CN108530322B

CN108530322B - Tyrosine semicarbazide hydrazone hydrochloride, its synthesis method and its use

Info

Publication number: CN108530322B
Application number: CN201810496509.8A
Authority: CN
Inventors: 赵新俊; 孙利权; 高汉荣; 陈有刚; 陈艳丽
Original assignee: Fule Ma Hongkai Dalian Medical Co ltd
Current assignee: Fule Ma Hongkai Dalian Medical Co ltd
Priority date: 2018-05-22
Filing date: 2018-05-22
Publication date: 2020-11-06
Anticipated expiration: 2038-05-22
Also published as: CN108530322A

Abstract

The invention relates to a method for synthesizing tyrosine semicarbazide hydrazone hydrochloride. Specifically, the process comprises a) esterifying tyrosine to give an esterified intermediate 2; b) protecting the amino group and the phenolic hydroxyl group in the esterification intermediate 2 to obtain a compound represented by formula 3; c) reducing the compound represented by formula 3 to obtain a compound represented by formula 4; d) oxidizing the compound represented by formula 4 to obtain a compound represented by formula 5; e) reacting the compound represented by formula 5 with semicarbazide to obtain a compound represented by formula 6; f) deprotecting the compound represented by formula 6 to obtain tyrosine semicarbazone hydrochloride represented by formula 7; wherein R represents a methyl group, an ethyl group or a propyl group; r₁Are protecting groups that can be used for both amino and phenolic hydroxyl groups. In addition, the invention also relates to the tyrosine semicarbazide hydrazone hydrochloride and application thereof.

Description

Tyrosine semicarbazide hydrazone hydrochloride, its synthesis method and its use

Technical Field

The invention relates to a protease stabilizer intermediate, in particular to a protease stabilizer intermediate tyrosine semicarbazone hydrochloride in a novel liquid detergent, and relates to a synthetic method of the intermediate tyrosine semicarbazone hydrochloride.

Background

In early liquid detergents, borides were typically added in order to maintain the protease activity in the detergent. Subsequent studies have shown boronThe compounds are degraded into boric acid, which is harmful to the environment. Later, danish novavissin (Novozymes) corporation developed a new protease stabilizer (WO 2013/004635, US 2014/0228274, and WO 2013/188344) in place of the boride. The novel protease stabilizer has a structure of X-B¹-B⁰-H ", wherein X represents an amino acid or peptide bearing an N-terminal protecting group, B¹Represents an amino acid, B⁰-H represents an aldehyde corresponding to an amino acid (abbreviated as aminoaldehyde).

It is well known that aminoaldehydes have been a difficult point of chemical synthesis. The chemical property of aldehyde is very active and easy to deteriorate, and the aldehyde can react with amino to generate Schiff base even under certain conditions. Particularly, the tyrosine corresponding aldehyde (casamino aldehyde for short) is an important intermediate of the novel protease stabilizer and shows better activity. However, there are few reports related to the synthesis of tyrosinal and its derivatives, and in the two prior art documents (BMCL,15(23),5176-5181, 2015; BMCL,18(1),95-98, 2008), the synthesis of tyrosinal and its derivatives was reported by the following route.

In the above route, it was mainly relied on to reduce the amide to the aldehyde at-78 ℃ with lithium aluminum hydride or diisobutylaluminum as the catalyst. Obviously, in the above-mentioned routes, the characteristics of flammability, high cost and necessity of low temperature use of the organoaluminum catalysts used greatly limit the industrial applicability of the existing reaction routes.

Further, firstly, the amino group of the amino aldehyde itself reacts with the aldehyde group and is difficult to be stably present. Secondly, the amino aldehyde derivatives obtained in the above routes have a protecting group at the amino group, which is not compatible with the protease stabilizer "X-B¹- "are partially bound. Therefore, even if the aminoaldehyde derivative is obtained by a known route, it is difficult to obtain a desired protease stabilizer by a condensation reaction.

Therefore, there is a need for aminoaldehyde derivatives and methods for their synthesis as appropriate intermediates,which is suitable for the synthesis of "X-B¹-B⁰-H "type protease stabilizers and are suitable for industrial production.

