WO2023016483A1 - 制备作为荧光示踪剂的吡嗪羧酸类衍生物的方法 - Google Patents

制备作为荧光示踪剂的吡嗪羧酸类衍生物的方法 Download PDF

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WO2023016483A1
WO2023016483A1 PCT/CN2022/111392 CN2022111392W WO2023016483A1 WO 2023016483 A1 WO2023016483 A1 WO 2023016483A1 CN 2022111392 W CN2022111392 W CN 2022111392W WO 2023016483 A1 WO2023016483 A1 WO 2023016483A1
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int
organic solvent
reaction
acid
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French (fr)
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郑骏浩
许旭华
皇甫港怀
夏雯蓉
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杭州中美华东制药有限公司
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Priority to CN202280054353.2A priority Critical patent/CN117769543A/zh
Publication of WO2023016483A1 publication Critical patent/WO2023016483A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/20—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D241/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with nitrogen atoms directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D241/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with nitrogen atoms directly attached to ring carbon atoms
    • C07D241/28—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with nitrogen atoms directly attached to ring carbon atoms in which said hetero-bound carbon atoms have double bonds to oxygen, sulfur or nitrogen atoms

Definitions

  • the present invention relates to a method for the preparation of 3,6-diamino-2,5-bis ⁇ N-(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine, an intermediate used in said preparation method Intermediate compounds and methods for preparing said intermediate compounds.
  • the amidation reaction uses D-serine benzyl ester hydrochloride as the reaction substrate to obtain intermediate 4, which is then catalyzed by palladium/carbon hydrogenation to generate compound 1.
  • D-serine benzyl ester hydrochloride and palladium/carbon Due to the high commercial price of D-serine benzyl ester hydrochloride and palladium/carbon, the production cost of the product is relatively high.
  • 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.
  • 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]amino A new approach to formyl ⁇ pyrazines.
  • the present inventors have prepared the intermediate of formula 1-Int for the first time by changing the amidation reaction substrate, specifically, using low-cost D-serine ester hydrochloride of formula A as the substrate, and by optimizing the reaction conditions compound.
  • 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.
  • the present invention provides a process for the preparation of 3,6-diamino-2,5-bis ⁇ N-(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine of formula 1, It is characterized in that the method comprises the following steps:
  • 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 D-serine ester hydrochloride of formula A, 3,6-diaminopyrazine-2,5-dicarboxylic acid and amide condensation agent carry out amidation reaction in the first organic solvent, to obtain formula 1- Intermediate of Int;
  • Step S2 Hydrolyze the intermediate of the formula 1-Int in a solvent under basic conditions, and then acidify the reaction mixture to obtain the 3,6-diamino-2,5-bis ⁇ N- [(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine, wherein the solvent is water, or a mixture of water and a second organic solvent.
  • 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.
  • the present invention provides a process for preparing said compound of formula 1-Int.
  • alkyl refers to a straight or branched chain saturated aliphatic hydrocarbon group.
  • C 1-8 alkyl refers to a linear or branched alkyl group having 1 to 8 carbon atoms.
  • C 1-6 alkyl refers to a straight or branched chain alkyl having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl).
  • 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.
  • C 2-6 alkenyl means a linear or branched monovalent hydrocarbon group containing one or more double bonds, having 2 to 8 carbon atoms (e.g. "C 2-6 alkenyl group” or "C 2-3 alkenyl”).
  • 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 .
  • 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”).
  • 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.
  • 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, for example 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).
  • each substituent is selected independently of the other. Accordingly, each substituent may be the same as or different from another (other) substituent.
  • one or more means 1 or more than 1 under reasonable conditions, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • room temperature means about 20 to 25°C.
  • the present invention provides a process for the preparation of 3,6-diamino-2,5-bis ⁇ N-(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine of formula 1, It is characterized in that the method comprises the following steps:
  • 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 D-serine ester hydrochloride of formula A, 3,6-diaminopyrazine-2,5-dicarboxylic acid and amide condensation agent carry out amidation reaction in the first organic solvent, to obtain formula 1- Intermediate of Int;
  • Step S2 Hydrolyze the intermediate of the formula 1-Int in a solvent under basic conditions, and then acidify the reaction mixture to obtain the 3,6-diamino-2,5-bis ⁇ N- [(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine, wherein the solvent is water, or a mixture of water and a second organic solvent.
  • each occurrence of R 1 is independently C 1-6 alkyl, preferably C 1-3 alkyl, more preferably methyl or ethyl.
  • the amidation reaction is carried out by adding the D-serine ester hydrochloride of the formula A, the 3,6-diaminopyrazine-2,5-di Formic 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 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 about 0°C to 2°C temperature.
  • 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.
  • 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.
  • DMF dimethylformamide
  • acetonitrile acetonitrile
  • methylene chloride methylene chloride
  • tetrahydrofuran tetrahydrofuran
  • ethanol two or more, preferably DMF.
  • the amide condensing agent used in the step S1 is not particularly limited, and it may be a commonly used amide condensing agent.
  • the amide condensing agent is triethylamine, 1-hydroxybenzotriazole (HOBt) and 1-(3-dimethylaminopropyl)-3 - A combination of ethylcarbodiimide hydrochloride (EDC.HCl).
  • 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 into DMF at about 0°C to 5°C, preferably at about 0°C to 2°C, and then stirred at room temperature to obtain the intermediate of formula 1-Int.
