Preparation method and application of an intermediate of 4-boron-10 acid-L-phenylalanine

Abstract

The present invention provides a preparation method and application of an intermediate of 4-boron-10-acid-L-phenylalanine. The structural formula of the intermediate is shown in Formula III. The compound of Formula III is prepared by contacting and reacting the compound of Formula II with the compound of Formula I under the action of a Grignard reagent. 4-boron-10-acid-L-phenylalanine is obtained by deprotecting the compound of Formula III, wherein R1 is a straight-chain or branched-chain alkyl having 1 to 10 carbon atoms, R2 is any one selected from H or an alkyl protecting group, and R3 and R4 are each independently selected from any one of H, tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, phthaloyl, p-toluenesulfonyl, and trityl. The preparation method of 4-boron-10-acid-L-phenylalanine in this application has the advantages of low cost, short reaction time, high yield, simple operation, controllable risk, and suitability for large-scale industrial production, etc.

Description

Technical Field

[0001] The present invention belongs to the field of organic chemistry, and particularly relates to a preparation method and application of an intermediate of 4-boron-10-acid-L-phenylalanine. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) is a new type of radiotherapy method. It utilizes the accumulation of boron-10 ( 10 10B) isotope in tumor cells, and then initiates a nuclear reaction through neutron irradiation to generate high-energy particles. These particles release energy within the cells, destroying the DNA of tumor cells, thereby achieving the therapeutic purpose. The advantage of BNCT lies in its precise killing of tumor cells and protection of normal cells, because the nuclear reaction mainly occurs in boron-containing tumor cells, causing less damage to surrounding normal tissues.

[0003] As a medicament for BNCT, a boron-containing compound in which a boron atom or a boron atom group is introduced into the basic skeleton is required. 4-boron-10-acid-L-phenylalanine ( 10 B-BPA) is the second-generation boron drug, which was launched in Japan in 2020. Its relative biological effectiveness coefficient is much higher than that of sodium dodecaborane disulfide ( 10 B-BSH), it is easy to cross the blood-brain barrier, and has fewer side effects. 10 B-BPA is taken up as a mimic of phenylalanine by LAT1, a type of amino acid transporter. In cancer cells, the expression of LAT1 is overexpressed, so 10 B-BPA is easily accumulated, and this property is utilized for the treatment of cancer.

[0004] In the prior art, limited by the supply situation of boron-10 reagents, currently 10 Most of the mainstream synthesis routes of B-BPA use boron-10 acid ester as the raw material, and use n-butyllithium or Grignard reagent to introduce the parent nucleus. For example, the following synthesis route was reported in CN110498810B:

[0005]

[0006] The reported reaction temperature is -30°C, and a large amount of Grignard reagent and tributyl borate are required during the reaction process, and the production cost is relatively high. In addition, many patent literatures have reported 10The preparation methods of B-BPA all have a series of problems such as high cost, complex reaction operation and post-treatment process, many side reactions, long reaction time, harsh reaction temperature, and low yield. Therefore, it is necessary to develop a preparation method of 4-boron-10-acid-L-phenylalanine with low cost, mild reaction conditions, short reaction time, high yield, stable productivity and conducive to industrial production. Summary of the Invention

[0007] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a preparation method and application of an intermediate of 4-boron-10-acid-L-phenylalanine, which is used to solve a series of problems in the prior art such as complex preparation process of 4-boron-10-acid-L-phenylalanine, many side reactions, long reaction time, harsh reaction temperature, and low yield.

[0008] To achieve the above purpose and other related purposes, the present invention is obtained through the following technical solutions.

[0009] The first aspect of the present invention provides a pinacol boron-10 acid ester derivative, and the structural formula of the pinacol boron-10 acid ester derivative is shown in Formula I:

[0010] ;

[0011] Wherein, R 1 is a straight-chain or branched-chain alkyl group with 1 to 10 carbon atoms; preferably, R 1 is a straight-chain or branched-chain alkyl group with 1 to 6 carbon atoms.

[0012] Further preferably, the R 1 is any one selected from methyl, ethyl, propyl, isopropyl, n-butyl, and isobutyl.

[0013] In some specific embodiments, the R 1 is isopropyl.

[0014] The second aspect of the present invention provides a preparation method of a pinacol boron-10 acid ester derivative, which is prepared by heating and refluxing boron-10 acid, pinacol and a monoalcohol in the presence of a reaction solvent; the monoalcohol is a straight-chain or branched-chain alkyl alcohol with 1 to 6 carbon atoms, and the reaction solvent is one or more selected from benzene series compounds with 6 to 10 carbon atoms or alkanes with 6 to 12 carbon atoms.

[0015] Preferably, the monoalcohol is any one of methanol, ethanol, isopropanol, propanol, n-butanol, 2-methyl-1-propanol, and 2-butanol.

[0016] Preferably, the reaction solvent is one or more of benzene, toluene, chlorobenzene, n-heptane, and n-dodecane. More preferably, the reaction solvent is an azeotrope of water.

[0017] Further preferably, the reaction solvent is toluene.

[0018] In some specific embodiments of the present application, toluene is selected as the reaction solvent. Toluene serves as a water separator in this reaction process. By heating under reflux, the water generated in the reaction is azeotropically distilled out, so that the water in the reaction reaches equilibrium, promoting the reaction and increasing the yield of the product.

