine-2-carboxamide is a compound that is widely available commercially.

[0011] Catalytic hydrogenation is typically carried out using hydrogen in the presence of a metal hydrogenation catalyst and a solvent.

[0012] Suitable metals in the metal hydrogenation catalyst are palladium or platinum, preferably palladium.

[0013] The metal is applied to an inert support, typically selected from carbon or aluminum oxide, preferably carbon. Typical metal loadings (w / w) on the support are 0.5% to 20%, preferably 3% to 15%, more preferably 8% to 12%. The most preferred metal hydrogenation catalyst is 10% palladium on carbon (10% Pd / C).

[0014] The metal hydrogenation catalyst is usually used in an amount of 3% to 20 w / w%, typically 10 w / w%, based on the pyrazine-2-carboxamide starting material.

[0015] The solvent may be an organic solvent selected from aliphatic alcohols such as methanol or ethanol, or water or a mixture thereof. The preferred solvent is water.

[0016] The catalytic hydrogenation is carried out at a reaction temperature of 20°C to the boiling point of the solvent, preferably 30°C to 60°C, more preferably 35°C to 45°C, under a hydrogen pressure of 5 bar to 50 bar, preferably 15 bar to 25 bar.

[0017] In the most preferred embodiment, catalytic hydrogenation is carried out using 10% Pd / C 10 w / w% catalyst in water at a reaction temperature of 40° C. and a hydrogen pressure of 20 bar.

[0018] The resulting piperazine-2-carboxamide can be isolated by procedures known to those skilled in the art, such as separation of the catalyst from the reaction mixture followed by removal of the solvent from the filtrate.

[0019] However, in a preferred embodiment, the piperazine-2-carboxamide is not isolated but is further processed in step b) after separating the catalyst from the reaction mixture.

[0020] In a further preferred embodiment, the catalyst can be reused several times, typically at least five times, after separation from the reaction mixture without significant loss of performance. If applicable, loss of catalyst performance can be compensated for by adding fresh catalyst.

[0021] Step b) Step b) involves the enzymatic conversion of piperazine-2-carboxamide of formula III with a hydrolase to form chiral piperazine-2-carboxylic acid of formula I.

[0022] Suitable hydrolases for the enzymatic conversion are typically peptidases, amidases or mixtures thereof.

[0023] In a preferred embodiment, the compound of formula Ia [ka] A hydrolase is selected that has the ability to form (S)-piperazine-2-carboxylic acid in an enantiomeric excess of at least 90%, preferably at least 95%, more preferably at least 98%.

[0024] Representative preferred hydrolases capable of forming (S)-piperazine-2-carboxylic acid of Formula Ia have amino acid sequences that are at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO:1.

[0025] SEQ ID NO: 1 MRSLLWASLL SGVLAGRALV SPDEFPEDIQ LEDLLEGSQQ LEDFAYAYPE RNRVFGGKAH 60 DDTVNYLYEE LKKTGYYDVY KQPQVHLWSN ADQTLKVGDE EIEAKTMTYS PSVEVTADVA 120 VVKNLGCSEA DYPSDVEGKV ALIKRGECPF GDKSVLAAKA KAAASIVYNN VAGSMAGTLG 180 AAQSDKGPYS AIVGISLEDG QKLIKLAEAG SVSVDLWVDS KQENRTTYNV VAQTKGGDPN 240 NVVALGGHTD SVEAGPGIND DGSGIISNLV IAKALTQYSV KNAVRFLFWT AEEFGLLGSN 300 YYVSHLNATE LNKIRLYLNF DMIASPNYAL MIYDGDGSAQ IEKLFEDYYD 360 SIDLPHIPTQ FDGRSDYEAF ILNGIPSGGL FTGAEGIMSE ENASRWGGQA GVAYDANYHA 420 AGDNMTNLNH EAFLINSKAT AFAVATYAND LSSIPKRNTT SSLHRRARTM RPFGKRAPKT 480 HAHVSGSGCW HSQVEA 496

[0026] Several enzymes or enzyme mixtures are commercially available, such as Flavorzyme® 1000L from Novozymes, a peptidase preparation derived from Aspergillus oryzae, Acylase Amano from Amano Enzyme Inc, and Acylase from Penicillium spp. from Fluka.

