CN104447823A - Preparation method of 4-dihydroxy boron-L-phenylalanine - Google Patents

Abstract

The invention provides a preparation method of 4-dihydroxy boron-L-phenylalanine, which comprises the following steps: -4-halophenylacrylic having amine terminal protected as shown in formula (I)Reacting alanine with a boronating agent and an organolithium compound to obtain a reaction mixture, wherein the reaction mixture comprises -4-dihydroxyborylphenylalanine with protected amine end shown in formula (II), and R is a protecting group;

Description

The preparation method of 4-dihydroxyboryl-L-type phenylalanine

technical field

The invention relates to a method for preparing borides for cancer treatment by using boron neutron capture therapy, in particular to a method for preparing 4-( ^10B ) dihydroxyboryl-L-type phenylalanine.

Background technique

4-( ¹⁰ B) dihydroxyboryl-L-phenylalanine (4-( ¹⁰ B) borono-L-phenylalanine, L- ¹⁰ BPA) is currently known to be able to use boron neutron capture therapy (boron neutron capture therapy) , BNCT) are important borides for the treatment of cancer.

Therefore, various synthesis methods of L-BPA have been developed at present. As shown in the following formula (A), the L-BPA synthetic method of the prior art includes two modes of forming bond (a) and bond (b):

Among them, the method for synthesizing L-BPA by forming the bond (a) is to try to introduce a substituent containing a dihydroxylboryl group (dihydroxylboryl group or borono group) into the skeleton of phenylalanine, thus in the pair of amide substituents L-BPA is produced by forming carbon-boron bonds. the

J.Org.Chem.1998,63,8019 discloses a method for cross-coupling reaction between (S)-4-iodophenylalanine and diboron compound (diboron compound) through protection of palladium catalyzed amine end. (S)-4-iodophenylalanine (such as (S)-N-tert-butoxycarbonyl-4-iodophenylalanine ((S)-N-Boc-4- iodophenylalanine)) and diboron compound (such as bis(pinacolato)diboron)) cross-coupling reaction to produce (S)-N-tert-butoxycarbonyl-4-pentanoylboryl phenylalanine acid ((S)-N-Boc-4-pinacolatoborono phenylalanine) protected (S)-4-boronyl ester group phenylalanine at the amine end; after that, remove the protecting group on the amine end and the boranol end Substituting group on, complete the preparation of L-BPA. the

However, since the selected bis-valeryl diboron is not a commercially available compound, this method requires additional pretreatment for the preparation of boronating agents, resulting in high complexity of the process and a long time spent, making it impossible to prepare High yield of L-BPA. In addition, the carboxylic acid group of (S)-4-iodophenylalanine protected at the amine end needs to be protected by a substituent to form a benzyl ester group (benzyl ester group) to increase the process yield to 88%; however, The preparation of L-BPA in this way also requires an additional step of deprotecting the carboxylic acid group, which virtually increases the complexity of the L-BPA process. the

Accordingly, the method provided in this document not only needs to involve the pretreatment of the preparation of the boronating agent, but also requires a lot of process time and synthesis steps to complete the steps of protecting and deprotecting the carboxylic acid group, which is not conducive to the industry. The main method of synthesizing L-BPA. the

On the other hand, the method for synthesizing L-BPA by forming a bond (b) is to couple an amino acid with a boron-containing benzyl fragment or a boron-containing benzaldehyde fragment, To synthesize L-BPA. Biosci.Biotech.Biochem.1996,60,683 reveals a kind of L-BPA enantioselective synthesis method (enantioselective synthesis), which makes boronic acid cyclic ethers (cyclic ethers of boronic acid) and L-valine Chiral derivatives from L-valine are coupled to produce L-BPA. However, this method needs to make boronic acid cyclic ether compounds from 4-dihydroxyboronyl benzyl bromide (4-boronobenzylbromide), and then perform coupling reaction with chiral derivatives of L-valine, and in the latter stage Undesirable racemization of amino acids tends to occur in the synthesis step, so that the method needs to carry out an enzymatic resolution step (enzymatic resolution step), which tends to reduce the yield, in order to obtain L-BPA with a certain optical purity. the

Accordingly, the method provided in this document still includes steps such as pretreatment for preparing boronating agent and posttreatment for enzymatic resolution, which makes the process involved in the method more complicated and takes a long time, and it is impossible to obtain High yield of L-BPA. the

In addition, it is currently known that 4-( ¹⁰ B) dihydroxyboryl-L-phenylalanine (4-( ¹⁰ B)borono-L-phenylalanine, ^{L- 10} BPA) containing 10 boron is accumulated in tumor cells The key factor of the treatment is to use thermal neutron beams to irradiate the boron accumulated in the tumor cells to kill the tumor cells by capturing the high-energy particles generated by the reaction, so as to achieve the purpose of treating cancer. Therefore, ¹⁰ boron can promote L- ¹⁰ BPA as a method of treating cancer through boron neutron capture therapy.

However, the boron element present in nature contains about 19.9% of ¹⁰ boron and about 80.1% of ¹¹ boron. Therefore, many researchers are still actively developing a method applicable to the synthesis of L-BPA, especially a method applicable to the synthesis of L-BPA rich in ¹⁰ boron.

J. Org. Chem. 1998, 63, 8019 additionally provides a method for synthesizing ¹⁰ boronating agent. Since the method involves multiple steps, it is easy to greatly reduce the ¹⁰ boron content of the ¹⁰ boron-rich material during the process. Therefore, the method provided by this document is not suitable for the synthesis of L-BPA rich in ¹⁰ boron.

As described in Biosci.Biotech.Biochem.1996,60,683, before the enzymatic resolution step, the method provided in this document cannot obtain L-BPA with a certain optical purity; and this method prepares ¹⁰ boronating agents Multiple steps are also involved, resulting in transformations of the ^10- boron-rich material during the process. Therefore, the method provided by this document is also not suitable for the synthesis of L-BPA rich in ¹⁰ boron.