[0019] Preferably, the molar ratio of boron-10 acid, pinacol and monoalcohol is 1:(0.9 - 1.5):(1 - 6).

[0020] Preferably, based on the addition amount of 1 g of boron-10 acid in the reaction, the addition amount of the reaction solvent is 1 - 50 mL.

[0021] Further preferably, the molar ratio of boron-10 acid, pinacol and monoalcohol is 1:(0.9 - 1.1):(3 - 6).

[0022] Further preferably, based on the addition amount of 1 g of boron-10 acid in the reaction, the addition amount of the reaction solvent is 4 - 6 mL.

[0023] Preferably, the reaction time is 2 - 24 h.

[0024] The third aspect of the present invention provides a method for preparing an intermediate compound. The compound of formula II reacts with the above-mentioned pinacol boron-10 acid ester derivative under the action of a Grignard reagent to prepare the intermediate compound, and the structure of the intermediate compound is as shown in formula III.

[0025] , ;

[0026] The synthetic route of the compound of formula III is as follows:

[0027]

[0028] The Grignard reagent is one or more selected from isopropylmagnesium chloride-lithium chloride, isopropylmagnesium bromide-lithium chloride, methylmagnesium chloride, and methylmagnesium bromide.

[0029] Preferably, the Grignard reagent is isopropylmagnesium chloride-lithium chloride.

[0030] The X is Br or I.

[0031] The R 2 is any one selected from H or an alkyl protecting group of C1 - C10.

[0032] The R 3 and R 4Each independently selected from any one of H, tert-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, phthaloyl, p-toluenesulfonyl, trityl.

[0033] Preferably, the R 2 is tert-butyl, and the R 3 and R 4 Each independently selected from any one of H or tert-butoxycarbonyl.

[0034] The molar ratio of the compound of formula II, boron-10 borate and Grignard reagent is 1:(0.9~2):(1~3).

[0035] Preferably, the molar ratio of the compound of formula II, boron-10 borate and Grignard reagent is 1:(0.9~1.05):(1~1.3).

[0036] The preparation method further includes a reaction solvent, and the reaction solvent is any one selected from tetrahydrofuran, methyltetrahydrofuran, dioxane, cyclopentyl methyl ether, methyl tert-butyl ether, petroleum ether, benzene or toluene.

[0037] The preparation method further includes an acid quenching step, and the acid is one or more selected from formic acid, acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, oxalic acid or tartaric acid.

[0038] Preferably, the acid used in the acid quenching step is glacial acetic acid, and the temperature of the acid quenching is 0~5°C.

[0039] Preferably, based on 1 mol of the compound of formula III, the addition amount of the acid is 3~6 mol.

[0040] Preferably, the compound of formula II is obtained by protecting the amino and carboxyl groups of L-phenylalanine and halogenating the H at its 4-position.

[0041] Preferably, the preparation method adopts the following one-step process or two-step process:

[0042] 1) One-step process: First add the compound of formula II, the reaction solvent and the pinacol boron-10 borate derivative, add the Grignard reagent at a reaction system temperature of -30~10°C, and react at this temperature for 1~5 h; finally quench with acid; preferably, the reaction system temperature is 0~5°C and the reaction time is 1~2 h;

[0043] 2) Two-step process: a) First, add the compound of formula II and a reaction solvent, add a Grignard reagent at a reaction system temperature of -30 to 10 °C, and react at this temperature for 0.5 to 2 h; b) Add the pinacol boronic acid-10 ester derivative at a reaction system temperature of -30 to 10 °C, and react at this temperature for 1 to 5 h; finally, quench with an acid; preferably, the reaction temperature for a) and b) is 0 to 5 °C, the reaction time for a) is 0.5 to 1 h, and the reaction time for b) is 1 to 2 h.

[0044] Preferably, the preparation method further includes a separation step, and the separation is to add water and a first organic solvent to the reaction solution, and obtain an oil-soluble substance through phase separation.

[0045] More preferably, the first organic solvent is dichloromethane.

[0046] Preferably, the preparation method further includes one or more impurity removal steps such as water washing, concentration under reduced pressure, drying, suction filtration, and distillation.

[0047] More preferably, the water washing is to wash the organic phase with water and / or saturated brine.

[0048] In some specific embodiments, the water washing is to wash the organic phase with water.

[0049] Preferably, the water washing can be carried out once or repeatedly.

[0050] Preferably, the preparation method further includes a purification step, and the purification is to carry out column chromatography purification on the organic phase after separation and impurity removal.

[0051] Gradient elution is used in the column chromatography, and the eluent is a mixed solution of n-heptane and ethyl acetate, and the volume ratio of n-heptane in the eluent decreases successively.

[0052] More preferably, the volume ratio of n-heptane to ethyl acetate in the gradient elution is 50:1 to 10:1.

[0053] The fourth aspect of the present invention provides a preparation method of 4-boron-10 acid-L-phenylalanine, further deprotecting the compound of formula III to prepare 4-boron-10 acid-L-phenylalanine, and the deprotection is carried out by an acid hydrolysis method.

[0054] In some specific embodiments, the deprotection is carried out on the compound of formula III without impurity removal and / or column chromatography purification.

[0055] In some specific embodiments, the deprotection is carried out on the compound of formula III after the above impurity removal and / or column chromatography purification steps.

[0056] Preferably, the method includes adding a second organic solvent, water and an acid to the compound of formula III, and reacting at 50-60 °C for 1-3 h to obtain.

