TW202321203A - Method for preparing pyrazine carboxylic acid derivative as fluorescent tracer - Google Patents

Abstract

The present application relates to a method for preparing a pyrazine derivative as a fluorescent tracer, and specifically relates to a method for preparing 3,6-diamino-2,5-bis{N-(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyrazine, an intermediate compound for the preparation method and a method for preparing the intermediate compound.

Description

Method for preparing pyrazine carboxylic acid derivatives as fluorescent tracers

The present invention relates to a method for preparing 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminoformyl}pyrazine, used for said Intermediate compounds of the methods of preparation and methods of preparing said intermediate compounds.

Pyrazine derivatives have potential applications as fluorescent tracers in the assessment of renal function. As a fluorescent tracer with great clinical application value, 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminoformyl }The research on pyrazine (compound 1) has received extensive attention, but there are very few reports on the synthesis route of this compound.

。其中，醯胺化反應以D-絲胺酸苄酯鹽酸鹽為反應受質，得到中間體4，再經鈀/碳催化氫化反應生成化合物1。由於D-絲胺酸苄酯鹽酸鹽和鈀/碳的商業價格昂貴，導致產品的生產成本較高。此外，氫化反應不僅對生產設備和場地有較高要求，而且氫氣是易燃易爆氣體，操作十分危險，存在極大的安全隱患。由於氫化反應使用了鈀/碳，處理不當會導致貴金屬殘留，因而存在藥物安全風險。 In 2011, Raghavan Rajagopalan et al. optimized the synthesis of compound 1 and reduced the number of reaction steps (J. Med. Chem. 2011, 54, 5048–5058). In this synthesis method, 3,6-diaminopyrazine-2,5-dicarboxylic acid as a raw material is subjected to amidation, palladium-catalyzed hydrogenation and other steps to obtain compound 1:

. Among them, the amidation reaction uses D-serine benzyl ester hydrochloride as a reaction substrate to obtain intermediate 4, which is then catalyzed by palladium/carbon hydrogenation to generate compound 1. Due to the high commercial price of D-serine benzyl ester hydrochloride and palladium/carbon, the production cost of the product is relatively high. In addition, the hydrogenation reaction not only has high requirements on production equipment and sites, but also hydrogen is a flammable and explosive gas, which is very dangerous to operate and poses a great safety hazard. Due to the use of palladium/carbon in the hydrogenation reaction, improper handling can lead to precious metal residues, thus posing a drug safety risk.

Therefore, there is still a need to develop suitable 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamate with high safety and high cost-effectiveness. Base} pyrazine industrial production method.

The purpose of the present invention is to overcome the problems in the prior art, to provide a method for preparing 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]amine A new approach to methylformyl}pyrazines.

The inventors of this case prepared the formula 1-Int for the first time by changing the substrate of the amidation reaction, specifically, using the low-cost D-serine ester hydrochloride of formula A as the substrate, and optimizing the reaction conditions. intermediate compounds. The intermediate compound can undergo ester hydrolysis reaction, and compound 1 can be obtained after acidification. Therefore, the preparation method of the present invention does not require palladium-catalyzed hydrogenation reaction, thereby greatly improving the safety of industrial production and avoiding the risk of precious metal residue caused by the palladium/carbon catalyst. The present inventors also found that chiral impurities can be produced in the ester hydrolysis reaction. The present inventors unexpectedly found that the chiral purity of Compound 1 can be improved by adjusting the conditions of the ester hydrolysis reaction, especially by selecting a suitable solvent. The preparation method of the invention has the advantages of lower cost, good safety, low risk of precious metal residue and high chiral purity of the product.

其中R ₁在每次出現時獨立地選自C _1-8烷基、C _2-8烯基、C _2-8炔基和C _3-8環烷基； 步驟S1. 使式A的D-絲胺酸酯鹽酸鹽、3,6-二胺基吡嗪-2,5-二甲酸和醯胺縮合劑在第一有機溶劑中進行醯胺化反應，以得到式1-Int的中間體； 步驟S2. 使所述式1-Int的中間體在鹼性條件下在溶劑中水解，然後酸化反應混合物，以得到所述式1的3,6-二胺基-2,5-雙{N-[(1R)-1-羧基-2-羥基乙基]胺基甲醯基}吡嗪，其中所述溶劑是水、或水和第二有機溶劑的混合物。 In a first aspect, the present invention provides for the preparation of 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyridine The method for oxazine, is characterized in that, described method comprises the following steps:

Wherein R ₁ is independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _3-8 cycloalkyl at each occurrence; Step S1. Make the D- of formula A Serine ester hydrochloride, 3,6-diaminopyrazine-2,5-dicarboxylic acid and amide condensing agent carry out amidation reaction in the first organic solvent to obtain the intermediate of formula 1-Int ; Step S2. hydrolyzing the intermediate of the formula 1-Int in a solvent under alkaline conditions, and then acidifying the reaction mixture to obtain the 3,6-diamino-2,5-bis{ N-[(1R)-1-carboxy-2-hydroxyethyl]aminoformyl}pyrazine, wherein the solvent is water, or a mixture of water and a second organic solvent.

其中每個R ₁可以相同或不同，且獨立地選自C _1-8烷基、C _2-8烯基、C _2-8炔基和C _3-8環烷基。所述式1-Int的化合物可以作為用於製備化合物1的中間體。 In a second aspect, the present invention provides compounds of formula 1-Int:

Wherein each R ₁ can be the same or different, and is independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _3-8 cycloalkyl. The compound of formula 1-Int can be used as an intermediate for preparing compound 1.