Furthermore, Bull.Chem.Soc.Jpn.2000,73,231 discloses a method utilizing palladium to catalyze 4-iodo-L-type phenylalanine and 4,4,5,5-tetramethyl-1,3,2 - A method for coupling dioxaborolane (common name: pinacolborane). However, this document does not mention how to use this method to prepare ^10- boron-rich L-BPA, and 4,4,5,5-tetramethyl- ^1,3,2 -dioxoborolane is not a commercial Therefore, the method provided by this document is also not suitable for the synthesis of L-BPA rich in ¹⁰ boron.

In addition, Synlett.1996, 167 disclosed a method for coupling reaction of iodophenylborate and L-serine zinc derivatives (L-serine zinc derivatives), which first involves the preparation of iodophenylborate Esters and steps such as the zinc derivative of preparation L-type serine, cause the productive rate of the L-BPA that makes is lower. In addition, since the ¹⁰ boron triiodide rich in ¹⁰ boron and 1,3-diphenylpropane-1,3-diol selected in this method are not commercially available compounds, the method provided in this document Still not suitable for the synthesis of L-BPA rich in ¹⁰ boron.

Contents of the invention

In view of the disadvantages of the prior art that the process time is too long, the process steps are cumbersome, and the boronating agent needs to be prepared in advance, the purpose of the present invention is to provide a kind of 4-dihydroxyboryl that does not need to go through complicated purification steps and is friendly to the environment. - A preparation method of L-type phenylalanine (4-borono-L-phenylalanine, L-BPA), thereby effectively saving time and cost, and improving process efficiency. Accordingly, the 4-dihydroxyboryl-L-phenylalanine prepared by the preparation method of the present invention also has the advantages of high chemical purity and high optical purity. the

Another object of the present invention is to provide a 4-( ¹⁰ B) dihydroxyboryl-L-phenylalanine (4-( ¹⁰ B) borono-L- phenylalanine, L- ¹⁰ BPA), thus effectively saving time and cost, and improving the process efficiency. The preparation method of the present invention is beneficial to the preparation of 4-( ¹⁰ B) dihydroxyboryl-L-type phenylalanine with high chemical purity, high optical purity and high isotopic purity.

Another object of the present invention is to provide a method suitable for preparing L-BPA and L- ¹⁰ BPA, which does not require complicated purification steps, and has the advantages of saving time and cost, high efficiency, environmental friendliness and convenience.

In order to achieve the aforementioned object, the preparation method of 4-dihydroxyboryl-L-type phenylalanine of the present invention comprises the following steps:

Reaction of (S)-4-halophenylalanine (N-protected (S)-4-halophenylalanine) with a boronating agent and an organolithium compound to obtain a reaction mixture, wherein The reaction mixture contains amine protected (S)-4-dihydroxyboryl phenylalanine (N-protected (S)-4-boronophenylalanine), the amine protected (S)-4-halo Phenylalanine has a structure shown in the following formula (I), and the protected (S)-4-dihydroxyboryl phenylalanine at the amine end has a structure shown in the following formula (II), and in formula ( In I) and formula (II), R is a protecting group;

The (S)-4-dihydroxyboryl phenylalanine protected at the amine end is isolated from the reaction mixture;

Deprotecting the protecting group of (S)-4-dihydroxyboryl phenylalanine protected at the amine end to obtain 4-dihydroxyboryl-L-type phenylalanine. According to the present invention, the obtained 4-dihydroxyboryl-L-type phenylalanine has a structure as shown in the following formula (III):

According to the present invention, the "borating agent" refers to any reaction that can make the amine end protected (S)-4-halophenylalanine react with the boronating agent and the organolithium compound. A reagent that replaces the X substituent (ie, X shown in formula (I)) of terminally protected (S)-4-halophenylalanine with a boron-containing substituent. the

According to the present invention, the boron element of the boronating agent can be any form of boron atom, such as ¹¹ boron, ¹⁰ boron or a combination thereof.

According to the present invention, the boron element of the boronizing agent is a combination of ¹¹ boron ( ¹¹ B) and ¹⁰ boron ( ¹⁰ B), and the content of ¹⁰ boron in the whole boron element can be 19.9%, but it is not limited thereto.

According to the present invention, the borating agent includes, but not limited to, trialkyl borate. The trialkyl borate includes: tributyl borate, triethyl borate, trimethyl borate, triisopropyl borate, tripropyl borate borate), tri-tert-butyl borate, or any applicable trialkyl borate, but not exclusively. the

The preparation method of the present invention has many advantages:

(1) The preparation method of the present invention reacts the (S)-4-halophenylalanine protected at the amine end with a boronating agent and an organolithium compound without pre-protecting the (S)-4 protected at the amine end. - the carboxylic acid group of halo-phenylalanine, the (S)-4-dihydroxyboryl-phenylalanine with protected amine end can be obtained. Therefore, the preparation method of the present invention also does not need the step of deprotecting the carboxylic acid group in the back-end process, so the process can be shortened;

(2) The preparation method of the present invention directly uses the boronating agent to participate in the reaction, so no additional pretreatment process for preparing the boronating agent is needed; and

(3) Since the preparation method of the present invention has the advantages of simple and convenient manufacturing process, the 4-dihydroxyboryl-L-type phenylalanine obtained through this preparation method can possess high chemical purity, high optical purity and excellent overall Advantages such as productivity. Therefore, the present invention can provide a kind of preparation method that saves time, cost and high efficiency. the

Preferably, in the (S)-4-halophenylalanine whose amine end is protected by formula (I), X is iodide or bromide; more preferably, in In the (S)-4-halophenylalanine whose amine end is protected by the formula (I), X is an iodine group. the

Preferably, the protected (S)-4-halophenylalanine at the amine end represented by formula (I) and the protected (S)-4-dihydroxyl group at the amine end represented by formula (II) In borylphenylalanine, the protecting group (R) is selected from the group consisting of: tert-butoxycarbonyl group (t-Boc or Boc), trityl group (trityl group, Trt), 3,5-dimethoxyphenylisopropoxycarbonyl group (3,5-dimethoxyphenylisopropoxycarbonyl group, Ddz), 2-(4-biphenyl)isopropoxycarbonyl group (2-(4-Biphenyl)isopropoxycarbonyl group, Bpoc) and 2-nitrophenylsulfenyl group (2-nitrophenylsulfenyl group, Nps); more preferably, (S)-4-halophenylalanine protected at the amine end represented by formula (I) And in the protected (S)-4-dihydroxyboryl phenylalanine shown in formula (II), the protecting group is tert-butoxycarbonyl. the

