3,6-DIAMINO-2,5-BIS{N-[(1R)-1-CARBOXY-2-HYDROXYETYL]CARBAMOYL}PYRAZIN, PREPARATIONS OF THIS PHARMACEUTICAL SUBSTANCE, METHODS OF QUALITY CONTROL OF THIS PHARMACEUTICAL SUBSTANCE OR ITS PREPARATIONS, AND METHODS OF PREPARATION OF COMPOUNDS RELATED TO THIS PHARMACEUTICAL SUBSTANCE

Abstract

The invention relates to a compound MB-102 and a method for preparing such compound. Specifically, the invention relates to compound (I), compound (X), compound (XI), and compound (XI), and a method for preparing such compounds. In addition, the invention relates to a method for controlling the quality of the MB-102 drug substance or preparation, including measuring the content of compound (I), compound (X), compound (XI), and / or compound (XI) in the drug substance or preparation, and optionally comparing this content with the specified content of each compound.

Description

MB-102 related compounds and their preparation methods and uses Technical Field

[0001] The present invention relates to pharmaceutical compounds, in particular to compounds related to 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine (MB-102), as well as preparation methods and uses thereof. Background Art

[0002] Glomerular Filtration Rate (GFR) is the best indicator for evaluating kidney function. 3,6-Diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine (MB-102) is a bis-serine aminopyrazine compound developed by MediBeacon and used in MediBeacon. TM Transdermal GFR measurement system has potential application prospects in evaluating renal function as a fluorescent tracer.

[0003] It is necessary to conduct quality control on MB-102's APIs, preparations and synthetic intermediates during the production and storage process.

[0004] Summary of the Invention

[0005] The present inventors have discovered that the compounds of Formula I, Formula VI, Formula X and Formula XI described in the present disclosure have the potential to be used for quality control of MB-102 drug substance and its formulations, for example, as quality control standards or reference substances.

[0006] In a first aspect, the present disclosure provides a compound of Formula I, and a compound of Formula II as a synthetic intermediate of the compound of Formula I:

[0007] in:

[0008] X1 is selected from

[0009] X2 is selected from H, substituted or unsubstituted C 1-10 Alkyl, and -(substituted or unsubstituted C 1-10 alkyl)-R 1 ;

[0010] X3 and X4 are each independently selected from substituted or unsubstituted C 1-10 Alkyl, and -(substituted or unsubstituted C 1-10 alkyl)-R 1 ;

[0011] R 1 Selected from: C 3-10Cycloalkyl, 4-10 membered heterocyclic group, C 6-10 aryl and 5-10 membered heteroaryl.

[0012] In a second aspect, the present disclosure provides a method for preparing the compound of formula I, characterized in that the method comprises the following steps:

[0013] a) subjecting the compound of formula II to a hydrogenation reaction with hydrogen gas or a hydrogen-borrowing agent in a solvent in the presence of a suitable metal catalyst to provide the compound of formula I.

[0014] In a third aspect, the present disclosure provides compounds of Formula VI, and compounds of Formula VII as synthetic intermediates of the compounds of Formula VI:

[0015] in:

[0016] X5 and X6 are each independently -(substituted or unsubstituted C 1-10 alkyl)-R 2 ;as well as

[0017] R 2 Selected from: C 3-10 Cycloalkyl, 4-10 membered heterocyclic group, C 6-10 aryl and 5-10 membered heteroaryl.

[0018] In a fourth aspect, the present disclosure provides a method for preparing the compound of formula VI, characterized in that the method comprises the following steps:

[0019] i) subjecting the compound of formula VII to a hydrogenation reaction in a solvent in the presence of a suitable metal catalyst to provide the compound of formula VI.

[0020] In a fifth aspect, the present disclosure provides compounds of formula X:

[0021] where R 3 is H, or substituted or unsubstituted C 1-4 Alkyl, preferably H or methyl.

[0022] In some preferred embodiments, R 3 For H.

[0023] In a sixth aspect, the present disclosure provides a method for preparing the compound of formula X, characterized in that the method comprises the following steps:

[0024] xi) providing a solution of a compound of formula A in a solvent;

[0025] x-ii) At elevated temperature, with acid R3 -COOH acylates the compound of formula A to provide the compound of formula X; and

[0026] x-iii) optionally, isolating and purifying the compound of formula X;

[0027] where R 3 is H, or substituted or unsubstituted C 1-4 Alkyl, preferably H or methyl, more preferably H.

[0028] In a seventh aspect, the present disclosure provides compounds of Formula XI:

[0029] In an eighth aspect, the present disclosure provides a method for preparing the compound of formula XI, characterized in that the method comprises the following steps:

[0030] xi-i) hydrolyzing the compound of formula A in water in the presence of a base at elevated temperature to provide the compound of formula XI or a salt thereof;

[0031] as well as

[0032] xi-ii) Optionally, isolating and purifying the compound of formula XI or a salt thereof.

[0033] In a ninth aspect, the present disclosure provides a method for quality control of a drug substance or a formulation of MB-102, comprising determining the content of the compound of formula I, preferably the compound of formula I-1, the compound of formula I-2 or the compound of formula I-3, the compound of formula VI, the compound of formula X and / or the compound of formula XI in the drug substance or formulation, and optionally comparing the content with a specified content of each of the compounds.

[0034] In a tenth aspect, the present disclosure provides the use of the compound of formula I, preferably the compound of formula I-1, the compound of formula I-2 or the compound of formula I-3, the compound of formula VI, the compound of formula X and / or the compound of formula XI as a standard or reference substance for quality control of the bulk drug or preparation of MB-102. DETAILED DESCRIPTION

[0035] definition

[0036] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. Reference to the technology used herein refers to the technology commonly understood in the art, including those changes in technology or equivalent technology substitutions that are obvious to those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0037] As used herein, the terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps (i.e., these terms also encompass the terms "consisting essentially of and "consisting of").

[0038] As used herein, the term "alkyl" is a linear or branched saturated monovalent hydrocarbon group. Preferably, the alkyl group has 1 to 10 carbon atoms (C 1-10 Alkyl), for example, having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl group includes C 1-8 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl and C 1-2 Alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. The alkyl group may be substituted or unsubstituted.

[0039] The term "alkenyl" as used herein is a linear or branched monovalent hydrocarbon group containing one or more double bonds (C=C). Preferably, the alkenyl group has 2 to 6 carbon atoms (C 2-6 The term "alkenyl" refers to a group consisting of, for example, 2, 3, 4, 5, or 6 carbon atoms and 1, 2, or 3 double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, hex-1-enyl, hex-2-enyl, and hex-3-enyl. The alkenyl group may be substituted or unsubstituted.

[0040] The term "alkynyl" as used herein is a linear or branched monovalent alkynyl group containing one or more triple bonds (C≡C). Preferably, the alkynyl group has 2 to 6 carbon atoms (C 2-6Alkynyl groups include, but are not limited to, ethynyl, propynyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, hex-1-ynyl, hex-2-ynyl, and hex-3-ynyl. The alkynyl group may be substituted or unsubstituted.

[0041] As used herein, the term "cycloalkyl" refers to a saturated (i.e., "cycloalkyl") or partially unsaturated (i.e., having one or more double bonds (cycloalkenyl) and / or triple bonds (cycloalkynyl) within the ring) monocyclic or polycyclic hydrocarbon ring having, for example, 3-10 (suitably 3-8, such as 5-7; more suitably 3-6, such as 5-6) ring carbon atoms, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclohexenyl, etc. The cycloalkyl group may be substituted or unsubstituted.

[0042] As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spirocyclic, fused or bridged systems, such as bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl or bicyclo[5.2.0]nonyl, decahydronaphthyl, etc.). Cycloalkyl has, for example, 3 to 10, for example, 3-7, 5-6 or 5-7 carbon atoms. The cycloalkyl may be substituted or unsubstituted.

[0043] As used herein, the term "heterocycle" or "heterocyclyl" means a saturated or partially unsaturated cyclic group having a ring heteroatom selected from N, O and S. Preferably, heterocycle is a 4 to 10-membered heterocycle having 1, 2, 3, 4, 5 or 6 ring heteroatoms selected from N, O and S. More preferably, heterocycle is a 4 to 7-membered saturated heterocycle having 1, 2, 3 or 4 ring heteroatoms selected from N, O and S, more preferably a 5 to 7-membered (e.g., 5 to 6-membered) saturated heterocycle having 1, 2 or 3 ring heteroatoms selected from N, O and S. Examples of heterocycles include, but are not limited to, azetidine, oxetanyl, tetrahydrofuran, pyrrolidine, imidazolidine, pyrazolidine, tetrahydropyran, piperidine, morpholine, thiomorpholine, piperazine. The heterocycle may be substituted or unsubstituted.

[0044] As used herein, the term “C 6-10 "Aryl" means phenyl or naphthyl, which may be substituted or unsubstituted.

[0045] As used herein, the term "heteroaryl" refers to a monocyclic heteroaryl or a bicyclic or polycyclic ring system containing at least one heteroaromatic ring (a heteroaromatic ring is an aromatic ring system containing at least one heteroatom), having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5, 6, 7, 8, 9 or 10 ring atoms, and which contains at least one heteroatom (e.g. oxygen, nitrogen or sulfur) which may be identical or different and which in each case may share two adjacent atoms with each other to form a paracyclic group with the aryl, heterocyclyl or cycloalkyl radicals, the point of attachment being on the heteroaromatic ring or on another ring. For example, as used herein, the term "5-10 membered heteroaryl" means a heteroaryl group containing 5 to 10 ring atoms, including 5-6 membered heteroaryl groups, examples of which include but are not limited to thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl, etc., or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their cyclic derivatives. The cycloalkyl derivatives are not limited to heteroaryl and heteroaryl, heteroaryl and aryl, heteroaryl and heterocyclyl or heteroaryl and cycloalkyl, in particular 5-6-membered heteroaryl and 5-6-membered heteroaryl, 5-6-membered heteroaryl and phenyl, 5-6-membered heteroaryl and 5-6-membered heterocyclyl, or 5-6-membered heteroaryl and C4-C6 cycloalkyl (in particular 5-6-membered heteroaryl and cyclobutyl, 5-6-membered heteroaryl and cyclopentyl, 5-6-membered heteroaryl and cyclohexyl), examples of which include but are not limited to indolyl, isoindolyl, indazolyl, benzimidazole, quinolinyl, isoquinolinyl, The heteroaryl group may be substituted or unsubstituted.

[0046] As used herein, the term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

[0047] As used herein, the term "substituted or unsubstituted" means that one or more hydrogen atoms attached to an atom or group are independently unsubstituted or substituted with one or more (e.g., 1, 2, 3, or 4) substituents. The substituents are independently selected from the group consisting of deuterium (D), halogen, -OH, mercapto, cyano, -CD3, C1-C6 alkyl (preferably C1-C3 alkyl), C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl (preferably C3-C8 cycloalkyl), aryl, heterocyclyl (preferably 3-8 membered heterocyclyl), heteroaryl, aryl C1-C6 alkyl-, heteroaryl C1-C6 alkyl, C1-C6 haloalkyl, -OC1-C6 alkyl (preferably -OC1-C3 alkyl), -OC2-C6 alkenyl, OC1-C6 alkylphenyl, C1-C6 alkyl-OH (preferably C1-C4 alkyl-OH), C1-C6 Alkyl-SH, C1-C6 alkyl-O-C1-C6 alkyl, OC1-C6 haloalkyl, NH2, C1-C6 alkyl-NH2 (preferably C1-C3 alkyl-NH2), -N(C1-C6 alkyl)2 (preferably -N(C1-C3 alkyl)2), -NH(C1-C6 alkyl) (preferably -NH(C1-C3 alkyl)), -N(C1-C6 alkyl)(C1-C6 alkylphenyl), -NH(C1-C6 alkylphenyl), nitro, -C(O)-OH, -C(O)OC1-C6 alkyl (preferably -C(O)OC1-C3 alkyl), -CONRiRii (wherein Ri and Rii are H, D and C1-C6 alkyl, preferably C1-C3 alkyl), -NHC(O)(C1-C6 alkyl), -NHC(O)(phenyl), -N(C1-C6 C(O) (C1-C6 alkyl), -N(C1-C6 alkyl)C(O)(phenyl), -C(O)C1-C6 alkyl, -C(O)heteroaryl (preferably -C(O)-5-7 membered heteroaryl), -C(O)C1-C6 alkylphenyl, -C(O)C1-C6 haloalkyl, -OC(O)C1-C6 alkyl (preferably -OC(O)C1-C3 alkyl), -S(O) -C1-C6 alkyl, -S(O)2-phenyl, -S(O)2-C1-C6 haloalkyl, -S(O)2NH2, -S(O)2NH(C1-C6 alkyl), -S(O)2NH(phenyl), -NHS(O)2(C1-C6 alkyl), -NHS(O)2(phenyl) and -NHS(O)2(C1-C6 haloalkyl). When an atom or group is substituted with multiple substituents, the multiple substituents may be the same or different.

[0048] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range "1 to 10" should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any individual value (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.) within the range of 1 to 10. This principle also applies to ranges that use only one value as a minimum or maximum value.

[0049] As used herein, the term "room temperature" refers to about 20 to 25°C.

[0050] As used herein, the term "elevated temperature" refers to a temperature greater than about 25°C. In some embodiments, an "elevated temperature" is not less than about 30°C, in some embodiments, not less than about 35°C, in some embodiments, not less than about 40°C, in some embodiments, not less than about 45°C, in some embodiments, not less than about 50°C, in some embodiments, not less than about 55°C, in some embodiments, not less than about 60°C, in some embodiments, not less than about 70°C, in some embodiments, not less than about 80°C, in some embodiments, not less than about 90°C, or in some embodiments, not less than about 100°C, or higher. In some embodiments, an "elevated temperature" is not more than about 100°C, in some embodiments, not more than about 90°C, in some embodiments, not more than about 80°C, in some embodiments, not more than about 70°C, in some embodiments, not more than about 60°C, in some embodiments, not more than about 55°C, in some embodiments, not more than about 50°C, in some embodiments, not more than about 45°C, or in some embodiments, not more than about 40°C.