[0057] The second organic solvent is any one or more selected from methanol, ethanol, isopropanol, acetone, butanone, methyl isobutyl ketone, acetonitrile, and preferably acetone.

[0058] The acid is any one of concentrated hydrochloric acid or trifluoroacetic acid, and preferably concentrated hydrochloric acid with a mass fraction of 36%-38%.

[0059] The molar ratio of the compound of formula III to the added acid is 1:(5-12).

[0060] More preferably, the molar ratio of the compound of formula III to the added acid is 1:(10-12).

[0061] Based on the amount of 1 g of the compound of formula III, the addition amount of the second organic solvent is 4-6 mL;

[0062] The volume ratio of the added water to the second organic solvent is 1:(5-10), and preferably 1:(8-9).

[0063] Preferably, the method further includes one or more impurity removal steps such as concentration under reduced pressure, washing with water, washing with an organic solvent, suction filtration, drying, etc.

[0064] In some specific embodiments, the method includes removing the second organic solvent in the reaction by distillation under reduced pressure.

[0065] In some specific embodiments, the method includes washing one or more times with dichloromethane under acidic and alkaline conditions to remove raw materials or by-products.

[0066] In some specific embodiments, the method includes adjusting the reactant solution after washing with the second organic solvent to neutral, and performing suction filtration, and the filter cake is washed with water and ethanol.

[0067] In some specific embodiments, the method includes drying the filter cake after washing to remove water and the second organic solvent.

[0068] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0069] 1. The preparation method of 4-boron-10-acid-L-phenylalanine in the present application only requires a small amount of boron source and Grignard reagent to achieve a high yield, and the reaction cost is low.

[0070] 2. The preparation method of 4-boron-10-acid-L-phenylalanine in this application has a short reaction time and does not require the reaction temperature to be lowered too much, avoiding the problem of large energy consumption in ultra-low temperature reactions.

[0071] 3. The 4-boron-10-acid-L-phenylalanine prepared by the preparation method of 4-boron-10-acid-L-phenylalanine in this application has a high yield, high purity, and stable quality, which is conducive to quality control.

[0072] 4. The preparation method of 4-boron-10-acid-L-phenylalanine in this application has the advantages of simple operation, stable process, controllable risk, and being suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 1H NMR spectrum of tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolborate)phenyl)propionate prepared in Example 5 of the present invention.

[0074] Figure 2 1H NMR spectrum of tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate prepared in Example 8 of the present invention.

[0075] Figure 3 1H NMR spectrum of 4-boronic acid-L-phenylalanine prepared in Example 12 of the present invention.

[0076] Figure 4 X-ray powder diffraction result of 4-boron-10-acid-L-phenylalanine prepared in Example 13 of the present invention.

[0077] Figure 5 TGA detection result of 4-boron-10-acid-L-phenylalanine prepared in Example 13 of the present invention.

[0078] Figure 6 Chemical structure formula and X-ray single crystal diffraction pattern of 4-boron-10-acid-L-phenylalanine hydrochloride dihydrate in Example 13 of the present invention, where (A) is the chemical structure formula of 4-boron-10-acid-L-phenylalanine hydrochloride dihydrate, and (B) is the X-ray single crystal diffraction pattern of 4-boron-10-acid-L-phenylalanine hydrochloride dihydrate. DETAILED DESCRIPTION OF THE INVENTION

[0079] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0080] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0081] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0082] Example 1

[0083] This example provides a preparation method of isopropyl pinacol borate, and the reaction synthesis route is as follows:

[0084]

[0085] The specific operation steps are as follows: Add boric acid (100 g, 1.0 eq), isopropyl alcohol (292 g, 3.0 eq), pinacol (201 g, 1.05 eq), and toluene (500 mL) to the reaction flask. Heat under reflux for water separation, and during this period, isopropyl alcohol (about 200 - 300 g) needs to be supplemented until the reaction ends (about 5 hours). After concentrating under reduced pressure to remove toluene, distill under reduced pressure to obtain 277.1 g of a nearly colorless liquid, with a yield of 92.1%.

[0086] Comparative Example 1

[0087] The only difference between this Comparative Example 1 and Example 1 is that toluene is not added, and the remaining steps are the same as those in Example 1.

[0088] Specifically: Add boric acid (100 g, 1.0 eq), isopropyl alcohol (292 g, 3.0 eq), and pinacol (201 g, 1.05 eq) to the reaction flask. Heat to boiling, and during this period, continuously separate the distillate and replenish isopropyl alcohol until the reaction reaches equilibrium. After concentrating under reduced pressure to remove isopropyl alcohol, distill under reduced pressure to obtain 223.3 g of a nearly colorless liquid, with a yield of 74.2%.

[0089] As can be seen from the results of Example 1 and Comparative Example 1 above, adding the solvent toluene during the reaction can greatly improve the yield of the product. This is because toluene acts as a water separator during this reaction process. Through heating and refluxing, the water generated in the reaction is azeotropically distilled out, bringing the water in the reaction to equilibrium, promoting the reaction to proceed, and increasing the yield of the product.