According to the present invention, using tert-butoxycarbonyl as protecting group has the following advantages:

(1) (S)-N-tert-butoxycarbonyl-4-halophenylalanine (N-Boc-(S)-4-halophenylalanine) is solid, so it is easy to operate in the process;

(2) One of the raw materials for the preparation of (S)-N tert-butoxycarbonyl-4-halophenylalanine is di-t-butyl dicarbonate, which is easy to obtain and cheap, etc. Advantages, so the prepared (S)-N-tert-butoxycarbonyl-4-halogenophenylalanine also has the advantages of being easy to obtain and cheap; and

(3) After the tert-butoxycarbonyl group of (S)-N-tert-butoxycarbonyl-4-dihydroxyboryl phenylalanine is deprotected, the tert-butoxycarbonyl group is decomposed into carbon dioxide and tert-butanol (tert- butanol), they are all low-risk chemical substances, so the use of (S)-N-tert-butoxycarbonyl-4-halophenylalanine to prepare 4-dihydroxyboryl-L-phenylalanine also has The advantages of safety and low risk. the

Preferably, the preparation method comprises, at a temperature of -100°C to -50°C, making the (S)-4-halophenylalanine with the amine end protected, the boronating agent and the organolithium compound reaction to obtain the reaction mixture; more preferably, the preparation method is at a temperature of -100°C to -70°C, the (S)-4-halophenylalanine with the amine end protected and the A boronating agent and the organolithium compound are reacted to obtain the reaction mixture. the

Preferably, the equivalent ratio of the boronating agent relative to the amine-terminally protected (S)-4-halophenylalanine is between 2 and 5. the

Preferably, the equivalent ratio of the organolithium compound relative to the protected (S)-4-halophenylalanine at the amine end is at least 3; more preferably, the organolithium compound is protected relative to the amine end The equivalent ratio of (S)-4-halophenylalanine is between 3 and 5. the

Preferably, the step of reacting the protected (S)-4-halophenylalanine at the amine end with the boronating agent and the organolithium compound to obtain the reaction mixture comprises:

Mixing the (S)-4-halophenylalanine protected at the amine end, the reaction solvent and the boronating agent to obtain a mixed solution; and

An inert organic solvent containing the organolithium compound is added to the mixed solution to obtain the reaction mixture. the

According to the present invention, in the inert organic solvent, the concentration of the organolithium compound is between 1 volume molarity (molarity, M) to 3M, but not limited thereto. Preferably, the concentration of the organolithium compound in the inert organic solvent is between 1M and 2M. the

Preferably, the step of reacting the (S)-4-halophenylalanine with the protected amine end with a boronating agent and an organolithium compound in the preparation method includes a temperature of -100°C to -50°C , adding an inert organic solvent containing the organolithium compound to the mixed solution to obtain the reaction mixture. In the aforementioned steps, the reaction temperature is more preferably between -100°C and -70°C; still more preferably between -85°C and -70°C. the

More preferably, since the organolithium compound will react violently after being mixed with the mixed solution, an inert organic solvent containing the organolithium compound is added dropwise to the mixed solution to obtain the reaction mixture; and in the mixed solution, gradually The length of time for dropping the inert organic solvent containing the organolithium compound depends on the amount of the inert organic solvent containing the organolithium compound, the concentration of the organolithium compound in the inert organic solvent, and the amount of the mixed solution, but is not limited thereto. For example, when the volume of the mixed solution is between 300 ml and 400 ml and the volume of the inert organic solvent containing the organolithium compound is between 50 ml and 100 ml, the inert organic solvent containing the organolithium compound is added dropwise to the mixed solution The time is as long as 2 to 3 hours. the

Since the organolithium compound will react violently after mixing with the mixed solution, the step of mixing the protected (S)-4-halophenylalanine at the amine end, the reaction solvent and the boronating agent is carried out in a nitrogen environment , to obtain the mixed solution; and the step of adding the inert organic solvent containing the organolithium compound to the mixed solution is also carried out in a nitrogen atmosphere to obtain the reaction mixture. the

Preferably, the step of separating and obtaining (S)-4-dihydroxyboryl phenylalanine protected at the amine end from the reaction mixture comprises:

adding an aqueous solution to the reaction mixture to obtain a first aqueous layer;

Extract the first water layer using an extraction solvent to obtain a second water layer, wherein the second water layer comprises (S)-4-dihydroxyboryl phenylalanine whose amine end is protected; and

Adjusting the pH value (pH value) of the second water layer to be lower than 4, thereby crystallizing (S)-4-dihydroxyboryl phenylalanine whose amine end is protected, to obtain from the second water layer The (S)-4-dihydroxyborylphenylalanine protected at the amine end was obtained by separation in the layers. the

Preferably, the pH value of the second aqueous layer is adjusted to be lower than 4, whereby the amine-terminally protected (S)-4-dihydroxyboryl phenylalanine crystallizes from the second aqueous layer The step of separating and obtaining (S)-4-dihydroxyboryl phenylalanine protected at the amine end comprises:

Adjusting the pH value of the second aqueous layer to be lower than 4, thereby crystallizing the (S)-4-dihydroxyboryl phenylalanine protected at the amine end to obtain (S)- Crystals of 4-dihydroxyborylphenylalanine; and

Filtering the crystals of (S)-4-dihydroxyboryl phenylalanine protected at the amine end, and drying the crystals of (S)-4-dihydroxyboryl phenylalanine protected at the amine end, to The amine-terminally protected (S)-4-dihydroxyborylphenylalanine is isolated from the second aqueous layer. the

Preferably, the step of adjusting the pH value of the second water layer to be lower than 4, thereby crystallizing (S)-4-dihydroxyboryl phenylalanine whose amine end is protected comprises: An acid solution is added to the water layer, thereby adjusting the pH value of the second water layer to be lower than 4, and crystallizing the (S)-4-dihydroxyboryl phenylalanine whose amine end is protected. More preferably, the pH value of the second water layer is adjusted to between 3 and 4 through the aforementioned steps. the

