Method for synthesizing (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid or its phosphonate derivative

Abstract

The present invention relates to a process for the synthesis of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid ("Compound"), a phosphonate derivative thereof, or a salt of any of these; to a crystalline form of the Compound; and to the use of the Compound (particularly a crystalline form of the Compound) or a phosphonate derivative or salt thereof for the preparation of 4-((R)-2-{[6-((S)-3-methoxy-pyrrolidin-1-yl)-2-phenyl-pyrimidine-4-carbonyl]-amino}-3-phosphono-propionyl)-piperazine-1-carboxylic acid butyl ester (also known as seratogrel) or a pharma- ceutically acceptable salt thereof. [Case 1] TIFF2025509711000039.tif62150

Description

[Technical field]

[0001] The present invention relates to (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid to a method for the synthesis of 4-((R)-2-{[6-((S)-3-methoxy-pyrrolidin-1-yl)-2-phenyl-pyrimidine-4-carbonyl]-amino}-3-phosphono-propionyl)-piperazine-1-carboxylic acid butyl ester (also known as selatogrel) or a pharma- ceutically acceptable salt thereof; to a crystalline form of the Compound; and to the use of the Compound (particularly a crystalline form of the Compound) or a phosphonate derivative or salt thereof for the preparation of 4-((R)-2-{[6-((S)-3-methoxy-pyrrolidin-1-yl)-2-phenyl-pyrimidine-4-carbonyl]-amino}-3-phosphono-propionyl)-piperazine-1-carboxylic acid butyl ester (also known as selatogrel) or a pharma- ceutically acceptable salt thereof.

[0002] [ka] [Background technology]

[0003] The preparation of seratogrel from the "compound" is described in WO2009 / 069100 and Caroff E et al., J.Med.Chem.(2015), 58, 9133-9153. Medical uses of seratogrel are described, for example, in WO2018 / 167139; Baldoni D et al., Clin Drug Investig(2014), 34(11), 807-818; Storey RF et al., European Heart Journal, ehz807, doi:10.1093 / eurheartj / ehz807; and Sinnaeve PR et al., J Am Coll Cardiol(2020), 75(20), 2588-97(doi.org / 10.1016 / j.jacc.2020.03.059). Seratogrel has been studied in a multi-center, double-blind, randomized, placebo-controlled, parallel-group study to evaluate the efficacy and safety of self-administered subcutaneous seratogrel in preventing all-cause death and treating acute myocardial infarction in patients with recent acute myocardial infarction (ClinicalTrials.gov Identifier: NCT04957). 719).

[0004] For example, "Compound" can be prepared by reacting commercially available (R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoic acid methyl ester (Boc-3-iodo-L-Ala-OMe) with triethyl phosphite and then saponifying with LiOH in a mixture of water and THF (WO2009 / 069100 and Caroff E et al., J. Med. Chem. (2015), 58, 9133-9153). The disadvantage of this method, especially for large-scale synthesis, is the use of a very expensive starting material, Boc-3-iodo-L-Ala-OMe, which is prone to racemization under the conditions of the Arbuzov reaction. Alternatively, "Compound" as a racemic mixture, together with its enantiomers, can be prepared according to the procedure shown in Scheme 1: Compound 2 can be prepared by reaction of ethyl 3-bromo-2-oxopropanoate with hydroxylamine hydrochloride. Reaction of compound 2 with triethyl phosphite gives ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)-propanoate (compound 3), which can be converted to compound 4 by hydrogenation in the presence of Pd / C as catalyst and protection of the amino group with Boc2O. The racemic mixture of "compound" and its enantiomers can be obtained from compound 4 by saponification with LiOH in a mixture of water and toluene at a temperature of about 20° C. (Stefan Abele, presentation at the 250th ACS meeting, Boston, August 17, 2015; Stefan Abele, presentation at the Swiss Industrial Chemistry Symposium, Basel, October 28, 2016). Chiral column chromatography or simulated moving bed chromatography can be used to obtain the chiral mixture of "compound" and its enantiomers. Even if separation of the enantiomers is possible at the stage of the ester (Compound 4), the final acid (racemate of Compound) or a later intermediate in the synthesis of Compound 5 (e.g. 4-[2-tert-butoxycarbonylamino-3-(diethoxy-phosphoryl)-propionyl]-piperazine-1-carboxylic acid butyl ester) by standard bed chromatography, such separation of the enantiomers is disadvantageous in that it is limited in scale and processing capacity as well as requires large amounts of solvent.

[0005] [ka]

[0006] Surprisingly, it has now been found that enzymatic resolution using hydrolases provides the Compounds in excellent yields and with high enantiomeric purity in large scale synthesis. [Brief description of the drawings]

[0007] [Figure 1]FIG. 1 shows the powder X-ray diffraction diagram of Compound in crystalline form (I), said powder X-ray diffraction diagram being measured using the XRPD method described in the experimental section and shown for Cu Kα radiation. The X-ray diffraction diagram shows peaks with the following percentage relative intensities (relative peak intensities are given in brackets) compared to the most intense peak in the diagram at the indicated refraction angles 2 theta (selected peaks with a relative intensity of greater than 10% from the 3-30° 2 theta range are reported): 9.8° (100%), 10.3° (37%), 12.5° (12%), 13.3° (16%), 16.2° (23%), 17.5° (34%), 19.8° (14%), 20.7° (24%), 22.4° (25%), 22.7° (15%), 24.2° (23%), 24.7° (14%), 26.8° (12%) and 27.3° (33%). In the X-ray diffraction diagram of Figure 1, the refraction angle 2 theta (2θ) is plotted on the horizontal axis and the counts on the vertical axis. For the avoidance of any doubt, the above peaks describe the experimental results of the powder X-ray diffraction shown in Figure 1. In contrast to the list of peaks above, it should be understood that only selected characteristic peaks are necessary to completely and unambiguously characterize each crystalline form of the "Compound" of the present invention. [Diagram 2] Figure 2 shows the gravimetric vapor sorption (GVS) diagram at 25°C for the crystalline form (I) of "Compound" obtained from Example 4. In the gravimetric vapor sorption diagram of Figure 2, relative humidity (%RH) is plotted on the horizontal axis and mass change (%dm) on the vertical axis. [Diagram 3] Figure 3 shows a differential scanning calorimetry (DSC) thermogram of Compound I. In the DSC thermogram of Figure 3, temperature (°C) is plotted on the horizontal axis and heat flow (mW) on the vertical axis. [Figure 4]FIG. 4 shows the powder X-ray diffraction diagram of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate in crystalline form (A), said powder X-ray diffraction diagram being measured using the XRPD method described in the experimental section and shown for Cu Kα radiation. The X-ray diffraction diagram shows peaks with the following percentage relative intensities (relative peak intensities are given in brackets) compared to the most intense peak in the diagram at the indicated refraction angles 2 theta (selected peaks with a relative intensity of more than 8% from the 3°-40° 2 theta range are reported): 10.2° (100%), 11.3° (99%), 13.2° (11%), 13.8° (9%), 15.2° (9%), 20.5° (13%), 22.7° (71%), 22.9° (33%), 25.3° (11%), 26.6° (18%) and 34.3° (49%). In the X-ray diffraction diagram of FIG. 4, refraction angles 2 theta (2θ) are plotted on the horizontal axis and counts on the vertical axis. For the avoidance of any doubt, the above peaks respectively describe the experimental results of the powder X-ray diffraction shown in FIG. 4. In contrast to the above list of peaks, it should be understood that only selected characteristic peaks are necessary to completely and unambiguously characterize each crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate of the present invention. [Diagram 5]FIG. 5 shows the powder X-ray diffraction diagram of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate in crystalline form (B), said powder X-ray diffraction diagram being measured using the XRPD method described in the experimental section and shown for Cu Kα radiation. The X-ray diffraction diagram shows peaks with the following percentage relative intensities (relative peak intensities are given in brackets) compared to the most intense peak in the diagram at the indicated refraction angle 2 theta (selected peaks with a relative intensity of more than 10% from the 3°-40° 2 theta range are reported): 10.9° (31%), 18.2° (23%), 19.5° (100%), 19.8° (24%), 25.3° (10%), 27.2° (91%), 28.7° (11%), 29.5° (65%), 33.0° (24%), 34.7° (22%) and 36.9° (30%). In the X-ray diffraction diagram of FIG. 5, refraction angle 2 theta (2θ) is plotted on the horizontal axis and counts on the vertical axis. For the avoidance of any doubt, the above peaks each describe the experimental results of the powder X-ray diffraction shown in Figure 5. In contrast to the above list of peaks, it should be understood that only selected characteristic peaks are necessary to completely and unambiguously characterize each crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate of the present invention. Summary of the Invention

[0008] Description of the invention The invention will now be described and various aspects of the invention will be illustrated.