In a third aspect, the present invention provides a process for preparing said compound of formula 1-Int.

definition

Unless defined otherwise hereinafter, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including those variations of the techniques or substitutions of equivalent techniques that are obvious to those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprises," "comprising," "has," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended, and do not Other unrecited elements or method steps are excluded although other unrecited elements or method steps are not necessarily present (ie, these terms also encompass the terms "consisting essentially of" and "consisting of").

As used herein, the term "alkyl" refers to a linear or branched saturated aliphatic hydrocarbon group. The term "C _1-8 alkyl" refers to a linear or branched alkyl group having 1 to 8 carbon atoms. For example, as used herein, the term "C _1-6 alkyl" refers to a straight or branched chain alkyl group (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl). The term "C _1-3 alkyl" refers to a linear or branched alkyl group having 1 to 3 carbon atoms, including methyl, ethyl, n-propyl and isopropyl.

As used herein, the term "C _2-6 alkenyl" means a linear or branched monovalent hydrocarbon group containing one or more double bonds, which has 2 to 8 carbon atoms (for example, "C _2-6 alkenyl group" or "C _2-3 alkenyl"). Examples of alkenyl groups that may be mentioned include, but are not limited to, e.g. vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, 4-methyl-3-pentenyl .

As used herein, the term "C _2-8 alkynyl" means a linear or branched monovalent hydrocarbon group containing one or more triple bonds, having 2 to 8 carbon atoms (e.g. "C _2-6 alkynyl " or "C _2-3 alkynyl"). Examples of alkynyl groups that may be mentioned include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl, 3-pentynyl , 4-pentynyl, 2-hexynyl, 3-hexynyl, etc.

As used herein, the term "C _3-8 cycloalkyl" refers to a saturated hydrocarbon ring having 3 to 8 ring carbon atoms. The cycloalkyl group suitably has 3 to 7 carbon atoms, such as 3 to 6 (“C _3-6 cycloalkyl”), 4 to 6 (“C _4-6 cycloalkyl”), or 5 to 6 ("C _5-6 cycloalkyl") carbon atoms. Examples of cycloalkyl that may be mentioned include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl (ring).

If substituents are described as being "independently selected from" a group, each substituent is selected independently of the other. Accordingly, each substituent may be the same as or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1 under reasonable conditions, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

As used herein, the term "room temperature" refers to about 20 to 25°C.

The term "about" means within ±10%, preferably within ±5%, more preferably within ±2% of the stated value.

其中R ₁在每次出現時獨立地選自C _1-8烷基、C _2-8烯基、C _2-8炔基和C _3-8環烷基； 步驟S1. 使式A的D-絲胺酸酯鹽酸鹽、3,6-二胺基吡嗪-2,5-二甲酸和醯胺縮合劑在第一有機溶劑中進行醯胺化反應，以得到式1-Int的中間體； 步驟S2. 使所述式1-Int的中間體在鹼性條件下在溶劑中水解，然後酸化反應混合物，以得到所述式1的3,6-二胺基-2,5-雙{N-[(1R)-1-羧基-2-羥基乙基]胺基甲醯基}吡嗪，其中所述溶劑是水、或水和第二有機溶劑的混合物。 In a first aspect, the present invention provides for the preparation of 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyridine The method for oxazine, is characterized in that, described method comprises the following steps:

Wherein R ₁ is independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _3-8 cycloalkyl at each occurrence; Step S1. Make the D- of formula A Serine ester hydrochloride, 3,6-diaminopyrazine-2,5-dicarboxylic acid and amide condensing agent carry out amidation reaction in the first organic solvent to obtain the intermediate of formula 1-Int ; Step S2. hydrolyzing the intermediate of the formula 1-Int in a solvent under alkaline conditions, and then acidifying the reaction mixture to obtain the 3,6-diamino-2,5-bis{ N-[(1R)-1-carboxy-2-hydroxyethyl]aminoformyl}pyrazine, wherein the solvent is water, or a mixture of water and a second organic solvent.

Preferably, each occurrence of R ₁ is independently C _1-6 alkyl, preferably C _1-3 alkyl, more preferably methyl or ethyl.

In some embodiments, in the step S1, the amidation reaction is carried out by adding the D-serine ester hydrochloride of the formula A, the 3,6-diaminopyrazine-2 , 5-dicarboxylic acid and the amide condensing agent are added dropwise to the first organic solvent at low temperature, and then mixed to obtain the intermediate of the formula 1-Int.

In some embodiments, in the step S1, the low temperature condition is about -10°C to 10°C, preferably about -5°C to 5°C, more preferably about 0°C to 5°C , particularly preferably at a temperature of about 0°C to 2°C.

In some embodiments, in the step S1, the mixing is performed at room temperature. In some embodiments, in the step S1, the mixing is stirring at room temperature for about 8 hours or longer, preferably about 24 hours or longer.

In some embodiments, in the step S1, the first organic solvent is a polar organic solvent, such as one selected from dimethylformamide (DMF), acetonitrile, methylene chloride, tetrahydrofuran and ethanol, Two or more, preferably DMF.

The amide condensing agent used in the step S1 is not particularly limited, and it can be a commonly used amide condensing agent. In some preferred embodiments, in the step S1, the amide condensing agent is triethylamine, 1-hydroxybenzotriazole (HOBt) and 1-(3-dimethylaminopropyl )-3-Ethylcarbodiimide hydrochloride (EDC.HCl) combination.