[0051] As used herein, the term "about" means within ±10%, preferably within ±5%, and more preferably within ±2% of the stated numerical value.

[0052] Compound and preparation method thereof

[0053] In a first aspect, the present disclosure provides a compound of Formula I, and a compound of Formula II as a synthetic intermediate of the compound of Formula I:

[0054] in:

[0055] X1 is selected from

[0056] X2 is selected from H, substituted or unsubstituted C 1-10 Alkyl, and -(substituted or unsubstituted C 1-10 alkyl)-R1 ;

[0057] X3 and X4 are each independently selected from substituted or unsubstituted C 1-10 Alkyl, and -(substituted or unsubstituted C 1-10 alkyl)-R 1 ;as well as

[0058] R 1 Selected from: C 3-10 Cycloalkyl, 4-10 membered heterocyclic group, C 6-10 aryl and 5-10 membered heteroaryl.

[0059] In some embodiments, R 1 Selected from: C 3-7 Cycloalkyl, preferably C 5-6 Cycloalkyl; 4-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; phenyl and 5-6 membered heteroaryl. Preferably, R 1 It is a phenyl group.

[0060] In some embodiments, X1 is In some such embodiments, X2 is selected from H, substituted or unsubstituted C 1-8 Alkyl, and -(substituted or unsubstituted C 1-8 alkyl)-R 1 In some embodiments, X2 is selected from H, substituted or unsubstituted C 1-6 Alkyl, and -(substituted or unsubstituted C 1-6 alkyl)-R 1 In some embodiments, X2 is selected from H, substituted or unsubstituted C 1-4 Alkyl, and -(substituted or unsubstituted C 1-4 alkyl)-R 1 In some embodiments, X2 is selected from H, unsubstituted C 1-2 Alkyl, and -(unsubstituted C 1-2 alkyl)-R 1 In some preferred embodiments, X2 is selected from H, ethyl and -CH2-R 1 More preferably, X2 is selected from H, ethyl and -CH2-phenyl.

[0061] In other embodiments, X1 is In some such embodiments, X3 is selected from substituted or unsubstituted C 1-8 Alkyl, and -(substituted or unsubstituted C 1-8 alkyl)-R 1 In some embodiments, X3 is selected from C 1-6 Alkyl, and -(substituted or unsubstituted C 1-6 alkyl)-R1 In some embodiments, X3 is selected from more preferably substituted or unsubstituted C 1-4 Alkyl, and -(substituted or unsubstituted C 1-4 alkyl)-R 1 In some embodiments, X3 is selected from the group consisting of more preferably unsubstituted C 1-2 Alkyl, and -(unsubstituted C 1-2 alkyl)-R 1 In some embodiments, X3 is more preferably selected from ethyl and -CH2-R 1 More preferably, X3 is selected from ethyl and -CH2-phenyl.

[0062] In some embodiments, X4 is selected from substituted or unsubstituted C 1-8 Alkyl, and -(substituted or unsubstituted C 1-8 alkyl)-R 1 In some embodiments, X4 is selected from C 1-6 Alkyl, and -(substituted or unsubstituted C 1-6 alkyl)-R 1 In some embodiments, X4 is selected from more preferably substituted or unsubstituted C 1-4 Alkyl, and -(substituted or unsubstituted C 1-4 alkyl)-R 1 In some embodiments, X4 is selected from the group consisting of more preferably unsubstituted C 1-2 Alkyl, and -(unsubstituted C 1-2 alkyl)-R 1 In some embodiments, X4 is more preferably selected from ethyl and -CH2-R 1 More preferably, X4 is selected from ethyl and -CH2-phenyl. Even more preferably, X4 is -CH2-phenyl.

[0063] In some embodiments, the compound of Formula I is a compound of Formula I-1:

[0064] In some embodiments, the compound of Formula I is a compound of Formula I-2:

[0065] In some embodiments, the compound of Formula I is a compound of Formula I-3:

[0066] In some embodiments, the compound of Formula II is a compound of Formula II-1:

[0067] In some embodiments, the compound of Formula II is a compound of Formula II-2:

[0068] In some embodiments, the compound of Formula II is a compound of Formula II-3:

[0069] In a second aspect, the present disclosure provides a method for preparing a compound of formula I as described above, characterized in that the method comprises the following steps:

[0070] a) subjecting the compound of formula II as described above to a hydrogenation reaction with hydrogen gas or a hydrogen-borrowing agent in a solvent in the presence of a suitable metal catalyst to provide the compound of formula I.

[0071] In some embodiments, in step a), the compound of formula II is subjected to the hydrogenation reaction with the hydrogen borrowing agent. In some such embodiments, the molar ratio of the compound of formula II to the hydrogen borrowing agent is about 1:2 to 1:20, preferably about 1:2 to 1:10, for example, about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8. In some embodiments, the hydrogen borrowing agent is selected from a combination of formic acid and an alkali metal formate, cyclohexadiene and isopropanol. In some preferred embodiments, the hydrogen borrowing agent is a combination of formic acid and an alkali metal formate. Preferably, the molar ratio of formic acid to the alkali metal formate is about 1:6 to 6:1, preferably about 1:1 to 5:1. In some embodiments, the alkali metal formate is sodium formate or potassium formate, preferably potassium formate. In some embodiments, the reaction of the compound of formula II with the hydrogen borrowing reagent is carried out at a temperature of about 20°C to 100°C, more preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C. In other embodiments, in step a), the compound of formula II is subjected to the hydrogenation reaction with the hydrogen. In some such embodiments, the pressure of the hydrogen is from about 0.05Mpa to about 0.8Mpa, preferably from about 0.1Mpa to about 0.4Mpa. In some embodiments, the reaction of the compound of formula II with the hydrogen is carried out at a temperature of about 15 to 25°C (e.g., about 20 to 25°C).

[0072] Reactions using hydrogen-borrowing reagents have many advantages. For example, they avoid the use of flammable and explosive hydrogen, greatly reducing safety risks in industrial production; they have short reaction times; and they use alkali metal formates rather than ammonium formate, avoiding the problem of pipeline blockage caused by sublimation when using ammonium formate.

[0073] In some embodiments, in step a), the volume ratio of the solvent to the compound of Formula II is about 10:1 to 80:1, preferably about 20:1 to 50:1. In some embodiments, the solvent is an aqueous alcohol solution. In some embodiments, the volume ratio of alcohol to water in the aqueous alcohol solution is about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1). In some embodiments, the alcohol in the aqueous alcohol solution is selected from methanol (MeOH), ethanol (EtOH), isopropyl alcohol, and any mixture thereof. Preferably, the alcohol is ethanol. In other embodiments, the solvent is a mixed solvent of an alcohol and an ester. In some embodiments, the volume ratio of the ester to the alcohol in the mixed solvent of an alcohol and an ester is about 1:1 to about 1:5, more preferably about 1:1 to about 1:2. In some embodiments, the alcohol in the mixed solvent of an alcohol and an ester is selected from methanol (MeOH), ethanol (EtOH), isopropyl alcohol, and any mixture thereof. Preferably, the alcohol is ethanol. In some embodiments, the ester in the mixed solvent of an alcohol and an ester is selected from ethyl acetate (EA), isopropyl acetate, or a mixture thereof. Preferably, the ester is ethyl acetate (EA). In some embodiments, the alcohol in the mixed solvent of alcohol and ester is ethanol, and / or the ester is ethyl acetate (EA).

[0074] In some preferred embodiments, the hydrogenation reaction of the compound of formula II with the hydrogen borrowing agent is carried out in the alcohol aqueous solution described above. In some preferred embodiments, the hydrogenation reaction of the compound of formula II with the hydrogen gas is carried out in the alcohol aqueous solution described above, or in a mixed solvent of the alcohol and ester described above.

[0075] In some embodiments, the mass ratio of the compound of Formula II to the metal catalyst is about 1:0.01 to 1:1, preferably about 1:0.2. In some embodiments, the metal catalyst is palladium on carbon (Pd / C). Preferably, the mass fraction of palladium in the Pd / C is about 5-20%, more preferably about 10% (for example, the Pd / C catalyst contains about 50 w / w% water).

[0076] In some embodiments, the hydrogenation reaction is carried out for a period of about 1 to 72 hours (h), preferably about 1 to 48 h, and more preferably about 6 to 12 h.

[0077] In some embodiments, the present disclosure provides a method as described above, comprising the following steps:

[0078] a) subjecting the compound of formula II to a hydrogenation reaction with the hydrogen gas or the hydrogen-borrowing reagent in an ethanol aqueous solution or a mixed solvent of ethanol and ethyl acetate under the catalysis of Pd / C for about 1 to 72 hours, for example, about 1 to 48 hours, about 1 to 12 hours, about 1 to 10 hours, about 2 to 5 hours, or about 6 to 12 hours to provide the compound of formula I,

[0079] in:

[0080] (1) In some embodiments, the pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa, and

[0081] The reaction of the compound of formula II with the hydrogen is carried out at a temperature of about 20 to 25° C.;

[0082] or

[0083] In other embodiments, the molar ratio of the compound of formula II to the hydrogen borrowing agent is about 1:2 to 1:10, for example, about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8,

[0084] The reaction of the hydrogen-borrowing reagent is carried out at a temperature of about 20°C to 100°C, preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C, and

[0085] The hydrogen borrowing reagent is a combination of formic acid and potassium formate, wherein the molar ratio of the formic acid to the potassium formate is about 1:1 to 5:1;

[0086] (2) the mass ratio of the compound of formula II to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2;

[0087] (3) the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate, to the compound of Formula II is about 10:1 to 80:1, preferably about 20:1 to 50:1; and

[0088] (4) the volume ratio of ethanol to water in the ethanol-water solution is from about 1:1 to about 8:1, for example, about 4:1 or about 6:1; and

[0089] The volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is about 1:1 to about 1:5, preferably about 1:1 to about 1:2.

[0090] In some embodiments of the methods described above according to the present disclosure, the compound of formula I is the compound of formula I-1, and the compound of formula II is the compound of formula II-1:

[0091] In some such embodiments, the method comprises the steps of:

[0092] a) subjecting the compound of formula II-1 to a hydrogenation reaction with a hydrogen-borrowing agent in the ethanol aqueous solution under the catalysis of Pd / C for about 1 to 12 hours to provide the compound of formula I-1.

[0093] In some preferred embodiments, the molar ratio of the compound of formula II-1 to the hydrogen borrowing agent is about 1:2 to 1:10, for example, about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8. In some embodiments, the reaction of the compound of formula II-1 with the hydrogen borrowing agent is carried out at a temperature of about 20°C to 100°C, preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C. In some embodiments, the hydrogen borrowing agent is a combination of formic acid and potassium formate, wherein the molar ratio of formic acid to potassium formate is about 1:1 to 5:1.

[0094] In other such embodiments, the method comprises the steps of:

[0095] a) subjecting the compound of formula II-1 to a hydrogenation reaction with the hydrogen gas in the ethanol aqueous solution or a mixed solvent of ethanol and ethyl acetate under the catalysis of the Pd / C for about 6 to 12 hours to provide the compound of formula I-1.

[0096] In some preferred embodiments, the pressure of the hydrogen is from about 0.05 MPa to about 0.8 MPa, preferably from about 0.1 MPa to about 0.4 MPa. In some embodiments, the reaction of the compound of formula II-1 with the hydrogen is carried out at a temperature of about 20 to 25 ° C. In some embodiments, the mass ratio of the compound of formula II-1 to the Pd / C is from about 1:0.01 to 1:1, preferably from about 1:0.2. In some embodiments, the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II-1 is from about 10:1 to 80:1, preferably from about 20:1 to 50:1. In some embodiments, the volume ratio of ethanol to water in the ethanol aqueous solution is from about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1). In other embodiments, the volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is from about 1:1 to about 1:5, preferably from about 1:1 to about 1:2.

[0097] In other embodiments of the methods according to the present disclosure described above, the compound of formula I is the compound of formula I-2, and the compound of formula II is the compound of formula II-2:

[0098] In some such embodiments, the method comprises the steps of:

[0099] a) subjecting the compound of formula II-2 to a hydrogenation reaction with the hydrogen gas in the ethanol aqueous solution or a mixed solvent of ethanol and ethyl acetate under the catalysis of Pd / C for about 6 to 12 hours to provide the compound of formula I-2.

[0100] In some preferred embodiments, the pressure of the hydrogen is from about 0.05 MPa to about 0.8 MPa, preferably from about 0.1 MPa to about 0.4 MPa. In some embodiments, the reaction of the compound of formula II-2 with the hydrogen is carried out at a temperature of about 20 to 25 ° C. In some embodiments, the mass ratio of the compound of formula II-2 to the Pd / C is from about 1:0.01 to 1:1, preferably from about 1:0.2. In some embodiments, the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II-2 is from about 10:1 to 80:1, preferably from about 20:1 to 50:1. In some embodiments, the volume ratio of ethanol to water in the ethanol aqueous solution is from about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1). In other embodiments, the volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is from about 1:1 to about 1:5, preferably from about 1:1 to about 1:2.

[0101] In other embodiments of the methods according to the present disclosure described above, the compound of formula I is the compound of formula I-3, and the compound of formula II is the compound of formula II-3:

[0102] In some such embodiments, the method comprises the steps of:

[0103] a) subjecting the compound of formula II-3 to a hydrogenation reaction with the hydrogen gas in the ethanol aqueous solution or the mixed solvent of ethanol and ethyl acetate under the catalysis of Pd / C for about 1 to 12 hours, for example, about 1 to 10 hours, about 2 to 5 hours, or about 6 to 12 hours to provide the compound of formula I-3.