Disclosure of Invention

Therefore, the present invention aims to provide an intermediate suitable for producing protease stabilizers, and a synthetic method thereof suitable for industrial production.

For the above purpose, considering the properties of aldehyde group, amino group and the use of the desired intermediate, and considering that the protecting group used should be conveniently introduced and removed and stable under various conditions, the present invention employs semicarbazide to protect aldehyde group to obtain tyrosine semicarbazide hydrazone hydrochloride (H-Tyr-sec. hcl) as an intermediate, first, the resulting derivative is stable under conventional acidic, basic and amino acid condensation conditions, and tyrosine aminoaldehyde can be easily obtained by exchange using formaldehyde or benzaldehyde; secondly, the intermediate is structurally stable, easy to store and transport, and capable of reacting with "X-B¹The condensation is carried out with high efficiency and no side reaction basically occurs.

Thus, according to one aspect of the present invention, there is provided a process for the synthesis of tyrosine semicarbazone hydrochloride, wherein the process is carried out according to the process scheme shown below, comprising:

a) esterifying tyrosine to obtain an esterified intermediate 2;

b) protecting the amino group and the phenolic hydroxyl group in the esterification intermediate 2 to obtain a compound represented by formula 3;

c) reducing the compound represented by formula 3 to obtain a compound represented by formula 4;

d) oxidizing the compound represented by formula 4 to obtain a compound represented by formula 5;

e) reacting the compound represented by formula 5 with semicarbazide to obtain a compound represented by formula 6;

f) deprotecting the compound represented by formula 6 to obtain tyrosine semicarbazone hydrochloride represented by formula 7;

wherein R represents a methyl group, an ethyl group or a propyl group; r₁Is tert-butyloxycarbonyl, benzyloxycarbonyl, methoxycarbonyl or ethoxycarbonyl.

According to one embodiment, in step a), thionyl chloride or hydrogen chloride, preferably thionyl chloride, is used as catalyst. The molar ratio of the catalyst to the tyrosine is 1-3, preferably 1.2-1.5. In this step, the solvent is not particularly limited, and the esterifying reagent used may be used together as a solvent, and may be, for example, a lower alcohol such as methanol, ethanol or isopropanol, preferably methanol. After the reaction is complete, the product obtained can be used directly in step b).

According to one embodiment, step b) is carried out in the presence of a base, using a protecting reagent capable of simultaneously protecting the amino group and the phenolic hydroxyl group, such as di-tert-butyl dicarbonate, benzyl chloroformate, methyl chloroformate or ethyl chloroformate, preferably di-tert-butyl dicarbonate, and using a molar ratio of the protecting reagent to tyrosine of 2 to 5, preferably 2.2 to 2.5. Tetrahydrofuran, methanol, toluene, dichloromethane or ethyl acetate are used as solvents; the base used may be an organic or inorganic base such as triethylamine, diisopropylethylamine, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate, preferably potassium carbonate. In a preferred embodiment, a di-tert-butyl dicarbonate, methylene chloride and potassium carbonate system is employed. The product obtained can be used directly in step c).

According to one embodiment, in step c), as reducing agent those compounds conventionally used in the art for reducing esters to alcohols may be used, such as borohydride, preferably sodium borohydride, potassium borohydride, zinc borohydride or calcium borohydride, more preferably sodium borohydride, wherein the molar ratio of reducing agent used to tyrosine is 1 to 4, preferably 1.8 to 2.2; the solvent used is tetrahydrofuran, methanol, ethanol, isopropanol or ethylene glycol dimethyl ether, preferably methanol. In a preferred embodiment, a sodium borohydride and methanol system is employed. The product obtained can be used directly in step d).

According to one embodiment, in step d), sodium hypochlorite is used as the oxidant, the molar ratio of the oxidant used to tyrosine being 1.5 to 3.0, preferably 1.8 to 2.2; using tetramethylpiperidine nitroxide and sodium bromide/potassium bromide as co-catalyst; ethyl acetate, methyl tert-butyl ether or dichloromethane, preferably ethyl acetate, is used as solvent. In a preferred embodiment, a sodium hypochlorite, tetramethylpiperidine nitroxide, sodium bromide and ethyl acetate system is employed. The product obtained can be used directly in step e).

