Method for preparation and purification of 4-Borono-L-phenylalanine

Abstract

This invention provides a method for preparing and purifying 4-borono-L-phenylalanine that reduces reaction steps, improves reaction efficiency, and maximizes yield. [Solution] A method for preparing 4-borono-L-phenylalanine is provided, comprising the step of reacting 4-halophenylalanine with a Grignard reagent in the presence of an amidine compound or a guanidine compound, without using a protecting group for the amino group of 4-halophenylalanine.

Description

[Technical Field]

[0001] This disclosure relates to a method for preparing and purifying 4-borono-L-phenylalanine. [Background technology]

[0002] 4-Borono-L-phenylalanine is used in boron neutron capture therapy (BNCT). Many methods for preparing 4-Borono-L-phenylalanine are currently known, including those disclosed in U.S. Patent No. US8765997B2 and U.S. Patent Publication No. US2023416280A1.

[0003] US8765997B2 discloses that a phenylcarbanion is formed by the reaction of N-protected-4-iodophenylalanine with n-butyllithium, and that the intermediate compound then attacks a boron ester to form a precursor of 4-borono-L-phenylalanine.

[0004] US2023416280A1 also describes a method for reacting N-protected 4-iodophenylalanine with an isopropylmagnesium chloride-lithium chloride complex (i-PrMgCl·LiCl, also known as the "Turbo Grignard reagent") to form a phenylcarbanion. This intermediate then attacks a boron ester to form a precursor of 4-borono-L-phenylalanine.

[0005] It is evident that conventional methods for preparing 4-borono-L-phenylalanine in the art involve initiating a reaction with at least one starting material having an amino group protecting group. Since such protecting groups must be removed after the initial reaction to produce the target compound, 4-borono-L-phenylalanine, the reaction time is prolonged and the yield is reduced.

[0006] Furthermore, in any reaction method, it is generally necessary to improve efficiency and input molar ratio. In the preparation of 4-boronono-L-phenylalanine, boronating reagents such as the aforementioned boron esters are usually the most expensive components in the preparation of 4-boronono-L-phenylalanine, and the reaction efficiency of the boronating reagent is particularly noteworthy. For example, US8765997B2 discloses that the reaction is carried out under conditions where the molar equivalent of the boronating reagent to N-protected-4-iodo-phenylalanine exceeds 3, and it is desirable to further reduce this molar ratio.

[0007] In conclusion, there exists an urgent problem to be solved in the prior art, which is to provide a method for preparing and purifying 4-boronono-L-phenylalanine that reduces reaction steps, improves reaction efficiency, and maximizes yield.

Summary of the Invention

[0008] Based on the above-mentioned drawbacks of the prior art, the present disclosure provides a method for preparing 4-boronono-L-phenylalanine that does not require a starting material with a protecting group, thereby omitting the deprotection step. For the above purpose, the present disclosure provides a method for preparing 4-boronono-L-phenylalanine including the following steps. a) Reacting 4-halo-phenylalanine with a Grignard reagent in the presence of an organic base to form a reaction mixture. b) Reacting the reaction mixture obtained in step a) with a boronating reagent.

[0009] In one embodiment of the method of the present disclosure, the halogen of the 4-halo-phenylalanine is fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0010] In one embodiment of the method of the present disclosure, the 4-halo-phenylalanine is 4-iodo-phenylalanine.

[0011] In one embodiment of the method of the present disclosure, the Grignard reagent is a linear or branched alkyl magnesium halide of C1~C 15 and is.

[0012] In one embodiment of the method of the present disclosure, the alkyl magnesium halide is isopropyl magnesium chloride, sec-butyl magnesium chloride or trimethylsilylmethyl magnesium chloride.

[0013] In one embodiment of the method of the present disclosure, the Grignard reagent is further combined with a metal halide to form a mixture.

[0014] In one embodiment of the method of the present disclosure, the metal halide is lithium chloride.

[0015] In one embodiment of the method of the present disclosure, the mixture of the Grignard reagent and the metal halide is a turbo Grignard reagent.

[0016] In one embodiment of the method of the present disclosure, the turbo Grignard reagent is an isopropyl magnesium chloride-lithium chloride complex (i-PrMgCl·LiCl).

[0017] In one embodiment of the method of the present disclosure, the organic base is an amidine compound or a guanidine compound.

[0018] In one embodiment of the method of the present disclosure, the amidine compound is represented by formula (I). [Chemical formula] Wherein, R1 to R4 are each independently H, a substituted or unsubstituted C1-C 20 alkyl group and a substituted or unsubstituted C1-C 20 cycloalkyl group, or any two of R1 to R4 are not H and are bonded to each other to form a substituted or unsubstituted ring.