In some embodiments, in the step S1, the D-serine ester hydrochloride of the formula A, the 2,5-dicarboxylic acid-3,6-diaminopyrazine, triethyl Amine, HOBt and EDC.HCl are added dropwise in DMF at about 0°C to 5°C, preferably about 0°C to 2°C, and then stirred at room temperature to obtain the formula 1-Int intermediate. For example, the agitation can be continued, eg, for about 24 hours.

（D-絲胺酸乙酯鹽酸鹽）及/或

（D-絲胺酸甲酯鹽酸鹽）。 In some embodiments, in the step S1, the D-serine ester hydrochloride of the formula A is in the form of a single compound represented by the formula A. In other embodiments, in the step S1, the D-serine ester hydrochloride of the formula A is in the form of a mixture of two or more different compounds each represented by the formula A. Preferably, the D-serine ester hydrochloride of the formula A is

(D-serine ethyl ester hydrochloride) and/or

(D-serine methyl ester hydrochloride).

The inventors of the present case prepared the intermediate compound of the formula 1-Int for the first time by using the D-serine ester hydrochloride of the formula A as the substrate of the amidation reaction and optimizing the reaction conditions. The inventors of the present case found that the operation of dropping the reactant and the amide condensing agent into the first organic solvent at low temperature is beneficial to the formation of the intermediate and high yield. Favorable low temperature conditions are temperatures of about 0°C to 5°C, temperatures of about 0°C to 2°C help to achieve higher yields. The present inventors also found that there may be a solvent effect in the amidation reaction, and the first organic solvent will affect the formation and yield of the intermediate compound of formula 1-Int. Preferably, the first organic solvent is DMF.

The prepared intermediate compound of formula 1-Int can be isolated by post-treatment. In some embodiments, the post-treatment includes: rotary evaporation to remove the first organic solvent, and then extracting the residue with water and a suitable solvent (such as ethyl acetate) multiple times; washing with saturated NaHCO ₃ aqueous solution and KHSO ₄ aqueous solution The combined organic phases were concentrated and then separated by column chromatography.

In some embodiments, in the step S2, the intermediate of the formula 1-Int is hydrolyzed in the solvent under basic conditions and at a suitable temperature, and then the reaction mixture is acidified to obtain 3, 6-diamino-2,5-di{N-[(1R)-1-carboxy-2-hydroxyethyl]aminoformyl}pyrazine, wherein the solvent is water, or water and a second organic mixture of solvents.

Preferably, the second organic solvent is a polar organic solvent, for example, selected from tetrahydrofuran, methanol, acetonitrile, ethanol, 1,4-dioxane and any combination thereof, more preferably tetrahydrofuran, acetonitrile or 1,4-diox Oxane.

The inventors of the present case found that chiral impurities would be generated in the ester hydrolysis reaction in step S2. The inventors of the present case unexpectedly found that, in some specific solvents, the ester hydrolysis reaction can provide more favorable chiral purity and yield of Compound 1. Chiral purity can be indicated by the dr value (ie, diastereomeric ratio). In step S2 of the method of the present invention, the 3,6-diamino-2,5-di{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl} of the formula 1 The pyrazines may have a chiral purity represented by a dr value of about 80:20 to about 94:6, preferably about 95:5 to about 99:1, for example about 96:4, about 97:3 or about 98 :2.

In some embodiments, in the step S2, the solvent is water. In some such embodiments, the hydrolysis is carried out at a suitable temperature, such as a temperature not exceeding about 20°C, such as about 1 to 15°C, especially about 5 to 10°C.

In other embodiments, in the step S2, the solvent is a mixture of water and the second organic solvent. In some such embodiments, in said step S2, said hydrolysis is carried out at a temperature not exceeding 20°C, such as about -10°C to 15°C, particularly about -5°C to 10°C C, for example a temperature of from -5°C to 0°C or from about 0°C to 5°C.

In other such embodiments, the solvent is a mixture of water and acetonitrile.

In other such embodiments, the solvent is a mixture of water and one selected from tetrahydrofuran, methanol, ethanol, and 1,4-dioxane. In some preferred embodiments, the solvent is a mixture of water and tetrahydrofuran or 1,4-dioxane. Preferably, the hydrolysis is carried out at a temperature of about -10°C to 10°C, preferably about -5°C to 5°C, more preferably about -5°C to 0°C.

In some of the embodiments described above, in the step S2, the solvent comprises an alkali metal hydroxide, for example, one, two or more selected from LiOH, NaOH and KOH, preferably LiOH .

The hydrolysis can be performed, eg, with agitated mixing (eg, for about 1 to 8 hours).

In some embodiments, in the step S2, the acidification is performed by adjusting the pH value of the reaction mixture with an acid after the hydrolysis is completed. Completion of the hydrolysis can be indicated, for example, by monitoring the progress of the reaction by TLC until the reaction substrate disappears.

In some embodiments, the pH is about 2 to 4, preferably about 3.

In some embodiments, in the step S2, after the acidification, the reaction mixture is mixed (for example by stirring) for an appropriate time, for example about 1 to 4 hours, preferably about 2 hours.

The present invention provides the method as described above, wherein in the step S2, the intermediate of the formula 1-Int is in water or a mixture of water and acetonitrile in the presence of an alkali metal hydroxide, in a range of not more than Hydrolysis at a temperature of about 20° C. After said hydrolysis is complete, the pH of the reaction mixture is adjusted to about 3 with acid and stirring is continued to obtain 3,6-diamino-2,5-bis{N-[( 1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyrazine.