[0027] In a preferred embodiment, the enzyme mixture Flavorzyme® 1000L from Novozymes or an enzyme preparation containing the hydrolase enzyme of SEQ ID NO: 1 as defined above can be used.

[0028] In another preferred embodiment, the hydrolase of SEQ ID NO: 1 can be obtained by expressing the enzyme in a suitable host, such as Pichia pastoris, and secreting it into the fermentation medium.

[0029] The hydrolase of SEQ ID NO: 1 is preferably leucine amido peptidase 2 (LAP2).

[0030] Alternatively, formula Ib [ka] A hydrolase can be selected that is capable of forming (R)-piperazine-2-carboxylic acid of the formula:

[0031] A commercially available enzyme that forms the (R)-enantiomer can be, for example, ADDZYME Bacillus subtilis protease from Advanced Enzyme Technologies.

[0032] The enzymatic conversion is carried out at a reaction temperature of 10°C to 50°C, preferably 20°C to 30°C, in the solvent used in the hydrogenation step, preferably in water.

[0033] The substrate load is generally kept below 30 w / v%, preferably between 1 w / v% and 25 w / v%.

[0034] Typically, the reaction does not require extra buffering agents because the amino acid buffers the reaction pH between 7.3 and 8.3.

[0035] Some enzymes, such as the hydrolase of SEQ ID NO: 1, may require the addition of a metal cofactor, such as zinc, which is added in the form of a suitable salt.

[0036] Once the enzymatic conversion is complete, the piperazine-2-carboxylic acid of formula I can be isolated according to procedures known to those skilled in the art.

[0037] However, in a preferred embodiment, the reaction mixture with chiral piperazine-2-carboxylic acid of formula I is converted to its hydrochloride salt by adding aqueous hydrochloric acid having an HCl concentration of 10% to 37% to the reaction mixture, such that the temperature of the reaction mixture is maintained in the range of 10°C to 30°C, preferably 15°C to 25°C.

[0038] It is more preferable to concentrate the reaction mixture under reduced pressure of 30 mbar to 120 mbar at a temperature of 30°C to 50°C before adding hydrochloric acid.

[0039] Under these conditions, the chiral piperazine-2-carboxylic acid of formula I, preferably piperazine-2-carboxylic acid of formula Ia, usually precipitates as the hydrochloride salt and can be obtained in crystalline form after filtering, washing the filter cake with aqueous hydrochloric acid and drying.

[0040] Process c) Step c) is optional and involves the introduction of an amino protecting group PG to give a compound of formula IV [ka] (wherein PG represents an amino protecting group) This requires the formation of chiral piperazine-2-carboxylic acid.

[0041] Suitable amino protecting groups are as defined above, the most preferred amino protecting group PG is Boc (tert-butoxycarbonyl).

[0042] For the introduction of the Boc group, a typical Boc-depositing agent such as di-tert-butyl dicarbonate (BocO) or 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (Boc ON) can be used, with BocO being preferred.

[0043] The reaction is usually carried out in the presence of a base selected from alkali carbonates such as potassium carbonate, sodium carbonate, or calcium carbonate, alkali bicarbonates such as sodium bicarbonate, alkali hydroxides such as sodium hydroxide, or tertiary amines such as triethylamine. Preferably, an alkali carbonate, more preferably potassium carbonate, is used. Suitable solvents are water, methanol, ethanol, acetone, acetonitrile, dioxane, or a mixture thereof. In a preferred embodiment, a mixture of water and acetone is used.

[0044] The reaction is carried out at a temperature of -15°C to 30°C, particularly 15°C to 30°C.

[0045] In a preferred embodiment, Formula IVa [ka] (2S)-4-tert-butoxycarbonylpiperazine-2-carboxylic acid is formed.