  • the agitation can be continued, eg, for about 24 hours.
  • the D-serine ester hydrochloride of the formula A 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 present inventors prepared the intermediate compound of formula 1-Int for the first time by using D-serine ester hydrochloride of formula A as the substrate of amidation reaction and optimizing the reaction conditions.
  • the present inventors found that the dropwise addition of reactants and 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 can affect the formation and yield of the intermediate compound of formula 1-Int.
  • the first organic solvent is DMF.
  • the prepared intermediate compound of formula 1-Int can be isolated by post-treatment.
  • 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.
  • 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-bis ⁇ N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine, wherein the solvent is water, or water and a second organic solvent mixture.
  • 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- Dioxane.
  • 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- Dioxane.
  • the present inventors found that chiral impurities would be produced in the ester hydrolysis reaction in step S2.
  • the present inventors 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).
  • step S2 of the method of the present invention the 3,6-diamino-2,5-bis ⁇ N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl ⁇ pyrazine of the formula 1
  • 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.
  • the solvent in the step S2, is water.
  • 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.
  • the solvent is a mixture of water and the second organic solvent.
  • said hydrolysis is carried out at a temperature not exceeding 20°C, for example about -10°C to 15°C, especially about -5°C to 10°C, for example -5°C °C to 0°C at or about 0°C to 5°C.
  • the solvent is a mixture of water and acetonitrile.
  • the solvent is a mixture of water and one selected from the group consisting of tetrahydrofuran, methanol, ethanol, and 1,4-dioxane.
  • the solvent is a mixture of water and tetrahydrofuran or 1,4-dioxane.
  • 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.
  • the solvent comprises an alkali metal hydroxide, such as one, two or more selected from LiOH, NaOH and KOH, preferably LiOH .
  • the hydrolysis may be performed, eg, with agitated mixing (eg, for about 1 to 8 hours).
  • 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.
  • the pH is about 2 to 4, preferably about 3.
  • the reaction mixture is mixed (for example by stirring) for a suitable 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 give 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 In a mixture of rings, hydrolyze at a temperature of about -10°C to 10°C (for example, about -5°C to 5°C, especially about -5°C or 0°C), and adjust the reaction mixture with an acid after the hydrolysis is completed. pH to about 3 and further stirring 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.
  • 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
  • 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.
  • 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.
  • the compound of Formula 1-Int is:
  • the present inventors prepared the intermediate compound of formula 1-Int for the first time.
  • Compound 1 can be obtained through the ester hydrolysis reaction of the intermediate compound and subsequent acidification, thereby avoiding the palladium catalytic hydrogenation reaction used in the prior art method, greatly improving the safety of industrial production, and avoiding the palladium/carbon Risk of precious metal residues from catalysts.
  • the present invention provides a method for the compound of the formula 1-Int, characterized in that the method comprises the following reaction steps:
  • R at each occurrence is independently selected from C 1-8 alkyl, C 2-8 alkenyl, C 2-8 alkynyl and C 1-8 cycloalkyl, 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-diaminopyrazine and amide condensation agent undergo amidation reaction in the first organic solvent to obtain the formula 1 - Compounds of Int.
  • each occurrence of R 1 is independently C 1-6 alkyl, preferably C 1-3 alkyl, more preferably methyl or ethyl.
  • the amidation reaction is carried out by adding the D-serine ester hydrochloride of the formula A, the 3,6-diaminopyrazine-2,5-di Formic 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 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 about 0°C to 2°C temperature.
  • 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.
  • 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.
  • DMF dimethylformamide
  • acetonitrile acetonitrile
  • methylene chloride methylene chloride
  • tetrahydrofuran tetrahydrofuran
  • ethanol two or more, preferably DMF.
  • the amide condensing agent used in the step S1 is not particularly limited, and it may be a commonly used amide condensing agent.
  • the amide condensing agent is triethylamine, 1-hydroxybenzotriazole (HOBt) and 1-(3-dimethylaminopropyl)-3 - A combination of ethylcarbodiimide hydrochloride (EDC.HCl).
  • 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 into 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 intermediate of formula 1-Int.
  • the D-serine ester hydrochloride of the formula A 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.
  • 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.
  • the present inventors 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.
  • 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; It can be prepared from known reagents by conventional methods.
  • D-serine methyl ester hydrochloride (18g), triethylamine (12g), 3,6-diaminopyrazine-2,5-dicarboxylic acid (20g), HOBt (14g) and EDC.HCl (19 g) were added dropwise to DMF (1000 mL), and the reaction was stirred at room temperature (rt) for 24 hours.
  • D-serine ethyl ester hydrochloride (19g) triethylamine (12g), 3,6-diaminopyrazine-2,5-dicarboxylic acid (20g), HOBt (14g) and EDC.HCl (19 g) was added dropwise to DMF (800 mL), and the reaction solution was stirred at room temperature for 24 hours.
  • D-serine ethyl ester hydrochloride (19g) triethylamine (12g), 3,6-diaminopyrazine-2,5-dicarboxylic acid (20g), HOBt (14g) and EDC.HCl (19 g) was added dropwise to DMF (800 mL), and the reaction solution was stirred at room temperature for 24 hours.