[0019] In one embodiment of the method of the present disclosure, the amidine compound is 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), diminazene, benzamidine, pentamidine, paraniline, or any combination thereof.

[0020] In one embodiment of the method of the present disclosure, the guanidine compound is represented by formula (II). [ka] In the formula, R1 to R5 are each independently H, substituted or unsubstituted C1 to C 20 Alkyl groups and substituted or unsubstituted C1-C 20 Either selected from the group consisting of cycloalkyl groups, or any two of R1 to R5 are not H atoms and are bonded to each other to form a substituted or unsubstituted ring.

[0021] In one embodiment of the method of the present disclosure, the guanidine compound is 1,1,3,3-tetramethylguanidine (TMG), 2-tert-butyl-1,1,3,3-tetramethylguanidine (Barton base), 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene (MTBD), 1,5,7-triazabicyclo[4.4.0]deca-5-ene (TBD), or any combination thereof.

[0022] In one embodiment of the method of the present disclosure, the reaction in step a) is carried out at a temperature in the range of -30°C to 10°C.

[0023] In one embodiment of the method of the present disclosure, the reaction in step a) is carried out such that the molar equivalent of the Grignard reagent to 4-halo-phenylalanine is at least 4.0 and the molar equivalent of the organic base to 4-halo-phenylalanine is at least 1.0.

[0024] In one embodiment of the method of this disclosure, the boronating reagent is a boric acid ester or a boric acid amide.

[0025] In one embodiment of the method of the present disclosure, the boric acid ester is triethyl borate or tributyl borate.

[0026] In one embodiment of the method of the present disclosure, the reaction in step b) is carried out at a temperature in the range of -50°C to 10°C.

[0027] In one embodiment of the method of the present disclosure, the reaction in step b) is carried out such that the molar equivalent of the borylation agent to 4-halo-phenylalanine is at least 0.9.

[0028] In one embodiment of the method disclosed herein, the method for preparing 4-borono-L-phenylalanine further comprises the step of quenching the reaction of step b) with an acid solution.

[0029] In one embodiment of the method of the present disclosure, the acid solution is a strong acid solution. In another embodiment of the method of the present disclosure, the acid solution is a hydrogen chloride or acetic acid solution.

[0030] In one embodiment of the method disclosed herein, the acid solution has a concentration in the range of 1 M to 6 M, and the quench reaction in step c) is carried out at a temperature in the range of 5°C to 25°C.

[0031] The disclosure also provides a method for purifying 4-borono-L-phenylalanine, comprising the step of purifying the 4-borono-L-phenylalanine obtained from the preparation method of the disclosure using a reversed-phase column.

[0032] Therefore, the method of this disclosure enables the use of starting materials that do not have amino group protecting groups in the preparation of 4-borono-L-phenylalanine, thereby reducing the number of reaction steps and improving the yield. [Modes for carrying out the invention]

[0033] The following embodiments are used to illustrate the present disclosure. Those skilled in the art will readily conceive of other advantages and effects of the present disclosure based on the disclosure herein. The present disclosure may also be implemented or applied as described in different embodiments. The present disclosure can be implemented by modifying or changing the following embodiments to suit different aspects and uses without departing from the scope of the present disclosure.

[0034] Furthermore, it should be noted that, as used in this disclosure, the singular forms “a,” “an,” and “the” include multiple subjects unless explicitly and clearly designated to be limited to a single subject. The term “or” is used interchangeably with the term “and / or” unless otherwise clearly indicated in the context.

[0035] As used herein, the terms “comprising,” “comprises,” “including,” and “includes” are used in reference to compositions, methods, and their respective components included in this disclosure and do not exclude the possibility of including elements not explicitly stated.

[0036] This disclosure provides a method for preparing 4-borono-L-phenylalanine, comprising the steps of: a) reacting 4-halo-phenylalanine with a Grignard reagent in the presence of an organic base to form a reaction mixture; and b) reacting the reaction mixture obtained in step a) with a boronating reagent.

[0037] The preparation of 4-Borono-L-phenylalanine involves the addition of a nucleophilic amino group (-NH2) and The amino acid phenylalanine, which has a carboxylic acid (-COOH) functional group, is involved, and the amino group is particularly nucleophilic. Therefore, to avoid off-target reactions, it is necessary to protect at least the amino group of phenylalanine during the nucleophilic reaction. For this reason, conventional methods for preparing 4-borono-L-phenylalanine always use N-protected phenylalanine as the starting material.

[0038] However, surprisingly, this disclosure reveals that the presence of an organic base is related to phenylalanine. We discovered that off-target reactions of boronating reagents to the amino group are significantly reduced. Although not theoretically bound, organic bases may form complexes with phenylalanine, thereby reducing the nucleophilicity of the amino group of phenylalanine and / or preventing the boronating reagent from interacting with the amino group of phenylalanine. As a result, phenylalanine without an amino group protecting group can be used as a starting material for the preparation of 4-borono-L-phenylalanine.