[0046] In a further embodiment of the present invention, the method of the present invention relates to a compound of formula X [ka] (In the formula, R 1 is a halogen or C 1-6 -alkyl, R 2 is hydrogen or halogen; R 3 is hydrogen or halogen; R 4 is C 1-6 - is alkyl; R 5 is hydrogen or carboxy; R 6 is hydrogen; R 7 is C 1-6 -Alkyl, C 3-7 -cycloalkyl, -C m H 2m -COOH, -C m H 2m -C 3-7 -cycloalkyl-COOH or carboxyphenyl; m is 1 to 6. or a pharmaceutically acceptable salt or enantiomer or diastereomer thereof. The compound of formula X is 1 ~R 7 and their processes are disclosed in PCT Publication WO 2015 / 132276, which is incorporated herein by reference. Formula X corresponds to formula IAA in WO 2015 / 132276 (page 22).

[0047] More preferred are compounds of formula XX, [ka] In the formula, R 1 ~R 4 and R 7 is as defined above.

[0048] An essential intermediate in the process for the preparation of compounds of formula XX is intermediate IX or an enantiomer or diastereomer thereof, [ka] In the formula, R 7 is as outlined above.

[0049] The preparation of compounds of formula X can be carried out by d) Formula IV [ka] where PG is an amino protecting group. or a salt thereof, by reacting an amine R 7 -NH2(wherein, R 7 is as defined above) in the presence of a coupling agent and a base to form a compound of formula V [ka] (Wherein PG and R 7 is as above) or a salt thereof to form a mixed urea of ​​formula IV; e) cyclizing the mixed urea of ​​formula V to give formula VI [ka] (Wherein PG and R 7 is as above) forming a hydantoin of f) reducing the hydantoin of formula VI to give the hydantoin of formula VII [ka] (Wherein PG and R 7 is as above) forming a cyclic urea of g) Deprotection and formation of a compound of formula IX Further includes:

[0050] In a preferred embodiment, intermediate IX has formula IXa or IXb [ka] (In the formula, R 7 is as defined herein) It may also be of the type:

[0051] In a preferred embodiment, intermediate IX may be of formula IXa:

[0052] Salts of chiral piperazine-2-carboxylic acid derivatives of formula IVb can be prepared by methods known to those skilled in the art.

[0053] The sodium salt can be prepared, for example, by reacting a chiral piperazine-2-carboxylic acid derivative of formula IV with aqueous methanolic sodium hydroxide solution, similar to example 4.3.6 in M. Laars et al, Tetrahedron: Asymmetry 21 (2010) 562-565.

[0054] Scheme 1 further illustrates the formation of intermediate IX.

[0055] Scheme 1 [ka]

[0056] Mixed urea (V) is amine R 7’ The cyclic urea (VI) can be prepared by coupling the cyclic urea (V) with chiral piperazine-2-carboxylic acid or its salt (IVb) in the presence of a coupling agent such as carbonyldiimidazole (CDI) and a suitable base such as triethylamine. Suitable chiral piperazine-2-carboxylic acid salts (IVb) are alkali metal salts such as sodium or potassium salts or ammonium salts such as triethylammonium salts. Suitable cyclization of the cyclic urea (V) with oxalyl chloride gives the hydantoin (VI). Subsequent reduction with a reducing agent selected from BH3·THF or NaBH4 in the presence of BF3·THF gives the cyclic urea (VII). 7’When is an ester group, saponification with aqueous sodium hydroxide or potassium hydroxide provides the corresponding acid (VIII). Boc deprotection of compound (VII) or (VIII) can be achieved using concentrated HCl in MIBK to form compound (IX). Compounds (IXa) or (IXb) with absolute configuration can be obtained by following the synthesis of Scheme 1 using the corresponding chiral starting material compound (IVb). [Example]

[0057] Abbreviation: a%=area% AOX I = alcohol oxidase I BMMY = Buffered Methanol Complex Medium (with yeast extract) df = film thickness GC = gas chromatography ID = Inner diameter L = length MeOH = methanol MIBK = methyl isobutyl ketone OD=optical density RCF = relative centrifugal force RRT = relative retention time XRF = X-ray fluorescence YPD = yeast extract peptone dextrose