[0009] 1) In a first aspect, the present invention relates to a process for preparing a compound of formula (I) or a salt thereof,

[0010] [ka]

[0011] The method comprises reacting a compound of formula (II)

[0012] [ka]

[0013] (In the formula, R 1 and R 2 are, independently of each other, (C 1-4 ) alkyl (especially ethyl), R 3 represents methyl, ethyl or n-propyl (particularly ethyl).) with a hydrolase to obtain a compound of formula (I) having an enantiomeric excess (ee) of at least 70%.

[0014] The definitions set forth herein apply uniformly throughout the specification and claims, unless a broader or narrower definition is given by a specific definition. It is to be understood that any definition or preferred definition of a term may define and replace the respective term independently of (and in conjunction with) any or all other terms or preferred definitions defined herein.

[0015] The term "equivalents" as used in the context of "The amount of a first compound is 'X' equivalents to the amount of a second compound" means that a mixture contains 'X' times the amount of a first compound (in arbitrary units relating to the number of molecules) relative to the amount of a second compound (expressed in the same units).

[0016] When not used in relation to temperature, the term "about" before a numerical value "X" means in this application between 10% of XX and 10% of X+X, in particular between 5% of XX and 5% of X+X, and especially between 2% of XX and 2% of X+X. In the specific case of temperature, the term "about" before a temperature "Y" means in this application between a temperature Y-10°C and Y+10°C, in particular between Y-5°C and Y+5°C, and especially between Y-3°C and Y+3°C. Room temperature means a temperature of about 25°C. Taste.

[0017] Whenever the words "between" or "to" are used to describe a numerical range, the endpoints of the stated range are expressly intended to be included in that range. For example: when a temperature range is described as being between 40° C. and 80° C. (or 40° C. to 80° C.), it is meant that the endpoints 40° C. and 80° C. are included in the range; or when a variable is defined as an integer between 1 and 4 (or 1 to 4), it is meant that the variable is the integer 1, 2, 3, or 4.

[0018] The expression %w / w means the percentage by weight relative to the total weight of the composition under consideration. Similarly, the expression v / v means the volume ratio of one component relative to the total volume (%v / v means the respective ratio expressed as a percentage).

[0019] The term "alkyl", used alone or in combination, means a straight or branched saturated hydrocarbon chain having 1 to 4 carbon atoms. x-y The term "alkyl" (x and y are each integers) refers to an alkyl group as defined above having x to y carbon atoms. For example, (C 1-4 ) The alkyl group has 1 to 4 carbon atoms. 1-4 Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and tert-butyl. 1 " is "(C 1-4 When expressing an "alkyl" group, "(C 1-4 The term "R alkyl" means methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec.-butyl and tert.-butyl, preferably methyl, ethyl and n-propyl, most preferably ethyl. 2 " is "(C 1-4 When expressing an "alkyl" group, "(C 1-4The term "alkyl" means methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec.-butyl and tert.-butyl, preferably methyl, ethyl and n-propyl, most preferably ethyl.

[0020] Any reference to a "hydrolase" that reacts with a compound of formula (II) to give a compound of formula (I) with a certain enantiomeric excess means that that hydrolase is also suitable for reacting with a compound of formula (II) to give a compound of formula (I) with that enantiomeric excess.

[0021] For the avoidance of doubt, an enantiomeric excess (ee) of a compound of formula (I) of at least 70% means that the ratio between the compound of formula (I) and its enantiomer in a mixture or product is 85:15 or higher; accordingly, an enantiomeric excess (ee) of a compound of formula (I) of at least 96% means that the ratio between the compound of formula (I) and its enantiomer in a mixture or product is 98:2 or higher.

[0022] The compound of formula (II) is understood to mean any mixture of the (S)-enantiomers with the (R)-enantiomers in a ratio of between 65:35 and 35:65, in particular in a ratio of between 55:45 and 45:55, and especially in a ratio of between 52:48 and 48:52. Most preferred is a racemic mixture having a 1:1 ratio of (S)-enantiomers to (R)-enantiomers.

[0023] The term "salt" as used in the context of "a compound of formula (I) or a salt thereof" means a compound of formula (I) in which the proton of the carboxylic acid function has been replaced with a suitable cation to form a salt of the compound of formula (I). Suitable cations are in particular cations having a weight of less than 110 g / mol, especially metal cations having a weight of 30 g / mol or less. Preferred are the alkali metal and alkaline earth metal salts of the compound of formula (I); in particular the salts of the compounds of formula (I) The sodium and potassium salts of compound (I), especially the sodium salt.

[0024] 2) A further embodiment is characterized in that the hydrolase is selected from the group consisting of AH002, AH008, AH012, AH016, AH017, AH018, AH019, AH022, AH023, AH025, AH027, AH028, AH032, AH034, AH035, AH036, AH037, AH041, AH042, AH044, AH045, AH047, AH048, AH051, AH052, AH055, AH056, AH057, AH059, AH060, AH061, AH062, CL055, CL067, Protease M, EU62, DSM-A1, DSM-A2, DSM-A3, DSM-A6, DSM-B1, DSM-B2, DSM-B3, DSM-B6, DSM-C1, DSM-C2, DSM-C3, DSM-D2 and DSM-D3.

[0025] AH002, AH008, AH012, AH016, AH017, AH018, AH019, AH022, AH023, AH025, AH027, AH028, AH032, AH034, AH035, AH036, AH037, AH041, AH042, AH044, AH045, AH047, AH048, AH051, AH052, AH055, AH056, AH057, AH059, AH060, AH061, AH062, CL055 and CL067 are commercially available enzymes and are available from Almac; Protease M is commercially available and is available from Amano Enzyme (in particular Amano Enzyme EU62 is commercially available and available from Eucodis Bioscience; DSM-A1, DSM-A2, DSM-A3, DSM-A6, DSM-B1, DSM-B2, DSM-B3, DSM-B6, DSM-C1, DSM-C2, DSM-C3, DSM-D2 and DSM-D3 are commercially available enzymes and available from DSM Innosyn / Innosyn. All enzymes are also available through Almac.

[0026] 3) In another aspect, the present invention relates to a process for preparing a compound of formula (I) or a salt thereof, comprising the steps of:

[0027] [ka]

[0028] The method comprises reacting a compound of formula (II)

[0029] [ka]

[0030] (In the formula, R 1 and R 2 are, independently of each other, (C 1-4 ) alkyl (especially ethyl), R 3 represents methyl, ethyl or n-propyl (particularly ethyl).) with a hydrolase, and the hydrolase may be any of AH002, AH008, AH012, AH016, AH017, AH018, AH019, AH022, AH023, AH025, AH027, AH028, AH032, AH034, AH035, AH036, AH037, AH041, AH042, AH044, AH045, AH047, AH048, AH051, AH052, AH055, AH056, AH057, AH059, AH060, AH061, AH062, CL055, CL067, Protease M, EU62, DSM-A1, DSM-A2, DSM-A3, DSM-A6, DSM-B1, DSM-B2, DSM-B3, DSM-B6, DSM-C1, DSM-C2, DSM-C3, DSM-D2 and DSM-D3.

[0031] 4) In another aspect, the present invention relates to a method for producing seratogrel,

[0032] [ka]

[0033] The method comprises reacting a compound of formula (II)

[0034] [ka]

[0035] (In the formula, R 1 and R 2 are, independently of each other, (C 1-4 ) alkyl (especially ethyl), R 3 represents methyl, ethyl or n-propyl (particularly ethyl).) is reacted with a hydrolase to give a compound of formula (I) or a salt thereof having an enantiomeric excess (ee) of at least 70%.

[0036] [ka]

[0037] The method includes a step of obtaining the above-mentioned

[0038] 5) A further embodiment relates to a method according to any one of embodiments 1), 3) or 4), wherein said hydrolase is selected from AH012, AH016, AH017, AH018, AH022, AH023, AH025, AH027, AH028, AH032, AH034, AH035, AH036, AH041, AH042, AH044, AH047, AH048, AH051, AH052, AH055, AH059, AH061 and Protease M.