[0104] In some preferred embodiments, the pressure of the hydrogen is from about 0.05 MPa to about 0.8 MPa, preferably from about 0.1 MPa to about 0.4 MPa. In some embodiments, the reaction of the compound of formula II-3 with the hydrogen is carried out at a temperature of about 20 to 25 ° C. In some embodiments, the mass ratio of the compound of formula II-3 to the Pd / C is from about 1:0.01 to 1:1, preferably from about 1:0.2. In some embodiments, the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II-3 is from about 10:1 to 80:1, preferably from about 20:1 to 50:1. In some embodiments, the volume ratio of ethanol to water in the ethanol aqueous solution is from about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1). In other embodiments, the volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is from about 1:1 to about 1:5, preferably from about 1:1 to about 1:2.

[0105] In some embodiments of the method according to the present disclosure described above, the method further comprises the following steps:

[0106] b) removing the metal catalyst from the reaction mixture of step a), and then drying the reaction mixture to obtain the compound of formula I; and

[0107] c) optionally, further purifying the compound of formula I obtained in step b).

[0108] In the step b), the metal catalyst can be removed by filtering through diatomaceous earth; the reaction mixture from which the metal catalyst has been removed can be concentrated to dryness under reduced pressure at an elevated temperature (eg, about 40-50° C., preferably about 45° C.).

[0109] In the step c), the purification can be performed in the following manner:

[0110] The compound of Formula I obtained in step b) is slurried with acetonitrile (eg, about 1 to 4 h), further slurried with water (about 1 to 4 h), then filtered, and the filter cake is dried.

[0111] For example, the drying can be performed by concentrating under reduced pressure at an elevated temperature (eg, about 40-50°C, preferably about 45°C).

[0112] In the step c), the purification can also be carried out in the following manner:

[0113] The compound of formula I obtained from step b) is passed through a chromatography column or high pressure preparative chromatography.

[0114] There is no particular limitation on the high pressure preparative chromatograph. Examples of the high pressure preparative chromatograph include, but are not limited to, LAB-ZB-05, medium and high pressure equipment from Beijing Aonuo Technology Co., Ltd., and the like.

[0115] In some embodiments of the method according to the present disclosure described above, wherein the compound of formula I is the compound of formula I-1, and the compound of formula II is the compound of formula II-1, the method includes performing step c). In some such embodiments, in step c), the compound of formula I-1 obtained from step b) is further purified by slurrying the compound of formula I-1 obtained from step b) with acetonitrile (e.g., about 1 to 4 h), further slurrying with water (about 1 to 4 h), then filtering, and drying the filter cake. The drying can be carried out by concentrating under reduced pressure at a higher temperature (e.g., about 40-50 ° C, preferably about 45 ° C).

[0116] In other embodiments, wherein the compound of formula I is the compound of formula I-2, and the compound of formula II is the compound of formula II-2, the method includes performing step c). In some such embodiments, in step c), the compound of formula I-2 obtained from step b) is further purified by slurrying the compound of formula I-2 obtained from step b) with acetonitrile (e.g., about 1 to 4 hours), further slurrying with water (about 1 to 4 hours), then filtering, and drying the filter cake; or passing the compound of formula I-2 from step b) through a chromatography column or a high-pressure preparative chromatograph. The drying can be performed by concentrating under reduced pressure at an elevated temperature (e.g., about 40-50°C, preferably about 45°C).

[0117] In yet other embodiments, wherein the compound of Formula I is the compound of Formula I-3 and the compound of Formula II is the compound of Formula II-3, the method comprises performing step c). In some such embodiments, in step c), the compound of Formula I-3 obtained from step b) is further purified by passing the compound of Formula I-3 from step b) through a chromatography column or high pressure preparative chromatography.

[0118] In some embodiments of the method according to the present disclosure described above, the method further comprises the following steps:

[0119] a') subjecting the compound of formula III, 3,6-diaminopyrazine-2,5-dioic acid, to an amide condensation reaction with a compound of formula IV or a salt thereof and a compound of formula V or a salt thereof in a suitable organic solvent in the presence of a suitable organic base, a suitable coupling agent and a suitable condensing agent to provide the compound of formula II,

[0120] wherein X1 and X4 are as defined above.

[0121] The salt of the compound of formula IV includes but is not limited to hydrochloride and hydrobromide, preferably hydrochloride; the salt of the compound of formula V includes but is not limited to hydrochloride and hydrobromide, preferably hydrochloride.

[0122] In some embodiments, wherein the compound of Formula II is the compound of Formula II-1, the method comprises:

[0123] The compound of formula III is reacted with the hydrochloride of the compound of formula IV having formula IV-1 and the hydrochloride of the compound of formula V having formula V-1 to provide the compound of formula II-1:

[0124] In other embodiments, wherein the compound of Formula II is the compound of Formula II-2, the method comprises:

[0125] The compound of formula III is reacted with the hydrochloride of the compound of formula IV having the formula IV-1 and the hydrochloride of the compound of formula V having the formula V-2 to provide the compound of formula II-2:

[0126] In yet other embodiments, wherein the compound of Formula II is the compound of Formula II-3, the method comprises:

[0127] The compound of formula III is reacted with the hydrochloride salt of the compound of formula IV-1 to provide the compound of formula II-3.

[0128] In some of the embodiments described above of the methods according to the present disclosure, the molar ratio of the compound of formula IV to the compound of formula III is about 0.5:1 to 8:1, preferably about 1:1 to 4:1.

[0129] In some embodiments, the molar ratio of the compound of formula V to the compound of formula III is about (0.5-8.0):1.0, preferably about 1:1 to 4:1.

[0130] In some embodiments, the volume ratio of the organic solvent to the compound of Formula III is about 10:1 to 80:1, preferably about 20:1 to 50:1.

[0131] In some embodiments, the molar ratio of the organic base to the compound of formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1.

[0132] In some embodiments, the molar ratio of the coupling agent to the compound of Formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1.

[0133] In some embodiments, the molar ratio of the condensing agent to the compound of formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1.

[0134] In some embodiments, the amide condensation reaction is first carried out at a suitable temperature, preferably about -5 to 20°C, more preferably about -5 to 10°C, more preferably about 0-10°C, for a suitable time, for example, about 10-60 minutes (min), and then at room temperature for a suitable time, preferably about 10-72h, more preferably about 18-36h.

[0135] In some embodiments, the organic solvent is N,N-dimethylformamide (DMF), DCM, DMSO, tetrahydrofuran, and / or 1,4-dioxane.

[0136] In some embodiments, the organic base is diisopropylethylamine (DIPEA), triethylamine, pyridine, and / or DMAP.

[0137] In some embodiments, the coupling agent is ethyl 2-oxime cyanoacetate (Oxyma), 1-hydroxybenzotriazole (HOBt), HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU) and / or O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU).

[0138] In some embodiments, the condensing agent is selected from: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), HOBt, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU), O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU) and any combination thereof.

[0139] In some embodiments of the methods described above according to the present disclosure, the methods include the following steps:

[0140] a') reacting the compound of formula III with the compound of formula IV and the compound of formula V in DMF at about -5 to 20°C, preferably about -5 to 10°C, more preferably about 0-10°C, in the presence of DIPEA, ethyl 2-oximecyanoacetate and EDC*HCl for about 10-60 min, and then reacting at room temperature for about 10-72 h, more preferably about 18-36 h.

[0141] In some embodiments, the method further comprises the following steps:

[0142] b') optionally, adding water to the reaction mixture in step a') to precipitate the compound of formula II; and

[0143] c') Optionally, further purifying the compound of formula II obtained in step b').

[0144] Preferably, in step b'), the volume ratio of water added to the reaction mixture is about 20:1 to 150:1, more preferably about 35:1 to 75:1.

[0145] Preferably, in step c'), the purification is carried out by passing the compound of formula II from step b') through a chromatography column or high pressure preparative chromatography.

[0146] In some embodiments of the methods described above according to the present disclosure, wherein the compound of Formula II is the compound of Formula II-1, the method comprises performing the step c'). In some such embodiments, in the step c'), the compound of Formula II-1 from the step b') is further purified by passing the compound of Formula II-1 through a chromatography column.

[0147] In other embodiments, wherein the compound of Formula II is the compound of Formula II-2, the method comprises performing step c'). In some such embodiments, in step c'), the compound of Formula II-2 from step b') is further purified by passing the compound of Formula II-2 through a chromatography column or high pressure preparative chromatography.

[0148] In yet other embodiments, wherein the compound of Formula II is the compound of Formula II-3, the method comprises performing step c'). In some such embodiments, in step c'), the compound of Formula II-3 from step b') is further purified by passing the compound of Formula II-3 through a chromatography column or high pressure preparative chromatography.

[0149] In a first particular embodiment, the method according to the present disclosure comprises the steps of:

[0150] (1) adding 3,6-diaminopyrazine-2,5-dioic acid (a compound of Formula III), D-serine benzyl ester hydrochloride, and L-serine benzyl ester hydrochloride to a container, adding DMF and stirring uniformly, wherein preferably, the molar ratio of D-serine benzyl ester hydrochloride to the compound of Formula III and the molar ratio of L-serine benzyl ester hydrochloride to the compound of Formula III are each about 0.5:1 to 8:1, preferably about 1:1 to 4:1; and the volume ratio of DMF to the compound of Formula III is about 10:1 to 80:1, preferably about 20:1 to 50:1;

[0151] (2) cooling to about -5°C to 20°C (preferably about -5 to 10°C, more preferably about 0-10°C), adding DIPEA, adding ethyl 2-oxime cyanoacetate after about 30 minutes, reacting for about 10 minutes to 60 minutes, then adding EDC*HCl, reacting for about 10 minutes to 60 minutes, then warming to room temperature and reacting for about 10 hours to 72 hours (preferably about 18-36 hours), wherein preferably, the volume ratio of DIPEA to the compound of formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1; and the molar ratio of ethyl 2-oxime cyanoacetate to the compound of formula III and the molar ratio of EDC*HCl to the compound of formula III are each about 0.5:1 to 9:1, preferably about 1:1 to 5:1;

[0152] (3) adding purified water at a volume ratio of about 20:1 to 150:1 (preferably about 35:1 to 75:1) relative to the compound of Formula III, filtering the precipitated solid to obtain a crude compound of Formula II-1;

[0153] (4) passing the crude product from step (3) through a chromatography column to obtain a purified compound of formula II-1;

[0154] (5) reacting the purified compound of formula II-1 with hydrogen at about 0.05 MPa to about 0.8 MPa (preferably about 0.1 MPa to about 0.4 MPa) in an alcohol aqueous solution under Pd / C catalysis for about 1 to 72 h (preferably about 1 to 48 h, more preferably about 6 to 12 h), removing Pd / C after the reaction (for example, filtering through diatomaceous earth), and drying the residue (for example, by concentrating the filtrate to dryness under reduced pressure at about 45° C.) to obtain a crude compound of formula I-1, wherein preferably , the mass ratio of the compound of formula II-1 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; the mass fraction of palladium in the Pd / C is about 5-20%, more preferably about 10% (for example, the Pd / C catalyst contains about 50 w / w% water); the volume ratio of alcohol to water in the alcohol aqueous solution is about 1:1 to about 8:1 (for example, about 4:1 or about 6:1); and the alcohol in the alcohol aqueous solution is selected from methanol, ethanol, isopropanol and any mixture thereof, preferably ethanol;

[0155] (6) Optionally, the crude product obtained from step (5) is first slurried with acetonitrile for about 1 to 4 hours, then slurried with water for about 1 to 4 hours, and then filtered, and the filter cake is dried under reduced pressure at an elevated temperature (e.g., about 45° C.) to obtain the compound of formula I-1 as the final product.

[0156] In a second special embodiment, the present disclosure provides a method as described in the first special embodiment, but L-serine benzyl ester hydrochloride in step (1) is replaced by D-serine ethyl ester hydrochloride; the compound of formula II-1 in steps (3), (4) and (5) is replaced by a compound of formula II-2; and the compound of formula I-1 in steps (5) and (6) is replaced by a compound of formula I-2.

[0157] In a third special embodiment, the present disclosure provides a method as described in the first special embodiment, but L-serine benzyl ester hydrochloride in step (1) is replaced by D-serine benzyl ester hydrochloride; the compound of formula II-1 in steps (3), (4) and (5) is replaced by a compound of formula II-3; and the compound of formula I-1 in steps (5) and (6) is replaced by a compound of formula I-3.

[0158] In a fourth special embodiment, the present disclosure provides a method as described in the first special embodiment, but the step (5) is replaced by the following step (5'):

[0159] (5') reacting the compound of formula II-1 in an alcohol aqueous solution in the presence of formic acid and potassium formate and under the catalysis of Pd / C at about 20°C to 100°C (preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C) for about 1 to 12 hours in a container. After the reaction, Pd / C is removed (for example, by filtering through diatomaceous earth), and the residue is dried (for example, by concentrating the filtrate to dryness under reduced pressure at about 40°C) to obtain a crude compound of formula I-1, wherein preferably, the compound of formula II-1 is reacted with formate (HCOO - ) is about 1:2 to 1:10 (e.g., about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8); the mass ratio of the compound of formula II-1 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; the mass fraction of palladium in the Pd / C is about 5-20%, more preferably about 10% (e.g., the Pd / C catalyst contains about 50w / w% water); the volume ratio of alcohol to water in the alcohol-aqueous solution is about 4:1 to about 8:1; and the alcohol in the alcohol-aqueous solution is selected from methanol, ethanol, isopropanol and any mixture thereof, preferably ethanol.

[0160] In a fifth special embodiment, the present disclosure provides a method as described in the fourth special embodiment, but L-serine benzyl ester hydrochloride in step (1) is replaced by D-serine ethyl ester hydrochloride; the compound of formula II-1 in steps (3), (4) and (5) is replaced by a compound of formula II-2; and the compound of formula I-1 in steps (5) and (6) is replaced by a compound of formula I-2.