[0058] Example 1 (S)-Piperazine-2-carboxylic acid a) Preparation of rac-piperazine-2-carboxamide Pyrazine-2-carboxamide (100 g, 812 mmol) was suspended in 300 mL of water in a pressure vessel and then inerted with argon. 10% Pd / C (anhydrous, 10.0 g) was added to the reaction mixture along with an additional 30 mL of water to rinse the reactor walls. The reactor was sealed, the atmosphere was replaced with hydrogen, and the reaction mixture was heated to 40 °C. The atmosphere was adjusted to 20 bar H2, and the mixture was stirred at 40 °C for 18 h while maintaining a constant hydrogen pressure of 20 bar in the vessel and recording gas consumption over time. The reactor was cooled to room temperature, the atmosphere was replaced with argon, and the progress of the reaction was confirmed by GC analysis (conversion >99%; 97% title compound). The mixture was filtered with an additional 170 mL of water to give an aqueous solution, and then the pH was adjusted to 7.8 by slowly adding concentrated (37%) aqueous HCl (55 mL, 655 mmol) while maintaining the temperature below 25° C. The resulting solution was used directly in the next step without product isolation.

[0059] GC method description: Stationary phase: Agilent HP-5 (L = 30 m, ID = 0.32 mm, df = 0.25 μm, maximum temperature 350 °C); Temperature program: Start at 100 °C, heating rate 10 °C / min to 350 °C, hold at 350 °C for 2 min, then cool at 40 °C / min to 100 °C, hold at 100 °C for 0.75 min; Run time: 34.0 min; Inlet mode: Constant pressure; Inlet initial pressure: 5.0 psi; Inlet initial flow rate: 0.727 m at 100 °C (starting oven temperature). L / min; initial velocity 16.05 cm / sec at 100 °C (starting oven temperature); split ratio, split flow 1:30, 41.38 mL / min; injection volume 1.0 μL; inlet temperature 280 °C; detector temperature 320 °C; detector H2 flow (fuel flow) 40 mL / min; detector air flow (oxidizer flow) 400 mL / min; detector N2 flow (constant configuration) 30 mL / min; retention time: pyrazine-2-carboxamide = 4.56 min (RRT 約 = 0.71), rac-piperazine-2-carboxamide = 6.46 min (RRT = 1.0).

[0060] b) Preparation of rac-piperazine-2-carboxamide (catalyst reuse) Pyrazine-2-carboxamide (10 g, 81.2 mmol) was suspended in 50 mL of water in a pressure vessel equipped with a deep-bottom tube with a 2 μm frit. The vessel was then inerted with argon. 10% Pd / C (anhydrous, 1.0 g) was added to the reaction mixture, the reactor was sealed, the atmosphere was replaced with hydrogen, and the reaction mixture was heated to 40 °C. The atmosphere was adjusted to 20 bar H2, and the mixture was stirred at 40 °C for 18 h while maintaining a constant hydrogen pressure of 20 bar in the vessel and recording gas consumption over time. The reactor was cooled to room temperature, the atmosphere was replaced with argon, and the reaction mixture was filtered from the reactor through a deep-bottom tube using an overpressure of Ar. The resulting solution was analyzed by GC and XRF spectroscopy to determine the presence of trace amounts of Pd. The vessel was depressurized and refilled with 10 g of pyrazine-2-carboxamide and water (50 mL), and the hydrogenation was repeated using the filtered catalyst again. This procedure was repeated five times, always achieving >98a% conversion and >92a% yield (determined by GC analysis), with Pd levels in solution always >2 ppm (determined by XRF spectroscopy).

[0061] c) Preparation of (S)-piperazine-2-carboxylic acid dihydrochloride To a pH-adjusted solution of the rac-piperazine-2-carboxamide solution from Example 1a (105 g; 812 mmol dissolved in approximately 555 mL of water at pH 7.8) was added an enzyme catalyst (Flavorzyme® 1000L (Novozyme), 50 mL) and the reaction was stirred at room temperature for 22 hours. The reaction was monitored by HPLC, which showed 47% acid formation after 20 hours.