[0039] In one embodiment of the method of the present disclosure, the halogen of the 4-halo-phenylalanine is fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0040] In one embodiment of the method of the present disclosure, the 4-halo-phenylalanine is 4-iodophenylalanine.

[0041] In one embodiment, the Grignard reagent is a group of compounds represented by the formula RMgX, where X is a halide and R is C1-C12. 31 alkyl groups, C1-C 31 These are organic groups such as cycloalkyl groups or aryl groups. Non-limiting examples of Grignard reagents include methylmagnesium chloride and phenylmagnesium bromide.

[0042] In one embodiment of the method disclosed herein, the Grignard reagent is C1-C 15is a linear or branched alkyl magnesium halide, for example, isopropyl magnesium chloride (i-PrMgCl), sec-butyl magnesium chloride and trimethylsilyl methyl magnesium chloride, but is not limited thereto.

[0043] In one embodiment of the method of the present disclosure, the Grignard reagent is further combined with a metal halide to form a mixture.

[0044] In one embodiment of the method of the present disclosure, the metal halide is lithium chloride.

[0045] In one embodiment of the method of the present disclosure, the mixture of the Grignard reagent and the metal halide is a turbo-Grignard reagent.

[0046] In one embodiment, the range of the number of carbon atoms in the present disclosure can extend from the lower limit to the upper limit. For example, C1~C 31 refers to the number of carbon atoms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31.

[0047] In one embodiment, the turbo-Grignard reagent is an isopropyl magnesium chloride-lithium chloride complex (i-PrMgCl·LiCl).

[0048] Other examples of the Grignard reagent are well known in the art, and those skilled in the art can easily select an appropriate Grignard reagent as needed.

[0049] In one embodiment of the method of the present disclosure, the organic base is an amidine compound or a guanidine compound.

[0050] In one embodiment, the amidine compound is represented by formula (I).

Chemical formula

[0051] In one embodiment, the guanidine compound is represented by formula (II). [ka] In the formula, R1 to R5 are each independently H, substituted or unsubstituted C1 to C 20 Alkyl groups, and substituted or unsubstituted C1-C 20 Either selected from the group consisting of cycloalkyl groups, or any two of R1 to R5 are not H atoms and are bonded to each other to form a substituted or unsubstituted ring.

[0052] In one embodiment, the substituent is selected from an alkoxy group, an azo group, an amino group, and any combination thereof.

[0053] In one embodiment, the substituted or unsubstituted ring is a 3- to 10-membered ring.

[0054] In one embodiment, the amidine compound includes, but is not limited to, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), diminazene, benzamidine, pentamidine, paraniline, or any combination thereof.

[0055] In one embodiment, the guanidine compound includes, but is not limited to, 1,1,3,3-tetramethylguanidine (TMG), 2-tert-butyl-1,1,3,3-tetramethylguanidine (Barton base), 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene (MTBD), 1,5,7-triazabicyclo[4.4.0]deca-5-ene (TBD), or any combination thereof.

[0056] In one embodiment of the method of the present disclosure, the reaction in step a) is carried out at a temperature in the range of -30°C to 10°C such that the molar equivalent of the Grignard reagent to 4-halo-phenylalanine is at least 4.0 and the molar equivalent of the organic base to 4-halo-phenylalanine is at least 1.0.

[0057] In one embodiment, the reaction of step a) is carried out at a temperature in the range of -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, or any two of these values.

[0058] In one embodiment, the reaction in step a) is carried out with a molar equivalent of the Grignard reagent to 4-halo-phenylalanine in the range of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or any two of these values.

[0059] In one embodiment, the reaction in step a) is carried out with a molar equivalent of the organic base to 4-halophenylalanine ranging from 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or any two of these values.

[0060] In one embodiment, step a) can be allowed to react for 10 minutes to 12 hours, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or any two of these values.

[0061] In one embodiment, the boronating reagent is a boric acid ester or a boric acid amide.

[0062] In one embodiment, the boric acid ester is a trialkyl borate such as triethyl borate or tributyl borate.

[0063] In one embodiment, the reaction in step b) is carried out at a temperature in the range of -50°C to 10°C, such that the molar equivalent of the borylation agent relative to 4-halo-phenylalanine is at least 0.9.

[0064] In one embodiment, the reaction in step b) is carried out at a temperature in the range of -50°C, -45°C, -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, or any two of these values.

[0065] In one embodiment, the reaction in step b) is carried out with a molar equivalent of the boronating agent relative to 4-halophenylalanine in the range of 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, or any two of these values.

[0066] In one embodiment, step b) can be made to react for a range of 5 minutes to 24 hours, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or any two of these values.