[0039] 6) A further embodiment relates to the method according to any one of embodiments 1), 3) or 4), wherein said hydrolases are selected from AH017, AH018, AH022, AH023, AH025, AH027, AH032, AH034, AH044, AH047, AH059, AH061 and Protease M.

[0040] 7) A further embodiment relates to the method according to any one of embodiments 1), 3) or 4), wherein said hydrolases are selected from AH018, AH022, AH023, AH027, AH034, AH044, AH047 and Protease M.

[0041] 8) A further embodiment relates to the method according to any one of embodiments 1), 3) or 4), in which said hydrolases are selected from AH018, AH023, AH034 and Protease M.

[0042] 9) A further embodiment relates to a method according to any one of embodiments 1), 3) or 4), in which said hydrolase is Protease M.

[0043] Protease M (especially Protease M-SD) is commercially available from Amano Enzyme (especially Amano Enzyme Manufacturing, Suqian, Jiangsu, China), which is obtained by fermentation from Aspergillus oryzae and has high protease and peptidase activity. Protease M-SD stands for spray-dried Protease M.

[0044] 10) A further embodiment relates to a process according to any one of embodiments 1) to 9), wherein said process gives a compound of formula (I) with an enantiomeric excess (ee) of at least 85%.

[0045] 11) A further embodiment relates to a process according to any one of embodiments 1) to 9), wherein said process gives a compound of formula (I) with an enantiomeric excess (ee) of at least 96%.

[0046] 12) A further embodiment relates to a process according to any one of embodiments 1) to 9), wherein said process gives a compound of formula (I) with an enantiomeric excess (ee) of at least 99%.

[0047] 13) A further aspect is R 1 and R 2are, independently of one another, methyl, ethyl or n-propyl (in particular methyl or ethyl).

[0048] 14) A further aspect is R 1 and R 2 represents the same alkyl group selected from methyl and ethyl.

[0049] 15) A further aspect is R 1 and R 2 and each of the formulas 1) to 12) above, represents ethyl.

[0050] 16) A further aspect is R 3 represents methyl or ethyl.

[0051] 17) A further aspect is R 3 represents ethyl.

[0052] 18) A further embodiment relates to a method according to any one of embodiments 1) to 17), in which the reaction (enzymatic resolution) is carried out in aqueous solution.

[0053] The enzymatic resolution may be carried out in water as the sole solvent (particularly purified water) or in a solvent mixture of water and a suitable organic solvent (particularly MTBE, DMSO or toluene), the addition of an organic solvent having the beneficial effect of reducing the viscosity of the solution.

[0054] 19) A further embodiment relates to the process according to any one of embodiments 1) to 17), wherein the reaction (enzymatic resolution) is carried out in a mixture of water and an organic solvent selected from MTBE, DMSO, toluene and any mixture thereof.

[0055] 20) A further embodiment relates to the process according to any one of embodiments 1) to 17), wherein the reaction (enzymatic resolution) is carried out in a mixture of water and an organic solvent selected from MTBE, DMSO and toluene.

[0056] 21) A further embodiment relates to the process according to any one of embodiments 1) to 17), wherein the reaction (enzymatic resolution) is carried out in a mixture of water and MTBE.

[0057] 22) A further embodiment relates to the method according to any one of embodiments 18) to 21), wherein the amount of said organic solvent is between 0% and 20% v / v.

[0058] The lower limits of the amount of organic solvent are 0% v / v, 3% v / v and 5% v / v, and the upper limits are 20% v / v, 15% v / v and 10% v / v. It is understood that each lower limit can be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein. The amount of organic solvent is preferably between 5% and 15% v / v.

[0059] 23) A further embodiment relates to the method according to any one of embodiments 18) to 22), in which the reaction (enzymatic resolution) is carried out in water at 25° C. and in the presence of a buffer having a pKa value between 5.2 and 8.4.

[0060] The lower limits of the pKa value of the buffer are 5.2, 5.5 and 6.2, and the upper limits are 8.4, 7.9 and 7.4. Each lower limit may be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein. Preferably, the buffer is selected from phosphate buffer and carbonate buffer. Particularly preferred is phosphate buffer.

[0061] 24) A further embodiment relates to the method according to any one of embodiments 18) to 23), in which the reaction (enzymatic resolution) is carried out at a pH value between 5.0 and 8.0.

[0062] The lower limits of the pH values ​​are 5.0, 6.0, 6.5 and 6.7, and the upper limits are 8.0, 7.4, 7.0 and 6.9. It is understood that each lower limit can be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein. Preferably, the pH values ​​are between 6.5 and 7.0, in particular between 6.7 and 6.9.

[0063] 25) A further embodiment relates to the method according to any one of embodiments 18) to 24), in which the pH value of the solution is maintained within a range of plus / minus 0.2 (preferably 0.1) during the reaction (enzymatic splitting) by addition of base.

[0064] The base may be added to the reaction mixture sequentially or continuously. Preferably, the base is added as an aqueous solution, in particular an aqueous solution of potassium carbonate. The pH value of the solution is preferably maintained within the range of between 6.6 and 7.0 (most preferably between 6.7 and 6.9).

[0065] 26) A further embodiment relates to the method according to any one of embodiments 1) to 25), in which the reaction (enzymatic resolution) is carried out at a temperature between 10° C. and 60° C.

[0066] The lower limits of the reaction temperature are 10°C, 15°C, 20°C and 25°C, and the upper limits are 60°C, 40°C, 33°C and 30°C. Each lower limit can be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein. Preferably, the reaction temperature is between 20°C and 33°C, particularly between 25°C and 30°C.

[0067] 27) A further embodiment relates to the method according to any one of embodiments 1) to 26), wherein the reaction (enzymatic cleavage) is carried out with an enzyme loading of between 0.05% and 100%.

[0068] The term "enzyme loading" refers to the weight ratio of the amount of enzyme to the amount of substrate used, i.e. the amount of compound of formula (II) used. For example, an enzyme loading of 1% means that 10 mg of enzyme is used in the reaction per 1 g of compound of formula (II). The lower limits of the enzyme loading are 0.05%, 0.2%, 0.5% and 0.8%, and the upper limits are 100%, 20%, 10% and 2%. It is understood that each lower limit can be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein. Preferably, the enzyme loading is between 0.2% and 10%, in particular between 0.5% and 2.0%.

[0069] 28) A further embodiment relates to the method according to any one of embodiments 1) to 27), wherein the reaction (enzymatic splitting) is carried out with a substrate loading in the reaction mixture of between 5.0 g substrate per liter (L) of water and 300 g substrate per liter (L) of water.

[0070] The term "substrate loading" refers to the amount of substrate used in grams, i.e. the amount of compound of formula (II) used in grams, per volume of water in liters in the reaction mixture. For the avoidance of doubt, only the volume of water at the start of the reaction is taken into account, not the volume of water added with base during the course of the reaction to keep the pH value constant. The lower limits of the substrate loading are 5.0 g substrate per liter of water, 20 g substrate per liter of water, and 100 g substrate per liter of water, and the upper limits are 300 g substrate per liter of water, 220 g substrate per liter of water, and 150 g substrate per liter of water. It is understood that each lower limit can be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein. Preferably, the substrate loading is between 20 g substrate per liter of water and 220 g substrate per liter of water.

[0071] 29) A further embodiment relates to the process according to any one of embodiments 1) to 28), wherein a salt of a divalent metal cation (in particular a chloride salt of a divalent metal cation) is added to the reaction mixture.

[0072] Preferred salts of divalent metal cations are ZnCl2, FeCl2, MnSO4, CoCl2, MgCl2, CaCl2 and NiCl2 (especially ZnCl2, FeCl2 and MnSO4). The above salts may be used at concentrations between 0.5 mM and 5.0 mM (especially between 1.0 mM and 3.0 mM).

[0073] 30) A further embodiment relates to the method according to any one of embodiments 1) to 29), wherein the reaction time of said reaction (enzymatic resolution) is between 5 hours and 36 hours.

[0074] The term "reaction time" refers to the time between the addition of the last reagent to the reaction mixture and the start of a work-up procedure (e.g., by extraction of said reaction mixture with an organic solvent). The lower reaction time limits are 5 hours, 10 hours, and 18 hours, and the upper reaction time limits are 36 hours, 28 hours, and 24 hours. It is understood that each lower limit can be combined with each upper limit. Thus, all combinations of lower and upper limits are specifically disclosed herein.

[0075] 31) A further embodiment relates to the method according to embodiment 30), wherein the reaction time of said reaction (enzymatic resolution) is between 10 hours and 28 hours.