[0161] In a sixth special embodiment, the present disclosure provides a method as described in the fourth special embodiment, except that the L-serine benzyl ester hydrochloride in step (1) is replaced by D-serine benzyl ester hydrochloride; the compound of formula II-1 in steps (3), (4) and (5) is replaced by a compound of formula II-3; and the compound of formula I-1 in steps (5) and (6) is replaced by a compound of formula I-3.

[0162] In a seventh particular embodiment, the method according to the present disclosure comprises the steps of:

[0163] (i) performing step (1) of the method described in any one of the first, second, third, fourth, fifth or sixth embodiments above;

[0164] (ii) performing step (2) of the method described in any one of the first, second, third, fourth, fifth or sixth embodiments above accordingly;

[0165] (iii) performing step (3) of the method described in any one of the first, second, third, fourth, fifth or sixth embodiments above accordingly;

[0166] (iv) performing step (5) of the method described in any of the first, second, third, fourth, fifth or sixth embodiments above accordingly;

[0167] (v) passing the product from step (iv) through a chromatography column or high pressure preparative chromatography; and

[0168] (vi) lyophilizing the product obtained from step (v).

[0169] In an eighth particular embodiment, the method according to the present disclosure comprises the steps of:

[0170] (i) performing step (5) of the method described in the third specific embodiment above;

[0171] (ii) purifying the product from step (ii) by passing it through a chromatography column or by high pressure preparative chromatography;

[0172] (iii) Lyophilizing the product obtained from step (ii) to obtain a compound of formula I-3.

[0173] In a third aspect, the present disclosure provides compounds of Formula VI, and compounds of Formula VII as synthetic intermediates of the compounds of Formula VI:

[0174] in:

[0175] X5 and X6 are each independently -(substituted or unsubstituted C 1-10 alkyl)-R 2 ;as well as

[0176] R 2 Selected from: C 3-10 Cycloalkyl, 4-10 membered heterocyclic group, C 6-10 aryl and 5-10 membered heteroaryl.

[0177] In some embodiments, R 2 Selected from: C 3-7 Cycloalkyl, preferably C 5-6 Cycloalkyl; 4-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; phenyl and 5-6 membered heteroaryl. Preferably, R 2 It is a phenyl group.

[0178] In some embodiments, X5 and X6 are each independently -(substituted or unsubstituted C 1-8 alkyl)-R 2 , preferably -(substituted or unsubstituted C 1-6 alkyl)-R 2 , more preferably -(substituted or unsubstituted C 1-4 alkyl)-R 2 , more preferably -(unsubstituted C 1-2 alkyl)-R 2 , more preferably -CH2-R 2 Preferably, X5 and X6 are each independently -CH2-phenyl.

[0179] In a fourth aspect, the present disclosure provides a method for preparing a compound of formula VI as described above, characterized in that the method comprises the following steps:

[0180] i) subjecting the compound of formula VII to a hydrogenation reaction in a solvent in the presence of a suitable metal catalyst to provide the compound of formula VI.

[0181] In some embodiments, in step i), the hydrogenation reaction is carried out under a hydrogen atmosphere. In some such embodiments, the pressure of the hydrogen is from about 0.05 MPa to about 0.8 MPa, preferably from about 0.1 MPa to about 0.4 MPa. In some embodiments, the reaction of the compound of Formula VII with the hydrogen is carried out at a temperature of about 20 to 30 ° C (e.g., about 20 to 25 ° C).

[0182] In some embodiments, the mass ratio of the compound of Formula VII to the metal catalyst is about 1:0.01 to 1:1, preferably about 1:0.2.

[0183] In some embodiments, the metal catalyst is palladium on carbon (Pd / C), wherein preferably, the mass fraction of palladium in the Pd / C is about 5-20%, more preferably about 10% (for example, the Pd / C catalyst contains about 50w / w% water).

[0184] In some embodiments, the volume ratio of the solvent to the compound of Formula VII is about 10:1 to 50:1, preferably about 10:1 to 30:1.

[0185] In some embodiments, the solvent is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane, preferably N,N-dimethylformamide.

[0186] In some embodiments, the hydrogenation reaction is performed for a period of about 5 to 72 hours, such as about 10 to 48 hours, or about 10 to 30 hours.

[0187] In some embodiments, the present disclosure provides a method for preparing a compound of Formula VI or a salt thereof as described above, the method comprising the following steps:

[0188] i) subjecting the compound of formula VII-1 to a hydrogenation reaction in N,N-dimethylformamide in the presence of about 10% Pd / C, under a hydrogen atmosphere of 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa, at a temperature of about 20 to 25° C. for about 5 to 72 hours, preferably about 10 to 30 hours, to provide the compound of formula VI,

[0189] in:

[0190] The mass ratio of the compound of formula VII-1 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; and

[0191] The volume ratio of the N,N-dimethylformamide to the compound of Formula VII-1 is about 10:1 to 50:1, preferably about 10:1 to 30:1.

[0192] In some embodiments, the method for preparing the compound of Formula VI or a salt thereof as described above further comprises the following steps:

[0193] i') subjecting the compound of formula VIII to an amide condensation reaction with the compound of formula IX or a salt thereof (preferably hydrochloride) under an inert atmosphere in a suitable organic solvent in the presence of a suitable organic base, a suitable coupling agent and a suitable condensing agent to provide the compound of formula VII,

[0194] wherein X5 and X6 are each as defined above; and

[0195] ii') optionally, filtering the reaction mixture in step i'), and adding water dropwise to the filtrate to crystallize the compound of formula VII; and

[0196] iii') Optionally, ethyl acetate is added to the isolated compound of formula VII for slurrying, followed by filtration and vacuum drying at elevated temperature (eg, about 50±5° C.) for more than 18 hours.

[0197] In some embodiments, in step i'), the inert atmosphere is under nitrogen protection. In some embodiments, the volume ratio of the organic solvent to the compound of formula IX or its salt is about 10:1 to 80:1, preferably about 20:1 to 50:1. In some embodiments, the molar ratio of the organic base to the compound of formula VIII is about 0.5:1 to 9:1, preferably about 1:1 to about 5:1. In some embodiments, the molar ratio of the condensing agent to the compound of formula VIII is about 0.5:1 to 9:1, preferably about 1:1 to about 5:1. In some embodiments, the molar ratio of the coupling agent to the compound of formula VIII is about 0.5:1 to 9:1, preferably about 1:1 to about 5:1. In some embodiments, the amide condensation reaction is carried out at a temperature of about 10 to 30°C, preferably about 20-30°C. In some embodiments, the amide condensation reaction lasts for about 10 to 72 hours, preferably about 18 to 36 hours. In some embodiments, the organic solvent is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane. In some embodiments, the organic base is diisopropylethylamine (DIPEA), triethylamine, pyridine and / or 4-dimethylaminopyridine (DMAP). In some embodiments, the coupling agent is ethyl 2-oxime cyanoacetate (Oxyma), HOBt, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU) and / or O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU). In some embodiments, the condensing agent is selected from: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCl), HOBt, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU), O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU) and any combination thereof.

[0198] In some embodiments, in step ii'), the volume ratio of the added water to the filtrate is about 20:1 to 150:1, preferably 35:1 to 75:1.

[0199] In some embodiments, in step iii'), the volume ratio of ethyl acetate to the compound of formula VII is about 5:1 to 30:1, preferably about 5:1 to 10:1.

[0200] In other embodiments, the compound of formula VIII is a compound of formula I-3:

[0201] The compound of formula IX is a compound of the following formula:

[0202] The organic solvent is DMF;

[0203] The organic base is diisopropylethylamine (DIPEA);

[0204] The coupling agent is HOBt; and

[0205] The condensing agent is: EDC*HCl.

[0206] In some embodiments, the method for preparing the compound of Formula VI or a salt thereof as described above further comprises the following steps:

[0207] ii) optionally, filtering the reaction mixture in step i), and adding ethyl acetate dropwise to the filtrate to crystallize the compound of formula VI; and

[0208] iii) Optionally, ethyl acetate is added to the isolated compound of formula VI for slurrying, followed by filtration and vacuum drying at an elevated temperature (eg, about 35±5° C.) for more than 12 hours.

[0209] In some such embodiments, in step ii), the volume ratio of the ethyl acetate to the filtrate is about 30:1 to 90:1, preferably about 30:1 to 50:1.

[0210] In some such embodiments, in step iii), the volume ratio of ethyl acetate to the compound of formula VI is about 1:1 to 10:1, preferably about 2:1 to 5:1.

[0211] In a fifth aspect, the present disclosure provides compounds of formula X:

[0212] where R 3 is H, or substituted or unsubstituted C 1-4 Alkyl, preferably H or methyl.

[0213] In some preferred embodiments, R 3 For H.

[0214] In a sixth aspect, the present disclosure provides a method for preparing the compound of formula X, characterized in that the method comprises the following steps:

[0215] xi) providing a solution of a compound of formula A in a solvent;

[0216] x-ii) At elevated temperature, with acid R 3 -COOH acylates the compound of formula A to provide the compound of formula X; and

[0217] x-iii) optionally, isolating and purifying the compound of formula X;

[0218] where R 3 As defined above.

[0219] In some preferred embodiments, R 3 For H.

[0220] In some embodiments, in step xi), the volume ratio of the solvent to the compound of formula A is about 5:1 to 30:1, preferably about 5:1 to 10:1. In some embodiments, the solvent in step xi) is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane, preferably DMF. In some embodiments, the elevated temperature in step x-ii) is about 55 to 75°C, preferably about 70°C. In some embodiments, in step x-ii), the acid R 3 The volume ratio of -COOH to the compound of formula A is about 0.5: 1 to 10: 1, preferably about 1: 1 to 5: 1. In some embodiments, in step x-ii), the reaction time is about 1 to 20 hours, preferably about 1 to 5 hours.

[0221] In some embodiments, step x-iii) comprises:

[0222] 1) concentrating the reaction mixture in step xi) until there is no fraction, and then adding ethyl acetate dropwise to the residue to crystallize the compound of formula X; and

[0223] 2) dissolving the isolated compound of formula X in the solvent used in step xi), then adding ethyl acetate dropwise to crystallize the compound of formula X, followed by filtering and drying in vacuo at an elevated temperature (e.g., about 30±5° C.);

[0224] 3) repeating the recrystallization of steps 1) and 2); and

[0225] 4) The dried compound of formula X obtained in step 3) is slurried with ethyl acetate, filtered, and dried under vacuum at an elevated temperature (eg, about 30±5° C.) for more than 16 hours.

[0226] In some such embodiments, in step 1), the volume ratio of ethyl acetate to the compound of formula A is about 15:1 to 90:1, preferably about 15:1 to 30:1. In some embodiments, in step 2), the volume ratio of the solvent to the compound of formula A is about 0.5:1 to 5:1, preferably about 1:1 to 2:1. In some embodiments, in step 2), the volume ratio of ethyl acetate to the compound of formula A is about 1:1 to 10:1, preferably about 2:1 to 4:1. In some embodiments, in step 4), the volume ratio of ethyl acetate to the compound of formula A is about 1:1 to 10:1, preferably about 2:1 to 5:1.

[0227] In a seventh aspect, the present disclosure provides compounds of Formula XI:

[0228] In an eighth aspect, the present disclosure provides a method for preparing the compound of formula XI, characterized in that the method comprises the following steps:

[0229] xi-i) hydrolyzing the compound of formula A in water in the presence of a base at elevated temperature to provide the compound of formula XI or a salt thereof;

[0230] as well as

[0231] xi-ii) Optionally, isolating and purifying the compound of formula XI or a salt thereof.

[0232] In some embodiments, in step xi-i), the volume ratio of water to the compound of formula A is about 5:1 to 30:1, preferably about 5:1 to 10:1. In some embodiments, in step xi-i), the molar ratio of the base to the compound of formula A is about 1:1 to 5:1, preferably about 2:1 to 5:1. In some embodiments, the base is an inorganic base, preferably potassium hydroxide, sodium hydroxide and mixtures thereof, more preferably potassium hydroxide. In some embodiments, in step xi-i), the elevated temperature is about 50 to 100°C, preferably about 80 to 90°C. In some embodiments, in step xi-i), the hydrolysis reaction is carried out for about 5 to 48 hours, preferably about 10 to 20 hours.

[0233] In some embodiments, step xi-ii) comprises:

[0234] 1) adding silica gel to the reaction mixture in step xi-ii) to concentrate the reaction mixture and prepare a silica gel powder, and subjecting the silica gel powder to silica gel column chromatography to obtain a solid crude product of the compound of formula XI or a salt thereof;

[0235] 2) The crude solid obtained from step xii-1) is slurried with methanol, filtered, and then vacuum dried at an elevated temperature (eg, about 50±5° C.) for more than 16 hours to obtain a purified compound of formula XI or a salt thereof.

[0236] In some such embodiments, in step 1), the mass ratio of the added silica gel to the compound of formula A is about 0.5:1 to 2:1, preferably about 2:1. In some embodiments, in step 1), the silica gel chromatography column is gradient eluted with EA / MeOH (1% trifluoroacetic acid (TFA)) as the eluent, wherein the volume ratio of EA:MeOH (1% TFA) is about 50:1 to 1:1, preferably about 30:1 to 10:1. In some embodiments, in step 2), the volume ratio of methanol to the compound of formula A is about 1:1 to 10:1, preferably about 2:1 to 5:1.

[0237] use

[0238] In a ninth aspect, the present disclosure provides a method for quality control of a drug substance or a formulation of MB-102, comprising determining the content of the compound of formula I (including the compound of formula I-1, the compound of formula I-2, the compound of formula I-3, and any combination thereof), the compound of formula VI, the compound of formula X, and / or the compound of formula XI in the drug substance or formulation, and optionally comparing the content with a specified content of each of the compounds.