Accordingly, the present invention can successfully separate and obtain the (S)-4-dihydroxyborylphenylalanine with the amine end protected from the reaction mixture through a simple process without complicated purification steps. Therefore, the preparation method of the present invention can avoid producing a large amount of waste solvents and silica gel, and thus has the advantage of being more environmentally friendly. the

Preferably, the step of deprotecting the protecting group of (S)-4-dihydroxyboryl phenylalanine protected at the amine end to obtain 4-dihydroxyboryl-L-phenylalanine comprises: acidifying the first An organic solution, wherein the first organic solution is a mixture containing (S)-4-dihydroxyboryl phenylalanine protected at the amine end and a first organic solvent, thereby deprotecting the protected amine end The protecting group of (S)-4-dihydroxyboryl phenylalanine, to make 4-dihydroxyboryl-L-type phenylalanine. the

According to the present invention, the preparation method comprises acidifying the first organic solution with an acidifying solution, thereby deprotecting the protecting group of the amine-terminally protected (S)-4-dihydroxyborylphenylalanine to obtain 4 -Dihydroxyboryl-L-phenylalanine. the

Preferably, the preparation method comprises acidifying the pH value of the first organic solution to below 3; more preferably, the preparation method comprises acidifying the pH value of the first organic solution to below 1, thereby deprotecting the The protecting group of (S)-4-dihydroxyboryl phenylalanine protected at the amine end in the first organic solution, so as to prepare 4-dihydroxyboryl-L-type phenylalanine. the

Preferably, the step of deprotecting the protecting group of the protected (S)-4-dihydroxyboryl phenylalanine at the amine end comprises:

Acidifying the first organic solution, wherein the first organic solution is a mixture containing (S)-4-dihydroxyboryl phenylalanine protected at the amine end and a first organic solvent, thereby deprotecting the amine end through The protecting group of (S)-4-dihydroxyboryl phenylalanine of protection, to obtain acidic mixture, wherein this acidic mixture comprises 4-dihydroxyboryl-L-type amphetamine; And

The pH value of the acidic mixture is adjusted to be greater than 1, thereby crystallizing 4-dihydroxyboryl-L-form amphetamine to obtain 4-dihydroxyboryl-L-form amphetamine from the acidic mixture. the

Preferably, the pH value of the acidic mixture is adjusted to be greater than 1, whereby 4-dihydroxyboryl-L-type amphetamine is crystallized to obtain the step of 4-dihydroxyboryl-L-type amphetamine from the acidic mixture include:

Adjusting the pH value of the acidic mixture to be greater than 1, thereby crystallizing 4-dihydroxyboryl-L-type amphetamine to obtain crystals of 4-dihydroxyboryl-L-type amphetamine; and

Filtrating the crystals of 4-dihydroxyboryl-L-amphetamine and drying the crystals of (S)-4-dihydroxyborylphenylalanine protected at the amine end, thereby obtaining 4-dihydroxyboryl phenylalanine from the acidic mixture Boryl-L-form amphetamine. the

More preferably, the step of adjusting the pH value of the acidic mixture to be greater than 1, thereby crystallizing 4-dihydroxyboryl-L-form amphetamine comprises: adjusting the pH value of the acidic mixture to between 1 and 3; and Continuously increasing the pH value of the acidic mixture to between 5 and 7.4, thereby crystallizing 4-dihydroxyboryl-L-phenylalanine in the acidic mixture. the

Still more preferably, the step of adjusting the pH value of the acidic mixture to greater than 1, thereby making 4-dihydroxyboryl-L-type amphetamine crystallization comprises: adjusting the pH value of the acidic mixture to 1.5; standing or stirring The acidic mixture for a period of time; and continuously increasing the pH value of the acidic mixture to 6.2, thus making the crystallization of 4-dihydroxyboryl-L-type phenylalanine in the acidic mixture more complete. the

According to the present invention, standing or stirring the acidic mixture can help to form more solids of 4-dihydroxyboryl-L-type phenylalanine, wherein the time of standing or stirring the acidic mixture can be as long as 0.5 hour or 1 hours, but not limited to that. the

Accordingly, the present invention does not need to go through complicated purification steps, and can complete the step of deprotecting the protecting group of (S)-4-dihydroxyboryl phenylalanine protected at the amine end only through a simple process, and thus Obtain 4-dihydroxyboryl-L-type phenylalanine with high chemical purity and high optical purity. Therefore, the preparation method of the present invention can avoid producing a large amount of waste solvents and silica gel, and thus has the advantage of being more environmentally friendly. the

Preferably, the boronating agent has a ^10- boron purity greater than or equal to 98%.

Preferably, the (S)-4-dihydroxyborylphenylalanine with the amine terminal protected is (S)-4-( ¹⁰ B)dihydroxyborylphenylalanine with the amine terminal protected.

Preferably, the (S)-4-dihydroxyboryl phenylalanine protected at the amine end as shown in formula (II) is (S)-4-( ¹⁰ B)dihydroxyboryl Phenylalanine (N-protected(S)-4-( ^10B )boronophenylalanine).

According to the present invention, the boronating agent comprises trialkyl ^{10 B} borate or any other suitable reagent with ¹⁰ boron purity of 98%. The ¹⁰ trialkyl borate includes tributyl borate (tributyl borate, ¹⁰ B(OBu) ₃ ), trimethyl borate (trimethyl borate, ¹⁰ B(OCH ₃ ) ₃ ) or any other ^{10 boron} with ^a purity of More than 98% applicable trialkyl borates, but not limited to them. More preferably, the boronating reagent having a ¹⁰ boron purity of greater than or equal to 98% is a commercially available reagent.

The preparation method of the present invention can complete the production of 4-( ¹⁰ B) dihydroxyboryl-L-type amphetamine without additional pretreatment process for preparing boronating agent. In addition, because the preparation method of the present invention has the advantages of short and relatively simple production process, the 4-( ^10B )dihydroxyboryl-L-phenylalanine obtained through this preparation method can have high chemical purity, high Optical purity, high isotopic purity and excellent total yield, and at the same time have the advantages of no complicated purification steps, time and cost saving, high efficiency and environmental friendliness as mentioned above.