[0090] Example 2

[0091] This example provides a preparation method of isopropyl pinacol boron-10 acid ester, and the reaction synthesis route is as follows:

[0092]

[0093] The specific operation steps are as follows: Add boric acid-10 (100 g, 1.0 eq), isopropyl alcohol (295 g, 3.0 eq), pinacol (203 g, 1.05 eq) and toluene (500 mL) to the reaction flask. Heat under reflux for water separation, and isopropyl alcohol needs to be replenished during this period until the reaction ends. After concentrating under reduced pressure to remove toluene, distillation under reduced pressure is carried out to obtain 274.4 g of a nearly colorless liquid, with a yield of 90.4%.

[0094] Example 3

[0095] Based on Example 2 above, in this Example 3, the influence of different monoalcohols on the yield of pinacol borate was investigated. The specific preparation method is the same as that of Example 2, and the obtained results are shown in the following table:

[0096] Table 1 Influence of different monoalcohol types on the yield of pinacol borate

[0097]

[0098] As can be seen from the results in Table 1, when isopropyl alcohol is used as the raw material, the yield of the prepared pinacol borate is the highest.

[0099] Example 4

[0100] This example provides a preparation method of tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacol borate)phenyl)propionate using a two-step process, and the reaction synthesis route is as follows:

[0101]

[0102] The specific operation steps are as follows:

[0103] 1) Prepare tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate using a method similar to Synthesis (2019), 51(3), 664-676.

[0104] 2) Add (S)-tert-butyl 2-(bis(tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate (10.00 g, 1.0 eq) and tetrahydrofuran (50 mL) to the reaction flask. After purging with nitrogen, cool the temperature to 0 °C. First, control the temperature at 0 - 5 °C and dropwise add a 1.3 mol / L solution of isopropylmagnesium chloride-lithium chloride in tetrahydrofuran (18.3 mL, 1.3 eq). React at 0 - 5 °C for 1 hour. Then, control the temperature at 0 - 5 °C and dropwise add isopropyl alcohol pinacol borate (3.57 g, 1.05 eq). Then, react at 0 - 5 °C for 2 hours. Finally, control the temperature at 0 - 5 °C and add glacial acetic acid (5.48 g, 5.0 eq) to quench the reaction. Then add water (30 mL) and dichloromethane (50 mL), and separate the layers. Wash the organic phase with water (10 mL). Concentrate the organic phase under reduced pressure to dryness to obtain a yellow oil. Column chromatography (n-heptane / ethyl acetate = 50:1 - 10:1) gives a nearly colorless oil of 6.89 g with a yield of 68.9%.

[0105] Example 5

[0106] This example provides a preparation method for (S)-tert-butyl 2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolborate)phenyl)propionate using a one-step process. The reaction synthesis route is as follows:

[0107]

[0108] The specific operation steps are as follows: Add the (S)-tert-butyl 2-(bis(tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate prepared in Example 4 above (10.00 g, 1.0 eq), isopropyl alcohol pinacol borate (3.57 g, 1.05 eq), and tetrahydrofuran (50 mL) to the reaction flask. After purging with nitrogen, cool the temperature to 0 °C. Control the temperature at 0 - 5 °C and dropwise add a 1.3 mol / L solution of isopropylmagnesium chloride-lithium chloride in tetrahydrofuran (18.3 mL, 1.3 eq). Then react at 0 - 5 °C for 2 hours. Control the temperature at 0 - 5 °C and add glacial acetic acid (5.48 g, 5.0 eq) to quench the reaction. Then add water (30 mL) and dichloromethane (50 mL), and separate the layers. Wash the organic phase with water (10 mL). Concentrate the organic phase under reduced pressure to dryness to obtain a yellow oil. Column chromatography (n-heptane / ethyl acetate = 50:1 - 10:1) gives a nearly colorless oil of 7.11 g with a yield of 71.1%.

[0109] Perform structural characterization on the (S)-tert-butyl 2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolborate)phenyl)propionate prepared in this example:

[0110] 1H-NMR (DMSOd6, 500MHz): δ7.57(d, J=11.9Hz, 2H), 7.17(d, J=11.9Hz,2H), 5.00(dd, J 1 =7.7Hz, J 2 =16.0Hz, 1H), 3.29(dd, J 1 =7.6Hz, J 2 =21.4Hz, 1H),3.09(dd, J 1 =16.1Hz, J 2 =20.8Hz, 1H), 1.42(s, 9H), 1.34 (s, 18H), 1.28 (s, 12H)

[0111] The 1H-NMR spectrum of tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-(pinacolboronate)phenyl)propionate prepared above is as Figure 1 shown.

[0112] It can be seen from the results of Example 4 and Example 5 that the preparation method of tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-(pinacolboronate)phenyl)propionate by a one-step process not only simplifies the operation process, saves time, but also enables the product to have a higher yield.

[0113] Example 6

[0114] Based on Example 5 above, in this example, the amounts of reactants, reaction temperature, and reaction time in the reaction process and their corresponding yields were further investigated. The specific results are shown in the following table.

[0115] Table 2 Effects of Different Parameters on the Yield of tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-(pinacolboronate)phenyl)propionate

[0116]

[0117] It can be seen from the results of Table 2 that in No. 2-1, when the amount of isopropyl alcohol pinacol borate is 1.05eq, the amount of isopropylmagnesium chloride-lithium chloride is 1.3eq, the reaction temperature is 0-5°C, and the reaction time is 2h, the yield can reach 71.1%; further increasing the amounts of isopropyl alcohol pinacol borate and isopropylmagnesium chloride-lithium chloride only results in a small increase in the yield; in addition, further reducing the reaction temperature to -10~0°C can slightly increase the yield, while reducing the temperature to -30~-20°C or increasing the temperature to 10~15°C will both lead to a significant decrease in the yield.