According to one embodiment, in step e), the molar ratio of semicarbazide to tyrosine is between 0.8 and 1.5, preferably between 0.95 and 1.05. The solvent is not particularly limited, but dichloromethane, methyl t-butyl ether, ethyl acetate or toluene is preferably used, and ethyl acetate is more preferably used. The product obtained can be used directly in step f).

According to one embodiment, in step f), trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid or hydrogen chloride is used as deprotecting reagent, wherein the molar ratio of deprotecting reagent to tyrosine used is 3 to 10, preferably 5 to 6; the solvent used is not particularly limited, and may be, for example, a lower alcohol such as methanol, ethanol, isopropanol, or dichloromethane. In a preferred embodiment, a hydrogen chloride and methanol system is employed.

In a particularly preferred embodiment of the invention, this is carried out by way of the following schematic representation:

a) carrying out esterification reaction on tyrosine and methanol under the action of thionyl chloride to obtain an esterification intermediate L-Tyr-OMe.HCl;

b) reacting the esterified intermediate L-Tyr-OMe.HCl with di-tert-butyl dicarbonate in the presence of potassium carbonate to obtain the di-Boc ester intermediate Boc-Tyr (Boc) -OMe;

c) reacting the double Boc ester intermediate Boc-tyr (Boc) -OMe with sodium borohydride to obtain the double Boc alcohol intermediate Boc-tyr (Boc) -olo;

d) reacting the double Boc alcohol intermediate Boc-tyr (Boc) -olo with sodium hypochlorite to obtain double Boc aldehyde intermediate Boc-tyr (Boc) -H;

e) reacting the double Boc aldehyde intermediate Boc-Tyr (Boc) -H with semicarbazide to give a double Boc semicarbazide hydrazone intermediate Boc-Tyr (Boc) -SEC;

f) reacting the double Boc semicarbazide hydrazone intermediate Boc-Tyr (Boc) -SEC with a strong acid to obtain tyrosine semicarbazide hydrazone hydrochloride H-Tyr-SEC.

In the process of the present invention, all of the raw materials are readily available on the market. Through six steps of high-yield reaction, except that the final product needs to be purified to obtain a solid, the intermediates in other steps do not need to be subjected to a fine purification step, the loss of the intermediates and the use of a large amount of purification solvents are reduced, and the process is simple and convenient and is easy for industrial continuous production. The final product can be used to produce protease stabilizers by a reaction that is easy to perform.

Drawings

FIG. 1 is a chart showing a HPLC chart of the reaction in step a) in preparation example 1 according to the present invention; wherein the retention time of L-tyrosine is 2.86 minutes, and the retention time of L-tyrosine methyl ester is 3.03 minutes;

FIG. 2 is a chart showing a reaction HPLC spectrum in step b) in preparative example 1 according to the present invention; wherein the retention time of the L-tyrosine methyl ester is 3.03 minutes; the retention time of Boc-Tyr (Boc) -OMe was 16.4 min;

FIG. 3 is a chart showing a reaction HPLC spectrum in step c) in preparative example 1 according to the present invention; wherein the Boc-Tyr (Boc) -OMe retention time is 16.4 minutes; the retention time of Boc-Tyr (Boc) -OLO was 13.5 min;

FIG. 4 is a chart showing a reaction HPLC spectrum in step d) in production example 1 according to the present invention; wherein the retention time of Boc-Tyr (Boc) -olo was 13.2 minutes; the retention time of Boc-Tyr (Boc) -H was 12.7 minutes;

FIG. 5 is a chart showing a reaction HPLC spectrum in step e) in preparative example 1 according to the present invention; wherein the retention time of Boc-Tyr (Boc) -H is 12.7 minutes, the retention time of Boc-Tyr (Boc) -SEC is 11.7 minutes, and the retention time of Boc-Tyr-SEC is 6.1 minutes;

FIG. 6 is a chart showing a reaction HPLC chart in step f) in production example 1 according to the present invention; wherein the retention time of Boc-Tyr (Boc) -H is 12.7 minutes, the retention time of Boc-Tyr (Boc) -SEC is 11.7 minutes, and the retention time of Boc-Tyr-SEC is 6.1 minutes;

FIG. 7 is an HPLC chromatogram showing tyrosine semicarbazone hydrochloride in step f) of preparation example 1 according to the present invention;

FIG. 8 is a diagram showing tyrosine semicarbazone hydrochloride in step f) of preparation example 1 according to the present invention¹H NMR spectrum.