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Abstract

本申请涉及制备作为荧光示踪剂的吡嗪类衍生物的方法，具体涉及制备3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的方法、用于所述制备方法的中间体化合物以及制备所述中间体化合物的方法。

Description

制备作为荧光示踪剂的吡嗪羧酸类衍生物的方法 技术领域
本发明涉及制备3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的方法、用于所述制备方法的中间体化合物以及制备所述中间体化合物的方法。
背景技术
吡嗪类衍生物作为荧光示踪剂在评估肾功能方面具有潜在的应用前景。作为一种极具临床应用价值的荧光示踪剂，3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(化合物1)的研究得到了广泛的关注，但是目前该化合物的合成路线报道极少。
2011年，Raghavan Rajagopalan等人对化合物1的合成进行了优化，减少了反应步骤(J.Med.Chem.2011,54,5048–5058)。在该合成方法中，作为原料的3,6-二氨基吡嗪-2,5-二甲酸经酰胺化、钯催化氢化等步骤得到化合物1：
Figure PCTCN2022111392-appb-000001
其中，酰胺化反应以D-丝氨酸苄酯盐酸盐为反应底物，得到中间体4，再经钯/碳催化氢化反应生成化合物1。由于D-丝氨酸苄酯盐酸盐和钯/碳的商业价格昂贵，导致产品的生产成本较高。此外，氢化反应不仅对生产设备和场地有较高要求，而且氢气是易燃易爆气体，操作十分危险，存在极大的安全隐患。由于氢化反应使用了钯/碳，处理不当会导致贵金属残留，因而存在药物安全风险。
因此，仍然需要开发具有高安全性和高成本效益的适合3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的工业化生产的方法。
发明内容
本发明的目的在于克服现有技术中存在的问题，提供一种用于制备3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的新方法。
本发明人通过改变酰胺化反应底物，具体而言，使用成本低廉的式A的D-丝氨酸酯类盐酸盐为底物，并通过优化反应条件，首次制备了式1-Int的中间体化合物。所述中间体化合物可以发生酯水解反应，经酸化后即可得到化合物1。因此，本发明的制备方法不需要钯催化氢化反应，从而极大地提高了工业生产的安全性，避免了钯/碳催化剂导致的贵金属残留的风险。本发明人还发现该酯水解反应中可以产生手性杂质。本发明人意外地发现，通过调整酯水解反应的条件，特别是选择合适的溶剂，可以提高化合物1的手性纯度。本发明的制备方法具有成本更低，安全性好，贵金属残留风险低以及产物手性纯度高的优势。
在第一方面，本发明提供制备式1的3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的方法，其特征在于，所述方法包括以下步骤：
Figure PCTCN2022111392-appb-000002
其中R 1在每次出现时独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 3-8环烷基；
步骤S1.使式A的D-丝氨酸酯盐酸盐、3,6-二氨基吡嗪-2,5-二甲酸和酰胺缩合剂在第一有机溶剂中进行酰胺化反应，以得到式1-Int的中间体；
步骤S2.使所述式1-Int的中间体在碱性条件下在溶剂中水解，然后酸化反应混合物，以得到所述式1的3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪，其中所述溶剂是水、或水和第二有机溶剂的混合物。
在第二方面，本发明提供式1-Int的化合物：
Figure PCTCN2022111392-appb-000003
其中每个R 1可以相同或不同，且独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 3-8环烷基。所述式1-Int的化合物可以作为用于制备化合物1的中间体。
在第三方面，本发明提供制备所述式1-Int的化合物的方法。
具体实施方式
定义
除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
如本文中所使用，术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤，尽管其它未列举的元素或方法步骤不一定存在(即，这些术语也涵盖术语“基本上由……组成”和“由……组成”)。
如本文中所使用，术语“烷基”是指直链或支链的饱和脂肪族烃基。术语“C 1-8烷基”指具有1至8个碳原子的直链或支链的烷基。例如，如本文中所使用，术语“C 1-6烷基”指具有1至6个碳原子的直链或支链的烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基或正己基)。术语“C 1-3烷基”指具有1至3个碳原子的直链或支链的烷基，包括甲基、乙基、正丙基和异丙基。
如本文中所使用，术语“C 2-8烯基”意指包含一个或多个双键的直链或支链的单价烃基，其具有2至8个碳原子(例如“C 2-6烯基”或“C 2-3烯基”)。可以提及的烯基的实例包括但不限于例如乙烯基、1-丙烯基、2-丙烯基、2-丁烯基、3-丁烯基、2-戊烯基、3-戊烯基、4-戊烯基、2-己烯基、3-己烯基、4-己烯基、5-己烯基、2-甲基-2-丙烯基、4-甲基-3-戊烯基。