The present invention also provides the method as described above, wherein in the step S2, the intermediate of the formula 1-Int is dissolved in water and tetrahydrofuran or 1,4-dioxane in the presence of an alkali metal hydroxide hydrolyzed at a temperature of about -10°C to 10°C (eg about -5°C to 5°C, especially about -5°C or 0°C), and after said hydrolysis is completed, use The pH of the reaction mixture was adjusted to about 3 with acid and stirred for about 2 hours to give 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl] Carbamoyl}pyrazine.

The acid may be one, two or more selected from acetic acid, trifluoroacetic acid, sulfuric acid, citric acid and hydrochloric acid.

及/或

。 In some embodiments, in the step S2, the intermediate of the formula 1-Int is in the form of a single compound represented by the formula 1-Int. In other embodiments, in the step S2, the intermediate of the formula 1-Int is in the form of a mixture of two or more different compounds each represented by the formula 1-Int. Preferably, the intermediate of the formula 1-Int is

and/or

.

其中每個R ₁可以相同或不同，且獨立地選自C _1-8烷基、C _2-8烯基、C _2-8炔基和C _1-8環烷基。 In a second aspect, the present invention provides compounds of formula 1-Int:

Wherein each R ₁ can be the same or different, and is independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _1-8 cycloalkyl.

In some preferred embodiments, each R ₁ can be the same or different, and is independently selected from C _1-6 alkyl, preferably C _1-3 alkyl, more preferably methyl or ethyl.

或

。 In some embodiments, the compound of Formula 1-Int is:

or

.

The inventors of the present case prepared the intermediate compound of the formula 1-Int for the first time. Compound 1 can be obtained by the ester hydrolysis reaction of the intermediate compound and subsequent acidification, thereby avoiding the palladium-catalyzed hydrogenation reaction used in the prior art method, greatly improving the safety of industrial production, and avoiding the palladium/carbon catalyst The resulting risk of precious metal residues.

其中R ₁在每次出現時獨立地選自C _1-8烷基、C _2-8烯基、C _2-8炔基和C _1-8環烷基，較佳為C _1-6烷基，更佳為C _1-3烷基，更佳為甲基或乙基； 步驟S1. 使式A的D-絲胺酸酯鹽酸鹽、2,5-二甲酸-3,6-二胺基吡嗪和醯胺縮合劑在第一有機溶劑中進行醯胺化反應，得到所述式1-Int的化合物。 In a third aspect, the present invention provides a method for the compound of the formula 1-Int, characterized in that the method comprises the following reaction steps:

Wherein _R is independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _1-8 cycloalkyl at each occurrence, preferably C _1-6 alkyl , more preferably C _1-3 alkyl, more preferably methyl or ethyl; Step S1. Make D-serine ester hydrochloride of formula A, 2,5-dicarboxylic acid-3,6-diamine Base pyrazine and amide condensing agent carry out amidation reaction in the first organic solvent to obtain the compound of the formula 1-Int.

In some preferred embodiments, each occurrence _{of R} is independently C _1-6 alkyl, preferably C _1-3 alkyl, more preferably methyl or ethyl.

In some embodiments, in the step S1, the amidation reaction is carried out by adding the D-serine ester hydrochloride of the formula A, the 3,6-diaminopyrazine-2 , 5-dicarboxylic acid and the amide condensing agent are added dropwise to the first organic solvent at low temperature, and then mixed to obtain the intermediate of the formula 1-Int.

In some embodiments, in the step S1, the low temperature condition is about -10°C to 10°C, preferably about -5°C to 5°C, more preferably about 0°C to 5°C , particularly preferably at a temperature of about 0°C to 2°C.

In some embodiments, in the step S1, the mixing is performed at room temperature. In some embodiments, in the step S1, the mixing is stirring at room temperature for about 8 hours or longer, preferably about 24 hours or longer.

In some embodiments, in the step S1, the first organic solvent is a polar organic solvent, such as one selected from dimethylformamide (DMF), acetonitrile, methylene chloride, tetrahydrofuran and ethanol, Two or more, preferably DMF.

The amide condensing agent used in the step S1 is not particularly limited, and it can be a commonly used amide condensing agent. In some preferred embodiments, in the step S1, the amide condensing agent is triethylamine, 1-hydroxybenzotriazole (HOBt) and 1-(3-dimethylaminopropyl )-3-Ethylcarbodiimide hydrochloride (EDC.HCl) combination.

In some embodiments, in the step S1, the D-serine ester hydrochloride of the formula A, the 2,5-dicarboxylic acid-3,6-diaminopyrazine, triethyl Amine, HOBt and EDC.HCl are added dropwise in DMF at about 0°C to 5°C, preferably at about 0°C to 2°C, and then stirred at room temperature for about 24 hours to obtain the formula 1 -Intermediate of Int.

（D-絲胺酸乙酯鹽酸鹽）及/或

（D-絲胺酸甲酯鹽酸鹽）。 In some embodiments, in the step S1, the D-serine ester hydrochloride of the formula A is in the form of a single compound represented by the formula A. In other embodiments, in the step S1, the D-serine ester hydrochloride of the formula A is in the form of a mixture of two or more different compounds each represented by the formula A. Preferably, the D-serine ester hydrochloride of the formula A is

(D-serine ethyl ester hydrochloride) and/or

(D-serine methyl ester hydrochloride).