[0118] Therefore, by adopting the reaction parameter conditions in No. 2-1, it is possible to save costs and simplify the operation steps while ensuring a high yield.

[0119] Example 7

[0120] This example provides a preparation method for tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate using a two-step process. The reaction synthesis route is as follows:

[0121]

[0122] The specific operation steps are as follows:

[0123] 1) Prepare tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-(4-iodophenyl)propionate using a method similar to US2018155368A1.

[0124] 2) Add tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-(4-iodophenyl)propionate (10.00 g, 1.0 eq) and tetrahydrofuran (50 mL) to the reaction flask. After purging with nitrogen, cool to -20 °C, and add 1.3 mol / L isopropylmagnesium chloride-lithium chloride tetrahydrofuran solution (36.1 mL, 2.1 eq) dropwise while maintaining the temperature at -20 to -10 °C. Then react at -20 to -10 °C for 1 hour, and then add isopropyl alcohol pinacol borate (4.35 g, 1.05 eq) dropwise and react at -20 to -10 °C for 2 hours. Quench by adding glacial acetic acid (6.71 g, 5.0 eq) while maintaining the temperature at -10 to 5 °C, then add water (30 mL) and dichloromethane (50 mL), separate the layers, wash the organic phase with water (10 mL), and concentrate the organic phase under reduced pressure to dryness to obtain a yellow oil. Column chromatography (n-heptane / ethyl acetate = 50:1 to 10:1) gives 7.15 g of a nearly colorless oil with a yield of 71.5%.

[0125] Example 8

[0126] This example provides a preparation method for tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate using a one-step process. The reaction synthesis route is as follows:

[0127]

[0128] The specific operation steps are as follows: Add (S)-tert-butyl 2-(tert-butoxycarbonylamino)-3-(4-iodophenyl)propionate (10.00 g, 1.0 eq), isopropanol pinacol borate (4.35 g, 1.05 eq) and tetrahydrofuran (50 mL) to the reaction flask. After purging with nitrogen, cool to 0 °C, and dropwise add 1.3 mol / L isopropylmagnesium chloride-lithium chloride tetrahydrofuran solution (36.1 mL, 2.1 eq) while controlling the temperature at -20~-10 °C. Then react for 2 hours while controlling the temperature at -20~-10 °C. Quench by adding glacial acetic acid (6.71 g, 5.0 eq) while controlling the temperature at 0~5 °C. Then add water (30 mL) and dichloromethane (50 mL), separate the layers, wash the organic layer with water (10 mL), and concentrate the organic layer under reduced pressure to dryness to obtain a yellow oil. Column chromatography (n-heptane / ethyl acetate = 50:1~10:1) gives a nearly colorless oil of 7.33 g with a yield of 73.3%.

[0129] The (S)-tert-butyl 2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate prepared in this example was structurally characterized as follows:

[0130] 1 H-NMR (DMSOd6, 500 MHz): δ 7.59 (d, J = 6.5 Hz, 2H), 7.25 (d, J = 6.5 Hz, 2H), 7.16 (d, J = 6.7 Hz, 1H), 4.01 (td, J 1 = 4.7 Hz, J 2 = 7.7 Hz, J 2 = 11.7 Hz, 1H), 2.97 (dd, J 1 = 4.4 Hz, J 2 = 11.5 Hz, 1H), 2.85 (dd, J 1 = 8.2 Hz, J 2 = 11.4 Hz, 1H), 1.36 (s, 9H), 1.34 (s, 9H), 1.29 (s, 12H)

[0131] LCMS (ESI) [M + H] = 448.1; [M + Na] = 470.1; [2M + H] = 895.4; [M + HCOOH - H] = 492.2

[0132] The hydrogen spectrum of the above-prepared (S)-tert-butyl 2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate is shown as Figure 2 follows.

[0133] From the results of Example 7 and Example 8, it can be seen that the preparation method of tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate by a one-step process not only simplifies the operation process, saves time, but also enables the product to have a higher yield.

[0134] Example 9

[0135] Based on Example 8 above, in this example, the amounts of reactants, reaction temperature, reaction time and other parameters during the reaction and the corresponding yields were further investigated. The specific results are shown in the following table.

[0136] Table 3 Effects of different parameters on the yield of tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate

[0137]

[0138] Comparing the results in Table 3 with those in Table 2, it can be seen that when the reactant changes from (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolborate)phenyl)propionate to tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate with single Boc protection, since the NHBoc position is not fully protected, the amount of Grignard reagent needs to be increased to ensure the yield of the product; in addition, the reaction temperature needs to be reduced to below -10 °C to keep the yield of the product above 70%.

[0139] The above results indicate that complete protection of the amino group and carboxyl group of L-phenylalanine can obtain a higher yield with the least amount of Grignard reagent and without reducing the reaction temperature too low.

[0140] Example 10

[0141] Based on Example 8 above, in this example, the effects of different boronic esters on the product yield during the reaction were further investigated. The specific results are shown in the following table.

[0142] Table 4 Effects of different pinacol borate substitutions on the yield of tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate

[0143]

[0144] The results in Table 4 show that under the same reaction conditions, when isopropyl alcohol pinacol borate is used as the boron source, the prepared tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolborate)phenyl)propionate has the highest yield.