Detailed Description

In the corresponding synthetic method provided by the invention, tyrosine which is a simple and cheap starting material is adopted, the tyrosine semicarbazone hydrochloride is produced with high yield under mild reaction conditions, the post-treatment method is simple, and an intermediate in each step is not required to be solidified and purified, so that the method is very suitable for commercial scale-up production.

As can be seen from the following specific examples, the method of the present invention has stable yield and high product purity through hundreds of gram-scale experiments. The process is easy to be amplified and is suitable for commercial amplification production.

The invention will be further illustrated below by particularly preferred synthetic routes. It should be understood by those skilled in the art that the present invention is not limited to the following specific embodiments. The reaction raw materials, catalysts, solvents and the like used in the following preparation examples are commercially available.

The reaction was monitored by HPLC during the reaction, wherein the peak numbers appearing in the table below were determined in terms of the order of appearance of the peaks. The HPLC (Agilent 1200) test conditions are as follows:

column: kromasil C18250X 4.6mm 5 μm

Column temperature: 25 deg.C

Detection wavelength: 210nm

Flow rate: 1mL/min

Sample introduction amount: 2 μ L

Operating time: 30min

Mobile phase A: 0.1% TFA/water (1 mL of TFA was added to 1000mL of water and mixed well)

Mobile phase B: acetonitrile

0min：70％A，30％B

10min：30％A，70％B

Preparation example 1: synthesis of tyrosine semicarbazide hydrazone hydrochloride

Step a): 136 g of tyrosine and 680 ml of anhydrous methanol are mixed under nitrogen protection, and 107 g of thionyl chloride is added dropwise at room temperature. Then the solution is heated to 57 ℃ to 63 ℃ and reacted for 2 hours. Then, the mixture was concentrated under reduced pressure at this temperature to obtain a suspension, and 100 ml of toluene was added to the suspension to obtain a suspension. This suspension was used directly in the next step.

The integration results of this step by HPLC are shown in Table 1 below.

TABLE 1

Step b): adding the suspension obtained in the step a) into 750 ml of dichloromethane, cooling to 0-5 ℃, adjusting the pH value of the system to 8-9 by using a potassium carbonate aqueous solution, and then dropwise adding 380 g of di-tert-butyl dicarbonate. Stirred and reacted at room temperature. After the reaction was completed, the mixture was allowed to stand, separated, and the organic phase was concentrated under reduced pressure at 40 ℃ to obtain 300 g of a pale yellow oil.

The integration results of this step by HPLC are shown in Table 2 below.

TABLE 2

Step c): 300 g of the yellow oil obtained in step b) are added to 500 ml of anhydrous methanol, and after stirring the mixture evenly, 57 g of sodium borohydride are added in portions. At this time, heat was released and a large amount of bubbles were generated. The reaction system is reacted for 1 hour at 30-35 ℃. After the reaction is finished, 500 ml of toluene is added, and then the citric acid aqueous solution is dropwise added to adjust the pH value of the reaction system to 3-4. After standing and separating, the toluene phase was concentrated under reduced pressure at 40 ℃ to give 380 g of an oil.

The integration results of this step by HPLC are shown in Table 3 below.

TABLE 3

Step d): 380 g of the yellow oil obtained in step c) are added to 2 l of ethyl acetate, and after stirring the mixture uniformly, 60 g of sodium bicarbonate, 800 ml of water, 8 g of sodium bromide and 2 g of tetramethylpiperidine nitroxide are added in turn. The system was cooled to 0 ℃ and 1.2 liters of an aqueous sodium hypochlorite solution was added dropwise while the system temperature was kept below 5 ℃. After the addition was complete, the mixture was stirred at a temperature of less than 5 ℃ for 1 hour. After the reaction is finished, standing and separating liquid. The organic phase was washed with 200 ml of saturated aqueous sodium bicarbonate solution and 200 ml of 5% aqueous citric acid solution in this order. The organic phase was concentrated under reduced pressure at 40 ℃ to give 280 g of an oil.