The prepared intermediate compound of formula 1-Int can be isolated by post-treatment. In some embodiments, the post-treatment includes: rotary evaporation to remove the first organic solvent, and then extracting the residue with water and a suitable solvent (such as ethyl acetate) multiple times; washing with saturated NaHCO ₃ aqueous solution and KHSO ₄ aqueous solution The combined organic phases were concentrated and then separated by column chromatography. Beneficial effect

The inventors of this case prepared the intermediate compound of formula 1-Int for the first time, which can obtain the target compound 1 after ester hydrolysis and acidification, avoiding the palladium-catalyzed hydrogenation reaction used in the prior art method. The preparation method of the invention has the advantages of lower cost, high safety, low risk of precious metal residue and high chiral purity of the product. Example

The present invention is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

The experimental method that does not indicate specific condition in the embodiment of the present invention, is conventional condition usually, or according to the condition suggested by raw material or commodity manufacturer; Known reagents are prepared by conventional methods.

(1) 醯胺化反應 在0°C條件下，分別將D-絲胺酸甲酯鹽酸鹽（18g）、三乙胺（12g）、3,6-二胺基吡嗪-2,5-二甲酸（20g）、HOBt（14g）和EDC.HCl（19g）滴加入DMF（1000mL）中，然後將反應液在室溫（r.t.）下攪拌24小時。反應結束後，旋蒸除去DMF，將剩餘物用水和乙酸乙酯多次萃取，以飽和NaHCO ₃水溶液和KHSO ₄水溶液洗滌合併的有機相，再將有機相濃縮，經柱層析分離，得到中間體6 (28g，收率為70%)。 LCMS: r.t. = 6.9 min, [M+H]+ = 401.1。 1H NMR (500 MHz, DMSO-d6) δ= 8.51 (d, J = 8.3 Hz, 2H), 6.76 (s, 4H), 5.32 (t, J = 5.5 Hz, 2H), 4.58 (dt, J = 7.9, 3.7 Hz, 2H), 3.90 (ddd, J = 11.0, 5.6, 3.9 Hz, 2H), 3.75 (ddd, J = 11.1, 5.4, 3.7 Hz, 2H), 3.69 (s, 6H)。 (2) 水解反應 將中間體6（20g）的四氫呋喃溶液與LiOH（5.0g）水溶液混合，在0°C下攪拌2小時。用TLC監測反應進程，當反應受質消失後，用三氟乙酸水溶液將反應液酸化（調節pH至約3），繼續攪拌2小時，得到紅色渾濁液。將該紅色液體過濾，洗滌濾餅並烘乾，得到目標化合物3,6-二胺基-2,5-雙{N-[(1R)-1-羧基-2-羥基乙基]胺基甲醯基}吡嗪（17.7g，收率為95%，dr=98:2）。 LCMS: r.t. = 2.9 min, [M+H]+ = 373.1。 1H NMR (500 MHz, DMSO-d6) δ= 8.47 (d, J = 8.2 Hz, 2H), 6.78 (s, 4H), 4.45 (dt, J = 7.8, 3.7 Hz, 2H), 3.88 (dd, J = 10.9, 3.9 Hz, 2H), 3.74 (dd, J = 11.0, 3.7 Hz, 2H)。 Embodiment 1 : Preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine

(1) Amidation reaction At 0°C, D-serine methyl ester hydrochloride (18g), triethylamine (12g), 3,6-diaminopyrazine-2,5 - Dicarboxylic acid (20 g), HOBt (14 g) and EDC.HCl (19 g) were added dropwise to DMF (1000 mL), and the reaction was stirred at room temperature (rt) for 24 hours. After the reaction, DMF was removed by rotary evaporation, the residue was extracted with water and ethyl acetate several times, the combined organic phase was washed with saturated NaHCO ₃ aqueous solution and KHSO ₄ aqueous solution, and then the organic phase was concentrated and separated by column chromatography to obtain intermediate Body 6 (28g, yield 70%). LCMS: rt = 6.9 min, [M+H]+ = 401.1. 1H NMR (500 MHz, DMSO-d6) δ= 8.51 (d, J = 8.3 Hz, 2H), 6.76 (s, 4H), 5.32 (t, J = 5.5 Hz, 2H), 4.58 (dt, J = 7.9 , 3.7 Hz, 2H), 3.90 (ddd, J = 11.0, 5.6, 3.9 Hz, 2H), 3.75 (ddd, J = 11.1, 5.4, 3.7 Hz, 2H), 3.69 (s, 6H). (2) Hydrolysis reaction The tetrahydrofuran solution of intermediate 6 (20 g) was mixed with LiOH (5.0 g) aqueous solution, and stirred at 0°C for 2 hours. The progress of the reaction was monitored by TLC. When the reaction substrate disappeared, the reaction solution was acidified with aqueous trifluoroacetic acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid solution. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (17.7g, 95% yield, dr=98:2). LCMS: rt = 2.9 min, [M+H]+ = 373.1. 1H NMR (500 MHz, DMSO-d6) δ= 8.47 (d, J = 8.2 Hz, 2H), 6.78 (s, 4H), 4.45 (dt, J = 7.8, 3.7 Hz, 2H), 3.88 (dd, J = 10.9, 3.9 Hz, 2H), 3.74 (dd, J = 11.0, 3.7 Hz, 2H).