[0145] Example 11

[0146] This example provides a preparation method for tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolboronate)phenyl)propionate by a one-step process. Compared with Example 5, the input amount of raw materials in this example is further increased, and the feasibility of subsequent industrial production using this method is further evaluated.

[0147] The specific operation steps are as follows: Add tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate (100.0 g, 1.0 eq), isopropyl pinacolboronate (35.7 g, 1.05 eq) and tetrahydrofuran (500 mL) to a reaction flask. After purging with nitrogen, cool to 0 °C, and dropwise add 1.3 mol / L isopropylmagnesium chloride-lithium chloride tetrahydrofuran solution (183.0 mL, 1.3 eq) while controlling the temperature at 0 - 5 °C. React at 0 - 5 °C for 2 hours. Control the temperature at 0 - 5 °C and add glacial acetic acid (54.8 g, 5.0 eq) to quench the reaction. Then add water (300 mL) and dichloromethane (500 mL), separate the layers, wash the organic phase with water (100 mL), and concentrate the organic phase under reduced pressure to dryness to obtain 123.6 g of a yellow oil.

[0148] Example 12

[0149] In this example, tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolboronate)phenyl)propionate obtained by column chromatography in Example 8 above is used as the raw material to further deprotect and prepare 4-borono-L-phenylalanine. The reaction synthesis route is as follows:

[0150]

[0151] The specific operation steps are as follows: tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-((4-pinacolboronate)phenyl)propionate (7.0 g, 1.0 eq), acetone (35 mL) and water (4 mL). After dissolution, add concentrated hydrochloric acid (15.8 g, 10.0 eq), heat to 55 - 60 °C and reflux for 3 hours. Concentrate the reaction solution under reduced pressure to remove acetone, add water (35 mL) to dissolve, wash with dichloromethane (2 × 35 mL), adjust the pH of the aqueous phase to 12 - 13 with 50% potassium hydroxide solution, wash with dichloromethane (2 × 35 mL) again, adjust the pH of the aqueous phase to 6 - 7 with concentrated hydrochloric acid, filter by suction, wash the filter cake successively with a small amount of water and ethanol, and dry at 60 °C under a blast until constant weight to obtain 3.02 g of a white solid with a yield of 92.3%.

[0152] Further structural characterization of the 4-borono-L-phenylalanine prepared in this example is as follows:

[0153] 1 H-NMR (D 2 O+CF 3 COOH, 500 MHz): δ 7.55 (d, J = 8.1 Hz, 2H), 7.14 (d, J = 7.6 Hz, 2H), 4.18 (dd, J 1 = 5.7 Hz, J 2 = 7.8 Hz, 1H), 3.17 (dd, J 1 = 5.7 Hz, J 2 = 14.6 Hz, 1H), 3.03 (dd, J 1 = 7.8 Hz, J 2 = 14.6 Hz, 1H)

[0154] LCMS (ESI) [M + H] = 210.1; [M - H] = 209.1

[0155] The 1H NMR spectrum of the 4-boronic acid-L-phenylalanine prepared above is as follows Figure 3 shown

[0156] Example 13

[0157] In this example, the tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolboronate)phenyl)propionate prepared in Example 11 without column chromatography purification was used as the raw material for further deprotection to prepare 4-boronic acid-L-phenylalanine. The reaction synthesis route is as follows

[0158]

[0159] The specific operation steps are as follows: Add the tert-butyl (S)-2-(bis(tert-butoxycarbonyl)amino)-3-((4-pinacolboronate)phenyl)propionate (theoretical amount 99.9 g, 1.0 eq) prepared in Example 11, acetone (500 mL), and water (50 mL) to the reaction flask. After dissolution, add concentrated hydrochloric acid (211.0 g, 11.4 eq), and heat to 55 - 60 °C for reflux reaction for 3 hours. The reaction solution was concentrated under reduced pressure to remove acetone, dissolved in water (500 mL), washed with dichloromethane (2 × 500 mL), the aqueous phase was adjusted to pH 12 - 13 with 50% potassium hydroxide solution, then washed with dichloromethane (2 × 500 mL), the aqueous phase was adjusted to pH 6 - 7 with concentrated hydrochloric acid, filtered by suction, the filter cake was rinsed successively with a small amount of water and ethanol, and then dried in a blast dryer at 60 °C to constant weight to obtain 29.2 g of white solid, with a yield of 76.7%.

[0160] The structural identification of 4-boron-10-acid-L-phenylalanine prepared in this example was carried out, and the specific detection items and detection results are shown in the following table.

[0161] Table 5 Identification Detection Items and Results of 4-boron-10-acid-L-phenylalanine

[0162]

[0163] Among the above detection items, the X-ray powder diffraction results are as Figure 4 shown, and the TGA detection results are as Figure 5 shown.

[0164] Furthermore, the prepared 4-boron-10-acid-L-phenylalanine finished product was dissolved in hydrochloric acid solution, and crystals were slowly precipitated by standing at room temperature. After filtration and drying, it was detected by X-ray single crystal diffraction method, and the absolute configuration was confirmed to be S configuration. The X-ray single crystal diffraction pattern and its chemical structural formula of the specifically precipitated 4-boron-10-acid-L-phenylalanine hydrochloride dihydrate are as Figure 6 shown, where (A) is the chemical structural formula of 4-boron-10-acid-L-phenylalanine hydrochloride dihydrate, and (B) is the X-ray single crystal diffraction pattern of 4-boron-10-acid-L-phenylalanine hydrochloride dihydrate.