[0239] In some embodiments, for the compound of formula I, the specified content is: based on the total weight of the drug substance, no more than about 0.9%, preferably no more than about 0.6%, and more preferably no more than about 0.45%.

[0240] In some embodiments, for the compound of formula I-1, the specified content is: no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15%, based on the total weight of the drug substance; or, in some embodiments, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20%, based on the total weight of the formulation.

[0241] In some embodiments, for the compound of formula I-2, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, and more preferably no more than about 0.15%.

[0242] In some embodiments, for the compound of formula I-3, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, and more preferably no more than about 0.15%.

[0243] In some embodiments, for the compound of formula VI, the specified content is no more than about 0.3%, preferably no more than about 0.25%, and more preferably no more than about 0.15%, based on the total weight of the drug substance.

[0244] In some embodiments, for the compound of formula X, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, and more preferably no more than about 0.15%.

[0245] In some embodiments, for the compound of formula XI, the specified content is: based on the total weight of the drug substance, no more than about 0.2%, preferably no more than about 0.15%, more preferably no more than about 0.10%; or, in some embodiments, based on the total weight of the formulation, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20%.

[0246] Methods for performing the above-mentioned assay are known in the art, such as HPLC method.

[0247] In a tenth aspect, the present disclosure provides the use of the compound of formula I (including the compound of formula I-1, the compound of formula I-2, the compound of formula I-3, and any combination thereof), the compound of formula VI, the compound of formula X, and / or the compound of formula XI as a standard or reference substance for quality control of the bulk drug or preparation of MB-102.

[0248] In another aspect, the present disclosure provides a 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine API having a low content of related compounds, characterized in that the related compounds are compounds of Formula I, preferably compounds of Formula I-1, compounds of Formula I-2, compounds of Formula I-3, compounds of Formula VI, compounds of Formula X, and / or compounds of Formula XI; and

[0249] The drug substance has one or more of the following contents:

[0250] Based on the total weight of the drug substance, no more than about 0.9%, preferably no more than about 0.6%, more preferably no more than about 0.45% of the compound of formula I,

[0251] Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula I-1,

[0252] Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula I-2,

[0253] Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula I-3,

[0254] Based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula VI,

[0255] Based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula X,

[0256] Based on the total weight of the drug substance, no more than about 0.2%, preferably no more than about 0.15%, more preferably no more than about 0.10% of the compound of formula XI.

[0257] In another aspect, the present disclosure provides a 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine formulation having a low content of related compounds, characterized in that the related compounds are selected from the compound of formula I-1 and the compound of formula XI; and

[0258] The formulation has one or both of the following contents:

[0259] Based on the total weight of the formulation, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20% of the compound of formula I-1;

[0260] Based on the total weight of the formulation, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20% of the compound of formula XI.

[0261] In some embodiments, the preparation described above is a solution for injection or a lyophilized powder for injection, preferably a solution for injection.

[0262] In an eleventh aspect, the present disclosure provides use of a compound of formula II as described above as a synthetic intermediate for a compound of formula I as described above.

[0263] In some embodiments, the present disclosure provides the use of a compound of formula II-1 as described above as a synthetic intermediate for a compound of formula I-1 as described above.

[0264] In some embodiments, the present disclosure provides the use of a compound of formula II-2 as described above as a synthetic intermediate for a compound of formula I-2 as described above.

[0265] In some embodiments, the present disclosure provides the use of a compound of formula II-3 as described above as a synthetic intermediate for a compound of formula I-3 as described above.

[0266] In a twelfth aspect, the present disclosure provides use of a compound of formula VII as described above as a synthetic intermediate for a compound of formula VI as described above.

[0267] In a thirteenth aspect, the present disclosure provides use of a compound of formula A as described above as a synthetic intermediate for compounds of formulas X and XI as described above. Beneficial effects

[0268] The present inventors provide compounds of Formula I, as described above, particularly compounds of Formula I-1, I-2, and I-3, as well as compounds of Formula VI, Formula X, and Formula XI, and methods for preparing the same, which have the advantages of ease of operation, high purity, and high yield. The present inventors have proposed for the first time the use of these compounds as standard substances or reference substances for quality control of the MB-102 drug substance or formulation, and have also provided corresponding quality control methods, which are of great significance for quality control during the synthesis and storage of MB-102.

[0269] Example

[0270] The present invention is further described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0271] The experimental methods in the examples of the present invention for which specific conditions are not specified are generally conventional conditions or conditions recommended by the raw material or product manufacturers; reagents for which the sources are not specified are generally conventional reagents available through commercial channels or can be prepared from known reagents by conventional methods.

[0272] Example 1-1: Preparation of Compound of Formula I-1

[0273] The compound of formula I-1 is prepared by a process comprising the following steps:

[0274] (1) In a three-necked jacketed flask, 3,6-diaminopyrazine-2,5-diacid (Compound III) (10 g, 1 eq), D-serine benzyl ester hydrochloride (12.86 g, 1.1 eq) and L-serine benzyl ester hydrochloride (12.86 g, 1.1 eq) were added. DMF (35 times the volume of Compound III) was added and stirred evenly.

[0275] (2) After cooling to 0°C, DIPEA (14.34 g, 2.2 eq) was added. After 30 min, ethyl 2-oximecyanoacetate (15.77 g, 2.2 eq) was added. After 20 min, EDC*HCl (21.28 g, 2.2 eq) was added. The mixture was reacted for another 40 min, then warmed to room temperature and reacted for 18 h.

[0276] (3) Purified water (70 times the volume of compound III) was added, and the solid precipitated and then filtered.

[0277] (4) Through the chromatography column (Xinweier C184806C sand plate chromatography column, ), and the eluate was concentrated under reduced pressure at 33°C to obtain pure compound of formula II-1 (4.8 g).

[0278] (5) Add the compound of formula II-1 (2 g, 1 eq) to a reaction flask, followed by ethanol (32 ml, 16 volumes), formic acid-potassium hydroxide aqueous solution (HCOOH: 1.0 g, 6 eq; KOH: 0.4 g, 6 eq; purified water: 8 ml, 4 volumes), and Pd / C (0.2 g, 0.1 volumes). React at 60°C for 2 h. After completion of the reaction, monitor the reaction by TLC. Remove the Pd / C by filtration through celite while hot. Wash the filter cake with ethanol aqueous solution (36% H2O, v / v) until the filtrate is light yellow. Combine the filtrates. Add 1 mol / L hydrochloric acid (7 ml), stir for 15 min, and concentrate to dryness under reduced pressure.

[0279] (6) The obtained solid was slurried with 20 ml of acetonitrile for 2 h, filtered, and then slurried with water for 2 h. After filtration, the filter cake was dried under reduced pressure at 40°C for 12 h to obtain a solid compound of formula II-1 (0.79 g): the overall molar yield was 10% (based on the compound of formula III) and the purity was 98.39%.

[0280] The chemical structure of the obtained product was determined to be as shown in Formula I-1 by H NMR analysis (Bruker AVANCE III HD). The obtained H NMR spectrum is shown in Table 1:

[0281] Table 1

[0282] Example 1-2

[0283] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to the compound of formula III is 0.5:1.0; the molar ratio of L-serine benzyl ester hydrochloride to the compound of formula III is 0.5:1.0; and the volume ratio of DMF to the compound of formula III is 10:1.

[0284] Examples 1-3

[0285] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to the compound of formula III is 1.0:1.0; the molar ratio of L-serine benzyl ester hydrochloride to the compound of formula III is 1.0:1.0; and the volume ratio of DMF to the compound of formula III is 20:1.

[0286] Examples 1-4

[0287] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to the compound of formula III is 4.0:1.0; the molar ratio of L-serine benzyl ester hydrochloride to the compound of formula III is 4.0:1.0; and the volume ratio of DMF to the compound of formula III is 50:1.

[0288] Examples 1-5

[0289] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to the compound of formula III is 8.0:1.0; the molar ratio of L-serine benzyl ester hydrochloride to the compound of formula III is 8.0:1.0; and the volume ratio of DMF to the compound of formula III is 80:1.

[0290] Examples 1-6

[0291] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (2), the molar ratio of DIPEA to the compound of formula III is 0.5:1.0; the molar ratio of EDC*HCl to the compound of formula III is 0.5:1.0; the temperature is lowered to -5°C; ethyl 2-oximecyanoacetate is added and the reaction is carried out for 10 minutes; EDC*HCl is added and the reaction is continued for another 10 minutes; and the reaction time at room temperature is 10 hours.

[0292] Examples 1-7

[0293] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (2), the molar ratio of DIPEA to the compound of formula III is 1.0:1.0; the molar ratio of EDC*HCl to the compound of formula III is 1.0:1.0; the temperature is lowered to 10°C; ethyl 2-oximecyanoacetate is added and the reaction is carried out for 60 minutes; EDC*HCl is added and the reaction is continued for another 60 minutes; and the reaction time at room temperature is 36 hours.

[0294] Examples 1-8

[0295] This example provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (2), the molar ratio of DIPEA to the compound of formula III is 5.0:1.0; the molar ratio of EDC*HCl to the compound of formula III is 5.0:1.0; the temperature is lowered to 15° C.; and the reaction time at room temperature is 72 h.

[0296] Examples 1-9

[0297] This example provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (2), the molar ratio of DIPEA to the compound of formula III is 9.0:1.0; the molar ratio of EDC*HCl to the compound of formula III is 9.0:1.0; and the temperature is lowered to 20°C.

[0298] Examples 1-10

[0299] This example provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that in step (3), the volume ratio of purified water to the compound of formula III is 20:1.

[0300] Examples 1-11

[0301] This example provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that in step (3), the volume ratio of purified water to the compound of formula III is 35:1.

[0302] Examples 1-12

[0303] This example provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that in step (3), the volume ratio of purified water to the compound of formula III is 75:1.

[0304] Examples 1-13

[0305] This example provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that in step (3), the volume ratio of purified water to the compound of formula III is 150:1.

[0306] Examples 1-14

[0307] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (5), the alcohol in the alcohol-water solution is selected from methanol, and the volume ratio of methanol to water is 1:1; the mass ratio of the compound of formula II-1 to Pd / C is 1:0.01; the mass fraction of palladium in Pd / C is 5%; and the reaction time is 1 h.

[0308] Examples 1-15

[0309] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (5), the alcohol in the alcohol-water solution is selected from ethanol, and the volume ratio of ethanol to water is 3:1; the mass ratio of the compound of formula II-1 to Pd / C is 1:0.2; the mass fraction of palladium in Pd / C is 10% (50% water); and the reaction time is 6 h.

[0310] Examples 1-16

[0311] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (5), the alcohol in the alcohol aqueous solution is selected from isopropanol, and the volume ratio of isopropanol to water is 5:1; the mass ratio of the compound of formula II-1 to Pd / C is 1:1; the mass fraction of palladium in Pd / C is 20%; and the reaction time is 12 h.

[0312] Examples 1-17

[0313] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of Example 1-1 in that: in step (5), the alcohol in the alcohol-water solution is selected from methanol, and the volume ratio of methanol to water is 8:1; the mass ratio of the compound of formula II-1 to Pd / C is 1:1; the mass fraction of palladium in Pd / C is 20%; and the reaction time is 48h.

[0314] Examples 1-18

[0315] This embodiment provides a method for preparing a compound of formula I-1, which differs from the method of embodiment 1-1 in that: in step (6), the crude product is slurried with acetonitrile for 1 hour and water for 1 hour, respectively, and then filtered, and the filter cake is placed at 45° C. and dried under reduced pressure to obtain the compound of formula I-1.

[0316] Examples 1-19

[0317] This example provides a method for preparing a compound of formula I-1, which differs from Example 1-1 in that: in step (6), the crude product is first slurried with acetonitrile for 4 hours, then slurried with water for 4 hours, filtered, and the filter cake is dried under reduced pressure at 45°C to obtain a compound of formula II-1.

[0318] The products of Examples 1-2 to 1-19 were all tested with a combined molar yield of approximately 10% (based on the compound of Formula III) and a purity of approximately 98%. The results indicate that the compound of Formula I-1 prepared under the described conditions has high purity and high yield.

[0319] Example 2-A: Preparation of Compounds of Formula I-2

[0320] The compound of formula I-2 is prepared by a process comprising the following steps:

[0321] (1) Compound III (10 g, 1 eq), D-serine benzyl ester hydrochloride (12.86 g, 1.1 eq) and D-serine ethyl ester hydrochloride (9.38 g, 1.1 eq) were added to a three-necked jacketed flask, and DMF (35 times the volume of compound III) was added and stirred evenly.

[0322] (2) After cooling to 0°C, DIPEA (14.34 g, 2.2 eq) was added. After 30 min, ethyl 2-oximecyanoacetate (15.77 g, 2.2 eq) was added. After 20 min, EDC*HCl (21.28 g, 2.2 eq) was added. The mixture was reacted for another 40 min, then warmed to room temperature and reacted for 18 h.

[0323] (3) Purified water (70 times the volume of compound III) was added, and the solid precipitated and then filtered to obtain a crude product (10 g) of the compound of formula II-2.

[0324] (4) Add the compound of formula II-2 (5 g) to a high-pressure hydrogenation autoclave and dissolve it in 175 ml of ethanol (36% H2O, v / v). Add Pd / C (0.5 g, 0.1 times the volume). Replace the atmosphere with nitrogen three times, then replace it with hydrogen three times (0.2 MPa), and react for 4 to 12 hours. After the reaction, remove the Pd / C by filtration through diatomaceous earth, and the filtrate is dried by spin drying or concentrated to dryness under reduced pressure.