如本文中所使用，术语“C 2-8炔基”表示包含一个或多个三键的直链或支链的单价烃基，其具有2至8个碳原子(例如“C 2-6炔基”或“C 2-3炔基”)。可以提及的炔基的实例包括但不限于乙炔基、1-丙炔基、2-丙炔基、2-丁炔基、3-丁炔基、2-戊炔基、3-戊炔基、4-戊炔基、2-己炔基、3-己炔基等。
如本文中所使用，术语“C 3-8环烷基”是指具有3至8个环碳原子的饱和烃环。所述环烷基适合地具有3至7个碳原子，例如3至6个(“C 3-6环烷基”)、4至6个(“C 4-6环烷基”)、或5至6个(“C 5-6环烷基”)碳原子。可以提及的环烷基的实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基和环辛基(环)。
如果取代基被描述为“独立地选自”一组基团，则各取代基独立于另一者被选择。因此，各取代基可与另一(其他)取代基相同或不同。
如本文中所使用，术语“一个或多个”意指在合理条件下的1个或超过1个，例如2个、3个、4个、5个、6个、7个、8个、9个或10个。
如本文中所使用，术语“室温”是指约20至25℃。
术语“约”是指在所述数值的±10％范围内，优选±5％范围内，更优选±2％范围内。
在第一方面，本发明提供制备式1的3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的方法，其特征在于，所述方法包括以下步骤：
Figure PCTCN2022111392-appb-000004
其中R 1在每次出现时独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 3-8环烷基；
步骤S1.使式A的D-丝氨酸酯盐酸盐、3,6-二氨基吡嗪-2,5-二甲酸和酰胺缩合剂在第一有机溶剂中进行酰胺化反应，以得到式1-Int的中间体；
步骤S2.使所述式1-Int的中间体在碱性条件下在溶剂中水解，然后酸化反应混合物，以得到所述式1的3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪，其中所述溶剂是水、或水和第二有机溶剂的混合物。
优选地，R 1在每次出现时独立地为C 1-6烷基，优选为C 1-3烷基，更优选为甲基或乙基。
在一些实施方案中，在所述步骤S1中，所述酰胺化反应是通过将所述式A的D-丝氨酸酯盐酸盐、所述3,6-二氨基吡嗪-2,5-二甲酸和所述酰胺缩合剂在低温条件下滴加到所述第一有机溶剂中，然后混合来进行的，以得到 所述式1-Int的中间体。
在一些实施方案中，在所述步骤S1中，所述低温条件为约-10℃至10℃，优选约-5℃至5℃，更优选约0℃至5℃，特别优选约0℃至2℃的温度。
在一些实施方案中，在所述步骤S1中，所述混合是在室温下进行。在一些实施方案中，在所述步骤S1中，所述混合为在室温下搅拌约8小时或更长时间，优选约24小时或更长时间。
在一些实施方案中，在所述步骤S1中，所述第一有机溶剂为极性有机溶剂，例如选自二甲基甲酰胺(DMF)、乙腈、二氯甲烷、四氢呋喃和乙醇中的一种、两种或更多种，优选为DMF。
对所述步骤S1中使用的酰胺缩合剂没有特别的限制，其可以为常用的酰胺缩合剂。在一些优选的实施方案中，在所述步骤S1中，所述酰胺缩合剂为三乙胺、1-羟基苯并三氮唑(HOBt)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC.HCl)的组合。
在一些实施方案中，在所述步骤S1中，将所述式A的D-丝氨酸酯盐酸盐、所述2,5-二甲酸-3,6-二氨基吡嗪、三乙胺、HOBt和EDC.HCl在约0℃至5℃、优选约0℃至2℃下滴加到DMF中，然后在室温下搅拌，以得到所述式1-Int的中间体。例如，所述搅拌可以持续例如约24小时。
在一些实施方案中，在所述步骤S1中，所述式A的D-丝氨酸酯盐酸盐是由所述式A表示的单一化合物的形式。在另一些实施方案中，在所述步骤S1中，所述式A的D-丝氨酸酯盐酸盐是各自由所述式A表示的两种或更多种不同化合物的混合物的形式。优选地，所述式A的D-丝氨酸酯盐酸盐为
Figure PCTCN2022111392-appb-000005
(D-丝氨酸乙酯盐酸盐)和/或
Figure PCTCN2022111392-appb-000006
(D-丝氨酸甲酯盐酸盐)。
本发明人通过使用式A的D-丝氨酸酯类盐酸盐为酰胺化反应的底物，并通过优化反应条件，首次制备了式1-Int的中间体化合物。本发明人发现在低温条件下进行滴加反应物和酰胺缩合剂到所述第一有机溶剂中的操作有利于所述中间体的形成和高收率。有利的低温条件是约0℃至5℃的温度，约0℃至2℃的温度有助于达到更高的收率。本发明人还发现在所述酰胺化反应中可能存在溶剂效应，所述第一有机溶剂会影响式1-Int的中间 体化合物的形成和收率。优选地，所述第一有机溶剂是DMF。
所制备的式1-Int的中间体化合物可以通过后处理分离。在一些实施方案中，所述后处理包括：旋蒸除去所述第一有机溶剂，然后用水和适合的溶剂(例如乙酸乙酯)多次萃取剩余物；以饱和NaHCO 3水溶液和KHSO 4水溶液洗涤合并的有机相，浓缩有机相，然后通过柱层析分离。
在一些实施方案中，在所述步骤S2中，使所述式1-Int的中间体在在碱性条件下并且在适合的温度下在所述溶剂中水解，然后酸化反应混合物，以得到3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨甲酰基}吡嗪，其中所述溶剂是水、或水和第二有机溶剂的混合物。
优选地，所述第二有机溶剂为极性有机溶剂，例如选自四氢呋喃、甲醇、乙腈、乙醇、1,4-二氧六环及其任意组合，更优选为四氢呋喃、乙腈或1,4-二氧六环。