According to the present invention, the reaction solvent includes ether solvent or any other applicable organic solvent, but is not limited thereto. The ether solvents that can be used in the present invention include tetrahydrofuran (tetrahydrofuran), 2-methyltetrahydrofuran (2-methyltetrahydrofuran), diethyl ether (diethyl ether) or any other applicable ether solvents. More preferably, the ether solvent includes tetrahydrofuran or 2-methyltetrahydrofuran. the

According to the present invention, organic lithium compounds include: n-butyl lithium, tert-butyl lithium, methyl lithium, sec-butyl lithium, Phenyl lithium or any other suitable organolithium compound. the

According to the present invention, the inert organic solvent refers to an organic material that can make the organolithium compound at least partially soluble therein, and the inert organic solvent is effective for the organolithium compound, the protected (S )-4-halophenylalanine and boronating agents are chemically inert. The inert organic solvents are for example but not limited to: alkanes, ether-type solvents or any other suitable organic solvents. The alkanes include: hexanes, heptane, cyclohexane, pentane or any other suitable alkanes, but not limited thereto. The ether solvents include: tetrahydrofuran, diethyl ether, diethoxymethane, dibutyl ether, 2-methyltetrahydrofuran or any other suitable organic solvents. the

According to the present invention, the extraction solvent refers to any solvent which is substantially immiscible with water or only slightly soluble in water. The extraction solvent includes: isobutyl alcohol, toluene, n-butyl alcohol, isopropyl acetate, ethyl acetate or any other suitable extraction solvent solvents, but not exclusively. the

According to the present invention, the acidic solution includes hydrochloric acid solution or any other suitable acidic solution, but is not limited thereto. the

According to the present invention, the first organic solvent includes: acetone, tetrahydrofuran, dioxane or any other suitable organic solvent, but not limited thereto. Preferably, the first organic solvent is acetone. the

According to the present invention, the acidifying solution comprises: hydrochloric acid in acetone, hydrochloric acid in tetrahydrofuran, hydrochloric acid in dioxane, trifluoro Trifluoroacetic acid in dichloromethane, methanesulfonic acid in dioxane, trimethylsilyl chloride in dichloromethane, but not only limited to this. Preferably, the acidifying solution is a solution containing hydrochloric acid. the

Detailed ways

Below, take (S)-N-tert-butoxycarbonyl-4-iodophenylalanine as a preferred embodiment of (S)-4-halophenylalanine protected at the amine end, and use (S )-N-tert-butoxycarbonyl-4-iodophenylalanine to prepare 4-dihydroxyboryl-L-type phenylalanine, thus illustrating the implementation of the present invention to overcome the technical defects of the prior art. Those skilled in the art can easily understand the advantages and effects that the present invention can achieve through the content of this specification, and make various modifications and changes without departing from the spirit of the present invention, so as to implement or apply the content of the present invention, and at the same time It should be understood that the examples listed here are not intended to limit the scope of rights claimed by the present invention. the

In this embodiment, the selected materials and their conditions are as follows:

a. (S)-4-halophenylalanine protected at the amine end: (S)-N-tert-butoxycarbonyl-4-iodophenylalanine ((S)-N-Boc-4- iodophenylalanine), the purity is higher than 96.8%;

b. Reaction solvent: 2-methyltetrahydrofuran;

c. Boronating agent: tributyl borate or ¹⁰ tributyl borate;

d. Inert organic solvent: hexane;

e. The concentration of organolithium compound in hexane is 1.6M;

f. Organolithium compound: n-butyllithium;

g. Extraction solvent: isobutanol;

h. Acidic solution: hydrochloric acid solution;

i. The equivalent ratio of boronating agent relative to (S)-N-tert-butoxycarbonyl-4-iodophenylalanine: 3.5;

j. The equivalent ratio of organolithium compound to (S)-N-tert-butoxycarbonyl-4-iodophenylalanine: 4.25;

k. First organic solvent: acetone; and

l. Acidification solution: a solution containing hydrochloric acid. the

Since the 2-methyltetrahydrofuran, isobutanol and acetone selected by the present invention are all nontoxic and environmentally friendly solvents, and 2-methyltetrahydrofuran can be recycled and reused, the present invention provides an environmentally friendly and The preparation method can greatly reduce the production cost. the

Example 1

(1) Preparation of (S)-N-tert-butoxycarbonyl-4-dihydroxyboryl-phenylalanine from (S)-N-tert-butoxycarbonyl-4-iodophenylalanine

Please refer to the following reaction formula (I), which is the reaction of (S)-N-tert-butoxycarbonyl-4-iodophenylalanine with tributyl borate to synthesize (S)-N-tert-butoxycarbonyl- The chemical reaction formula of 4-dihydroxyborylphenylalanine. the

First, a 1-liter three-necked bottle equipped with a mechanical stirrer, a thermometer, and a nitrogen gas inlet joint sealed with a rubber gasket was prepared. Inject 150 milliliters of 2-methyltetrahydrofuran into the three-necked flask, and then add 24.7 millimoles (10.0 grams, purity 96.8%) of (S)-N-tert-butoxycarbonyl-4-iodophenylalanine, The two were uniformly stirred to form a solution; then, 77.8 mmol (17.9 g, 21 ml) of tributyl borate was added to the solution to form a mixed solution. the

Next, the mixed solution was cooled to between -85°C and -76°C, and a 1.6M (109 mmol, 68 ml) hexane solution of n-butyllithium was added dropwise to the mixed solution over 2.5 hours to A reaction mixture is obtained. After adding the hexane solution of n-butyllithium, take a small amount of the reaction mixture sample and quickly add water, and analyze it with high performance liquid chromatography (HPLC) to determine the starting material (S)-N-tert-butoxycarbonyl - The content of 4-iodophenylalanine has been reduced to less than 0.5%. the

Then, 180 ml of cold water were slowly added to the reaction mixture over 30 minutes. the