[0076] 32) A further embodiment relates to a process according to any one of embodiments 1) to 31), comprising a work-up of the reaction mixture by a process comprising the steps of: adding an aqueous solution of a base (especially an aqueous solution of potassium carbonate) to adjust the pH value to between 7.4 and 9.0 (especially between 7.5 and 8.5, especially between 7.6 and 8.0); adding an organic solvent (especially MTBE); separating the organic and aqueous layers; adding an aqueous solution of an acid (especially hydrochloric acid) to the aqueous layer to adjust the pH value to between 1.0 and 4.0 (especially between 1.2 and 2.0, especially between 1.4 and 1.6); and isolating the precipitated compound of formula (I).

[0077] 33) A further embodiment relates to a process according to any one of embodiments 1) to 32), comprising the further step of recrystallizing the compound of formula (I) from 2-propanol.

[0078] (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)-propanoic acid may be recrystallized from a solvent selected from methanol, ethanol, 2-propanol or any mixture thereof. The recrystallization solution may contain up to 20% w / w water. Preferably, the recrystallization solution contains less than 5% w / w water. The water content in the recrystallization is preferably as low as possible, since the presence of water reduces the yield. Recrystallization has the advantage of reducing the protein (enzyme) content.

[0079] The phrase "a solvent selected from methanol, ethanol, 2-propanol or any mixture thereof" means that said solvent is methanol, ethanol, 2-propanol or a mixture of two or three of methanol, ethanol and 2-propanol. When said solvent is a mixture, it is preferred that said solvent is a mixture of two of methanol, ethanol and 2-propanol.

[0080] 34) A further embodiment relates to a process according to any one of embodiments 1) to 33), which gives (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)-propanoic acid in crystalline form (I).

[0081] 35) A further embodiment relates to the process according to embodiment 34), wherein the crystalline form (I) of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid is characterized by the presence of peaks at the following refraction angles 2θ: 9.8°, 10.3° and 17.5° in the powder X-ray diffraction diagram.

[0082] 36) A further embodiment relates to the process according to embodiment 34), wherein the crystalline form (I) of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid is characterized by the presence of peaks at the following refraction angles 2θ: 9.8°, 10.3°, 16.2°, 17.5° and 27.3° in the powder X-ray diffraction diagram.

[0083] 37) A further embodiment relates to the process according to embodiment 34), wherein the crystalline form (I) of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid is characterized by the presence of peaks at the following refraction angles 2θ in the X-ray powder diffraction diagram: 9.8°, 10.3°, 12.5°, 16.2°, 17.5°, 20.7°, 22.4°, 24.2°, 24.7° and 27.3°.

[0084] 38) A further embodiment is characterized in that the crystalline form (I) of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid essentially exhibits a gravimetric vapor sorption profile (adsorption cycle) as shown in FIG. 2, The method according to any one of embodiments 34) to 37), wherein the vapor sorption profile is measured at 25° C.

[0085] 39) A further embodiment is a compound of formula (III)

[0086] [ka]

[0087] is reacted with hydrogen in the presence of palladium on charcoal and BocO to give a compound of formula (II)

[0088] [ka]

[0089] (In the formula, R1 and R 2 are, independently of each other, (C 1-4 ) alkyl (especially ethyl), R 3 represents methyl, ethyl or n-propyl (particularly ethyl).

[0090] 40) A further aspect is R 1 , R 2 and R 3 represents ethyl.

[0091] 41) A further embodiment relates to a process according to any one of embodiments 39) or 40), which is carried out in a solvent selected from methanol, ethanol and 2-propanol, in particular ethanol.

[0092] 42) A further embodiment is that the hydrogenation is carried out at a temperature between 50° C. and 70° C. (particularly between 55° C. and 65° C. The method according to any one of embodiments 39) to 41), wherein the process is carried out at a temperature between

[0093] 43) A further embodiment is a compound of formula (IV)

[0094] [ka]

[0095] with hydroxylamine hydrochloride and trialkyl phosphite (particularly triethyl phosphite) to give a compound of formula (III)

[0096] [ka] (In the formula, R 1 and R 2 are, independently of each other, (C 1-4 ) alkyl (especially ethyl), R 3represents methyl, ethyl or n-propyl (particularly ethyl).

[0097] 44) A further aspect is R 1 , R 2 and R 3 represents ethyl.

[0098] 45) A further embodiment is a compound of formula (V)

[0099] [ka] with a base (especially NaOEt) to give a compound of formula (II)

[0100] [ka] (In the formula, R 1 and R 2 are, independently of each other, (C 1-4 ) alkyl (especially ethyl), R 3 represents methyl, ethyl or n-propyl (especially ethyl); The compound of formula (V) has an enantiomeric excess (ee) of at least 80% and the compound of formula (II) has an enantiomeric excess (ee) of less than 10%, in particular 2% or less; the process according to any one of embodiments 1) to 44).

[0101] The enzymatic resolution of the compound of formula (II), as disclosed herein, in particular in embodiments 1) to 31), produces (before working up of the crude reaction mixture) a mixture of the compound of formula (I), in particular (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid, and the compound of formula (V), in particular (S)-ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate. Said crude mixture of acids and esters may be separated by any suitable method, such as extraction; precipitation of one component or chromatography. In particular, said crude mixture of acids and esters may be separated by partition between an aqueous layer (containing said acids) and an organic layer (containing said esters) having a pH value higher than 7.4. A preferred organic solvent for fractionation / extraction is an ether, in particular MTBE. The ester (particularly (S)-ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)-propanoate) may be isolated by removal of the organic solvent from the extract and used in a base-based recycling procedure to give the racemic compound of formula (II) (particularly ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate) which may be used again in the enzymatic resolution.

[0102] 46) A further embodiment is a compound of formula (V) having an enantiomeric excess (ee) of at least 90%. (particularly at least 96%).

[0103] 47) A further embodiment relates to a process according to any one of embodiments 45) or 46), which gives a compound of formula (II) with an enantiomeric excess (ee) of less than 4%, in particular 2% or less.

[0104] 48) A further aspect is R 1 and R 2 are, independently of one another, methyl, ethyl or n-propyl (in particular methyl or ethyl).

[0105] 49) A further aspect is R 1 and R 2 represents the same alkyl group selected from methyl and ethyl.

[0106] 50) A further aspect is R 1 and R 2 and each of R 1 to R 2 represent ethyl.

[0107] 51) A further aspect is R 3 represents methyl or ethyl.

[0108] 52) A further aspect is R 3 represents ethyl.

[0109] 53) A further embodiment relates to the process according to any one of embodiments 45) to 52), wherein the reaction (racemization) is carried out in a solvent selected from diethyl ether, MTBE, THF or 2-methyl-tetrahydrofuran.

[0110] 54) A further embodiment relates to the process according to any one of embodiments 45) to 52), wherein the reaction (racemization) is carried out in MTBE.

[0111] 55) A further embodiment relates to a process according to any one of embodiments 45) to 54), wherein the base is selected from sodium ethoxide (NaOEt) and potassium tert-butoxide (KOtBu), in particular sodium ethoxide.

[0112] 56) A further embodiment relates to the process according to any one of embodiments 45) to 54), wherein the base is a solution of sodium ethoxide (NaOEt) in ethanol.

[0113] 57) A further embodiment relates to the process according to any one of embodiments 55) to 56), wherein the amount of base is between 0.1 eq and 1.0 eq (in particular between 0.4 eq and 0.8 eq, in particular between 0.5 eq and 0.7 eq) relative to the amount of compound of formula (V).

[0114] 58) A further embodiment relates to the process according to any one of embodiments 45) to 57), wherein the reaction (racemization) is carried out at a reaction temperature between 0° C. and 30° C. (particularly between 0° C. and 10° C., more particularly between 0° C. and 5° C.).

[0115] 59) A further embodiment relates to the process according to any one of embodiments 45) to 58), wherein the water content in the reaction mixture of said reaction (racemization) is ≦0.5% v / v (particularly ≦0.2% v / v, especially ≦0.1% v / v).

[0116] 60) Another embodiment of the present invention is characterized by the presence of peaks in the powder X-ray diffraction diagram at the following refraction angles 2θ: 9.8°, 10.3° and 17.5°, (R The present invention relates to a crystalline form of 2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid (crystalline form (I)).