本发明人发现在步骤S2的酯水解反应中会产生手性杂质。本发明人意外地发现，在一些特定的溶剂中，所述酯水解反应能够提供更有利的化合物1的手性纯度和收率。手性纯度可以由dr值(即，非对映体比例)来表示。在本发明方法的步骤S2中，所述式1的3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨甲酰基}吡嗪可以具有由如下的dr值表示的手性纯度：约80:20至约94:6，优选约95:5至约99:1，例如约96:4、约97:3或约98:2。
在一些实施方案中，在所述步骤S2中，所述溶剂为水。在一些此类实施方案中，所述水解在合适的温度下进行，例如不超过约20℃，例如约1至15℃，特别是约5至10℃的温度。
在另一些实施方案中，在所述步骤S2中，所述溶剂为水和所述第二有机溶剂的混合物。在一些此类实施方案中，在所述步骤S2中，所述水解在不超过20℃的温度下进行，例如约-10℃至15℃，特别是约-5℃至10℃，例如-5℃至0℃或约0℃至5℃的温度。
在另一些此类实施方案中，所述溶剂为水和乙腈的混合物。
在另一些此类实施方案中，所述溶剂为水和选自四氢呋喃、甲醇、乙醇和1,4-二氧六环中的一种的混合物。在一些优选地实施方案中，所述溶剂为水和四氢呋喃或1,4-二氧六环的混合物。优选地，所述水解在约-10℃至10℃，优选约-5℃至5℃，更优选约-5℃至0℃的温度下进行。
在一些上文所述的实施方案中，在所述步骤S2中，所述溶剂包含碱金 属氢氧化物，例如选自LiOH、NaOH和KOH中的一种、两种或更多种，优选LiOH。
所述水解可以例如搅拌的混合(例如持续约1至8小时)下进行。
在一些实施方案中，在所述步骤S2中，所述酸化是在所述水解完成后，通过用酸调节反应混合物的pH值来进行的。例如，可以通过用TLC监测反应进程，直至反应底物消失，来指示所述水解的完成。
在一些实施方案中，所述pH值为约2至4，优选约3。
在一些实施方案中，在所述步骤S2中，在所述酸化之后，再将反应混合物混合(例如通过搅拌)适当的时间，例如约1至4小时，优选约2小时。
本发明提供如上文所述的方法，其中在所述步骤S2中，使所述式1-Int的中间体在碱金属氢氧化物的存在下在水或水和乙腈的混合物中，在不超过约20℃的温度下水解，在所述水解完成后用酸调节反应混合物的pH值至约3并继续搅拌，以得到3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨甲酰基}吡嗪。
本发明还提供如上文所述的方法，其中在所述步骤S2中，使所述式1-Int的中间体在碱金属氢氧化物的存在下在水和四氢呋喃或1,4-二氧六环的混合物中，在约-10℃至10℃(例如约-5℃至5℃，特别是约-5℃或0℃)的温度下水解，在所述水解完成后用酸调节反应混合物的pH值至约3并再搅拌约2小时，以得到3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨甲酰基}吡嗪。
所述酸可以是选自醋酸、三氟乙酸、硫酸、柠檬酸和盐酸中的一种、两种或更多种。
在一些实施方案中，在所述步骤S2中，所述式1-Int的中间体为由所述式1-Int表示的单一化合物的形式。在另一些实施方案中，在所述步骤S2中，所述式1-Int的中间体为各自由所述式1-Int表示的两种或更多种不同化合物的混合物的形式。优选地，所述式1-Int的中间体为
Figure PCTCN2022111392-appb-000007
和/或
Figure PCTCN2022111392-appb-000008
在第二方面，本发明提供式1-Int的化合物：
Figure PCTCN2022111392-appb-000009
其中每个R 1可以相同或不同，且独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 1-8环烷基。
在一些优选的实施方案中，每个R 1可以相同或不同，且独立地选自C 1- 6烷基，优选为C 1-3烷基，更优选为甲基或乙基。
在一些实施方案中，所述式1-Int的化合物为：
Figure PCTCN2022111392-appb-000010
本发明人首次制备了所述式1-Int的中间体化合物。通过所述中间体化合物的酯水解反应和随后的酸化即可得到化合物1，从而避免了现有技术方法所使用的钯催化氢化反应，极大地提高了工业生产的安全性，避免了钯/碳催化剂导致的贵金属残留的风险。
在第三方面，本发明提供所述式1-Int的化合物的方法，其特征在于，所述方法包括以下反应步骤：
Figure PCTCN2022111392-appb-000011
其中R 1在每次出现时独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 1-8环烷基，优选为C 1-6烷基，更优选为C 1-3烷基，更优选为甲基或乙基；
步骤S1.使式A的D-丝氨酸酯盐酸盐、2,5-二甲酸-3,6-二氨基吡嗪和酰胺缩合剂在第一有机溶剂中进行酰胺化反应，得到所述式1-Int的化合物。
在一些优选的实施方案中，R 1在每次出现时独立地为C 1-6烷基，优选为C 1-3烷基，更优选为甲基或乙基。
在一些实施方案中，在所述步骤S1中，所述酰胺化反应是通过将所述 式A的D-丝氨酸酯盐酸盐、所述3,6-二氨基吡嗪-2,5-二甲酸和所述酰胺缩合剂在低温条件下滴加到所述第一有机溶剂中，然后混合来进行的，以得到所述式1-Int的中间体。
在一些实施方案中，在所述步骤S1中，所述低温条件为约-10℃至10℃，优选约-5℃至5℃，更优选约0℃至5℃，特别优选约0℃至2℃的温度。
在一些实施方案中，在所述步骤S1中，所述混合是在室温下进行。在一些实施方案中，在所述步骤S1中，所述混合为在室温下搅拌约8小时或更长时间，优选约24小时或更长时间。
在一些实施方案中，在所述步骤S1中，所述第一有机溶剂为极性有机溶剂，例如选自二甲基甲酰胺(DMF)、乙腈、二氯甲烷、四氢呋喃和乙醇中的一种、两种或更多种，优选为DMF。