[0067] In one embodiment of the method disclosed herein, the method for preparing 4-borono-L-phenylalanine further comprises the step of quenching the reaction of step b) with an acid solution.

[0068] In one embodiment of the method disclosed herein, the acid solution is an acetic acid solution.

[0069] In one embodiment of the method disclosed herein, the acid solution is a strong acid solution such as an aqueous solution of hydrogen chloride, i.e., an HCl(aq.) solution.

[0070] In one embodiment of the method disclosed herein, the aqueous hydrogen chloride solution has a concentration in the range of 1 M to 6 M, and the quench reaction in step c) is carried out at a temperature in the range of 5°C to 25°C.

[0071] In one embodiment, the concentration of the aqueous hydrogen chloride solution is in the range of 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M, 4.5 M, 5.0 M, 5.5 M, 6.0 M, or any two of these values.

[0072] In one embodiment, the quench reaction in step c) is carried out at a temperature in the range of 5°C, 10°C, 15°C, 20°C, 25°C, or any two of these values.

[0073] The disclosure also provides a method for purifying 4-borono-L-phenylalanine, comprising the step of purifying the 4-borono-L-phenylalanine obtained from the preparation method of the disclosure using a reversed-phase column.

[0074] As used herein, the term “reverse-phase column” refers to a column having a non-polar stationary phase and used with a polar mobile phase. Non-limiting examples of reverse-phase columns include reverse-phase C18 columns such as the Acclaim® 120C18 column and columns containing SEPABEADS® SP850 resin. In the context of this disclosure, columns containing activated carbon are also considered reverse-phase columns.

[0075] As used herein, the term "bed" refers to the volume of eluate relative to the amount of chromatographic resin used. For example, passing 100 mL of resin through 100 mL of eluate and collecting the resulting volume is defined as one bed. Therefore, the chromatographic flow rate can be defined as a specific number of beds per time.

[0076] Boron exists in nature as an isotope. 10 B and 11 It exists as B. In this disclosure, for demonstration purposes, in the preparation of 4-borono-L-phenylalanine, a naturally occurring boron-containing compound and essentially 10 We utilize both compounds containing only B (i.e., boron with 5 neutrons). 10 The term "B" essentially refers to boron isotopes. 10 This term is used to refer to compounds containing only B.

[0077] This disclosure shows that an unprotected starting material, such as 4-halo-phenylalanine, reacts with a Grignard reagent in the presence of an amidine or guanidine compound to form an intermediate having a carbanion group, which then reacts with a boron ester to obtain 4-borono-L-phenylalanine. [Examples]

[0078] Various characteristics and effectiveness will be explained by the following examples. The examples described are used to illustrate the characteristics of this disclosure and are not limited to those described in any particular example.

[0079] Materials and Reagents 4-IL-phenylalanine, manufactured by Combi-Blocks. THF, manufactured by MACRON Corporation. DBU, manufactured by MACKLIN. DBN, manufactured by Angene Corporation. Burton base, manufactured by Angene Corporation. MTBD, manufactured by Angene. 1.3M iPrMgCl·LiCl THF solution, manufactured by Aladdin 2.0M iPrMgCl THF solution, manufactured by Acros. Tributyl borate, manufactured by TCI. Triethyl borate, manufactured by TCI. SepaBeads (trademark) SP850 resin, manufactured by Mitsubishi Chemical Group. NaOH, manufactured by Honeywell. HCl, manufactured by ALL-IN-LINE-CHEMICALS Na2CO3, manufactured by Nature's Workshop. Boric acid - 10 B, manufactured by Aladdin. 1-Butanol, manufactured by Kanto Chemical Co., Ltd. Toluene, manufactured by JTbaker. Other materials and reagents were purchased from Sigma-Aldrich or equivalent suppliers.

[0080] Tributyl borate - 10 Synthesis of B (RD202414-250205)

[0081] Isotopes 10 Tributyl borate containing B (i.e., tributyl borate- 10 B) is synthesized using conventional methods. Simply put, a Dean-Stark trap and capacitor are set up. Boric acid -10 B (21.4 g, 0.35 mol), 1-butanol (80.4 g, 1.09 mol), and toluene (35 g) were placed in a 250 mL reactor and heated under reflux for 0.5 hours. After distillation, water was collected. After removing the water to complete the reaction, the residue remaining in the reactor was distilled under reduced pressure to obtain tributyl borate as a clear liquid. 10 58.2 g of B was obtained (yield 72.5%). The purity was confirmed to be 99.0% by GC (gas chromatography).