[0165] The above results show that when 4-boron-10-acid-L-phenylalanine is prepared by the method in this application, even without the step of column chromatography purification, the total yield of the two steps is still as high as 76.7%, and the purity of the product is as high as 97.8%; therefore, this method in this application simplifies the complex post-treatment steps in the reaction process while ensuring the product yield and stability, saving costs and time, and can be applied to large-scale industrial production.

[0166] Comparative Example 2

[0167] This Comparative Example 2 is a comparative example of the above Example 4 and Example 12. The boron source was replaced with tributyl borate, the reaction temperature was further reduced, the reaction time was prolonged, and trifluoroacetic acid was used during the deprotection of amino and carboxyl groups to investigate the influence on the product yield. The reaction synthesis route is as follows:

[0168]

[0169] The specific operation steps are as follows: Add (S)-tert-butyl 2-(bis(tert-butoxycarbonyl)amino)-3-(4-iodophenyl)propionate (10.00 g, 1.0 eq) and tetrahydrofuran (40 mL) to a reaction flask. After purging with nitrogen, cool the mixture to -20 °C, and dropwise add a 1.3 mol / L solution of isopropylmagnesium chloride-lithium chloride in tetrahydrofuran (17.1 mL, 1.2 eq) while maintaining the temperature at -20 to -15 °C. Then, react the mixture at -20 to -15 °C for 0.5 hour. Next, dropwise add tributyl borate (5.52 g, 1.3 eq) while maintaining the temperature at -20 to -15 °C, and then react the mixture at -20 to -15 °C for 16 hours. Add a 3% (w / w) hydrochloric acid solution (33.0 g, 1.5 eq) to adjust the pH to 4 - 5 while maintaining the temperature at -20 to 10 °C, and then react the mixture at 0 to 20 °C for 0.5 hour. Extract the reaction mixture with ethyl acetate (2 × 450 mL). Wash the combined organic phases once with saturated brine (300 mL), dry the organic phases with anhydrous sodium sulfate (50 g), filter, wash the filter cake with ethyl acetate (50 mL), and concentrate the filtrate under reduced pressure until no more liquid distills off to obtain 12.24 g of a yellow oil. Perform column chromatography (n-heptane / ethyl acetate = 50:1 to 10:1) to obtain 4.43 g of a nearly colorless oil with a yield of 52.1%.

[0170] Add trifluoroacetic acid (27.5 g, 30.0 eq) to 4.40 g (1.0 eq) of the above oil, react at 20 - 30 °C for 24 h, concentrate to dryness under reduced pressure, then add toluene (3 × 90 mL) and concentrate under reduced pressure three times. Pulp and wash the resulting viscous substance with dichloromethane (45 mL), filter by suction, and dry the filter cake in a blast dryer at 60 °C to constant weight to obtain 1.44 g of an off-white solid with a yield of 85.5%.

[0171] Comparative Example 3

[0172] This Comparative Example 3 is a comparative example of the above Example 11 and Example 13. Replace the boron source with tributyl borate, further lower the reaction temperature, extend the reaction time, and use (S)-tert-butyl 2-(tert-butoxycarbonylamino)-3-((4-pinacolboronate)phenyl)propionate without column chromatography purification to investigate the effect on the total yield of the two steps. The reaction synthesis route is as follows:

[0173]

[0174] The specific operation steps are as follows: Add tert-butyl (S)-2-(tert-butoxycarbonylamino)-3-(4-iodophenyl)propionate (10.00 g, 1.0 eq), toluene (56 mL), and tetrahydrofuran (56 mL) into a reaction flask. After purging with nitrogen, cool the mixture to -20 °C, and dropwise add a 1.3 mol / L solution of isopropylmagnesium chloride-lithium chloride in tetrahydrofuran (36.6 mL, 2.1 eq) while controlling the temperature at -20 to -15 °C. Then, react at -20 to -15 °C for 5 hours. Next, dropwise add tributyl borate (5.66 g, 1.1 eq) while controlling the temperature at -20 to -15 °C, and then react at -20 to -15 °C for 16 hours. Add a 3% (w / w) hydrochloric acid solution (136.0 g, 5.0 eq) while controlling the temperature at -20 to 10 °C to adjust the pH to about 1, and then maintain the temperature at 0 to 20 °C and react for 0.5 h. Concentrate the reaction solution under reduced pressure to remove tetrahydrofuran, then extract with ethyl acetate (2 × 75 mL). Wash the combined organic phase once with saturated brine (45 mL), dry the organic phase with anhydrous sodium sulfate (10 g), filter, wash the filter cake with ethyl acetate (50 mL), and concentrate the filtrate under reduced pressure until no more liquid comes out to obtain 9.63 g of a yellow oil. The theoretical yield is 8.17 g.

[0175] Add isopropanol (8 mL), water (32 mL), and concentrated hydrochloric acid (22.6 g, 10.0 eq) to 9.62 g of the above oil (theoretical amount 8.17 g, 1.0 eq), heat up to 55 to 60 °C and react for 5 h. Cool to 20 to 30 °C, add water (70 mL), then wash with ethyl acetate (70 mL). Adjust the pH of the aqueous phase to 6 to 7 with 20% sodium hydroxide solution, filter by suction. Wash the filter cake successively with water and acetone, and then dry at 60 °C under a blast of air until a constant weight is obtained to obtain 2.06 g of a yellow solid. The combined yield of the two steps is 44.1%.