[0062] The resulting reaction mixture was concentrated under reduced pressure (30–120 mbar, 45° C.) to approximately 400 mL, followed by the addition of concentrated (37%) aqueous HCl to the reaction mixture (190 mL, 2.28 mol) over 45 min to precipitate (S)-piperazine-2-carboxylic acid dihydrochloride and stirring for 4 h to ensure complete product precipitation.

[0063] The resulting crystals were filtered off, washed with HCl (3N, 120 mL, 360 mmol), and dried under reduced pressure (5 mbar, 45 °C, 24 h) to give the desired product in 38% yield (62 g) with a purity of 87 a% and >99% ee.

[0064] LC Chiral Method Description (for ee-determination) - Stationary phase: Astec Chirobiotic T (L=25 cm, ID=4.6 mm, particle size=5 μm).

[0065] Eluent: A) potassium phosphate 50 mM pH 7.0 B) methanol; pump program: isocratic 90A:10B, run time 13 min, flow rate: 1.0 mL / min; column oven temperature: 10 °C; injection volume: 2 μL; detection: DAD 198 nm.

[0066] Retention times: (S)-piperazine-2-carboxylic acid = 4.42 min, (R)-piperazine-2-carboxylic acid = 4.82 min, rac-piperazine-2-carboxamide = 10.43 min. c) NMR data of (S)-piperazine-2-carboxylic acid dihydrochloride 1 H NMR(600 MHz,D2O)δ ppm:4.17(dd,J=11.3,3.9 Hz,1 H),3.91(dd,J=14.1,3.8 Hz,1 H),3.73(dt,J=14.0,3.3 Hz,1 H),3.69-3.63(m,1 H),3.48-3.41(m,2 H),3.39-3.33(m,1 H).

[0067] d) Preparation of (S)-piperazine-2-carboxylic acid using a fraction of SEQ ID NO: 1. d1) Enzyme preparation of SEQ ID NO: 1: The enzyme DNA sequence was inserted into a Pichia pastoris expression / integration plasmid, linearized, and stably integrated into the genome of a Mut+ wild-type Pichia pastoris strain at the AOX I locus by homologous recombination. Recombinant strains incorporating the expression cassette were selected using the Zeocin antibiotic resistance marker. Target protein expression was under the control of the endogenous inducible AOX I promoter, and the expressed protein was secreted into the culture supernatant via a cleavable N-terminal fusion with the S. cerevisiae α-mating factor secretion signal peptide.

[0068] Overnight cultures of single colonies of recombinant strains were grown in YPD medium (Sigma Aldrich Y1375, ready-made media powder) without antibiotic selection.

[0069] To produce the enzyme, expression cultures in BMMY medium (110 mL) were inoculated with the corresponding YPD overnight cultures using 500 mL shake flasks to a final OD600 of 1. Target protein expression in the cultures was induced by activation of the AOX promoter by the addition of 1% MeOH (v / v). Additional 1.5% (v / v) methanol was added twice 24 h over the course of 3 days of expression (a total of 6 × 1.5 mL of 100% MeOH over 3 days), and the cultures were shaken at 28 °C and 180 rpm.

[0070] After 3 days, the expression culture supernatants were clarified by centrifugation (RCF 12000xg, 15 min), frozen at -80°C and then lyophilized (-80°C / 100 μbar) without further processing steps.

[0071] The resulting lyophilized powder was used without further purification steps.

[0072] d2) Preparation of (S)-piperazine-2-carboxylic acid Rac-piperazine-2-carboxamide (2 g, 15 mmol) was dissolved in 2N HCl (6 mL, 12 mmol) and water was added (2 mL) to give a 20% (w / v) solution of rac-2-piperazinecarboxamide at pH 7.8.

[0073] To an aliquot of this solution (1 mL) was added an enzyme preparation containing SEQ ID NO:1 (100 mg, lyophilized powder) and ZnCl2 solution (1 M, 20 μL).