[0325] (5) The obtained solid was prepared as follows: chromatographic column: self-assembled spring column (θ30*250mm, filled with Huapu C18 particles); flow rate: 30ml / min; column temperature: 30℃; gradient as follows:

[0326] Time (min) 0-12-29-29.1-33

[0327] A(%)20-85-30-90-90

[0328] B(%)80-15-70-10-10

[0329] Mobile phase A: 0.1% TFA·water

[0330] Mobile phase B: acetonitrile (MeCN)

[0331] The effluent from 19 min to 22 min was collected.

[0332] (6) The MeCN in the effluent was removed by rotary evaporation and then freeze-dried.

[0333] 1 g of the compound of formula I-2 was obtained with a purity of 98.5% and a comprehensive molar yield of 11% (based on the compound of formula III).

[0334] The chemical structure of the obtained product was determined to be as shown in Formula I-2 by H NMR analysis (Bruker AVANCE III HD). The obtained H NMR spectrum is shown in Table 2:

[0335] Table 2

[0336] Example 2-B: Preparation of Compounds of Formula I-2

[0337] The compound of formula I-2 is prepared by a process comprising the following steps:

[0338] (1) Compound III (10 g, 1 eq), D-serine benzyl ester hydrochloride (12.86 g, 1.1 eq) and D-serine ethyl ester hydrochloride (9.38 g, 1.1 eq) were added to a three-necked jacketed flask, and DMF (35 times the volume of compound III) was added and stirred evenly.

[0339] (2) After cooling to 0°C, DIPEA (14.34 g, 2.2 eq) was added. After 30 min, ethyl 2-oximecyanoacetate (15.77 g, 2.2 eq) was added. After 20 min, EDC*HCl (21.28 g, 2.2 eq) was added. The mixture was reacted for another 40 min, then warmed to room temperature and reacted for 18 h.

[0340] (3) Purified water (70 times the volume of compound III) was added, and the solid precipitated and then filtered to obtain a crude product (10 g) of the compound of formula II-2.

[0341] Through the chromatography column (Hinwell C184806C sand plate chromatography column, ) or high pressure preparative chromatography (LAB-ZB-05, domestic Aonuo medium and high pressure equipment) to obtain pure compound of formula II-2.

[0342] (4) Add the compound of formula II-2 (5 g) to a high-pressure hydrogenation autoclave and dissolve it in 175 ml of ethanol (36% H2O, v / v). Then add Pd / C (0.5 g, 0.1 times the volume). After nitrogen displacement three times, the atmosphere was replaced with hydrogen three times (0.2 MPa) and the reaction was continued for 4 to 12 hours. After the reaction, the Pd / C was removed by filtration through diatomaceous earth, and the filtrate was spin-dried or concentrated to dryness under reduced pressure.

[0343] (5) The obtained crude product was first slurried with acetonitrile for 1 hour and then slurried with water for 1 hour. After filtering, the filter cake was dried under reduced pressure at 45°C to obtain the compound of formula I-2.

[0344] Example 2-1

[0345] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to compound III is 0.5:1.0; the molar ratio of D-serine ethyl ester hydrochloride to compound III is 0.5:1.0; and the volume ratio of DMF to compound III is 10:1.

[0346] Example 2-2

[0347] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to compound III is 1.0:1.0; the molar ratio of D-serine ethyl ester hydrochloride to compound III is 1.0:1.0; and the volume ratio of DMF to compound III is 20:1.

[0348] Example 2-3

[0349] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to compound III is 4.0:1.0; the molar ratio of D-serine ethyl ester hydrochloride to compound III is 4.0:1.0; and the volume ratio of DMF to compound III is 50:1.

[0350] Examples 2-4

[0351] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (1), the molar ratio of D-serine benzyl ester hydrochloride to compound III is 8.0:1.0; the molar ratio of D-serine ethyl ester hydrochloride to compound III is 8.0:1.0; and the volume ratio of DMF to compound III is 80:1.

[0352] Examples 2-5

[0353] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (2), the molar ratio of DIPEA to compound III is 0.5:1.0; the molar ratio of EDC*HCl to compound III is 0.5:1.0; the temperature is lowered to -5°C; ethyl 2-oximecyanoacetate is added and the reaction is carried out for 10 minutes; EDC*HCl is added and the reaction is continued for another 10 minutes; and the reaction time at room temperature is 10 hours.

[0354] Examples 2-6

[0355] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (2), the molar ratio of DIPEA to compound III is 1.0:1.0; the molar ratio of EDC*HCl to compound III is 1.0:1.0; the temperature is lowered to 10°C; ethyl 2-oximecyanoacetate is added and the reaction is carried out for 60 minutes; EDC*HCl is added and the reaction is continued for another 60 minutes; and the reaction time at room temperature is 36 hours.

[0356] Examples 2-7

[0357] This example provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (2), the molar ratio of DIPEA to compound III is 5.0:1.0; the molar ratio of EDC*HCl to compound III is 5.0:1.0; the temperature is lowered to 15°C; and the reaction time at room temperature is 72 h.

[0358] Examples 2-8

[0359] This example provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (2), the molar ratio of DIPEA to compound III is 9.0:1.0; the molar ratio of EDC*HCl to compound III is 9.0:1.0; and the temperature is lowered to 20°C.

[0360] Examples 2-9

[0361] This embodiment provides a method for preparing a compound of formula I-2, which differs from embodiments 2-A and 2-B in that in step (3), the volume ratio of purified water to the compound of formula III is 20:1.

[0362] Example 2-10

[0363] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that in step (3), the volume ratio of purified water to compound III is 35:1.

[0364] Example 2-11

[0365] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that in step (3), the volume ratio of purified water to compound III is 75:1.

[0366] Example 2-12

[0367] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that in step (3), the volume ratio of purified water to compound III is 150:1.

[0368] Example 2-13

[0369] This embodiment provides a method for preparing a compound of formula I-2, which differs from embodiments 2-A and 2-B in that: in step (4), the alcohol in the alcohol-water solution is selected from methanol, and the volume ratio of methanol to water is 1:1; the mass ratio of the compound of formula II-2 to Pd / C is 1:0.01; the mass fraction of palladium in Pd / C is 5%; the pressure of hydrogen is 0.05 MPa; and the reaction time is 1 h.

[0370] Examples 2-14

[0371] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (4), the alcohol in the alcohol-water solution is selected from ethanol, and the volume ratio of ethanol to water is 3:1; the mass ratio of the compound of formula II-2 to Pd / C is 1:0.2; the mass fraction of palladium in Pd / C is 10% (50% water), and the hydrogen pressure is 0.1 MPa; and the reaction time is 6 h.

[0372] Example 2-15

[0373] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (4), the alcohol in the alcohol-water solution is selected from isopropanol, and the volume ratio of isopropanol to water is 5:1; the mass ratio of the compound of formula II-2 to Pd / C is 1:1; the mass fraction of palladium in Pd / C is 20%; the pressure of hydrogen is 0.4 MPa; and the reaction time is 12 h.

[0374] Example 2-16

[0375] This embodiment provides a method for preparing a compound of formula I-2, which differs from Examples 2-A and 2-B in that: in step (4), the alcohol in the alcohol-water solution is selected from methanol, and the volume ratio of methanol to water is 8:1; the mass ratio of the compound of formula II-2 to Pd / C is 1:1; the mass fraction of palladium in Pd / C is 20%; the pressure of hydrogen is 0.8 MPa; and the reaction time is 48 h.

[0376] Example 2-17

[0377] This example provides a method for preparing a compound of formula I-2, which differs from Example 2-B in that: in step (5), the crude product is first slurried with acetonitrile for 4 hours and then slurried with water for 4 hours. After filtering, the filter cake is dried under reduced pressure at 45°C to obtain a compound of formula I-2.

[0378] The combined molar yield of the products of Examples 2-B and 2-1 to 2-17 was about 11% (based on the compound of Formula III) and the purity was about 98%.

[0379] The results show that the compound of formula I-2 prepared by the reaction under the conditions has high purity and high yield.

[0380] Example 3-1: Preparation of the compound of formula I-3

[0381] The compound of formula I-3 is prepared by a process comprising the following steps:

[0382] (1) Add the compound of formula II-3 (5 g) to a high-pressure hydrogenation autoclave and dissolve it in 175 ml of ethanol (36% H2O, v / v). Add Pd / C (0.5 g, 0.1 volume). Replace the atmosphere with nitrogen three times, then replace it with hydrogen three times (0.2 MPa), and react for 4 to 12 hours. After the reaction, remove the Pd / C by filtration through diatomaceous earth, and spin-dry the filtrate.

[0383] (2) The obtained solid was prepared as follows: chromatographic column: self-assembled spring column (θ30*250mm, filled with Huapu C18 particles); flow rate: 30ml / min; column temperature: 30℃; gradient as follows:

[0384] Time(min)0-20-21-25-26-28

[0385] A(%)70-70-10-10-70-70

[0386] B(%)30-30-90-90-30-30

[0387] Mobile phase A: 0.1% TFA·water

[0388] Mobile phase B: acetonitrile

[0389] The effluent from 13 min to 19 min was collected.

[0390] (3) The MeCN in the effluent was removed by rotary evaporation and then freeze-dried.

[0391] 1 g of the compound of formula I-3 was obtained with a purity of 98.7% and a comprehensive molar yield of 29% (based on the compound of formula II-3).

[0392] The chemical structure of the obtained product was determined to be as shown in Formula I-3 by H NMR analysis (Bruker AVANCE III HD). The obtained H NMR spectrum is shown in Table 3:

[0393] Table 3

[0394] Example 3-2

[0395] This embodiment provides a method for preparing a compound of formula I-3, which differs from Example 3-1 in that: in step (1), the mass ratio of the compound of formula II-3 to Pd / C is 1:0.01, and the mass fraction of palladium in Pd / C is 5%; the alcohol in the alcohol-water solution is selected from methanol, and the volume ratio of methanol to water is 1:1; the pressure of hydrogen is 0.05Mp; and the reaction time is 1h.

[0396] Example 3-3

[0397] This embodiment provides a method for preparing a compound of formula I-3, which differs from Example 3-1 in that: in step (1), the mass ratio of the compound of formula II-3 to Pd / C is 1:1, and the mass fraction of palladium in Pd / C is 10% (50% water); the alcohol in the alcohol-water solution is selected from isopropanol, and the volume ratio of isopropanol to water is 3:1; the hydrogen pressure is 0.1 MPa; and the reaction time is 6 h.

[0398] Examples 3-4

[0399] This embodiment provides a method for preparing a compound of formula I-3, which differs from Example 3-1 in that: in step (1), the mass ratio of the compound of formula II-3 to Pd / C is 1:0.1, and the mass fraction of palladium in Pd / C is 20%; the alcohol in the alcohol-water solution is selected from methanol, and the volume ratio of methanol to water is 5:1; the pressure of hydrogen is 0.4 MPa; and the reaction time is 12 h.

[0400] Examples 3-5

[0401] This embodiment provides a method for preparing a compound of formula I-3, which differs from Example 3-1 in that: in step (1), the mass ratio of the compound of formula II-3 to Pd / C is 1:1, and the mass fraction of palladium in Pd / C is 10% (50% water); the alcohol in the alcohol-water solution is selected from methanol; the volume ratio of methanol to water is 8:1; the pressure of hydrogen is 0.8 MPa; and the reaction time is 48 h.

[0402] The combined molar yield of the products of Examples 3-2 to 3-5 was about 29% (based on the compound of Formula II-3) and the purity was about 98%. The results show that the compound of Formula I-3 prepared under the above conditions has high purity and high yield.

[0403] Example 4: Preparation of the compound of formula VI

[0404] To a three-necked jacketed flask, add the purified compound of Formula II-3 (40.0 g, 1.0 eq) and ethyl acetate / ethanol (952 mL / 952 mL, 23.8 vol / 23.8 vol). Stir and dissolve, then add 10% Pd / C (4.0 g, 10 wt%). Seal the reactor and replace the atmosphere with nitrogen. After replacement, replace the atmosphere with hydrogen three times. Maintain the reaction temperature at 25±5°C and the hydrogen pressure at 0.1-0.3 MPa for 2 hours. Filter and rinse the filter cake with ethanol. Combine the filtrates and concentrate until no significant fraction remains. Then, add silica gel (40 g) to prepare silica gel powder. The crude solid powder is separated by column chromatography to obtain the compound of Formula I-3 (4.6 g) as a solid (eluted with EA, EA / MeOH (1% TFA) (50 / 1), and EA / MeOH (1% TFA) (30 / 1).

[0405] Under nitrogen, add DMF (147.2 mL, 32.0 vol) to a three-necked flask. Add D-serine benzyl ester hydrochloride (2.3 g, 1.0 eq) with stirring and stir for at least 15 minutes while maintaining the internal temperature at 0-10°C. Add N,N-diisopropylethylamine (1.29 g, 1.0 eq) dropwise, maintaining the internal temperature below 10°C. After the addition is complete, maintain the temperature at 0-10°C and stir for 5-15 minutes. Subsequently, raise the internal temperature to 20-30°C and add the compound of Formula I-3 (4.6 g, 1.0 eq), 1-hydroxybenzotriazole monohydrate (1.52 g, 1.0 eq), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.91 g, 1.0 eq) in sequence. After the additions are complete, stir the reaction at 20-30°C for 4 hours. The filtrate was filtered, and purified water (294.4 g, 64.0 vol) was added dropwise to the filtrate at 10-20°C to induce crystallization. The filter cake was rinsed with water and then slurried in ethyl acetate (46 mL, 10.0 vol). The filtrate was filtered, and the filter cake was dried under vacuum at 50°C for 22 hours to obtain 2.6 g of crude benzyl (3,6-diamino-5-(((S)-1-(((S)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl)amino)-3-hydroxy-1-oxopropan-2-yl)carbamoyl)pyrazine-2-carbonyl)-L-serine ester.