(1) 醯胺化反應 在2°C下，分別將D-絲胺酸乙酯鹽酸鹽（19g）、三乙胺（12g）、3,6-二胺基吡嗪-2,5-二甲酸（20g）、HOBt（14g）和EDC.HCl（19g）滴加入DMF（800mL）中，然後將反應液在室溫下攪拌24小時。反應結束後，旋蒸除去DMF，將剩餘物用水和乙酸乙酯多次萃取，以飽和NaHCO ₃水溶液和KHSO ₄水溶液洗滌合併的有機相，再將有機相濃縮，經柱層析分離，得到中間體5 (32.5g，收率為75%)。 LCMS: r.t. = 6.6 min, [M+H]+ = 429.2。 1H NMR (500 MHz, DMSO-d6) δ= 8.50 (d, J = 8.2 Hz, 2H), 6.76 (s, 4H), 5.31 (t, J = 5.4 Hz, 2H), 4.55 (dt, J = 7.9, 3.8 Hz, 2H), 4.15 (q, J = 7.1 Hz, 4H), 3.89 (ddd, J = 11.1, 5.7, 4.0 Hz, 2H), 3.75 (ddd, J = 11.0, 5.3, 3.6 Hz, 2H), 1.21 (t, J = 7.1 Hz, 6H)。 (2) 水解反應 將中間體5（21g）的四氫呋喃溶液與LiOH（5.0g）水溶液混合，在0°C下攪拌2小時。用TLC監測反應進程，當反應物消失後，用醋酸水溶液將反應液酸化（調節pH至約3），繼續攪拌2小時，得到紅色渾濁液。將該紅色液體過濾，洗滌濾餅並烘乾，得到目標化合物3,6-二胺基-2,5-二{N-[(1R)-1-羧基-2-羥基乙基]胺基甲醯基}吡嗪(18g，收率為96%，dr=98:2)。其特徵資料同實施例1步驟(2)所記載。 Embodiment 2 : Preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine

(1) Amidation reaction At 2°C, D-serine ethyl ester hydrochloride (19g), triethylamine (12g), 3,6-diaminopyrazine-2,5- Diformic acid (20 g), HOBt (14 g) and EDC.HCl (19 g) were added dropwise into DMF (800 mL), and the reaction solution was stirred at room temperature for 24 hours. After the reaction, DMF was removed by rotary evaporation, the residue was extracted with water and ethyl acetate several times, the combined organic phase was washed with saturated NaHCO ₃ aqueous solution and KHSO ₄ aqueous solution, and then the organic phase was concentrated and separated by column chromatography to obtain intermediate Body 5 (32.5g, yield 75%). LCMS: rt = 6.6 min, [M+H]+ = 429.2. 1H NMR (500 MHz, DMSO-d6) δ= 8.50 (d, J = 8.2 Hz, 2H), 6.76 (s, 4H), 5.31 (t, J = 5.4 Hz, 2H), 4.55 (dt, J = 7.9 , 3.8 Hz, 2H), 4.15 (q, J = 7.1 Hz, 4H), 3.89 (ddd, J = 11.1, 5.7, 4.0 Hz, 2H), 3.75 (ddd, J = 11.0, 5.3, 3.6 Hz, 2H) , 1.21 (t, J = 7.1 Hz, 6H). (2) Hydrolysis reaction The tetrahydrofuran solution of intermediate 5 (21g) was mixed with LiOH (5.0g) aqueous solution, and stirred at 0°C for 2 hours. The progress of the reaction was monitored by TLC. When the reactants disappeared, the reaction solution was acidified with aqueous acetic acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid solution. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (18g, yield 96%, dr=98:2). Its characteristic data are recorded with embodiment 1 step (2).

(1) 醯胺化反應 在5°C下，分別將D-絲胺酸乙酯鹽酸鹽（19g）、三乙胺（12g）、3,6-二胺基吡嗪-2,5-二甲酸（20g）、HOBt（14g）和EDC.HCl（19g）滴加入DMF（800mL）中，然後將反應液在室溫下攪拌24小時。反應結束後，旋蒸除去DMF，將剩餘物用水和乙酸乙酯多次萃取，以飽和NaHCO ₃水溶液和KHSO ₄水溶液洗滌合併的有機相，再將有機相濃縮，經柱層析分離，得到中間體5 (28g，收率為65%)。其特徵資料同實施例2步驟(1)所記載。 (2) 水解反應 將中間體5（21g）的甲醇溶液與LiOH（5g）混合，在10°C下攪拌2小時。用TLC監測反應進程，當反應物消失後，用三氟乙酸水溶液將反應液酸化（調節pH至約3），繼續攪拌2小時，得到紅色渾濁液。將該紅色液體過濾，洗滌濾餅並烘乾，得到目標化合物3,6-二胺基-2,5-二{N-[(1R)-1-羧基-2-羥基乙基]胺基甲醯基}吡嗪(11g，收率為60%，dr=80:20)。其特徵資料同實施例1步驟(2)所記載。 Embodiment 3 : Preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine

(1) Amidation reaction At 5°C, D-serine ethyl ester hydrochloride (19g), triethylamine (12g), 3,6-diaminopyrazine-2,5- Diformic acid (20 g), HOBt (14 g) and EDC.HCl (19 g) were added dropwise into DMF (800 mL), and the reaction solution was stirred at room temperature for 24 hours. After the reaction, DMF was removed by rotary evaporation, the residue was extracted with water and ethyl acetate several times, the combined organic phase was washed with saturated NaHCO ₃ aqueous solution and KHSO ₄ aqueous solution, and then the organic phase was concentrated and separated by column chromatography to obtain intermediate Body 5 (28g, yield 65%). Its characteristic data are recorded with embodiment 2 step (1). (2) Hydrolysis reaction The methanol solution of Intermediate 5 (21g) was mixed with LiOH (5g), and stirred at 10°C for 2 hours. The progress of the reaction was monitored by TLC. When the reactants disappeared, the reaction liquid was acidified with aqueous trifluoroacetic acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid liquid. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (11 g, yield 60%, dr=80:20). Its characteristic data are recorded with embodiment 1 step (2).