对所述步骤S1中使用的酰胺缩合剂没有特别的限制，其可以为常用的酰胺缩合剂。在一些优选的实施方案中，在所述步骤S1中，所述酰胺缩合剂为三乙胺、1-羟基苯并三氮唑(HOBt)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC.HCl)的组合。
在一些实施方案中，在所述步骤S1中，将所述式A的D-丝氨酸酯盐酸盐、所述2,5-二甲酸-3,6-二氨基吡嗪、三乙胺、HOBt和EDC.HCl在约0℃至5℃、优选约0℃至2℃下滴加到DMF中，然后在室温下搅拌约24小时，以得到所述式1-Int的中间体。
在一些实施方案中，在所述步骤S1中，所述式A的D-丝氨酸酯盐酸盐是由所述式A表示的单一化合物的形式。在另一些实施方案中，在所述步骤S1中，所述式A的D-丝氨酸酯盐酸盐是各自由所述式A表示的两种或更多种不同化合物的混合物的形式。优选地，所述式A的D-丝氨酸酯盐酸盐为
Figure PCTCN2022111392-appb-000012
(D-丝氨酸乙酯盐酸盐)和/或
Figure PCTCN2022111392-appb-000013
(D-丝氨酸甲酯盐酸盐)。
所制备的式1-Int的中间体化合物可以通过后处理分离。在一些实施方案中，所述后处理包括：旋蒸除去所述第一有机溶剂，然后用水和适合的溶剂(例如乙酸乙酯)多次萃取剩余物；以饱和NaHCO 3水溶液和KHSO 4水溶液洗涤合并的有机相，浓缩有机相，然后通过柱层析分离。
有益效果
本发明人首次制备了式1-Int的中间体化合物，其通过酯水解反应和酸化后可得到目标化合物1，避免了现有技术方法所使用的钯催化氢化反应。本发明的制备方法具有成本更低，安全性高，贵金属残留风险低以及产物手性纯度高的优势。
实施例
以下结合实施例进一步描述本发明，但提供这些实施例并非意图限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常为常规条件，或按照原料或商品制造厂商所建议的条件；未注明来源的试剂，通常为通过商业途径可购得的常规试剂或者可以由已知的试剂通过常规方法制备得到。
实施例1：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
Figure PCTCN2022111392-appb-000014
(1)酰胺化反应
在0℃条件下，分别将D-丝氨酸甲酯盐酸盐(18g)、三乙胺(12g)、3,6-二氨基吡嗪-2,5-二甲酸(20g)、HOBt(14g)和EDC.HCl(19g)滴加入DMF(1000mL)中，然后将反应液在室温(r.t.)下搅拌24小时。反应结束后，旋蒸除去DMF，将剩余物用水和乙酸乙酯多次萃取，以饱和NaHCO 3水溶液和KHSO 4水溶液洗涤合并的有机相，再将有机相浓缩，经柱层析分离，得到中间体6(28g，收率为70％)。
LCMS:r.t.＝6.9min,[M+H]+＝401.1。
1H NMR(500MHz,DMSO-d6)δ＝8.51(d,J＝8.3Hz,2H),6.76(s,4H),5.32(t,J＝5.5Hz,2H),4.58(dt,J＝7.9,3.7Hz,2H),3.90(ddd,J＝11.0,5.6,3.9Hz,2H),3.75(ddd,J＝11.1,5.4,3.7Hz,2H),3.69(s,6H)。
(2)水解反应
将中间体6(20g)的四氢呋喃溶液与LiOH(5.0g)水溶液混合，在0℃下搅拌2小时。用TLC监测反应进程，当反应底物消失后，用三氟乙酸水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(17.7g，收率为95％，dr＝98:2)。
LCMS:r.t.＝2.9min,[M+H]+＝373.1。
1H NMR(500MHz,DMSO-d6)δ＝8.47(d,J＝8.2Hz,2H),6.78(s,4H),4.45(dt,J＝7.8,3.7Hz,2H),3.88(dd,J＝10.9,3.9Hz,2H),3.74(dd,J＝11.0,3.7Hz,2H)。
实施例2：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
Figure PCTCN2022111392-appb-000015
(1)酰胺化反应
在2℃下，分别将D-丝氨酸乙酯盐酸盐(19g)、三乙胺(12g)、3,6-二氨基吡嗪-2,5-二甲酸(20g)、HOBt(14g)和EDC.HCl(19g)滴加入DMF(800mL)中，然后将反应液在室温下搅拌24小时。反应结束后，旋蒸除去DMF，将剩余物用水和乙酸乙酯多次萃取，以饱和NaHCO 3水溶液和KHSO 4水溶液洗涤合并的有机相，再将有机相浓缩，经柱层析分离，得到中间体5(32.5g，收率为75％)。
LCMS:r.t.＝6.6min,[M+H]+＝429.2。
1H NMR(500MHz,DMSO-d6)δ＝8.50(d,J＝8.2Hz,2H),6.76(s,4H),5.31(t,J＝5.4Hz,2H),4.55(dt,J＝7.9,3.8Hz,2H),4.15(q,J＝7.1Hz,4H),3.89(ddd,J＝11.1,5.7,4.0Hz,2H),3.75(ddd,J＝11.0,5.3,3.6Hz,2H),1.21(t,J＝7.1Hz,6H)。
(2)水解反应
将中间体5(21g)的四氢呋喃溶液与LiOH(5.0g)水溶液混合，在0℃ 下搅拌2小时。用TLC监测反应进程，当反应物消失后，用醋酸水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(18g，收率为96％，dr＝98:2)。其表征数据同实施例1步骤(2)所记载。
实施例3：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
Figure PCTCN2022111392-appb-000016