[0074] The reaction was incubated at room temperature with shaking in an Eppendorf ThermoMixer C for 2 days.

[0075] The reaction mixture formed the desired product (S)-piperazine-2-carboxylic acid in 22 a% and over 98% ee.

[0076] Example 2 (S)-Piperazinecarboxylic acid dihydrochloride a) Preparation of (S)-piperazinecarboxylic acid dihydrochloride (Flavorzyme) To a pH-adjusted solution of the rac-2-piperazinecarboxamide solution from Example 1a (105 g; 812 mmol dissolved in approximately 555 mL of water at pH 7.8) was added an enzyme catalyst (100 g, Flavorzyme® 1000L (Novozymes)) and the reaction was stirred at room temperature for 21 hours. The reaction was monitored by HPLC, which showed 52% acid formation.

[0077] The resulting reaction mixture was concentrated under reduced pressure (30-120 mbar, 45°C) to approximately 530 g, followed by cooling (ice cooling) to 20-23°C. Concentrated (37%) aqueous HCl (190 mL, 2.28 mol, 2.8 equiv) was then added over 30 min to precipitate (S)-piperazinecarboxylic acid dihydrochloride. Stirring for an additional 4.5 h at room temperature ensured complete product precipitation.

[0078] The resulting crystals were filtered off, washed with HCl (3N, 120 mL, 360 mmol), and dried under high vacuum overnight to give the desired product in 40% yield (69 g) with a purity of 97a% and 99.1% ee.

[0079] b1) Enzyme preparation of leucine amido peptidase 2 (LAP2) The enzyme was produced as in Example 1, d1), with the modification that expression was carried out in fermentors at a 10 L scale, as known to those skilled in the art.

[0080] Two parallel cultures of 10 L LAP2 fed-batch Pichia pastoris bioreactors were grown in fed-batch mode for 26 hours using glycerol as the carbon source. After exhaustion of the glycerol feed, recombinant LAP2 protein expression was induced by adding a pulse of 100% methanol at 3% of the culture volume. This was repeated approximately 26 times after exhaustion of the previous pulse, for a total runtime of 96 hours. The supernatants were combined and filtered using a 0.2 μM PES Repligen hollow fiber membrane (12 L / min feed flow, 0.07 MPa transmembrane pressure); the remaining 1.2 L retentate was washed with 4 x 500 mL of dH2O and then discarded. The resulting filtrate was concentrated to approximately 2 L using a 10 kDa mPES Repligen hollow fiber membrane (12 L / min feed flow, 0.12 MPa transmembrane pressure). The concentrate was buffer exchanged at constant volume with 10 L of 25 mM sodium acetate, 100 mM NaCl, 0.5 mM ZnCl, pH 5.6 buffer at a feed flow of 12 L / min and a transmembrane pressure of 0.12 MPa. The resulting solution was then concentrated to approximately 1.25 L and filter-sterilized using a 0.2 μm mPES bottle-top filter.

[0081] b2) Preparation of (S)-piperazinecarboxylic acid dihydrochloride (LAP2) To a pH-adjusted solution of the rac-2-piperazinecarboxamide solution from Example 1a (105 g; 812 mmol dissolved in approximately 555 mL of water at pH 7.8), an enzyme catalyst was added (25 mL of leucinamide peptidase 2 (LAP2) preparation; SEQ ID NO: 1), and the reaction was stirred at room temperature for 19 hours. The reaction was monitored by HPLC, which showed 53% acid formation after 20 hours. The resulting reaction mixture was concentrated to approximately 500 g under reduced pressure (30-120 mbar, 45°C) and subsequently cooled (ice-cooled) to 20-23°C. Concentrated (37%) aqueous HCl (190 mL, 2.28 mol, 2.8 equiv.) was then added over 30 minutes to precipitate (S)-piperazinecarboxylic acid dihydrochloride. Stirring for an additional 4.5 hours at room temperature ensured complete product precipitation.