Afterwards, the reaction mixture after adding water was warmed back to between 5°C and 10°C, and stirred uniformly for 10 to 20 minutes, and then the insoluble matter in the reaction mixture was removed by filtration, washed with 20 ml of water, and then The cleaning solution and the filtrate were combined and put into a separatory funnel, and allowed to stand for stratification. the

Next, separate the lower water layer in the separatory funnel to obtain the first water layer; then use isobutanol to extract the first water layer in the separatory funnel, and separate the lower water layer to obtain the second water layer layer. the

Next, adjust the temperature of the second water layer to between 20°C and 25°C, and use 37% hydrochloric acid solution to adjust its pH value to between 3.0 and 4.0, thereby making (S)-N-tert Butoxycarbonyl-4-dihydroxyborylphenylalanine started to precipitate in the second aqueous layer. Continue to stir the second water layer for 30 minutes, and adjust its pH value to 3.0, and then continue to stir for 2 hours between 20°C and 25°C to facilitate more (S)-N-tert-butoxycarbonyl- 4-Dihydroxyboryl phenylalanine solid precipitated out. the

Finally, filter the second water layer to obtain solid crystals; then wash the solid crystals with 20 ml of water, and dry them in a vacuum oven at 50°C for at least 5 hours until they lose weight on drying (loss on drying, LOD) was less than 0.5% to obtain 5.1 g of (S)-N-tert-butoxycarbonyl-4-dihydroxyborylphenylalanine as a white solid. the

The chemical purity was 98.8% by HPLC analysis, and the yield was 66%. the

The melting point (instrument: Electrothermal9100), specific rotation (specific rotation, ), ¹ hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) and ¹³ carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR), infrared spectroscopy (IR) and electrospray-mass spectrometry (ESI-MS) analysis results are as follows:

Melting point: 150°C (decomposition temperature);

+13.5° (c=0.5, methanol);

¹ H-NMR (500MHz, DMSO-d ₆ ): δ8.0(s, 2H), 7.7(d, J=7.8Hz, 2H), 7.2(d, J=7.8Hz, 2H), 7.0(d, J=8.4Hz,2H), 4.1(m,1H), 3.0(dd,J=13.8,4.5Hz,1H), 2.8(dd,J=13.7,10.3Hz,1H) and 1.3(s,9H);

¹³ C-NMR (125MHz, DMSO-d ₆ ): δ173.63, 155.48, 139.96, 134.06, 131.96, 128.18, 78.13, 55.06, 36.53 and 28.19;

_and ^_

ESI(+)-MS m/z=332.0 (M+Na) ⁺ .

In this specification, 25 in the specific rotation analysis result means that the temperature at the time of measurement is 25°C; D means D sodium light; c means the sample concentration, and the unit is g/ml. In this specification, s in the NMR spectrum analysis results stands for singlet; d stands for doublet; dd stands for doublet of doublets; m stands for multiplet; J stands for Coupling constants in Hertz (Hz); DMSO-d ₆ is dimethyl sulfoxide with protons replaced by deuterium. In this specification, ν in the result of infrared spectroscopic analysis represents a wave number, and the unit is 1/cm (cm ^-1 ). In this specification, the m/z of the mass spectrometry results represents the ratio of mass to charge; (M+Na) ⁺ represents the fragment ions formed by the sample and sodium ions.

(2) Preparation of 4-dihydroxyboryl-L-type phenylalanine from (S)-N-tert-butoxycarbonyl-4-dihydroxyborylphenylalanine

17.9 mmol (5.63 g, purity 98.5%) of (S)-N-tert-butoxycarbonyl-4-dihydroxyboryl phenylalanine was added to a mixed solution containing 34 ml of acetone and 3.8 ml of water, And make it stir evenly, then add 3.8 ml of 37% concentrated hydrochloric acid to form an acidic mixture. Stirring of the acidic mixture was continued at 55°C for 1.5 hours, and the reaction was confirmed to be complete by HPLC. the

Afterwards, the fully reacted acidic mixture was cooled to room temperature, and its pH value was adjusted to 1.5 using aqueous sodium hydroxide solution, and then stirred for 30 minutes, thereby making 4-dihydroxyboryl-L-phenylalanine in acidic Precipitation started in the mixture. Afterwards, the pH value of the acidic mixture was adjusted to 6.2 using aqueous sodium hydroxide solution, and the mixture was continuously stirred at room temperature overnight to facilitate the precipitation of more 4-dihydroxyboryl-L-type phenylalanine solids. the

Finally, filter the acidic mixture to obtain solid crystals; then wash the solid crystals with water and 50% acetone aqueous solution, and dry them in a vacuum oven at 80°C for at least 6 hours to constant weight to obtain 3.51 grams of white solids 4-Dihydroxyboryl-L-phenylalanine. the

Its chemical purity is 99.6% through HPLC analysis, and yield rate is 93.2%; And this white crystal is determined after analyzing through chiral high-performance liquid chromatography (chiral HPLC), and the prepared 4-dihydroxyboryl-L-type phenylpropanoid The ratio of the L-enantiomer to the D-enantiomer of L-BPA is 100:0 (ie, 100% enantiomeric excess). the

The melting point, specific rotation, ¹ H-NMR, ¹³ C-NMR, IR and liquid phase-electrospray-mass spectrometry (LC-ESI-MS) analysis results of the prepared L-BPA are as follows:

Melting point: 275°C to 280°C (decomposition temperature);

-4.7° (c=0.5, 1M hydrochloric acid);

¹ H-NMR (500MHz, D ₂ O, CF ₃ COOD): δ7.2(d, J=7.9Hz, 2H), 6.8(d, J=8.0Hz, 2H), 3.9(dd, J=7.8, 5.7Hz, 1H), δ2.9(dd, J=14.6, 5.6Hz, 1H), 2.7(dd, J=14.6, 7.9Hz, 1H);

¹³ C-NMR (125MHz, D ₂ O, CF ₃ COOD): δ171.81, 137.31, 135.16, 132.42, 129.65, 54.64 and 36.32;

_and ^_

LC-ESI(+)-MS (M+Na) ⁺ = m/z 232.0.