[0117] The crystalline form according to embodiment 60) is intended to include the "compound" in free form (non-salt form). Moreover, the crystalline form may contain non-coordinating and / or coordinating solvents (particularly non-coordinating and / or coordinating water). Coordinating solvents (particularly coordinating water) are used herein as a term for crystalline solvates (particularly crystalline hydrates). For the avoidance of doubt, in this application, the term "crystalline hydrate" includes stoichiometric and non-stoichiometric hydrates (particularly stoichiometric hydrates). Similarly, non-coordinating solvents are used herein as a term for physically adsorbed or physically trapped solvents (as defined by Polymorphism in the Pharmaceutical Industry (Ed. R. Hilfiker, VCH, 2006), Chapter 8: UJ Griesser: The Importance of Solvates).

[0118] 61) Another embodiment of the invention relates to a crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid according to embodiment 60) (crystalline form (I)), characterized by the presence of peaks at the following refraction angles 2θ in the X-ray powder diffraction diagram: 9.8°, 10.3°, 16.2°, 17.5° and 27.3°.

[0119] 62) Another embodiment of the invention relates to a crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid according to embodiment 60) (crystalline form (I)), characterized by the presence of peaks at the following refraction angles 2θ in the X-ray powder diffraction diagram: 9.8°, 10.3°, 12.5°, 16.2°, 17.5°, 20.7°, 22.4°, 24.2°, 24.7° and 27.3°.

[0120] 63) Another embodiment of the present invention relates to a crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid according to any one of embodiments 60) to 62) (crystalline form (I)) having an X-ray powder diffraction pattern essentially as shown in FIG.

[0121] 64) Another embodiment of the present invention relates to a crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid according to any one of embodiments 60) to 63) (crystalline form (I)) characterized by an endothermic peak at about 180° C. (in particular at 180° C.±1° C., especially at 180° C.) as measured by DSC.

[0122] 65) Another embodiment of the present invention relates to a crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid (crystalline form (I)) according to any one of embodiments 60) to 64), having a gravimetric vapor sorption profile essentially as shown in FIG. 2, said gravimetric vapor sorption profile being measured at 25° C.

[0123] 66) Another embodiment of the present invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate (crystalline form (A)), characterized by the presence of peaks at the following refraction angles 2θ: 10.2°, 11.3° and 22.7° in the powder X-ray diffraction diagram.

[0124] The crystalline form according to embodiment 66) is intended to include ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate in free form (non-salt form). Furthermore, the crystalline form may contain non-coordinating and / or coordinating solvents (in particular non-coordinating and / or coordinating water). Coordinating solvents (in particular coordinating water) are herein referred to as crystalline solvates (in particular crystalline hydrates). For the avoidance of doubt, in this application, the term "crystalline hydrate" includes stoichiometric and non-stoichiometric hydrates, particularly stoichiometric hydrates. Similarly, non-coordinating solvent is used herein as a term for physically adsorbed or physically trapped solvents (see Polymorphism in the Pharmaceutical Industry (Ed. R. Hilfiker, VCH, 2006), Chapter 8: Definition by UJ Griesser: The Importance of Solvates).

[0125] 67) Another embodiment of the invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate according to embodiment 66) (crystalline form (A)), characterized by the presence of peaks at the following refraction angles 2θ in the X-ray powder diffraction diagram: 10.2°, 11.3°, 13.2°, 22.7° and 22.9°.

[0126] 68) Another embodiment of the invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate according to embodiment 66) (crystalline form (A)), characterized by the presence of peaks at the following angles of refraction 2θ in the X-ray powder diffraction diagram: 10.2°, 11.3°, 13.2°, 13.8°, 15.2°, 20.5°, 22.7°, 22.9°, 25.3° and 26.6°.

[0127] 69) Another embodiment of the present invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate (crystalline form (A)) according to any one of embodiments 66) to 68), having an X-ray powder diffraction pattern essentially as shown in FIG.

[0128] 70) Another embodiment of the present invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate (crystalline form (B)), characterized by the presence of peaks at the following refraction angles 2θ: 10.9°, 19.5° and 27.2° in the powder X-ray diffraction diagram.

[0129] The crystalline form according to embodiment 70) is intended to include ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate in free form (non-salt form). Furthermore, the crystalline form may contain non-coordinating and / or coordinating solvents (particularly non-coordinating and / or coordinating water). Coordinating solvents (particularly coordinating water) are used herein as a term for crystalline solvates (particularly crystalline hydrates). For the avoidance of doubt, in this application the term "crystalline hydrate" includes stoichiometric and non-stoichiometric hydrates (particularly stoichiometric hydrates). Similarly, non-coordinating solvents are used herein as a term for physically adsorbed or physically trapped solvents (Polymorphism in the Pharmaceutical Industry (Ed. R. Hilfiker, VCH, 2006), Chapter 8: Definition by UJ Griesser: The Importance of Solvates).

[0130] 71) Another embodiment of the invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate according to embodiment 70) (crystalline form (B)), characterized by the presence of peaks at the following refraction angles 2θ in the X-ray powder diffraction diagram: 10.9°, 18.2°, 19.5°, 27.2° and 29.5°.

[0131] 72) Another embodiment of the invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate according to embodiment 70) (crystalline form (B)), characterized by the presence of peaks at the following angles of refraction 2θ in the X-ray powder diffraction diagram: 10.9°, 18.2°, 19.5°, 19.8°, 25.3°, 27.2°, 29.5°, 33.0°, 34.7° and 36.9°.

[0132] 73) Another embodiment of the present invention relates to a crystalline form of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate according to any one of embodiments 70) to 72) (crystalline form (B)), having an X-ray powder diffraction pattern essentially as shown in FIG.

[0133] For the avoidance of any doubt, whenever one of the above embodiments refers to "peaks at the following refraction angles 2θ in a powder X-ray diffraction diagram", it is to be understood that the powder X-ray diffraction diagram was obtained using combined Cu Kα1 and Kα2 radiation without removing Kα2; and the accuracy of the 2θ values ​​provided herein is within the range of + / -0.1 to 0.2°. In particular, when specifying a refraction angle 2θ for a peak in the embodiments and claims of the present invention, the 2θ values ​​stated are to be understood as being between said value minus 0.2° and said value plus 0.2° (2θ+ / -0.2°); and preferably between said value minus 0.1° and said value plus 0.1° (2θ+ / -0.1°).

[0134] For example, when defining the presence of a peak in an X-ray powder diffraction diagram, a common way is to do this in terms of the S / N ratio (S=signal, N=noise). According to this definition, when stating that a peak must be present in an X-ray powder diffraction diagram, it is understood that the peak in the X-ray powder diffraction diagram is defined by having a S / N ratio (S=signal, N=noise) greater than x (x being a number greater than 1), usually greater than 2, in particular greater than 3.

[0135] In the context of the statement that the crystalline form essentially exhibits the X-ray powder diffraction pattern represented in Figure 1, 4 or 5, respectively, the term "essentially" means that at least the main peaks of the diagram represented in said figure, i.e. peaks having a relative intensity of more than 20%, in particular more than 10%, compared to the most intense peak in the diagram, must be present. However, a person skilled in the art of X-ray powder diffraction should be aware that the relative intensities of X-ray powder diffraction diagrams can be subject to strong intensity variations due to preferred orientation effects.

[0136] Abbreviations and Terms Used herein Abbreviation: The following abbreviations are used throughout the specification and examples: Ac Acetyl AcCN Acetonitrile AcOH Acetic acid (L-)Ala (L-)Alanine aq Water-based Boc tert.-Butyloxycarbonyl DSC Differential Scanning Calorimetry ee enantiomeric excess eq. equivalent weight Et Ethyl EtOH Ethanol GVS Gravimetric Vapor Sorption h time HPLC High Performance Liquid Chromatography IPA 2-Propanol JT Jacket temperature M molar concentration Me Methyl min MTBE Methyl tert-butyl ether NaOEt Sodium ethoxide PDA Photodiode Array Detector RBF Round Bottom Flask rpm revolutions per minute rt retention time RT room temperature THF Tetrahydrofuran TFA Trifluoroacetic acid Vol 1 vol means 1 L of solvent per kg of relevant starting material Percentage determined by %a / a area ratio

[0137] Experimental section X-ray powder diffraction analysis (XRPD) Powder X-ray diffraction patterns were collected on a Bruker D8 Advance X-ray diffractometer equipped with a Lynxeye detector operating in reflection mode (coupled 2theta / theta). Typically, a Cu-X-ray tube was scanned at 40 kV / 40 mA. A step size of 0.02° (2θ) and a step time of 0.04 seconds per step were applied over a scan range of 3-50° 2θ. The divergence slit was set to fixed sample illumination (variable slit size) and the antiscatter slit was set to 0.3°. The powder was pressed slightly into a silicon single crystal sample holder with a depth of 0.5 mm and the sample was rotated in its own plane during the measurement. Diffraction data are reported using Cu Kα (λ=1.5418 Å) radiation. As is typical for previously recorded powder X-ray diffraction patterns, the accuracy of the 2θ values ​​provided herein is within + / - 0.1 to 0.2 degrees.