(1)酰胺化反应
在5℃下，分别将D-丝氨酸乙酯盐酸盐(19g)、三乙胺(12g)、3,6-二氨基吡嗪-2,5-二甲酸(20g)、HOBt(14g)和EDC.HCl(19g)滴加入DMF(800mL)中，然后将反应液在室温下搅拌24小时。反应结束后，旋蒸除去DMF，将剩余物用水和乙酸乙酯多次萃取，以饱和NaHCO 3水溶液和KHSO 4水溶液洗涤合并的有机相，再将有机相浓缩，经柱层析分离，得到中间体5(28g，收率为65％)。其表征数据同实施例2步骤(1)所记载。
(2)水解反应
将中间体5(21g)的甲醇溶液与LiOH(5g)混合，在10℃下搅拌2小时。用TLC监测反应进程，当反应物消失后，用三氟乙水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(11g，收率为60％，dr＝80:20)。其表征数据同实施例1步骤(2)所记载。
实施例4：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
Figure PCTCN2022111392-appb-000017
(1)酰胺化反应
在5℃下，分别将D-丝氨酸乙酯盐酸盐(19g)、三乙胺(12g)、3,6-二氨基吡嗪-2,5-二甲酸(20g)、HOBt(14g)和EDC.HCl(19g)滴加入二氯甲烷(1200mL)中，然后将反应液在室温下搅拌24小时。反应结束后，旋蒸除去二氯甲烷，将剩余物用水和乙酸乙酯多次萃取，以饱和NaHCO 3水溶液和KHSO 4水溶液洗涤合并的有机相，再将有机相浓缩，经柱层析分离，得到中间体5(5.2g，收率为12％)。其表征数据同实施例2步骤(1)所记载。
(2)水解反应
将中间体5(43g)的乙醇溶液与LiOH(12g)混合，在20℃下搅拌2小时。用TLC监测反应进程，当反应物消失后，用三氟乙酸水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(14g，收率为38％，dr＝82:18)。其表征数据同实施例1步骤(2)所记载。
实施例5：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
将按照实施例1步骤(1)制备的中间体6(20g)的乙腈溶液与NaOH(6.0g)水溶液混合，在20℃下搅拌2小时。用TLC监测反应进程，当反应底物消失后，用醋酸水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(16.7g，收率为90％，dr＝92:8)。其表征数据同实施例1步骤(2)所记载。
实施例6：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
将按照实施例2步骤(1)制备的中间体5(20g)的1,4-二氧六环溶液与 KOH(14g)水溶液混合，在-5℃下搅拌8小时。用TLC监测反应进程，当反应底物消失后，用盐酸将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(15.3g，收率为88％，dr＝95:5)。其表征数据同实施例1步骤(2)所记载。
实施例7：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
将按照实施例3步骤(1)制备的中间体5(21g)的甲醇溶液与LiOH(5g)混合，在0℃下搅拌2小时。用TLC监测反应进程，当反应物消失后，用三氟乙水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(11g，收率为60％，dr＝86:14)。其表征数据同实施例1步骤(2)所记载。
实施例8：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
将按照实施例2步骤(1)制备的中间体5(20g)的水溶液与KOH(14g)水溶液混合，在10℃下搅拌3小时。用TLC监测反应进程，当反应底物消失后，用三氟乙酸水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(16g，收率为92％，dr＝98:2)。其表征数据同实施例1步骤(2)所记载。
实施例9：3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的制备
将按照实施例2步骤(1)制备的中间体5(20g)的水溶液与KOH(14g)水溶液混合，在20℃下搅拌3小时。用TLC监测反应进程，当反应底物消失后，用三氟乙酸水溶液将反应液酸化(调节pH至约3)，继续搅拌2小时，得到红色浑浊液。将该红色液体过滤，洗涤滤饼并烘干，得到目标化合物3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪(16g， 收率为92％，dr＝95:5)。其表征数据同实施例1步骤(2)所记载。
除本文中描述的实施方案外，根据前述描述，本发明的多种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。
本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (22)

  1. 制备式1的3,6-二氨基-2,5-双{N-(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪的方法，其特征在于，所述方法包括以下步骤：
    Figure PCTCN2022111392-appb-100001
    其中R 1在每次出现时独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 3-8环烷基，优选为C 1-6烷基，更优选为C 1-3烷基，更优选为甲基或乙基；