[0082] Example 1 (RD202414-250404) [ka]

[0083] Under an inert nitrogen atmosphere at 25°C, 29.1 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.1 mol) was added to 100 g of tetrahydrofuran (THF). Next, 45.6 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 0.3 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0084] 295.0 g of i-PrMgCl·LiCl solution (1.3 M, 0.4 mol in THF) was added to the primary reactor, cooled to -5°C to 5°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -40°C to -30°C, and then tributyl borate was added to the reaction mixture. 10 23.0 g (0.1 mol) of B was added, and the reaction was continued for another 2 hours to obtain an intermediate mixture.

[0085] 256.0 g of a 10% HCl (aq.) solution was added to a secondary reactor and cooled to 5°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 13.1 g (yield 62.9%) of crude 4-vorono-L-phenylalanine as a pale yellow solid. The purity was confirmed to be 98.1% by UPLC (ultrahigh performance liquid chromatography). 1 H NMR(600MHz,D2O,CF3COOH): δ7.68(d,J=8.0Hz,2H), 7.28(d,J=8.0Hz,2H), 4.29(dd,J= 7.7,5.7Hz,1H), 3.30(dd,J=14.6,5.7Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 10 Calculated value for BNO4 [M+H] + 209.09, measured value 209.56.

[0086] Example 2 (RD202414-250507) [ka]

[0087] Under an inert nitrogen atmosphere at 25°C, 29.1 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.1 mol) was added to 99.8 g of tetrahydrofuran (THF). Next, 45.6 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 0.3 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0088] 292.0 g of i-PrMgCl·LiCl solution (1.3 M, 0.4 mol in THF) was added to the primary reactor, cooled to -5°C to 5°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -45°C to -35°C, and then 23.0 g (0.1 mol) of tributyl borate was added to the reaction mixture, and the reaction was continued for a further 2 hours to obtain an intermediate mixture.

[0089] 248.0 g of a 10% HCl (aq.) solution was added to a secondary reactor and cooled to 5°C-10°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 13.1 g (yield 62.6%) of crude 4-vorono-L-phenylalanine as a pale yellow solid. The purity was confirmed to be 96.9% by UPLC. 1 H NMR(600MHz,D2O,CF3COOH): δ7.68(d,J=8.0Hz,2H), 7.28(d,J=8.0Hz,2H), 4.29(dd,J= 7.7,5.7Hz,1H), 3.30(dd,J=14.6,5.7Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 Calculated value for BNO4 [M+H] + 210.09, measured value 210.56.

[0090] By comparing Example 1 and Example 2 described above, 10 It has been shown that the reaction using a borylation reagent for the B isotope and the reaction using a borylation reagent for natural boron are interchangeable. In other words, in the method of this disclosure, the preparation of 4-borono-L-phenylalanine 10 No observable differences were observed whether B isotope or natural boron was used.

[0091] Example 3 (RD202414-250607)

[0092] Under an inert nitrogen atmosphere at 25°C, 8.7 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.03 mol) was added to 30 g of tetrahydrofuran (THF). Next, 14.0 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 0.09 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0093] 60.0 g of i-PrMgCl solution (2.0 M, 0.12 mol in THF) was added to the primary reactor, cooled to -5°C to 5°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -45°C to -30°C, and then 6.9 g (0.03 mol) of tributyl borate was added to the reaction mixture, and the reaction was continued for a further 2 hours to obtain an intermediate mixture.

[0094] 83.0 g of a 10% HCl (aq.) solution was added to a secondary reactor and cooled to 5°C-10°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 2.7 g (yield 43.8%) of crude 4-borono-L-phenylalanine as a pale yellow solid. The purity was confirmed to be 96.3% by UPLC. 1 H NMR(600MHz,D2O,CF3COOH): δ7.68(d,J=8.0Hz,2H), 7.28(d,J=8.0Hz,2H), 4.29(dd,J= 7.7,5.8Hz,1H), 3.30(dd,J=14.6,5.7Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 Calculated value for BNO4 [M+H] + 210.09, measured value 210.54.

[0095] Example 4 (RD202414-7250609)

[0096] Under an inert nitrogen atmosphere at 25°C, 8.7 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.03 mol) was added to 30 g of tetrahydrofuran (THF). Next, 15.4 g of 2-tert-butyl-1,1,3,3-tetramethylguanidine (Barton's base, 0.09 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0097] 100.5 g of i-PrMgCl·LiCl solution (1.3 M, 0.12 mol in THF) was added to the primary reactor, cooled to -5°C to 5°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -40°C to -30°C, and then 6.9 g (0.03 mol) of tributyl borate was added to the reaction mixture, and the reaction was continued for a further 2 hours to obtain an intermediate mixture.

[0098] 83.0 g of a 10% HCl (aq.) solution was added to a secondary reactor and cooled to 5°C-10°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 2.0 g (yield 32.3%) of crude 4-vorono-L-phenylalanine as a pale yellow solid. The purity was confirmed to be 94.0% by UPLC. 1 H NMR (600MHz, D2O, CF3COOH): δ7.68(d,J=8.0Hz,2H), 7.28(d,J=8.0Hz,2H), 4.29(dd,J= 7.7,5.8Hz,1H), 3.30(dd,J=14.6,5.7Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 Calculated value for BNO4 [M+H] + 210.09, measured value 210.50.