[0138] Differential Scanning Calorimetry (DSC) DSC data were collected on a Mettler Toledo STARe System (DSC3 module, measuring cell with ceramic sensor, and STAR software version 16.00b) equipped with a 34-position autosampler. The instrument was calibrated for energy and temperature using indium standards. Typically, 1-5 mg of each sample was heated in an automatically drilled aluminum pan from -20 to 250 °C for 10 °C min, unless otherwise noted. -1 The sample was heated at 200 K. A nitrogen purge was applied over the sample at 20 mL min. -1 The melting point was maintained at 100° C. The peak temperature is reported for the melting point.

[0139] Gravimetric Vapor Sorption (GVS) Analysis Measurements were performed on a multisample instrument SPS-100n (ProUmid GmbH, Ulm, Germany) operating in stepping mode at 25 °C. Samples were equilibrated at 40% RH before starting a predefined humidity program (40-0-95-40% RH, 5% ΔRH steps were applied with a maximum equilibration time of 24 h for each step). Approximately 20-30 mg of each sample was used. Classification of hygroscopicity was performed according to the European Pharmacopeia Technical Guide (1999, p. 86), e.g., non-hygroscopic: mass increase less than 0.2% mass / mass; slightly hygroscopic: mass increase less than 2% and more than 0.2% mass / mass; hygroscopic: mass increase less than 15% and more than 2% mass / mass. The 40% relative humidity in the first adsorption scan and Consider the mass change between 0.25 and 80% relative humidity.

[0140] High-performance liquid chromatography (HPLC) HPLC (Method 1): HPLC system: Agilent 1260 series system Flow rate: 1.0mL / min Column temperature: 30℃ Autosampler temperature: RT Injection volume: 5μL Column: Zorbax SB-Aq, 150x4.6mm, 3.5μm Detection wavelength (UV): 210nm Solvent A: 10 mM K2HPO4 aqueous solution (adjusted to pH 7.0 with 85% phosphoric acid) Solvent B: AcCN

[0141] [Table 1]

[0142] HPLC (Method 2): HPLC system: Agilent 1260 series system Flow rate: 1.0mL / min Column temperature: 30℃ Autosampler temperature: RT Injection volume: 5μL Column: Zorbax SB-Aq, 150x4.6mm, 3.5μm Detection wavelength (UV): 210nm Solvent A: 10 mM K2HPO4 aqueous solution (adjusted to pH 7.0 with 85% phosphoric acid) Solvent B: AcCN

[0143] [Table 2]

[0144] HPLC (Method 3): HPLC system: Agilent 1260 series system Flow rate: 1.0mL / min Column temperature: 40℃ Autosampler temperature: RT Injection volume: 5μL Column: Atlantis T3, 150x4.6mm, 3μm Detection wavelength (UV): 215nm Solvent A: H3PO4 in water (1% v / v) Solvent B: AcCN

[0145] [Table 3]

[0146] Chiral HPLC: Chiral HPLC (Method 1): HPLC system: Agilent 1260 series system Flow rate: 0.9mL / min Column temperature: 27℃ Autosampler temperature: RT Injection volume: 20μL Column: Chiralpak IC-3, 250x4.6mm, 3μm Detection wavelength (UV): 215nm Runtime 45min Mobile phase: 0.05% TFA-AcCN / 0.05% TFA-H2O=30 / 70 Chiral HPLC (Method 2): Flow rate: 1mL / min Column temperature: 35℃ Sample temperature: 35℃ Injection volume: 30μL Column: Chiralpak IC, 250x4.6mm, 5μm Detection wavelength (UV): 220nm Runtime 40min Mobile phase: 20% EtOH in heptane (+0.1% TFA) Chiral HPLC (Method 3): Flow rate: 1mL / min Column temperature: 25℃ Sample temperature: 25℃ Injection volume: 30μL Column: Chiralpak IC-3, 250x4.6mm, 5μm Detection wavelength (PDA): 215nm Runtime 32min Mobile phase 70:30 Water (0.05% TFA):CH3CN (0.05% TFA) Example 1: Synthesis of ethyl 3-bromo-2-(hydroxyimino)propanoate: Hydroxylamine hydrochloride (81.4 kg) and water (460 kg) were charged into the reactor and ethyl 3-bromo-2-(hydroxyimino)propanoate seeds (69.0 g) were added. The mixture was stirred at 20-25°C for 0.5 h and ethyl 3-bromo-2-oxopropanoate (230 kg, 1.0 eq.) was added over 1 h at 20-25°C. The reaction mixture was stirred for 2-3 h, toluene (905 kg) was added and the mixture was stirred at 20-25°C for 4-5 h. The layers were separated and water (368 kg) was added to the organic layer and the mixture was stirred at 20-25°C for 0.5 h and the layers were separated. The organic layer was concentrated to 1.0-2.0 vol. at 45-50°C (jacket temperature) and warmed to 45-50°C (internal temperature). Seeds of ethyl 3-bromo-2-(hydroxyimino)propanoate (138 g) were added, the mixture was stirred at 45-50° C. for 0.5 h and n-heptane (840 kg) was added to the mixture at 45-50° C. within 2-4 h. The mixture was cooled to 0-5° C. within 6-8 h, stirred at 0-5° C. for 4-5 h and centrifuged. The solid was washed with n-heptane (88.4 kg) and dried under vacuum at 30-35° C. for 24 h to give the product (136 kg).

[0147] Example 2: Synthesis of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate: Triethyl phosphite (129 kg, 1.2 eq.) and n-heptane (223 kg) were charged into the reactor and the internal temperature was adjusted to 70-75°C. Ethyl 3-bromo-2-(hydroxyimino)propanoate (136 kg, 1.0 eq.) was dissolved in isopropyl acetate (285 kg) at 20-25°C and this solution was added slowly to the reaction mixture over 2-4 h at 70-75°C. The reaction mixture was stirred at 70-75°C for 10-12 h and cooled to 20-25°C. Seeds of ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate (81.3 g) were added to the mixture at 20-25°C and the mixture was stirred for 1-2 h and n-heptane was added to the reaction mixture. n-heptane (461 kg) was added at 20-25° C. over 1-2 h. The mixture was cooled to 0-5° C., stirred for 2-3 h and centrifuged. The solid obtained was washed with n-heptane (184 kg) and dried under vacuum at 20-25° C. (JT) for 24 h to obtain the product (150 kg). Depending on the exact amount of the solvent, the temperature profile during the crystallization process and seeding, the product is obtained either in crystalline form (A) or in crystalline form (B).

[0148] [Table 4]