Example 2

(1) Preparation of (S)-N-tert-butoxycarbonyl-4-( ¹⁰ B)dihydroxyboryl-phenylalanine from (S)-N-tert-butoxycarbonyl-4-iodophenylalanine

First, a 3-liter three-necked bottle equipped with a mechanical stirrer, a thermometer, and a nitrogen inlet joint sealed with a rubber gasket was prepared. Inject 750 milliliters of 2-methyltetrahydrofuran into the three-necked flask, then add 128 millimoles (50.0 grams, 100% purity) of (S)-N-tert-butoxycarbonyl-4-iodophenylalanine, The two were uniformly stirred to form a solution; then, 393 mmol (90.1 g, 106 ml) of ¹⁰ tributyl borate was added to the solution to form a mixed solution.

Next, the mixed solution was cooled to between -85°C and -76°C, and a 1.6M (600 mmol, 375 ml) hexane solution of n-butyllithium was added dropwise to the mixed solution over 3 hours to A reaction mixture is obtained. the

Then, the reaction mixture was stirred at -80°C for 0.5 hours, and a small amount of the reaction mixture sample was quickly added to water and analyzed by HPLC to determine the starting material (S)-N-tert-butoxycarbonyl-4-iodobenzene The content of alanine has been reduced to less than 0.5%. the

Afterwards, 900 ml of cold water was slowly added dropwise to the reaction mixture within 15 to 20 minutes, after that, the reaction mixture after adding water was returned to the temperature between 5°C and 10°C, and stirred uniformly for 10 to 20 minutes, and then filtered through The insoluble matter in the reaction mixture was removed by means, and washed with 100 ml of water, and then the filtrate and washings were combined into a separatory funnel for liquid phase extraction. the

Next, separate the lower water layer in the separatory funnel to obtain the first water layer; then use isobutanol to extract the first water layer in the separatory funnel, and separate the lower water layer to obtain the second water layer layer. the

Next, adjust the temperature of the second water layer to between 20°C and 25°C, and use 37% hydrochloric acid solution to adjust its pH value to between 3.0 and 4.0, thereby making (S)-N-tert Butoxycarbonyl-4-( ¹⁰ B)dihydroxyborylphenylalanine started to precipitate in the second aqueous layer. Continue to stir the second water layer for 30 minutes, and adjust its pH value to 3.0, and then continue to stir at 20°C to 25°C for 2 hours to facilitate the precipitation of more (S)-N-tert-butoxycarbonyl groups -4-( ¹⁰ B) dihydroxyboryl phenylalanine solid.

Finally, filter the solid product in the second water layer; wash the solid twice with water, and place it in a vacuum drying oven at 50° C. for at least 4 hours until its loss on drying is less than 0.5%, obtaining 25.8 grams of (S )-N-tert-butoxycarbonyl-4-( ^10B )dihydroxyborylphenylalanine as a white solid.

The chemical purity was 98.6% by HPLC analysis, and the yield was 65.1%. the

Melting ^point , specific optical rotation, ¹ H-NMR, ¹³ C-NMR, IR and high-resolution Analysis results such as rate mass spectrometry (HRMS) are as follows:

Melting point: 150°C (decomposition temperature);

: +14° (c=0.5, methanol);

¹ H-NMR (500MHz, DMSO-d ₆ ): δ8.0(s, 2H), 7.7(d, J=7.7Hz, 2H), 7.2(d, J=7.6Hz, 2H), 7.0(d, J=8.4Hz,2H), 4.1(m,1H), 3.0(dd,J=13.8,4.5Hz,1H), 2.8(dd,J=13.7,10.3Hz,1H), 1.3(s,9H);

¹³ C-NMR (125MHz, DMSO-d ₆ ): δ173.63, 155.48, 139.96, 134.06, 131.94, 128.18, 78.13, 55.06, 36.53, 28.19;

_and ^_

HRMS (ESI): Anal. Calcd. for C ₁₄ H ₂₀ ¹⁰ BNO ₆ 307.1420 ([MH] ⁻ ), found 307.1333.

(2) Preparation of 4-( ¹⁰ B) dihydroxyboryl-L-type phenylalanine from (S)-N-tert-butoxycarbonyl-4-( ¹⁰ B) dihydroxyboryl phenylalanine

Add 66.2 mmol (20.5 g, purity 99.6%) of (S)-N-tert-butoxycarbonyl-4-( ¹⁰ B) dihydroxyboryl phenylalanine to a solution containing 122 ml of acetone and 14 ml of water In the mixture, and make it evenly stirred, then add 14 ml of 37% concentrated hydrochloric acid to form an acidic mixture. Stirring of the acidic mixture was continued at 55°C for 1.5 to 2 hours, and the reaction was confirmed to be complete by HPLC.

Afterwards, the fully reacted acidic mixture is cooled to room temperature, and its pH value is adjusted to 1.5 using aqueous sodium hydroxide solution, thereby making 4-( ¹⁰ B) dihydroxyboryl-L-type phenylalanine in the acidic mixture Started to precipitate out, and continued to stir for 50 minutes. Afterwards, the pH value of the acidic mixture was adjusted to 6.2 using aqueous sodium hydroxide solution and stirred at room temperature for at least 25 minutes to ensure the precipitation of more 4-( ¹⁰ B)dihydroxyboryl-L-phenylalanine solid.

Finally, filter the acidic mixture to obtain solid crystals; then wash the solid crystals with water and 50% acetone aqueous solution, and dry them in a vacuum oven at 80°C for at least 6 hours to constant weight to obtain 13.3 grams of 4- ( ¹⁰ B) Dihydroxyboryl-L-type phenylalanine white solid.

Its chemical purity is 99.9% through HPLC analysis, and productive rate is 96.4%; And this white crystal is determined after chiral HPLC analysis, and the prepared 4-( ¹⁰ B) dihydroxyboryl-L-type phenylalanine (L - ¹⁰ BPA) in a 100:0 ratio of the L-enantiomer to the D-enantiomer (ie, 100% enantiomeric excess).