[0082] The resulting crystals were filtered off, washed with HCl (3N, 120 mL, 360 mmol), and dried under high vacuum overnight to give the desired product in 41% yield (69 g) with a purity of 98 a% and >99% ee.

[0083] Example 3 a) Preparation of (2S)-4-tert-butoxycarbonylpiperazine-2-carboxylic acid (S)-Piperazine-2-carboxylic acid dihydrochloride (10.8 g; 50 mmol) and potassium carbonate (7.3 g; 53 mmol) were combined in aqueous acetone (17 g acetone and 85 g water). The resulting solution was filtered through Celite (1 g) to remove any remaining enzyme residue from the previous step. A solution of Boc anhydride (12 g) in acetone (17 g) was charged to the solution over a 4-hour period, during which time the product gradually crystallized. After charging was complete, the pH of the reaction mixture was adjusted back to pH 7 from pH 6 using potassium bicarbonate (0.42 g) in water (2 g). Stirring was continued overnight to ensure complete product precipitation. The residue was filtered and washed with aqueous acetone (11 g acetone and 1 g water) and acetone (12 g), and the wet cake was dried overnight at 45°C / 12 mbar to yield 8.8 g of the title compound.

[0084] NMR and MS data for (2S)-4-tert-butoxycarbonylpiperazine-2-carboxylic acid. 1 H NMR(600 MHz,D2O)δ ppm:4.30(ddd,J=14.6,4.1,1.0 Hz,1H),4.04(br s,1H),3.78(dd,J=10.0,4.0 Hz,1H),3.45(br d,J=12.8 Hz,1H),3.33(ddd,J=14.5,10.9,3.3 Hz,1H),3.39(br s,1H),3.15(ddd,J=12.9,10.7,3.8 Hz,1H),1.48(s,9H) MS [MH] - At m / z=229.1.

[0085] b) Preparation of (2S)-4-tert-butoxycarbonylpiperazine-2-carboxylic acid To a mixture of (S)-piperazinecarboxylic acid dihydrochloride (50 g; 246 mmol), acetone (77.9 g), and water (347 g) was slowly added a solution of potassium carbonate (34 g; 246 mmol) in water (47.2 g). The resulting solution was stirred with Celite (5 g) for 10 minutes and filtered to remove any remaining enzyme residue from the previous step. The filtered residue was washed with an aqueous acetone solution (13 g acetone and 34 g water). At 20 °C, a solution of Boc anhydride (56.4 g, 259 mmol) in acetone (77.9 g) was added to the solution over a 4-hour period, during which time the product gradually crystallized. After the addition was complete, the pH of the reaction mixture was adjusted from pH 5.5 to pH 7 using 15 mL of a preparative solution consisting of potassium bicarbonate (12.3 g) and water (50 g). Stirring was continued overnight to ensure complete product precipitation. The residue was filtered, washed with aqueous acetone (44 g acetone and 4 g water) and acetone (40 g) and the wet cake was dried at 43° C. / 5 mbar for 6 hours to give 38.25 g of the title compound. 1H NMR(600 MHz,D2O)δ ppm:4.30(ddd,J=14.6,4.1,1.0 Hz,1H),4.04(br s,1H),3.78(dd,J=10.0,4.0 Hz,1H),3.45(br d,J=12.8 Hz,1H),3.33(ddd,J=14.5,10.9,3.3 Hz,1H),3.39(br s,1H),3.15(ddd,J=12.9,10.7,3.8 Hz,1H),1.48(s,9H) MS [M-H] - m / z=229.1にて。

Claims

1. Formula I 【Chemistry 1】 1. A process for the preparation of chiral piperazine-2-carboxylic acid of formula (I) or a salt thereof, comprising: a) Formula II 【Chemistry 2】 by catalytic hydrogenation of pyrazine-2-carboxamide of formula III using hydrogen in the presence of a metal hydrogenation catalyst and water 【Transformation 3】 forming a piperazine-2-carboxamide of and b) enzymatically converting the piperazine-2-carboxamide of formula III with a hydrolase having an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1 in water to form a chiral piperazine-2-carboxylic acid of formula I or a salt thereof. A method comprising:

2. join 【Chemistry 4】 but, 【Transformation 5】 The method of claim 1 , wherein

3. 3. A process according to claim 1 or 2, wherein the metal hydrogenation catalyst is Pd or Pt, preferably Pd, with a metal loading on the support of 0.5 w / w% to 20 w / w%, preferably 3 w / w% to 15%, more preferably 8 to 12 w / w%, and an inert support selected from carbon or aluminium oxide, more preferably carbon.