[0176] From the results of Comparative Example 2 and Comparative Example 3 above, it can be seen that when the boron source is replaced with tributyl borate, even if the reaction temperature is lowered and the reaction time is greatly extended, the yield of the product will still be significantly reduced. This is because when tributyl borate is used as the boronating reagent, its reaction activity is relatively low, resulting in a higher reaction difficulty. At this time, a large amount of boronating reagent and Grignard reagent, as well as a lower reaction temperature and a longer reaction time, are often required to achieve a higher product yield.

[0177] In summary, the present application provides a new method for preparing 4-boron-10-acid-L-phenylalanine. Using the method of the present application, with less use of boron source and Grignard reagent, the reaction time is further shortened, and it is not necessary to lower the temperature too much to obtain a product with a higher yield. Moreover, the purity of the product is above 97%. While reducing costs, it ensures the yield and purity of the product, and is suitable for large-scale industrial production.

[0178] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing an intermediate compound, characterized in that: The compound of formula II is contacted and reacted with a pinacol boron-10 acid ester derivative under the action of a Grignard reagent, wherein the structure of the intermediate compound is shown in formula III, and the structure of the pinacol boron-10 acid ester derivative is shown in formula I. ， ， ； The pinacol boron-10 acid ester derivative is prepared by heating and refluxing boron-10 acid, pinacol and isopropanol in the presence of toluene; the molar ratio of the boron-10 acid, pinacol and isopropanol is 1:(0.9-1.5):(1-6); based on the addition amount of 1g of boron-10 acid in the reaction, the addition amount of toluene is 1-50mL; the reaction time is 2-5h; The Grignard reagent is isopropylmagnesium chloride-lithium chloride; Said X is Br or I; The R2 is a tert-butyl group, and the R3 and R4 are tert-butyloxycarbonyl groups; The preparation method adopts the following one-step process: firstly add the compound of formula II, the reaction solvent and the pinacol boron-10 ester derivative, add the Grignard reagent at a reaction system temperature of 0-5°C, and react at the temperature for 1-2h; finally, add acid to quench; The molar ratio of the compound of formula II, boron-10 ester and Grignard reagent is 1:(0.9-1.05):(1-1.3).

2. The preparation method according to claim 1, characterized in that: The molar ratio of the boric acid-10, pinacol and isopropanol is 1:(0.9-1.1):(3-6).

3. The preparation method according to claim 1, characterized in that: Based on the addition amount of 1 g of boric acid-10 in the reaction, the addition amount of toluene is 4-6 mL.

4. The preparation method according to claim 1, characterized in that: The reaction solvent is any one selected from tetrahydrofuran, methyltetrahydrofuran, dioxane, cyclopentyl methyl ether, methyl tert-butyl ether, petroleum ether, benzene or toluene.

5. The preparation method according to claim 1, characterized in that: The acid is one or more selected from formic acid, acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, oxalic acid or tartaric acid.

6. The preparation method according to claim 1, characterized in that: The compound of formula II is prepared by protecting the amino and carboxyl groups of L-phenylalanine and halogenating the H at position 4 thereof.

7. The preparation method according to claim 1, characterized in that: The preparation method further comprises a separation step, wherein water and a first organic solvent are added to the solution after the reaction, and the oil-soluble substance is obtained by phase separation.

8. The preparation method according to claim 7, characterized in that: The first organic solvent is dichloromethane.

9. The preparation method according to claim 1, characterized in that: The preparation method further comprises one or more impurity removal steps of water washing, reduced pressure concentration, drying, suction filtration and distillation.

10. The preparation method according to claim 9, characterized in that: The water washing is to use water and / or saturated brine to wash the organic phase.

11. The preparation method according to claim 1, characterized in that: The preparation method further comprises a purification step, wherein the purification is to perform column chromatography purification on the separated and impurity-free organic phase.

12. The preparation method according to claim 11, characterized in that: The eluent in the column chromatography is a mixed solution of n-heptane and ethyl acetate, and the volume proportion of n-heptane in the eluent decreases successively.

13. The preparation method according to claim 12, characterized in that: The volume ratio of n-heptane to ethyl acetate in the gradient elution is 50:1-10:

1.

14. A method for preparing 4-boronic acid-10-L-phenylalanine, characterized in that: The method of claim 1 is used to prepare a compound of formula III, and the compound of formula III is further deprotected to prepare 4-boronic acid-10-L-phenylalanine, wherein the deprotection is carried out by an acid hydrolysis method; the method comprises adding a second organic solvent, water and an acid to the compound of formula III, and reacting at 50-60° C. for 1-3 hours to obtain; The second organic solvent is acetone; The acid is concentrated hydrochloric acid with a mass fraction of 36% to 38%; The molar ratio of the compound of formula III to the added acid is 1:(5-12); Based on 1 g of the compound of formula III, the amount of the second organic solvent added is 4 to 6 mL; The volume ratio of the added water to the second organic solvent is 1:(8-9).

15. The preparation method according to claim 14, characterized in that: The method further comprises one or more impurity removal steps of reduced pressure concentration, water washing, organic solvent washing, suction filtration and drying.

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