[0149] Example 3: Synthesis of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoate: Ethyl 3-(diethoxyphosphoryl)-2-(hydroxyimino)propanoate (105 kg, 1.0 eq.), EtOH (158 kg) and 90% Boc2O in THF (116 kg, 1.2 eq.) were charged into a 2000 L autoclave. A mixture of 20% Pd / C (2.62 kg) and EtOH (131 kg) was prepared in a vessel and added to the autoclave. The vessel was washed with EtOH (105 kg) and this EtOH was added to the autoclave. The pressure in the autoclave was reduced to 50-100 mbar and increased to 0.2 MPa with nitrogen, this process was repeated three times. The pressure in the autoclave was reduced to 50-100 mbar. The pressure in the autoclave was then increased to 0.2 MPa with hydrogen and reduced to 0.02 MPa, this process was repeated three times. The mixture was warmed to 55-65°C by pressurizing the autoclave to 0.4-0.5 MPa within 0.5-1.0 h. The autoclave was pressurized to 1.4-1.5 MPa and the mixture was stirred at 55-65°C for 1.0 h. The hydrogen supply was stopped and the mixture was cooled to 20-25°C. The pressure in the autoclave was reduced to 0.02 MPa. The pressure in the autoclave was then increased to 0.2 MPa with nitrogen and reduced to 0.02 MPa, and this process was repeated three times. The pressure in the autoclave was increased to 0.2 MPa with hydrogen and reduced to 0.02 MPa, and this process was repeated three times. The mixture was warmed to 55-65°C by pressurizing the autoclave to 0.4-0.5 MPa within 0.5-1.0 h. The autoclave was pressurized to 1.4-1.5 MPa and the mixture was stirred at 55-65°C for 6.0 h. The hydrogen supply was stopped and the mixture was cooled to 20-25°C. The pressure in the autoclave was reduced to 0.02 MPa. The pressure in the autoclave was then increased to 0.2 MPa with nitrogen and reduced to 0.02 MPa, and this process was repeated three times. The pressure in the autoclave was increased to 0.2 MPa with hydrogen and reduced to 0.02 MPa, and this process was repeated three times. The mixture was warmed to 55-65°C by pressurizing the autoclave to 0.4-0.5 MPa within 0.5-1.0 h. The autoclave was pressurized to 1.4-1.5 MPa and the mixture was stirred at 55-65°C for 4.0 h. The hydrogen supply was stopped and the mixture was cooled to 20-25°C.The pressure in the autoclave was reduced to 0.02 MPa and increased to 0.2 MPa with nitrogen, and this process was repeated three times. The pressure was released and the mixture was filtered through celite. The cake was washed with EtOH (105 kg) and the solution was concentrated to dryness at 45-55°C (jacket temperature). MTBE (210 kg) was added to the residue and the mixture was concentrated to dryness at 45-55°C (jacket temperature), and this process was repeated two times. The residue was dissolved in MTBE (68.3 kg) and the solution was stirred at 20-30°C for 0.5 h. The solution of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoate in MTBE was used in the next step. HPLC (Method 2): rt=13.6 min.

[0150] Example 4: Synthesis of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid: In a 3000 L reactor, KH2PO4 (19.4 kg) was dissolved in water (1403 kg) at 25-30 °C. Aqueous K2CO3 solution (20%) was added to the mixture at 25-30 °C to adjust the pH value to 6.7-6.9. In a separate vessel, 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)ethyl propanoate (281 kg) was dissolved in MTBE (135 kg) at 25-30 °C and added to the reactor. The vessel was washed with MTBE (30.9 kg) and the washings were added to the reaction mixture. Protease M-SD (3.1 kg, spray-dried form of Protease M, Amano) was dissolved in water (31.0 kg) and added to the mixture at 25-30 °C. The reaction mixture was stirred at 25-30°C for 24-36 h, and the pH value was maintained between 6.7 and 6.9 by the addition of aqueous K2CO3 (20%). Then, aqueous K2CO3 (20%) was added to the mixture to adjust the pH to 7.6-8.0. MTBE (1044 kg) and Celite (6.2 kg) were added to the mixture at 25-30°C, and the mixture was stirred for 0.5 hr. The resulting suspension was filtered, and the filter cake was washed with MTBE (64.5 kg) and water (84.2 kg). The layers were separated, and the organic layer (containing the remaining ester) was set aside. The aqueous layer was extracted twice with MTBE (1044 kg and 519 kg, respectively), cooled to 10-20°C, acidified by dropwise addition of aqueous HCl (18.2%), and the pH was adjusted to 1.4-1.6. The resulting suspension was cooled to -2 to +2°C over 1-2 h, stirred for an additional 0.5 h, and centrifuged. Water (421 kg) was added to the resulting solid, and the suspension was stirred at 0-10°C for 0.5-1.0 h. After centrifugation, the wet solid was washed with cold water (211 kg) and dissolved in 2-propanol (IPA) (842 kg) at 70-75°C. The resulting suspension was filtered, the reactor was washed with hot IPA (421 kg), and the filtrate was combined and concentrated to about 2 vol. at 40-50°C. The filtrate was warmed to 70-75°C, stirred for 0.5 h, cooled to -10 to -15°C over 2-3 h, and stirred for 1 h. The resulting suspension was centrifuged to obtain a white solid, which was dried under vacuum at 35-40°C (JT) for 24 h and cooled to 20-30°C under nitrogen atmosphere to obtain the product (105 kg) in crystalline form (I).

[0151] [Table 5]

[0152] Example 5: Racemization of (S)-ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (by-product of Example 4) ("recycle"): A solution of (S)-ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (approximately 276 kg crude starting material) in MTBE (total weight of starting material and MTBE: 570 kg) was charged into a reactor and concentrated to 1.0-1.5 vol. at 40-50° C. under vacuum. MTBE (1381 kg) was added and concentrated to 1.0-1.5 vol. at 40-50° C. under vacuum. MTBE (1381 kg) was added again and concentrated to 1.0-1.5 vol. at 40-50° C. under vacuum. MTBE (262 kg) was added to the mixture and stirred at 0-5° C. for 15 min. A 20% solution of NaOEt in EtOH (146 kg) was added dropwise within 1-2 h at 0-5° C. and the solution was stirred for another 0.5-1 h at 0-5° C. AcOH (30.4 kg) was added slowly and the mixture was stirred for 0.5 h, MTBE (829 kg) and saturated aqueous NaHCO3 (829 kg) were added at 10-20° C. and the mixture was stirred for 0.5 h. The layers were separated, the organic layer was concentrated to dryness and the residue was dissolved in MTBE (140 kg) to give a solution of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (201 kg crude product) in MTBE, which can be used in the enzymatic racemic resolution described in Example 4.

[0153] Example 6: Enzyme Screening

[0154] [ka]

[0155] 193 hydrolase enzymes were screened according to the following procedure: Each enzyme (10 mg lyophilized enzyme powder or 30 μL liquid enzyme or 10 mg immobilized enzyme) was dissolved in phosphate buffer (500 μL, 0.1 M, pH 7.0) and a solution of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (10 mg) in MTBE (37.5 μL) was added. The mixture was shaken at 25 °C and 210 rpm for 20 h, adjusted to pH 1-2 with aqueous HCl (60 μL, 2 M) and extracted with MTBE (3x0.7 mL). The solution was concentrated in vacuo and the residue was taken up in EtOH (0.8 mL), filtered and analysed by chiral HPLC (Method 2); Retention times: (R)-acid: rt=9.1 min; (S)-acid: rt=23.8 min; (R)-ester: rt=13.7 min; (S)-ester: rt=29.8 min.

[0156] [Table 6]

[0157] [Table 7]

[0158] Example 7: Optimization of enzyme loading and use of co-solvents Condition A : Each enzyme (5 mg of lyophilized enzyme powder) was dissolved in phosphate buffer (250 μL, 0.1 M, pH 7.0) and a solution of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (5 mg) in MTBE (12.5 μL) was added. The mixture was shaken at 250 rpm for 20 h at 30° C. and further treated according to the work-up conditions.

[0159] Condition B : A stock solution of each enzyme (0.5 mg) in phosphate buffer (119 μL, 0.1 M, pH 7.0) was diluted with phosphate buffer (119 μL, 0.1 M, pH 7.0) and a solution of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (5 mg) in MTBE (12.5 μL) was added. The mixture was shaken at 250 rpm for 20 h at 30° C. and further treated according to the work-up conditions.

[0160] Condition C : Stock solutions of each enzyme (0.5 mg) in phosphate buffer (119 μL, 0.1 M, pH 7.0) were prepared by dissolving ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (5 mg) in phosphate buffer (131 μL, 0.1 M, The mixture was shaken at 250 rpm for 20 h at 30° C. and further treated according to the post-treatment conditions.

[0161] Condition D : Solutions of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (25 mg / mL solvent) and each enzyme (1% by weight relative to the ester) in phosphate buffer (0.1 M, pH 7.0) and MTBE (5% total volume) were shaken at 250 rpm for 18 h at 30 °C and further treated according to the work-up conditions.

[0162] Condition E : Solutions of ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (100 mg / mL solvent) and each enzyme (1% by weight relative to the ester) in phosphate buffer (0.1 M, pH 7.0) and MTBE (5% total volume) were shaken at 250 rpm for 18 h at 30 °C and further treated according to the work-up conditions.

[0163] Post-processing conditions : To each vial was added MTBE (1 mL), followed by aqueous HCl (20 μL, 2 M). The mixture was shaken at 200 rpm at 25° C. for 30 min and then centrifuged to obtain separate layers. A portion of the organic layer (0.8 mL) was removed and dried through a MgSO4 pipette into a clean Eppendorf vial. The MTBE was then removed in a Genevac rotary vacuum pump, taken up in EtOH (200 μL) and analyzed by chiral HPLC (Method 2); retention times: (R)-acid: rt=9.1 min; (S)-acid: rt=23.8 min; (R)-ester: rt=13.7 min; (S)-ester: rt=29.8 min.