[0099] Example 5 (RD202414-250615)

[0100] Under an inert nitrogen atmosphere at 25°C, 8.7 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.03 mol) was added to 30.0 g of tetrahydrofuran (THF). Next, 11.3 g of 1,5-diazabicyclo(4.3.0)non-5-ene (DBU, 0.09 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0101] 96.8 g of i-PrMgCl·LiCl solution (1.3 M, 0.12 mol in THF) was added to the primary reactor, cooled to -5°C to 10°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -45°C to -30°C, and then 6.9 g (0.03 mol) of tributyl borate was added to the reaction mixture, and the reaction was continued for a further 2 hours to obtain an intermediate mixture.

[0102] 83.0 g of a 10% HCl (aq.) solution was added to a secondary reactor and cooled to 5°C-10°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 3.5 g (yield 55.9%) of crude 4-vorono-L-phenylalanine as a pale yellow solid. The purity was confirmed to be 96.7% by UPLC. 1 H NMR (600MHz, D2O, CF3COOH): δ7.68(d,J=8.0Hz,2H), 7.28(d,J=8.0Hz,2H), 4.30(dd,J= 7.7,5.8Hz,1H), 3.30(dd,J=14.6,5.8Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 Calculated value for BNO4 [M+H] + 210.09, measured value 210.53.

[0103] Example 6 (RD202414-250618)

[0104] Under an inert nitrogen atmosphere at 25°C, 4.4 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.015 mol) was added to 15 g of tetrahydrofuran (THF). Next, 6.91 g of 7-methyl-1,5,7-triazabicyclo(4.4.0)deca-5-ene (MTBD, 0.045 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0105] 48.9 g of i-PrMgCl·LiCl solution (1.3 M, 0.06 mol in THF) was added to the primary reactor, cooled to -10°C to 5°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -40°C to -30°C, and then 3.4 g (0.015 mol) of tributyl borate was added to the reaction mixture, and the reaction was continued for a further 2 hours to obtain an intermediate mixture.

[0106] 42.0 g of a 10% HCl (aq.) solution was added to a secondary reactor and cooled to 5°C-10°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 1.1 g (yield 33.8%) of crude 4-vorono-L-phenylalanine as a pale yellow solid. The purity was confirmed to be 95.2% by UPLC. NMR (600 MHz, D2O, CF3COOH): δ 7.68 (dd, J=8.1 Hz, 2 H), 7.28 (d, J=8.0 Hz, 2 H), 4.29 (dd, J=7.7, 5.7 Hz, 1 H), 3.30 (dd, J=14.6, 5.8 Hz, 1 H), 3.17 (dd, J=14.6, 7.7 Hz, 1 H), ESI-MS: C9H 13 Calculated value for BNO4 [M+H] + 210.09, measured value 210.53.

[0107] Example 7 (RD202414-240812)

[0108] Under an inert nitrogen atmosphere at 25°C, 8.7 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 0.03 mol) was added to 30 g of tetrahydrofuran (THF). Next, 6.8 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 0.045 mol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0109] 97.4 g of i-PrMgCl·LiCl solution (1.3 M, 0.06 mol in THF) was added to the primary reactor, cooled to -10°C to 10°C, and then the solution mixture was added dropwise to the primary reactor to start the reaction. After 0.5 hours, the primary reactor was cooled to -40°C to -30°C, and then tributyl borate was added to the reaction mixture. 10 6.9 g (0.03 mol) of B was added, and the reaction was continued for another 2 hours to obtain an intermediate mixture.

[0110] 66.0 g of 10% HCl (aq.) solution was added to the secondary reactor and cooled to 5°C-10°C. The intermediate mixture was then added dropwise to the secondary reactor to quench the reaction. The pH was adjusted to the isoelectric point, and the product was precipitated. The mixture was filtered to obtain 4.1 g (yield 65.4%) of crude 4-vorono-L-phenylalanine as a pale yellow solid. 10 We obtained sample B. UPLC confirmed its purity to be 98.7%. 1 H NMR(600MHz,D2O,CF3COOH): δ7.68(d,J=8.1Hz,2H), 7.28(d,J=8.0Hz,2H), 4.30(dd,J= 7.7,5.8Hz1H), 3.30(dd,J=14.6,5.8Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 10 The calculated value for BNO4 is [M+H] + 209.09, and the measured value is 209.49.