[0406] Crude benzyl (3,6-diamino-5-(((S)-1-(((S)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-yl)amino)-3-hydroxy-1-oxopropan-2-yl)carbamoyl)pyrazine-2-carbonyl)-L-serine ester (4.5 g, 1.0 eq), 10% Pd / C (0.9 g, 20 wt%), and DMF (45 mL, 10.0 vol) were added to a hydrogenation reactor. The reactor was sealed and the atmosphere was purged with nitrogen. After purging, the atmosphere was purged with hydrogen three times, maintaining the reaction temperature at 25±5°C and the hydrogen pressure at 0.1-0.3 MPa. The reaction was continued for 2 hours. The reaction mixture was filtered, and the filter cake was rinsed with DMF. Ethyl acetate (135 mL, 30.0 vol) was added dropwise to the filtrate to induce crystallization. The filtered cake was slurried in ethyl acetate (22.5 mL, 5.0 vol), then filtered and dried under vacuum at 35°C for 24 hours to obtain the compound of Formula VI (2.1 g) as a solid. The overall molar yield was 3.7% (based on the compound of Formula II-3) with a purity of 92.7%.

[0407] Table 4. H NMR spectra of compounds of formula VI

[0408] Example 5: Preparation of (2R,2'R)-2,2'-((3-amino-6-formamidopyrazine-2,5-dicarbonyl)bis(azadiyl))bis(3-hydroxypropionic acid)

[0409] MB-102 (15 g, 1.0 eq) and DMF (150 mL, 10.0 vol) were added to a three-necked jacketed flask. After stirring to dissolve, the temperature was raised to 70°C. Formic acid (37.5 mL, 2.5 vol) was added and the mixture was kept at 70°C for 1 hour. Most of the DMF was removed by concentration, and ethyl acetate (300 mL, 20.0 vol) was added dropwise to crystallize. Filter the mixture to obtain the crude product of Formula X. This crude product was added to DMF (15 mL, 1.0 vol) and dissolved at room temperature. Ethyl acetate (30 mL, 2.0 vol) was then slowly added dropwise to crystallize. The mixture was slurried, filtered, and dried under vacuum at 30°C. The recrystallization process was repeated. After recrystallization, the product was filtered and the filter cake was slurried in ethyl acetate (30 mL, 2.0 vol), filtered again, and dried under vacuum at 35°C for 16 hours to obtain (2R,2'R)-2,2'-((3-amino-6-formamidopyrazine-2,5-dicarbonyl)bis(azadiyl))bis(3-hydroxypropionic acid) (3.0 g) as a solid. The overall molar yield was 18.6% (based on MB-102), and the purity was 83.4%.

[0410] Table 5. H NMR Spectrum of (2R,2'R)-2,2'-((3-amino-6-formamidopyrazine-2,5-dicarbonyl)bis(azadiyl))bis(3-hydroxypropionic acid)

[0411] Example 6: Preparation of Compounds of Formula XI

[0412] Purified water (300 mL, 10.0 vol), potassium hydroxide (11.3 g, 2.5 eq), and MB-102 (30 g, 1.0 eq) were added to a three-necked jacketed flask. The reaction solution was heated to 80°C and stirred for 16 hours, then cooled to 25°C. Silica gel (60 g) was added to the reaction solution and concentrated to a silica gel powder. The powder was then purified by silica gel column chromatography to obtain a crude solution of the compound of Formula XI. The solution was concentrated to precipitate a solid, which was then slurried in methanol (150 mL, 5.0 vol) at 50°C, cooled, and filtered. The wet product was dried under vacuum at 35°C to obtain the compound of Formula XI (1.3 g) as a solid. The overall molar yield was 11.3% (based on MB-102), with a purity of 99.3%.

[0413] Table 6. H NMR spectra of compounds of formula XI

[0414] Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0415] 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

1. A method for preparing a compound of formula I or a salt thereof, characterized in that The method comprises the following steps: a) subjecting the compound of formula II to a hydrogenation reaction with hydrogen or a hydrogen-borrowing agent in a solvent in the presence of a suitable metal catalyst to provide the compound of formula I, in: X1 is selected from X2 is selected from H, substituted or unsubstituted C 1-10 Alkyl, and -(substituted or unsubstituted C 1-10 Alkyl)-R 1 ; X3 and X4 are each independently selected from substituted or unsubstituted C 1-10 Alkyl, and -(substituted or unsubstituted C 1-10 Alkyl)-R 1 ;as well as R 1 Selected from: C 3-10 Cycloalkyl, 4-10 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl.

2. The method according to claim 1, characterized in that: R 1 Selected from: C 3-7 Cycloalkyl, preferably C 5-6 Cycloalkyl; 4-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; phenyl and 5-6 membered heteroaryl; preferably phenyl.

3. The method according to claim 1 or 2, characterized in that: X1 is 4. The method according to any one of claims 1 to 3, characterized in that X2 is selected from: H, substituted or unsubstituted C 1-8 Alkyl, and -(substituted or unsubstituted C 1-8 Alkyl)-R 1 ; Preferably H, substituted or unsubstituted C 1-6 Alkyl, and -(substituted or unsubstituted C 1-6 Alkyl)-R 1 ; More preferably, H, substituted or unsubstituted C 1-4 Alkyl, and -(substituted or unsubstituted C 1-4 Alkyl)-R 1 ; More preferably H, unsubstituted C 1-2 Alkyl, and -(unsubstituted C 1-2 Alkyl)-R 1 ; More preferably H, ethyl and -CH2-R 1 ; More preferred are H, ethyl and -CH2-phenyl.

5. The method according to claim 1 or 2, characterized in that: X1 is 6. The method according to claim 1, 2 or 5, characterized in that: X3 is selected from: Substituted or unsubstituted C 1-8 Alkyl, and -(substituted or unsubstituted C 1-8 Alkyl)-R 1 ; Preferably substituted or unsubstituted C 1-6 Alkyl, and -(substituted or unsubstituted C 1-6 Alkyl)-R 1 ; More preferably, substituted or unsubstituted C 1-4 Alkyl, and -(substituted or unsubstituted C 1-4 Alkyl)-R 1 ; More preferably, unsubstituted C 1-2 Alkyl, and -(unsubstituted C 1-2 Alkyl)-R 1 ; More preferably, ethyl and -CH2-R 1 ; More preferred are ethyl and -CH2-phenyl.

7. The method according to any one of claims 1 to 6, characterized in that X4 is selected from: Substituted or unsubstituted C 1-8 Alkyl, and -(substituted or unsubstituted C 1-8 Alkyl)-R 1 ; Preferably substituted or unsubstituted C 1-6 Alkyl, and -(substituted or unsubstituted C 1-6 Alkyl)-R 1 ; More preferably, substituted or unsubstituted C 1-4 Alkyl, and -(substituted or unsubstituted C 1-4 Alkyl)-R 1 ; More preferably, unsubstituted C 1-2 Alkyl, and -(unsubstituted C 1-2 Alkyl)-R 1 ; More preferably, ethyl and -CH2-R 1 ; More preferably ethyl and -CH2-phenyl; More preferred is -CH2-phenyl.

8. The method according to any one of claims 1 to 7, characterized in that (1) In the step a), the compound of formula II is subjected to the hydrogenation reaction with the hydrogen-borrowing reagent, Preferably, the molar ratio of the compound of formula II to the hydrogen borrowing reagent is about 1:2 to 1:20, preferably about 1:2 to 1:10, for example about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8; and / or Preferably, the hydrogen borrowing agent is selected from a combination of formic acid and an alkali metal formate, cyclohexadiene and isopropanol, more preferably a combination of formic acid and an alkali metal formate, wherein preferably, the molar ratio of the formic acid to the alkali metal formate is about 1:6 to 6:1, more preferably about 1:1 to 5:1; and / or Preferably, the alkali metal formate is sodium formate or potassium formate, preferably potassium formate; and / or Preferably, the reaction of the compound of formula II with the hydrogen borrowing agent is carried out at a temperature of about 20°C to 100°C, more preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C; or In the step a), the compound of formula II is subjected to the hydrogenation reaction with the hydrogen gas. Preferably, the pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, more preferably about 0.1 MPa to about 0.4 MPa; and / or Preferably, the reaction of the compound of formula II with the hydrogen is carried out at a temperature of about 15 to 25°C; and / or (2) the volume ratio of the solvent to the compound of formula II is about 10:1 to 80:1, preferably about 20:1 to 50:1; and / or (3) The solvent is an alcohol aqueous solution, Preferably, the volume ratio of alcohol to water in the alcohol aqueous solution is about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1); and / or Preferably, the alcohol in the alcohol aqueous solution is selected from methanol, ethanol, isopropanol and any mixture thereof, more preferably ethanol; or The solvent is a mixed solvent of alcohol and ester, Preferably, the volume ratio of the ester to the alcohol in the mixed solvent of the alcohol and the ester is about 1:1 to about 1:5, more preferably about 1:1 to about 1:2; and / or Preferably, the alcohol in the mixed solvent of alcohol and ester is methanol, ethanol, isopropanol or any mixture thereof, and / or the ester is ethyl acetate (EA), isopropyl acetate or a mixture thereof; and / or (4) the hydrogenation reaction of the compound of formula II and the hydrogen-borrowing reagent is carried out in the alcohol aqueous solution; or The hydrogenation reaction of the compound of formula II and the hydrogen is carried out in the alcohol aqueous solution or a mixed solvent of the alcohol and the ester; and / or (5) The mass ratio of the compound of formula II to the metal catalyst is about 1:0.01 to 1:1, preferably about 1:0.2; and / or (6) The metal catalyst is palladium on carbon (Pd / C), wherein preferably, the mass fraction of palladium in the Pd / C is about 5-20%, more preferably about 10% (for example, the Pd / C catalyst contains about 50 w / w% of water); and / or (7) The hydrogenation reaction is carried out for about 1 to 72 hours, such as about 1 to 48 hours, about 1 to 12 hours, about 1 to 10 hours, about 2 to 5 hours, or about 6 to 12 hours.

9. The method according to any one of claims 1 to 8, characterized in that The method comprises the following steps: a) subjecting the compound of formula II to a hydrogenation reaction with the hydrogen gas or the hydrogen-borrowing reagent in an ethanol aqueous solution or a mixed solvent of ethanol and ethyl acetate under the catalysis of Pd / C for about 1 to 72 hours, for example, about 1 to 48 hours, about 1 to 12 hours, about 1 to 10 hours, about 2 to 5 hours, or about 6 to 12 hours to provide the compound of formula I, in: (1) The pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4Mpa, and The reaction of the compound of formula II with the hydrogen is carried out at a temperature of about 20 to 25° C.; or The molar ratio of the compound of formula II to the hydrogen-borrowing agent is about 1:2 to 1:10, for example, about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8, The reaction of the compound of formula II with the hydrogen-borrowing agent is carried out at a temperature of about 20°C to 100°C, preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C, and The hydrogen borrowing reagent is a combination of formic acid and potassium formate, wherein the molar ratio of the formic acid to the potassium formate is about 1:1 to 5:1; (2) the mass ratio of the compound of formula II to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; (3) the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II is about 10:1 to 80:1, preferably about 20:1 to 50:1; and (4) the volume ratio of ethanol to water in the ethanol aqueous solution is about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1); and The volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is about 1:1 to about 1:5, preferably about 1:1 to about 1:

2.

10. The method according to any one of claims 1 to 4 and 7 to 9, characterized in that The compound of formula I is a compound of formula I-1, and the compound of formula II is a compound of formula II-1: Preferably, the method comprises the following steps: a) subjecting the compound of formula II-1 to a hydrogenation reaction with the hydrogen-borrowing reagent in the ethanol aqueous solution under the catalysis of the Pd / C for about 1 to 12 hours to provide the compound of formula I-1, Preferably, the molar ratio of the compound of formula II-1 to the hydrogen borrowing reagent is about 1:2 to 1:10, such as about 1:2.5, about 1:4, about 1:5, about 1:6 or about 1:8, and / or The reaction of the compound of formula II-1 with the hydrogen-borrowing agent is carried out at a temperature of about 20°C to 100°C, preferably about 20°C to 80°C, more preferably room temperature or about 50°C to 70°C, more preferably about 60°C. and / or The hydrogen borrowing reagent is a combination of formic acid and potassium formate, wherein the molar ratio of the formic acid to the potassium formate is about 1:1 to 5:1; or a) subjecting the compound of formula II-1 to a hydrogenation reaction with the hydrogen gas in the ethanol aqueous solution or the mixed solvent of ethanol and ethyl acetate under the catalysis of the Pd / C for about 6 to 12 hours to provide the compound of formula I-1, Preferably, the pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa; and / or Preferably, the reaction of the compound of formula II-1 with the hydrogen is carried out at a temperature of about 20 to 25°C; and / or Preferably, the mass ratio of the compound of formula II-1 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; and / or Preferably, the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II-1 is about 10:1 to 80:1, preferably about 20:1 to 50:1; and / or Preferably, the volume ratio of ethanol to water in the ethanol aqueous solution is about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1); and / or Preferably, the volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is about 1:1 to about 1:5, preferably about 1:1 to about 1:

2.

11. The method according to any one of claims 1 to 2 and 5 to 9, characterized in that The compound of formula I is a compound of formula I-2, and the compound of formula II is a compound of formula II-2: Preferably, the method comprises the following steps: a) subjecting the compound of formula II-2 to a hydrogenation reaction with the hydrogen gas in the ethanol aqueous solution or the mixed solvent of ethanol and ethyl acetate under the catalysis of the Pd / C for about 6 to 12 hours to provide the compound of formula I-2; Preferably, the pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa; and / or Preferably, the reaction of the compound of formula II-2 with the hydrogen is carried out at a temperature of about 20 to 25°C; and / or Preferably, the mass ratio of the compound of formula II-2 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; and / or Preferably, the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II-2 is about 10:1 to 80:1, preferably about 20:1 to 50:1; and / or Preferably, the volume ratio of ethanol to water in the ethanol aqueous solution is about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1); and / or Preferably, the volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is about 1:1 to about 1:5, preferably about 1:1 to about 1:

2.