(1) 醯胺化反應 在5°C下，分別將D-絲胺酸乙酯鹽酸鹽（19g）、三乙胺（12g）、3,6-二胺基吡嗪-2,5-二甲酸（20g）、HOBt（14g）和EDC.HCl（19g）滴加入二氯甲烷（1200mL）中，然後將反應液在室溫下攪拌24小時。反應結束後，旋蒸除去二氯甲烷，將剩餘物用水和乙酸乙酯多次萃取，以飽和NaHCO ₃水溶液和KHSO ₄水溶液洗滌合併的有機相，再將有機相濃縮，經柱層析分離，得到中間體5 (5.2g，收率為12%)。其特徵資料同實施例2步驟(1)所記載。 (2) 水解反應 將中間體5（43g）的乙醇溶液與LiOH（12g）混合，在20°C下攪拌2小時。用TLC監測反應進程，當反應物消失後，用三氟乙酸水溶液將反應液酸化（調節pH至約3），繼續攪拌2小時，得到紅色渾濁液。將該紅色液體過濾，洗滌濾餅並烘乾，得到目標化合物3,6-二胺基-2,5-二{N-[(1R)-1-羧基-2-羥基乙基]胺基甲醯基}吡嗪(14g，收率為38%，dr=82:18)。其特徵資料同實施例1步驟(2)所記載。 Embodiment 4 : Preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine

(1) Amidation reaction At 5°C, D-serine ethyl ester hydrochloride (19g), triethylamine (12g), 3,6-diaminopyrazine-2,5- Diformic acid (20 g), HOBt (14 g) and EDC.HCl (19 g) were added dropwise into dichloromethane (1200 mL), and the reaction solution was stirred at room temperature for 24 hours. After the reaction was completed, dichloromethane was removed by rotary evaporation, and the residue was extracted with water and ethyl acetate several times, and the combined organic phase was washed with saturated NaHCO ₃ aqueous solution and KHSO ₄ aqueous solution, and then the organic phase was concentrated and separated by column chromatography. Intermediate 5 was obtained (5.2 g, 12% yield). Its characteristic data are recorded with embodiment 2 step (1). (2) Hydrolysis reaction The ethanol solution of Intermediate 5 (43g) was mixed with LiOH (12g), and stirred at 20°C for 2 hours. The progress of the reaction was monitored by TLC. When the reactants disappeared, the reaction liquid was acidified with aqueous trifluoroacetic acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid liquid. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (14g, yield 38%, dr=82:18). Its characteristic data are recorded with embodiment 1 step (2).

Embodiment 5 : The preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine will be carried out according to Example 1 The acetonitrile solution of Intermediate 6 (20 g) prepared in step (1) was mixed with NaOH (6.0 g) aqueous solution, and stirred at 20° C. for 2 hours. The progress of the reaction was monitored by TLC. When the reaction substrate disappeared, the reaction solution was acidified with aqueous acetic acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid solution. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (16.7g, 90% yield, dr=92:8). Its characteristic data are recorded with embodiment 1 step (2).

Embodiment 6 : The preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine will be carried out according to Example 2 The 1,4-dioxane solution of intermediate 5 (20 g) prepared in step (1) was mixed with KOH (14 g) aqueous solution and stirred at -5°C for 8 hours. The progress of the reaction was monitored by TLC. When the reaction substrate disappeared, the reaction solution was acidified with hydrochloric acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid solution. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (15.3g, 88% yield, dr=95:5). Its characteristic data are recorded with embodiment 1 step (2).

Embodiment 7 : The preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine will be carried out according to Example 3 The methanol solution of Intermediate 5 (21g) prepared in step (1) was mixed with LiOH (5g), and stirred at 0°C for 2 hours. The progress of the reaction was monitored by TLC. When the reactants disappeared, the reaction liquid was acidified with aqueous trifluoroacetic acid (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid liquid. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (11 g, yield 60%, dr=86:14). Its characteristic data are recorded with embodiment 1 step (2).

Embodiment 8 : The preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine will be carried out according to Example 2 The aqueous solution of Intermediate 5 (20 g) prepared in step (1) was mixed with KOH (14 g) aqueous solution, and stirred at 10° C. for 3 hours. The reaction progress was monitored by TLC. When the reaction substrate disappeared, the reaction solution was acidified with trifluoroacetic acid aqueous solution (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid solution. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (16g, yield 92%, dr=98:2). Its characteristic data are recorded with embodiment 1 step (2).

Embodiment 9 : The preparation of 3,6- diamino -2,5- bis {N-[(1R)-1- carboxy -2- hydroxyethyl ] aminoformyl } pyrazine will be carried out according to Example 2 The aqueous solution of Intermediate 5 (20 g) prepared in step (1) was mixed with an aqueous KOH (14 g) solution, and stirred at 20° C. for 3 hours. The reaction progress was monitored by TLC. When the reaction substrate disappeared, the reaction solution was acidified with trifluoroacetic acid aqueous solution (adjusting the pH to about 3), and stirring was continued for 2 hours to obtain a red turbid solution. The red liquid was filtered, the filter cake was washed and dried to obtain the target compound 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminomethyl Acyl}pyrazine (16g, yield 92%, dr=95:5). Its characteristic data are recorded with embodiment 1 step (2).