[0111] Example 8 (RD202414-240922) [ka]

[0112] Under an inert nitrogen atmosphere at 25°C, 0.58 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 2 mmol) was added to 5.0 g of tetrahydrofuran (THF). Next, 0.94 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 6.15 mmol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0113] After cooling the solution mixture to -30°C, 7.7 mL (10 mmol) of i-PrMgCl·LiCl solution (1.3 M in THF) was added dropwise to the solution mixture to initiate the reaction. After 1.5 hours, 0.32 g (2.2 mmol) of triethyl borate was added to the reaction mixture, and the reaction was continued for a further 20 minutes. A sample of the reaction mixture was analyzed using UPLC, and 4-Borono-L-phenylalanine (BPA) was identified. The UPLC results are shown in Table 1 below.

[0114] [Table 1]

[0115] The reaction mixture was heated again to 25°C, and then 1M acetic acid was added to the reaction mixture to adjust the pH to 7, thereby quenching the reaction.

[0116] The reaction mixture, with its pH adjusted, was concentrated under reduced pressure and extracted with water and ethyl acetate solution. The aqueous phase containing the target product was recovered.

[0117] The pH of the recovered aqueous phase was adjusted to its isoelectric point, and the product was precipitated. The precipitate was then filtered and dried under reduced pressure. A total of 0.15 g (0.7 mmol, 35% yield) of 4-vorono-L-phenylalanine product was isolated.

[0118] Example 9 (RD202414-240927)

[0119] Under an inert nitrogen atmosphere at 25°C, 0.58 g of 4-iodo-L-phenylalanine (4-IL-phenylalanine, 2 mmol) was added to 5 g of tetrahydrofuran (THF). Next, 0.94 g of 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU, 6.15 mmol) was added dropwise to the THF solution of 4-iodo-L-phenylalanine to obtain a solution mixture.

[0120] 6.2 mL (8 mmol) of i-PrMgCl·LiCl solution (1.3 M in THF) was added to the reactor, cooled to -10°C, and then the solution mixture was added dropwise to the reactor to initiate the reaction. After 2 hours, 0.66 g (2.2 mmol) of tributyl borate was added to the reaction mixture and the reaction was continued for another 24 hours. A sample of the reaction mixture was analyzed using UPLC, and 4-Borono-L-phenylalanine (BPA) was identified. The UPLC results are shown in Table 2 below.

[0121] [Table 2]

[0122] Example 10

[0123] Amino acids are typically purified using ion exchange resins following absorption, washing, and elution procedures. Simply put, a crude solution containing amino acids is passed through a column packed with ion exchange resin, causing the amino acids to bind to charged functional groups on the resin. The column is then washed to remove impurities and unbound molecules. Next, the eluate is passed through the column to release the amino acids from the resin, allowing the purified amino acids to flow out and be recovered.

[0124] Accordingly, in order to find the optimal method for purifying the crude 4-borono-L-phenylalanine product obtained by the 4-borono-L-phenylalanine preparation method of this disclosure, various combinations of Relite® JA310 (weakly basic anionic resin), Diaion® CRB05 (boron chelate resin), Diaion® SK1B (strongly acidic cationic resin), Relite® WK60L (weakly acidic cationic resin), and SEPABEADS® SP850 (non-polar resin) were investigated. The purification results are shown in Table 3 below.

[0125] Specifically, batch numbers RD202414-241220, RD202414-240204, and RD202414-240604 of the crude 4-vorono-L-phenylalanine product were produced according to the procedure described in Example 2 (RD202414-250507), but the amounts of 4-iodo-L-phenylalanine (4-IL-PA), tributyl borate (B(OBu)3), tetrahydrofuran (THF), 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU), and i-PrMgCl·LiCl reagent were adjusted as shown in Table 3. These crude products were separated into an organic phase (ORG) and an aqueous phase (AQ), and each phase was recovered and concentrated for purification using different column combinations.

[0126] The inputs and products of each purification step were examined using UPLC (223 nm), and the recovery rates after each purification step were calculated for various chemicals, including 4-Borono-L-phenylalanine (BPA) and impurities such as L-phenylalanine (Phe), DBU, and 4-iodo-L-phenylalanine (4-IL-PA). Recovery rates may exceed 100% due to quantitative differences before and after the purification step.