The integration results of this step by HPLC are shown in Table 4 below.

TABLE 4

Step e): 280 g of the oil obtained in step d) are added to 1.5 l of ethyl acetate and, after stirring to homogeneity, the semicarbazide hydrochloride and the aqueous solution of sodium bicarbonate prepared beforehand are added. The system was allowed to react at 20 ℃ to 25 ℃ for 2 hours. After the reaction was completed, the mixture was allowed to stand, separated, and the organic phase was concentrated under reduced pressure to obtain 260 g of a pale yellow oil.

The integration results of this step by HPLC are shown in Table 5 below.

TABLE 5

Step f): 260 g of the oil obtained in step e) are added to 800 ml of anhydrous methanol, and 118 g of hydrogen chloride gas are then introduced. The system was allowed to react at 20 ℃ to 25 ℃ for 24 hours. After the reaction was complete, the reaction was concentrated at 40 ℃ under reduced pressure to give an oil. Then, 800 ml of methyl t-butyl ether was added and a large amount of solid was precipitated. Filtration and drying in vacuo at 40 ℃ gave 112 g of a pale yellow solid. By HPLC and¹the product was identified by H NMR as tyrosine semicarbazide hydrazone hydrochloride in 99.7% chemical purity. The total yield of the six steps is 57.8 percent by calculation.

The integration results of this step by HPLC are shown in Table 6 below.

TABLE 6

Preparation example 2: synthesis of tyrosine semicarbazide hydrazone hydrochloride

Step a): 272 g of tyrosine and 1.4 l of anhydrous methanol are mixed under nitrogen and 200 g of thionyl chloride are added dropwise at room temperature. The resulting solution was then heated to 57 ℃ to 63 ℃ and reacted for 2 hours. Then, the mixture was concentrated under reduced pressure at this temperature to obtain a suspension, and 200 ml of toluene was added thereto, followed by concentration again to obtain a suspension. This suspension was used directly in the next step.

Step b): adding the suspension obtained in the step a) into 1.5L of toluene, cooling to 0-5 ℃, adjusting the pH value of the system to 8-9 by using a potassium carbonate aqueous solution, and then dropwise adding 765 g of di-tert-butyl dicarbonate. Stirred and reacted at room temperature. After the reaction was completed, the mixture was allowed to stand, separated, and the organic phase was concentrated under reduced pressure at 40 ℃ to obtain 610 g of a pale yellow oil.

Step c): 610 g of the yellow oil obtained in step b) are added to 1 l of absolute ethanol and, after stirring to homogeneity, 110 g of sodium borohydride are added in portions. At this time, heat was released and a large amount of bubbles were generated. The reaction system is reacted for 1 hour at 30-35 ℃. After the reaction is finished, 1 liter of toluene is added, and then a citric acid aqueous solution is dropwise added to adjust the pH value of the reaction system to 3-4. After standing and separating the phases, the toluene phase was concentrated under reduced pressure at 40 ℃ to give 780 g of an oil.

Step d): 780 g of the yellow oil obtained in step d) are added to 4 l of methyl tert-butyl ether, and after stirring the mixture to homogeneity, 120 g of sodium bicarbonate, 1.6 l of water, 16 g of sodium bromide and 5 g of tetramethylpiperidine nitroxide are added in this order. The system was cooled to 0 ℃ and 2.5 liters of an aqueous sodium hypochlorite solution were added dropwise while the system temperature was kept below 5 ℃. After the addition was complete, the mixture was stirred at a temperature of less than 5 ℃ for 1 hour. After the reaction is finished, standing and separating liquid. The organic phase was washed successively with 400 ml of saturated aqueous sodium bicarbonate solution and 400 ml of 5% aqueous citric acid solution. The organic phase was concentrated under reduced pressure at 40 ℃ to give 580 g of an oil.