In addition to the embodiments described herein, various modifications of the invention will be apparent to those skilled in the art to which the invention pertains from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Each reference cited in this application, including all patents, patent applications, journal articles, books, and any other publications, is hereby incorporated by reference in its entirety.

Claims (22)

A method for preparing 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]aminoformyl}pyrazine of formula 1, characterized in that The method comprises the steps of:

Wherein _R is independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _3-8 cycloalkyl at each occurrence, preferably C _1-6 alkyl , more preferably C _1-3 alkyl, more preferably methyl or ethyl; Step S1. Make D-serine ester hydrochloride of formula A, 3,6-diaminopyrazine-2,5 -diformic acid and amide condensing agent carry out amidation reaction in the first organic solvent, to obtain the intermediate of formula 1-Int; Step S2. make the intermediate of described formula 1-Int in solvent under alkaline condition hydrolysis in medium, and then acidify the reaction mixture to obtain the 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamate of the formula 1 Base} pyrazine, wherein the solvent is water, or a mixture of water and a second organic solvent. The method of claim 1, characterized in that in the step S1, the amidation reaction is carried out by adding the D-serine ester hydrochloride of the formula A, the 3,6-diamine Pyrazine-2,5-dicarboxylic acid and the amide condensing agent are added dropwise into the first organic solvent at low temperature, and then mixed to obtain the intermediate of the formula 1-Int. The method of claim 2, characterized in that in the step S1, the low temperature condition is about -10°C to 10°C, preferably about -5°C to 5°C, more preferably about 0°C to 5°C, particularly preferably a temperature of about 0°C to 2°C. The method according to claim 2 or 3, characterized in that in the step S1, the mixing is performed at room temperature. The method according to any one of claims 2 to 4, characterized in that in the step S1, the mixing is stirred at room temperature for about 8 hours or longer, preferably about 24 hours or longer. The method according to any one of claims 1 to 5, characterized in that in the step S1, the first organic solvent is a polar organic solvent, for example selected from dimethylformamide (DMF), acetonitrile, di One, two or more of methyl chloride, tetrahydrofuran and ethanol, preferably DMF. The method according to any one of claims 1 to 6, characterized in that in the step S1, the amide condensing agent is triethylamine, 1-hydroxybenzotriazole (HOBt) and 1-(3 - combination of dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl). The method according to any one of claims 1 to 7, characterized in that in the step S1, the D-serine ester hydrochloride of the formula A, the 2,5-dicarboxylic acid-3, 6-diaminopyrazine, triethylamine, HOBt and EDC.HCl are added dropwise in DMF at about 0°C to 5°C, preferably about 0°C to 2°C, and then stirred at room temperature , to obtain the intermediate of the formula 1-Int. The method according to any one of claims 1 to 8, characterized in that in the step S1, the D-serine ester hydrochloride of the formula A is in the form of a single compound represented by the formula A, Or in the form of a mixture of two or more different compounds each represented by said formula A, preferably

and/or

. The method according to any one of claims 1 to 9, characterized in that in the step S2, the intermediate of the formula 1-Int is hydrolyzed in the solvent under basic conditions and at a suitable temperature, The reaction mixture was then acidified to give 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyrazine. The method according to any one of claims 1 to 10, characterized in that the second organic solvent is a polar organic solvent, especially selected from tetrahydrofuran, methanol, acetonitrile, ethanol, 1,4-dioxane and any combination thereof , preferably tetrahydrofuran, acetonitrile, 1,4-dioxane. The method according to any one of claims 1 to 11, characterized in that in the step S2, the solvent is water. The method according to any one of claims 1 to 11, characterized in that in the step S2, the solvent is a mixture of water and the second organic solvent. The method according to any one of claim items 1 to 13, characterized in that in the step S2, the solvent comprises an alkali metal hydroxide, such as one, two or more selected from LiOH, NaOH and KOH species, preferably LiOH. The method according to any one of claims 1 to 14, characterized in that in the step S2, the acidification is carried out by adjusting the pH value of the reaction mixture with an acid after the hydrolysis is completed. The method according to claim 15, characterized in that the pH value is about 2 to 4, preferably about 3. The method according to any one of claims 1 to 16, characterized in that in the step S2, after the acidification, the reaction mixture is mixed for about 1 to 4 hours, preferably about 2 hours. The method according to any one of claims 15 to 17, characterized in that the acid is one, two or more selected from acetic acid, trifluoroacetic acid, sulfuric acid, citric acid and hydrochloric acid. The method according to any one of claim items 1 to 18, characterized in that in the step S2, the intermediate of the formula 1-Int is in the form of a single compound represented by the formula 1-Int, or each is represented by The form of the mixture of two or more different compounds represented by the formula 1-Int, preferably

and/or

. A compound of formula 1-Int:

Wherein each R ₁ can be the same or different, and independently selected from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl and C _3-8 cycloalkyl, preferably C _{1-8 6} alkyl, more preferably C _1-3 alkyl, more preferably methyl or ethyl. As the compound of claim 20, it is:

or

. A method for preparing the compound of claim 20 or 21, characterized in that the method comprises the following steps:

Step S1. making D-serine ester hydrochloride of formula A, 2,5-dicarboxylic acid-3,6-diaminopyrazine and amide condensing agent carry out amidation reaction in the first organic solvent, The compound of the formula 1-Int is obtained; preferably, the step S1 is as described in any one of claims 2-9.