    步骤S1.使式A的D-丝氨酸酯盐酸盐、3,6-二氨基吡嗪-2,5-二甲酸和酰胺缩合剂在第一有机溶剂中进行酰胺化反应，以得到式1-Int的中间体；
    步骤S2.使所述式1-Int的中间体在碱性条件下在溶剂中水解，然后酸化反应混合物，以得到所述式1的3,6-二氨基-2,5-双{N-[(1R)-1-羧基-2-羟基乙基]氨基甲酰基}吡嗪，其中所述溶剂是水、或水和第二有机溶剂的混合物。
  2. 根据权利要求1所述的方法，其特征在于，在所述步骤S1中，所述酰胺化反应是通过将所述式A的D-丝氨酸酯盐酸盐、所述3,6-二氨基吡嗪-2,5-二甲酸和所述酰胺缩合剂在低温条件下滴加到所述第一有机溶剂中，然后混合来进行的，以得到所述式1-Int的中间体。
  3. 根据权利要求2所述的方法，其特征在于，在所述步骤S1中，所述低温条件为约-10℃至10℃，优选约-5℃至5℃，更优选约0℃至5℃，特别优选约0℃至2℃的温度。
  4. 根据权利要求2或3所述的方法，其特征在于，在所述步骤S1中，所述混合是在室温下进行。
  5. 根据权利要求2至4中任一项所述的方法，其特征在于，在所述步骤S1中，所述混合为在室温下搅拌约8小时或更长时间，优选约24小时或更长时间。
  6. 根据权利要求1至5中任一项所述的方法，其特征在于，在所述步骤S1中，所述第一有机溶剂为极性有机溶剂，例如选自二甲基甲酰胺(DMF)、乙腈、二氯甲烷、四氢呋喃和乙醇中的一种、两种或更多种，优选为DMF。
  7. 根据权利要求1至6中任一项所述的方法，其特征在于，在所述步骤S1中，所述酰胺缩合剂为三乙胺、1-羟基苯并三氮唑(HOBt)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC.HCl)的组合。
  8. 根据权利要求1至7中任一项所述的方法，其特征在于，在所述步骤S1中，将所述式A的D-丝氨酸酯盐酸盐、所述2,5-二甲酸-3,6-二氨基吡嗪、三乙胺、HOBt和EDC.HCl在约0℃至5℃、优选约0℃至2℃下滴加到DMF中，然后在室温下搅拌，以得到所述式1-Int的中间体。
  9. 根据权利要求1至8中任一项所述的方法，其特征在于，在所述步骤S1中，所述式A的D-丝氨酸酯盐酸盐是由所述式A表示的单一化合物的形式，或者各自由所述式A表示的两种或更多种不同化合物的混合物的形式，优选为
    Figure PCTCN2022111392-appb-100002
    和/或
    Figure PCTCN2022111392-appb-100003
  10. 根据权利要求1至9中任一项所述的方法，其特征在于，在所述步骤S2中，使所述式1-Int的中间体在碱性条件并且在适合的温度下在所述溶剂中水解，然后酸化反应混合物，以得到3,6-二氨基-2,5-二{N-[(1R)-1-羧基-2-羟基乙基]氨甲酰基}吡嗪。
  11. 根据权利要求1至10中任一项所述的方法，其特征在于，所述第二有机溶剂为极性有机溶剂，特别是选自四氢呋喃、甲醇、乙腈、乙醇、1,4-二氧六环及其任意组合，优选为四氢呋喃、乙腈、1,4-二氧六环。
  12. 根据权利要求1至11中任一项所述的方法，其特征在于，在所述步骤S2中，所述溶剂为水。
  13. 根据权利要求1至11中任一项所述的方法，其特征在于，在所述步骤S2中，所述溶剂为水和所述第二有机溶剂的混合物。
  14. 根据权利要求1至13中任一项所述的方法，其特征在于，在所述步骤S2中，所述溶剂包含碱金属氢氧化物，例如选自LiOH、NaOH和KOH中的一种、两种或更多种，优选为LiOH。
  15. 根据权利要求1至14中任一项所述的方法，其特征在于，在所述步骤S2中，所述酸化是在所述水解完成后，通过用酸调节反应混合物的pH值来进行的。
  16. 根据权利要求15所述的方法，其特征在于，所述pH值为约2至4，优选约3。
  17. 根据权利要求1至16中任一项所述的方法，其特征在于，在所述步骤S2中，在所述酸化之后，再将反应混合物混合约1至4小时，优选约2小时。
  18. 根据权利要求15至17中任一项所述的方法，其特征在于，所述酸为选自醋酸、三氟乙酸、硫酸、柠檬酸和盐酸中的一种、两种或更多种。
  19. 根据权利要求1至18中任一项所述的方法，其特征在于，在所述步骤S2中，所述式1-Int的中间体为由所述式1-Int表示的单一化合物的形式，或者各自由所述式1-Int表示的两种或更多种不同化合物的混合物的形式，优选为
    Figure PCTCN2022111392-appb-100004
    和/或
    Figure PCTCN2022111392-appb-100005
  20. 式1-Int的化合物：
    Figure PCTCN2022111392-appb-100006
    其中每个R 1可以相同或不同，且独立地选自C 1-8烷基、C 2-8烯基、C 2-8炔基和C 3-8环烷基，优选为C 1-6烷基，更优选为C 1-3烷基，更优选为甲基或乙基。
  21. 根据权利要求20所述的化合物，其为：
    Figure PCTCN2022111392-appb-100007
  22. 制备权利要求20或21的式1-Int的化合物的方法，其特征在于，所述方法包括以下步骤：
    Figure PCTCN2022111392-appb-100008
    步骤S1.使式A的D-丝氨酸酯盐酸盐、2,5-二甲酸-3,6-二氨基吡嗪和酰胺缩合剂在第一有机溶剂中进行酰胺化反应，得到所述式1-Int的化合物；
    优选地，其中所述步骤S1如权利要求2至9中任一项所述。
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