Step e): 580 g of the oil obtained in step d) are added to 3 l of ethyl acetate and, after stirring to homogeneity, the semicarbazide hydrochloride and the aqueous solution of sodium bicarbonate prepared beforehand are added. The system was allowed to react at 20 ℃ to 25 ℃ for 2 hours. After the reaction was completed, the mixture was allowed to stand, separated, and the organic phase was concentrated under reduced pressure to obtain 560 g of a pale yellow oil.

Step f): 560 g of the oil obtained in step e) are added to 1.6 l of anhydrous methanol, and 218 g of hydrogen chloride gas are then introduced. The system was allowed to react at 20 ℃ to 25 ℃ for 24 hours. After the reaction was complete, the reaction was concentrated at 40 ℃ under reduced pressure to give an oil. Then, 1.6L of methyl t-butyl ether was added, and a large amount of solid was precipitated. Filtration and drying in vacuo at 40 ℃ gave 212 g of a pale yellow solid. The obtained product is tyrosine semicarbazone hydrochloride through verification, and the chemical purity is 99.6%. The total yield of the six steps is 54.6 percent.

Claims

1. A method of synthesizing tyrosine semicarbazone hydrazone hydrochloride, wherein the method comprises:

a) esterifying tyrosine to obtain an esterified intermediate 2;

2. The process according to claim 1, wherein in step a), thionyl chloride or hydrogen chloride is used as catalyst; wherein the molar ratio of the catalyst to the tyrosine is 1-3.

3. The process according to claim 2, wherein in step a) thionyl chloride is used as catalyst.

4. The process according to claim 2, wherein in step a) the molar ratio of catalyst to tyrosine used is from 1.2 to 1.5.

5. The method according to claim 1, wherein step b) is carried out in the presence of a base, wherein di-tert-butyl dicarbonate, benzyl chloroformate, methyl chloroformate or ethyl chloroformate is used as a protecting reagent, and the molar ratio of the protecting reagent to tyrosine used is 2-5; the base used is triethylamine, diisopropylethylamine, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate.

6. The process according to claim 5, wherein in step b) di-tert-butyl dicarbonate is used as protecting agent.

7. The method according to claim 5, wherein in step b) the molar ratio of the protecting reagent to tyrosine used is 2.2-2.5.

8. The method according to claim 1, wherein in step c), borohydride is used as a reducing agent, and the molar ratio of the reducing agent to tyrosine is 1-4; tetrahydrofuran, methanol, ethanol, isopropanol or ethylene glycol dimethyl ether is used as a reaction solvent.

9. The process according to claim 8, wherein in step c) sodium borohydride, potassium borohydride, zinc borohydride or calcium borohydride is used as reducing agent.

10. The process according to claim 8, wherein in step c) the molar ratio of reducing agent to tyrosine used is between 1.8 and 2.2.

11. The process according to claim 8, wherein in step c) methanol is used as reaction solvent.

12. The method according to claim 1, wherein in step d), sodium hypochlorite is used as the oxidant, and the molar ratio of the oxidant to tyrosine is 1.5-3.0; using tetramethylpiperidine nitroxide and sodium bromide/potassium bromide as co-catalyst; ethyl acetate, methyl tert-butyl ether or dichloromethane was used as solvent.

13. The process according to claim 12, wherein in step d) the molar ratio of oxidant to tyrosine used is between 1.8 and 2.2.

14. The process according to claim 1, wherein in step e) the molar ratio of semicarbazide to tyrosine is between 0.8 and 1.5.

15. The process according to claim 14, wherein in step e) the molar ratio of semicarbazide to tyrosine is between 0.95 and 1.05.

16. The process according to claim 1, wherein in step f), trifluoroacetic acid, methanesulfonic acid, p-toluenesulfonic acid or hydrogen chloride is used as deprotecting reagent; the molar ratio of the deprotection reagent to tyrosine is 3-10.

17. The process of claim 1, wherein in step f), the molar ratio of deprotecting reagent to tyrosine is 5-6.

18. The method of claim 1, wherein R is methyl and R is₁Is tert-butyloxycarbonyl.