[0127] [Table 3]

[0128] Example 11 (RD202414-250411)

[0129] However, further trials surprisingly showed that purification using only one column yielded better results than any of the purification methods shown in Example 10. Briefly, a purification column containing SEPABEADS® SP850 resin was prepared by packing a glass column with 100 mL of SP850 resin and washing it with purified water. 8.0 g of 4-Borono-L-phenylalanine- 10 Crude product B (RD202414-250404) was dissolved in 392.0 g of 2% HCl solution. Then, 4-Borono-L-phenylalanine-10 Solution B was passed through a purification column packed with SEPABEADS (trademark) SP850 resin, followed by washing with purified water. The flow rate was approximately 1-3 beds / hour. The recovery standard was determined by UPLC, and 516.5 g of eluate was collected in a 1 L glass bottle. 10% Na2CO3 was added to the eluate to adjust the pH. The pH was adjusted to the isoelectric point to precipitate the target product, and the mixture was stirred at room temperature for 16 hours. The resulting mixture was filtered to obtain 6.4 g (80.3% yield) of 4-Borono-L-phenylalanine as a white solid. 10 We obtained sample B. UPLC confirmed its purity to be 99.7%. 1 H NMR (600MHz, D2O, CF3COOH): δ7.68(d,J=8.1Hz,2H), 7.28(d,J=8.0Hz,2H), 4.29(dd,J= 7.7,5.8Hz,1H), 3.30(dd,J=14.6,5.7Hz,1H), 3.17(dd,J=14.6,7.7Hz,1H), ESI-MS:C9H 13 10 Calculated value for BNO4 [M+H] + 209.09, measured value 209.56. Melting point: 275~280℃ (decomposition).

[0130] While some embodiments of this disclosure have been described in detail above, those skilled in the art can make various modifications and changes to the specific embodiments shown without substantially departing from the teachings and merits of this disclosure. Such modifications and changes are included within the scope of this disclosure as set forth in the appended claims.

Claims

1. a) A step of reacting 4-halo-phenylalanine with a Grignard reagent in the presence of an organic base to form a reaction mixture, b) A step of reacting the reaction mixture obtained in step a) with a boronating reagent, including, A method for preparing 4-Borono-L-phenylalanine.

2. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the halogen of 4-halo-phenylalanine is fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

3. The Grignard reagent is C １ ~C １５ A method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the linear or branched alkylmagnesium halide is...

4. Said C １ ~C １５ The method for preparing 4-borono-L-phenylalanine according to claim 3, wherein the linear or branched alkylmagnesium halide is isopropylmagnesium chloride, sec-butylmagnesium chloride, or trimethylsilylmethylmagnesium chloride.

5. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the Grignard reagent is further combined with a metal halide.

6. The method for preparing 4-borono-L-phenylalanine according to claim 5, wherein the metal halide is lithium chloride.

7. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the organic base is an amidine compound or a guanidine compound.

8. The aforementioned amidine compound is represented by formula (I), 【Chemistry 1】 wherein, R １ ~R ４ each independently represents H, a substituted or unsubstituted C １ ~C ２０ alkyl group, or a substituted or unsubstituted C １ ~C ２０ cycloalkyl group, or any two of said R １ ~R ４ are not H and are bonded to each other to form a substituted or unsubstituted ring The method for preparing 4-borono-L-phenylalanine according to claim 7.

9. The method for preparing 4-borono-L-phenylalanine according to claim 8, wherein the amidine compound is 1,8-diazabicyclo[5.4.0]undeca-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, diminazene, benzamidine, pentamidine, paraniline, or any combination thereof.

10. The guanidine compound is represented by formula (II), 【Chemistry 2】 In the formula, R １ ~R ５ Each of these independently represents H, a substituted or unsubstituted C. １ ~C ２０ Alkyl groups and substituted or unsubstituted C １ ~C ２０ Selected from the group consisting of cycloalkyl groups, or the R １ ~R ５ Any two of these are not H, and they bond to each other to form a substituted or unsubstituted ring. The method for preparing 4-borono-L-phenylalanine according to claim 7.

11. The method for preparing 4-vorono-L-phenylalanine according to claim 10, wherein the guanidine compound is 1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 7-methyl-1,5,7-triazabicyclo[4.4.0]deca-5-ene, 1,5,7-triazabicyclo[4.4.0]deca-5-ene, or any combination thereof.

12. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the reaction in step a) is carried out at a temperature in the range of -30°C to 10°C.

13. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the reaction in step a) is carried out such that the molar equivalent of the Grignard reagent to the 4-halo-phenylalanine is at least 4.0 and the molar equivalent of the organic base to the 4-halo-phenylalanine is at least 1.

0.

14. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the boronating reagent is a boric acid ester or a boric acid amide.

15. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the reaction in step b) is carried out at a temperature in the range of -50°C to 10°C.

16. The method for preparing 4-borono-L-phenylalanine according to claim 1, wherein the reaction in step b) is carried out such that the molar equivalent of the boronating reagent to the 4-halo-phenylalanine is at least 0.

9.

17. c) A method for preparing 4-borono-L-phenylalanine according to claim 1, further comprising the step of quenching the reaction of step b) with an acid solution.

18. A method for purifying 4-borono-L-phenylalanine, comprising the step of purifying 4-borono-L-phenylalanine obtained from any one of claims 1 to 17 using a reversed-phase column.

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