The melting point, specific rotation, ¹ H-NMR, ¹³ C-NMR, IR, inductively coupled plasma mass spectrometry (ICP-MS) and HRMS analysis results of the prepared L- ¹⁰ BPA are as follows:

Melting point: 275°C to 280°C (decomposition temperature);

-5.4° (c=0.5, 1M hydrochloric acid);

¹ H-NMR (500MHz, D ₂ O, CF ₃ COOD): δ7.2(d, J=8.0Hz, 2H), 6.8(d, J=8.0Hz, 2H), 3.9(dd, J=7.8, 5.7Hz, 1H), 2.8(dd, J=14.6, 5.6Hz, 1H), 2.7(dd, J=14.6, 7.9Hz, 1H);

¹³ C-NMR (125MHz, D ₂ O, CF ₃ COOD): δ171.80, 137.31, 135.16, 132.37, 129.65, 54.64, 36.32;

IR(KBr) _νmax : 3585, 3148, 3038, 2923, 1636, 1610, 1507, 1410, 1398, 1345, 1085, 716cm ^-1 ;

ICP-MS: The content of ¹⁰ B relative to ¹¹ B (the isotopic purity of ¹⁰ B) is 99.4% (w/w%); and

HRMS (ESI): Anal. Calcd. for C ₉ H ₁₃ ¹⁰ BNO ₄ 209.0974 ([M+H] ⁺ ), found 209.0970.

The above-mentioned embodiments are only examples for convenience of description, and the scope of rights claimed by the present invention should be determined by the claims, rather than limited to the above-mentioned embodiments. the

Claims (11)

1. a preparation method of 4-dihydroxyboryl-L-type phenylalanine, which comprises the following steps: reacting the amine-terminally protected (S)-4-halophenylalanine with a boronating agent and an organolithium compound to obtain a reaction mixture comprising amine-terminally protected (S)-4-di Hydroxyboryl phenylalanine, the protected (S)-4-halophenylalanine at the amine end has a structure shown in the following formula (I), the protected (S)-4-di Hydroxyboryl phenylalanine has a structure shown in the following formula (II), and in formula (I) and formula (II), R is a protecting group; The (S)-4-dihydroxyboryl phenylalanine protected at the amine end is isolated from the reaction mixture; The protecting group of the amine-terminal protected (S)-4-dihydroxyborylphenylalanine is deprotected to produce 4-dihydroxyboryl-L-phenylalanine. 2. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1, wherein the boronating agent has ¹⁰ boron purity greater than or equal to 98%, and the amine end is protected ( S)-4-dihydroxyboryl phenylalanine is (S)-4-( ¹⁰ B)dihydroxyboryl phenylalanine protected at the amine end, 4-dihydroxyboryl-L-type phenylalanine The acid is 4-( ¹⁰ B) dihydroxyboryl-L-phenylalanine. 3. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein in formula (I), X is iodo or bromo. 4. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein in formula (I) and formula (II), the protecting group is selected from the following groups: The group consisting of: tert-butoxycarbonyl, trityl, 3,5-dimethoxyphenylisopropoxycarbonyl, 2-(4-biphenyl)isopropoxycarbonyl and 2-nitrophenylsulfinyl Acyl. 5. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein the protected (S)-4-halophenylalanine at the amine end is combined with the The boronating agent and the organolithium compound react to obtain the step of the reaction mixture comprising: Reacting the amine-terminally protected (S)-4-halophenylalanine with the boronating agent and the organolithium compound at a temperature of -100°C to -50°C to obtain the reaction mixture. 6. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein the boronating agent is protected (S)-4-halobenzene with respect to the amine end The equivalence ratio of alanine is between 2 and 5. 7. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1, wherein the organolithium compound is protected (S)-4-halophenylalanine with respect to the amine end The equivalence ratio of the acids is at least 3. 8. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein the protected (S)-4-halophenylalanine and the boron The step of reacting the reacting agent and the organolithium compound to obtain the reaction mixture comprises: mixing the (S)-4-halophenylalanine whose amine end is protected, the reaction solvent and the boronating agent to obtain a mixed solution; and An inert organic solvent containing the organolithium compound is added to the mixed solution at a temperature of -100°C to -50°C to obtain the reaction mixture. 9. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein the (S)-4-dihydroxyl group protected at the amine end is isolated from the reaction mixture The steps for borylphenylalanine include: adding an aqueous solution to the reaction mixture to obtain a first aqueous layer; Extracting the first aqueous layer with an extraction solvent to obtain a second aqueous layer, wherein the second aqueous layer comprises (S)-4-dihydroxyborylphenylalanine whose amine end is protected; and adjusting the pH value of the second aqueous layer to be lower than 4, thereby crystallizing the (S)-4-dihydroxyboryl phenylalanine protected at the amine end to separate from the second aqueous layer to obtain (S)-4-dihydroxyborylphenylalanine protected at the amine end. 10. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein the protected (S)-4-dihydroxyborylphenylalanine at the amine end is deprotected The protecting group of acid comprises: Acidifying the first organic solution, which is a mixture containing (S)-4-dihydroxyborylphenylalanine protected at the amine end and a first organic solvent, thereby deprotecting the protected amine end The protecting group of (S)-4-dihydroxyboryl phenylalanine to obtain 4-dihydroxyboryl-L-type phenylalanine. 11. The preparation method of 4-dihydroxyboryl-L-type phenylalanine according to claim 1 or 2, wherein the protected (S)-4-dihydroxyborylphenylalanine of the amine end is deprotected The protecting group of acid, to make the step of 4-dihydroxyboryl-L-type phenylalanine comprises: Acidifying the first organic solution, which is a mixture containing (S)-4-dihydroxyborylphenylalanine protected at the amine end and a first organic solvent, thereby deprotecting the protected amine end The protecting group of (S)-4-dihydroxyboryl phenylalanine, to obtain an acidic mixture, wherein the acidic mixture comprises 4-dihydroxyboryl-L-type phenylalanine; and adjusting the pH value of the acidic mixture to be greater than 1, thereby crystallizing 4-dihydroxyboryl-L-phenylalanine to obtain 4-dihydroxyboryl-L-phenylalanine from the acidic mixture .