[0164] [Table 8]

[0165] [Table 9]

[0166] [Table 10]

[0167] [Table 11]

[0168] [Table 12]

[0169] Example 8: Synthesis of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid (1 g scale): To a 50 mL 3-necked round bottom flask (RBF) was added ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxy-phosphoryl)propanoate (1 g, 2.83 mmol) in MTBE (0.5 mL + 0.1 mL MTBE for inlet wash). To this solution was added phosphate buffer (0.1 M, pH 7.0, 9 mL) and the biphasic mixture was heated to 30°C under stirring. To this biphasic mixture was added Amano's Protease M (10 mg in 0.5 mL phosphate buffer) and the solution was stirred for 18 h. The pH of the reaction was maintained between pH 6.8-7.0 by addition of 20% K2CO3(aq). The reaction was adjusted to pH 8.0 and extracted with MTBE (2x10 mL). The aqueous portion was returned to the reaction vessel and adjusted to pH 1.5 with 2 M HCl, at which point rapid precipitation occurred. The suspension was cooled to 0° C. in an ice-water bath and stirred at this temperature for 30 min. The suspension was filtered and diluted with 0.1 M phosphate buffer (pH 1.5, 2 mL) and 0.1 M HCl (4 mL). and dried for 1 h. (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid was obtained as a white solid (420 mg, 45.6%) in 99% ee (Chiral HPLC (Method 3); Retention times: (R)-acid: rt=5.7; (S)-acid: rt=6.6 min; (R)-ester: rt=25.3 min; (S)-ester: rt=28.1 min.). Example 9: Synthesis of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid: Water (3.6 L; 6.6 vol) was added to a 10 L reactor and stirred vigorously. Potassium dihydrogen phosphate (49.0 g; 360 mmol) was added and the pH of the reactor contents was adjusted to pH 6.8 (± 0.2) with 20% (w / v) aqueous potassium carbonate. Ethyl 2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoate (1.55 mmol) was dissolved in MTBE (360 mL) in a 1 L glass bottle and added to the reactor. The glass bottle was washed with MTBE (90 mL) and this solution was added to the reactor. In a 100 mL bottle, Amano's Protease M (6.0 g) was dissolved in purified water (55 mL) and added to the reactor in one go. The bottle was washed with purified water (27.5 mL) and this solution was added to the reactor. The reaction mixture was stirred at 28±2° C. for 8 hours and maintained at pH 6.8 by addition of 20% (w / v) aqueous potassium carbonate. External heating was turned off and the mixture was stirred for an additional 15 hours. The pH was adjusted to 7.8±0.2 with 20% (w / v) aqueous potassium carbonate. Celite (12.0 g) and MTBE (4.1 L) were charged to the reactor and after 10 minutes the reactor contents were filtered through a Büchner funnel. The two-phase mixture was returned to the reactor and the layers were allowed to separate for at least 5 minutes. The organic layer (containing the remaining ester) was stored in a 25 L HDPE drum and the aqueous layer was returned to the reactor. MTBE (4.1 L) was added, the mixture was stirred for 5 minutes, the layers were separated and the aqueous layer was drained. This organic layer was combined with the previous organic layer. The aqueous layer was returned to the reactor and MTBE (2.05 L) was added, the mixture was stirred for 5 minutes, the layers were separated and the aqueous layer was drained. This organic layer was combined with the previous organic layer. The aqueous layer was transferred back to the reactor and adjusted to pH 1.5 by dropwise addition of 18.5% aqueous HCl. The reactor contents were cooled to 3° C.±2° C., gently stirred for 30 min, and filtered through a Buchner funnel. The filter cake was washed with chilled aqueous HCl (0.1 M, 820 mL) and dried on the funnel for 1 h. The cake was further dried under vacuum at 50° C. to constant weight to give (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid in 46% yield, 98% purity (NMR) and 99.9% ee (chiral HPLC (Method 3)).

Claims

1. A method for producing a compound of formula (I) or a salt thereof, comprising the steps of: 【Chemistry 1】 Compound of formula (II) 【Chemistry 2】 (In the formula, R １ and R ２ are, independently of each other, (C １－４ ) alkyl, R ３ represents methyl, ethyl, or n-propyl) with a hydrolase to obtain a compound of formula (I) having an enantiomeric excess (ee) of at least 70%; A method for producing a compound of formula (I) or a salt thereof.

2. The hydrolase is AH002, AH008, AH012, AH016, AH017, AH018, AH019, AH022, AH023, AH025, AH027, AH028, AH032, AH034, AH035, AH036, AH037, AH041, AH042, AH044, AH045, AH047, AH048, AH051, AH052, AH055, AH056, AH057, AH059, AH060, AH061, AH062, CL055, CL067, Protease 2. The method of claim 1, wherein the SM is selected from DSM, EU62, DSM-A1, DSM-A2, DSM-A3, DSM-A6, DSM-B1, DSM-B2, DSM-B3, DSM-B6, DSM-C1, DSM-C2, DSM-C3, DSM-D2 and DSM-D3.

3. A method for producing a compound of formula (I) or a salt thereof, comprising the steps of: 【Transformation 3】 Compound of formula (II) 【Chemistry 4】 (In the formula, R １ and R ２ are, independently of each other, (C １－４ ) alkyl, R ３ represents methyl, ethyl, or n-propyl) with a hydrolase; The hydrolase is AH002, AH008, AH012, AH016, AH017, AH018, AH019, AH022, AH023, AH025, AH027, AH028, AH032, AH034, AH035, AH036, AH037, AH041, AH042, AH044, AH045, AH047, AH048, AH051, AH052, AH055, AH056, AH057, AH059, AH060, AH061, AH062, CL055, CL067, Protease M, EU62, DSM-A1, DSM-A2, DSM-A3, DSM-A6, DSM-B1, DSM-B2, DSM-B3, DSM-B6, DSM-C1, DSM-C2, DSM-C3, DSM-D2 and DSM-D3; A method for producing a compound of formula (I) or a salt thereof.

4. Seratogrel 【Transformation 5】 A method for producing Compound of formula (II) 【Transformation 6】 (In the formula, R １ and R ２ are, independently of each other, (C １－４ ) alkyl, R ３ represents methyl, ethyl or n-propyl.) with a hydrolase to give a compound of formula (I) or a salt thereof having an enantiomeric excess (ee) of at least 70%. 【Transformation 7】 A method for producing seratogrel, comprising the step of obtaining

5. 5. The method of claim 1, 3 or 4, wherein the hydrolase is selected from AH018, AH022, AH023, AH027, AH034, AH044, AH047 and Protease M.

6. 5. The method of claim 1, 3 or 4, wherein the hydrolase is Protease M.

7. 5. The process of claim 1, 3 or 4, which provides a compound of formula (I) having an enantiomeric excess (ee) of at least 96%.

8. R １ and R ２ and each represent ethyl.

9. R ３ 5. The method of claim 1, 3 or 4, wherein represents methyl or ethyl.

10. 5. The method according to claim 1, 3 or 4, wherein the reaction (enzymatic resolution) is carried out in a mixture of water and an organic solvent selected from MTBE, DMSO, toluene and any mixture thereof.

11. 5. The method according to claim 1, 3 or 4, wherein the reaction (enzymatic resolution) is carried out at a pH value between 5.0 and 8.

0.

12. 5. The method of claim 1, 3 or 4, wherein the reaction (enzymatic splitting) is carried out at an enzyme loading of between 0.2% and 10%.

13. Compound of formula (V) 【Transformation 8】 with a base to give a compound of formula (II) 【Chemistry 9】 (In the formula, R １ and R ２ are, independently of each other, (C １－４ ) alkyl, R ３ represents methyl, ethyl or n-propyl; 5. The method of claim 1, 3 or 4, wherein the compound of formula (V) has an enantiomeric excess (ee) of at least 80% and the compound of formula (II) has an enantiomeric excess (ee) of less than 10%.

14. A crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid, characterized by the presence of peaks at the following refraction angles 2θ: 9.8°, 10.3° and 17.5° in the powder X-ray diffraction diagram.

15. 15. The crystalline form of (R)-2-((tert-butoxycarbonyl)amino)-3-(diethoxyphosphoryl)propanoic acid according to claim 14, characterized by an endothermic peak at about 180°C as measured by DSC.

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