4. 4. The process according to any one of claims 1 to 3, wherein the catalytic hydrogenation is carried out at a reaction temperature of from 20°C to the boiling point of the solvent, preferably from 30°C to 60°C, and at a hydrogen pressure of from 5 bar to 50 bar, preferably from 15 bar to 25 bar.

5. The process according to any one of claims 1 to 4, wherein the piperazine-2-carboxamide of formula III obtained in step a) is not isolated but is further treated in situ in step b).

6. The process according to any one of claims 1 to 5, wherein the hydrogenation catalyst is reused.

7. 2. The method of claim 1, wherein the hydrolase is the enzyme mixture Flavorzyme® or an enzyme preparation containing the hydrolase of SEQ ID NO:

1.

8. The method according to claim 1 or 7, wherein the enzymatic conversion is carried out at a reaction temperature of 10°C to 50°C, preferably 20°C to 30°C.

9. 9. The process of any one of claims 1, 7 and 8, wherein after the enzymatic conversion, the chiral piperazine-2-carboxylic acid of formula I is converted to its hydrochloride salt by adding aqueous hydrochloric acid to the reaction mixture so that the reaction temperature is maintained in the range of -15°C to 30°C.

10. c) introducing an amino protecting group PG to form a compound of formula IV 【Transformation 6】 (wherein PG represents an amino protecting group) 2. The method of claim 1, further comprising forming a chiral piperazine-2-carboxylic acid of formula:

11. When PG is Boc (tert-butoxycarbonyl), the chiral piperazine-2-carboxylic acid of formula IV is 【Transformation 7】 The method according to claim 10, wherein the compound is (2S)-4-tert-butoxycarbonylpiperazine-2-carboxylic acid.

12. Formula IX 【Transformation 8】 (In the formula, R ７ But C １－６ -Alkyl, C ３－７ -cycloalkyl, -C ｍ H ２ｍ -COOH, -C ｍ H ２ｍ -C ３－７ -cycloalkyl-COOH or carboxyphenyl, and m is 1 to 6. or an enantiomer or stereoisomer thereof, d) Formula IV 【Chemistry 9】 where PG is an amino protecting group. or a salt thereof, by converting an amine R ７ -NH ２ (In the formula, R ７ is as defined above) in the presence of a coupling agent and a base to form a compound of formula V 【Chemistry 10】 (Wherein PG and R ７ is as above) or salt thereof to form a mixed urea of ​​formula IV; e) cyclizing the mixed urea of ​​formula V to give formula VI 【Chemistry 11】 (Wherein PG and R ７ is as above) forming a hydantoin of formula (I); f) reducing the hydantoin of formula VI to give the hydantoin of formula VII 【Chemistry 12】 (Wherein PG and R ７ is as above) forming a cyclic urea of g) Deprotection and formation of the compound of formula IX 12. The method of claim 10 or 11, comprising:

13. Formula X 【Chemistry 13】 (In the formula, R １ is a halogen or C １－６ - alkyl, R ２ is hydrogen or halogen; R ３ is hydrogen or halogen; R ４ is C １－６ - alkyl; R ５ is hydrogen or carboxy; R ６ is hydrogen; R ７ is C １－６ -Alkyl, C ３－７ -cycloalkyl, -C ｍ H ２ｍ -COOH, -C ｍ H ２ｍ -C ３－７ -cycloalkyl-COOH or carboxyphenyl; m is 1 to 6. Use of the method according to any one of claims 1 to 12 for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt or enantiomer or diastereomer thereof.

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