12. The method according to any one of claims 1 to 2 and 5 to 9, characterized in that The compound of formula I is a compound of formula I-3, and the compound of formula II is a compound of formula II-3: Preferably, the method comprises the following steps: a) subjecting the compound of formula II-3 to a hydrogenation reaction with the hydrogen in the ethanol aqueous solution or the mixed solvent of ethanol and ethyl acetate under the catalysis of the Pd / C for about 1 to 12 hours, for example, about 1 to 10 hours, about 2 to 5 hours, or about 6 to 12 hours, to provide the compound of formula I-3; Preferably, the pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa; and / or Preferably, the reaction of the compound of formula II-3 with the hydrogen is carried out at a temperature of about 20 to 25°C; and / or Preferably, the mass ratio of the compound of formula II-3 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; and / or Preferably, the volume ratio of the ethanol aqueous solution, or the mixed solvent of ethanol and ethyl acetate to the compound of formula II-3 is about 10:1 to 80:1, preferably about 20:1 to 50:1; and / or The volume ratio of ethanol to water in the ethanol aqueous solution is about 1:1 to about 8:1 (e.g., about 4:1 or about 6:1); and / or Preferably, the volume ratio of ethyl acetate to ethanol in the mixed solvent of ethanol and ethyl acetate is about 1:1 to about 1:5, preferably about 1:1 to about 1:

2.

13. The method according to any one of claims 1 to 12, characterized in that The method further comprises the following steps: b) removing the metal catalyst from the reaction mixture of step a), and then drying the reaction mixture to obtain the compound of formula I; and c) optionally, further purifying the compound of formula I obtained in step b), preferably by the following means: The compound of formula I obtained in step b) is slurried with acetonitrile (eg, about 1 to 4 h), further slurried with water (about 1 to 4 h), then filtered, and the filter cake is dried; or The compound of formula I obtained from step b) is passed through a chromatography column or high pressure preparative chromatography.

14. The method according to any one of claims 1 to 13, characterized in that When the compound of formula I is the compound of formula I-1, and the compound of formula II is the compound of formula II-1, the method comprises performing step c), preferably further purifying the compound of formula I-1 obtained from step b) by slurrying the compound of formula I-1 obtained from step b) with acetonitrile (e.g., about 1 to 4 h), further slurrying with water (about 1 to 4 h), then filtering, and drying the filter cake; or When the compound of formula I is the compound of formula I-2, and the compound of formula II is the compound of formula II-2, the method comprises performing step c), preferably further purifying the compound of formula I-2 obtained from step b) by the following method: slurrying the compound of formula I-2 obtained from step b) with acetonitrile (for example, about 1 to 4 hours), further slurrying with water (about 1 to 4 hours), then filtering, and drying the filter cake; or passing the compound of formula I-2 from step b) through a chromatography column or a high pressure preparative chromatograph; or When the compound of formula I is the compound of formula I-3, and the compound of formula II is the compound of formula II-3, the method includes performing step c), preferably further purifying the compound of formula I-3 obtained from step b) by passing the compound of formula I-3 from step b) through a chromatography column or a high-pressure preparative chromatograph.

15. The method according to any one of claims 1 to 14, characterized in that The method further comprises the following steps: a') reacting the compound 3,6-diaminopyrazine-2,5-dioic acid of formula III with the compound of formula IV or its salt (preferably hydrochloride) and the compound of formula V or its salt (preferably hydrochloride) in a suitable organic solvent; In the presence of a suitable organic base, a suitable coupling agent and a suitable condensing agent, an amide condensation reaction is carried out to provide a compound of formula II, wherein X1 and X4 are as defined in any one of claims 1 to 14.

16. The method according to claim 15, characterized in that The compound of formula II is the compound of formula II-1, and the method comprises: The compound of formula III is reacted with the hydrochloride of the compound of formula IV having formula IV-1 and the hydrochloride of the compound of formula V having formula V-1 to provide the compound of formula II-1: or The compound of formula II is the compound of formula II-2, and the method comprises: The compound of formula III is reacted with the hydrochloride of the compound of formula IV having the formula IV-1 and the hydrochloride of the compound of formula V having the formula V-2 to provide the compound of formula II-2: or The compound of formula II is the compound of formula II-3, and the method comprises: The compound of formula III is reacted with the hydrochloride salt of the compound of formula IV-1 to provide the compound of formula II-3.

17. The method according to claim 15 or 16, characterized in that The molar ratio of the compound of formula IV to the compound of formula III is about 0.5:1 to 8:1, preferably about 1:1 to 4:1; and / or The molar ratio of the compound of formula V to the compound of formula III is about (0.5-8.0):1.0, preferably about 1:1 to 4:1; The volume ratio of the organic solvent to the compound of formula III is about 10:1 to 80:1, preferably about 20:1 to 50:1; and / or The molar ratio of the organic base to the compound of formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1; and / or The molar ratio of the coupling agent to the compound of formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1; and / or The molar ratio of the condensing agent to the compound of formula III is about 0.5:1 to 9:1, preferably about 1:1 to 5:1; and / or The amide condensation reaction is first carried out at a suitable temperature, preferably about -5 to 20°C, more preferably about -5 to 10°C, more preferably about 0-10°C for a suitable time, such as about 10-60 min, and then at room temperature for a suitable time, preferably about 10-72 h, more preferably about 18-36 h; and / or The organic solvent is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane; and / or The organic base is diisopropylethylamine (DIPEA), triethylamine, pyridine and / or 4-dimethylaminopyridine (DMAP); and / or The coupling agent is ethyl 2-oxime cyanoacetate (Oxyma), 1-hydroxybenzotriazole (HOBt), HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU) and / or O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU); and / or The condensing agent is selected from: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCl), HOBt, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU), O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU) and any combination thereof.

18. The method according to any one of claims 15 to 17, characterized in that The method comprises the following steps: a') mixing the compound of formula III with the compound of formula IV and the compound of formula V The reaction is carried out in DMF at about -5 to 20°C, preferably about -5 to 10°C, more preferably about 0-10°C, in the presence of DIPEA, ethyl 2-oximecyanoacetate and EDC*HCl for about 10-60 min, and then at room temperature for about 10-72 h, more preferably about 18-36 h.

19. The method according to any one of claims 15 to 18, characterized in that The method further comprises the following steps: b') optionally, adding water to the reaction mixture in step a') to precipitate the compound of formula II, wherein preferably, the volume ratio of the added water to the compound of formula III is about 20:1 to 150:1, more preferably about 35:1 to 75:1; and c') Optionally, further purifying the compound of formula II obtained from step b'), preferably by passing the compound of formula II from step b') through a chromatography column or high pressure preparative chromatography.

20. The method according to any one of claims 15 to 19, characterized in that When the compound of formula II is the compound of formula II-1, the method comprises performing the step c'), wherein the compound of formula II-1 obtained from the step b') is further purified by passing the compound of formula II-1 through a chromatography column; or When the compound of formula II is the compound of formula II-2, the method comprises performing the step c'), wherein the compound of formula II-2 obtained from the step b') is further purified by passing the compound of formula II-2 through a chromatography column or a high pressure preparative chromatography; or When the compound of formula II is the compound of formula II-3, the method comprises performing the step c'), wherein the compound of formula II-3 obtained from the step b') is further purified by passing the compound of formula II-3 through a chromatography column or high pressure preparative chromatography.

21. A process for preparing a compound of formula VI or a salt thereof, It is characterized in that The method comprises the following steps: i) subjecting the compound of formula VII to a hydrogenation reaction in a solvent in the presence of a suitable metal catalyst to provide the compound of formula VI, in: X5 and X6 are each independently -(substituted or unsubstituted C 1-10 Alkyl)-R 2 ;as well as R 2 Selected from: C 3-10 Cycloalkyl, 4-10 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl.

22. The method according to claim 21, wherein: R 2 Selected from: C 3-7 Cycloalkyl, preferably C 5-6 Cycloalkyl; 4-7 membered heterocyclic group, preferably 5-6 membered heterocyclic group; phenyl and 5-6 membered heteroaryl; preferably phenyl.

23. The method according to claim 21 or 22, characterized in that X5 and X6 are each independently -(substituted or unsubstituted C 1-8 Alkyl)-R 2 , preferably -(substituted or unsubstituted C 1-6 Alkyl)-R 2 , more preferably -(substituted or unsubstituted C 1-4 Alkyl)-R 2 , more preferably -(unsubstituted C 1-2 Alkyl)-R 2 , more preferably -CH2-R 2 , more preferably -CH2-phenyl.

24. The method according to any one of claims 21 to 23, characterized in that (1) In step i), the hydrogenation reaction is carried out under a hydrogen atmosphere, Preferably, the pressure of the hydrogen is about 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa; and / or Preferably, the reaction of the compound of formula VII with the hydrogen is carried out at a temperature of about 20 to 30° C.; and / or (2) the mass ratio of the compound of formula VII to the metal catalyst is about 1:0.01 to 1:1, preferably about 1:0.2; and / or (3) The metal catalyst is palladium on carbon (Pd / C), wherein preferably, the mass fraction of palladium in the Pd / C is about 5-20%, more preferably about 10% (for example, the Pd / C catalyst contains about 50 w / w% of water); and / or (4) the volume ratio of the solvent to the compound of formula VII is about 10:1 to 50:1, preferably about 10:1 to 30:1; and / or (5) The solvent is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane, preferably N,N-dimethylformamide; and / or (6) The hydrogenation reaction is carried out for about 5 to 72 hours, such as about 10 to 48 hours, or about 10 to 30 hours.

25. The method according to any one of claims 21 to 24, characterized in that The method comprises the following steps: i) subjecting the compound of formula VII-1 to a hydrogenation reaction in N,N-dimethylformamide in the presence of about 10% Pd / C, under a hydrogen atmosphere of 0.05 MPa to about 0.8 MPa, preferably about 0.1 MPa to about 0.4 MPa, at a temperature of about 20 to 25° C. for about 5 to 72 hours, preferably about 10 to 30 hours, to provide the compound of formula VI, in: The mass ratio of the compound of formula VII-1 to the Pd / C is about 1:0.01 to 1:1, preferably about 1:0.2; and The volume ratio of the N,N-dimethylformamide to the compound of formula VII-1 is about 10:1 to 50:1, preferably about 10:1 to 30:

1.

26. The method according to any one of claims 1 to 25, characterized in that The method further comprises the following steps: i') subjecting the compound of formula VIII to an amide condensation reaction with the compound of formula IX or a salt thereof (preferably hydrochloride) under an inert atmosphere in a suitable organic solvent in the presence of a suitable organic base, a suitable coupling agent and a suitable condensing agent to provide the compound of formula VII, wherein X5 and X6 are each as defined in any one of claims 21 to 25; and ii') optionally, filtering the reaction mixture in step i'), and adding water dropwise to the filtrate to crystallize the compound of formula VII, wherein preferably, the volume ratio of the added water to the filtrate is about 20:1 to 150:1, preferably 35:1 to 75:1; and iii') Optionally, ethyl acetate is added to the separated compound of formula VII for slurrying, followed by filtering and vacuum drying at elevated temperature for more than 18 hours, wherein preferably, the volume ratio of ethyl acetate to the compound of formula VII is about 5:1 to 30:1, preferably about 5:1 to 10:

1.

27. The method according to claim 26, characterized in that The inert atmosphere is under nitrogen protection; and / or The volume ratio of the organic solvent to the compound of formula IX or its salt is about 10:1 to 80:1, preferably about 20:1 to 50:1; and / or The molar ratio of the organic base to the compound of formula VIII is about 0.5:1 to 9:1, preferably about 1:1 to about 5:1; and / or The molar ratio of the condensing agent to the compound of formula VIII is about 0.5:1 to 9:1, preferably about 1:1 to about 5:1; and / or The molar ratio of the coupling agent to the compound of formula VIII is about 0.5:1 to 9:1, preferably about 1:1 to about 5:1; and / or The amide condensation reaction is carried out at a temperature of about 10 to 30°C, preferably about 20 to 30°C; and / or The amide condensation reaction lasts for about 10 to 72 hours, preferably about 18 to 36 hours; and / or The organic solvent is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane; and / or The organic base is diisopropylethylamine (DIPEA), triethylamine, pyridine and / or 4-dimethylaminopyridine (DMAP); and / or The coupling agent is ethyl 2-oxime cyanoacetate (Oxyma), HOBt, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazole-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU) and / or O-(benzotriazole-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate. acid salt (HBPyU); and / or The condensing agent is selected from: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCl), HOBt, HATU, HBTU, HCTU, TBTU, TSTU, TNTU, O-(7-azabenzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HAPyU), O-(benzotriazol-1-yl)-bis(tetrahydropyrrolyl)carbonium hexafluorophosphate (HBPyU) and any combination thereof.

28. The method according to claim 26 or 27, characterized in that The compound of formula VIII is a compound of formula I-3: The compound of formula IX is a compound of the following formula: The organic solvent is DMF; The organic base is diisopropylethylamine (DIPEA); The coupling agent is HOBt; and The condensation agent is: EDC*HCl.

29. The method according to any one of claims 21 to 28, characterized in that The method further comprises the following steps: ii) optionally, filtering the reaction mixture in step i), and adding ethyl acetate dropwise to the filtrate to crystallize the compound of formula VI, wherein preferably, the volume ratio of the ethyl acetate to the filtrate is about 30:1 to 90:1, preferably about 30:1 to 50:1; and iii) Optionally, ethyl acetate is added to the separated compound of formula VI for slurrying, followed by filtering and vacuum drying at elevated temperature for more than 12 hours, wherein preferably, the volume ratio of ethyl acetate to the compound of formula VI is about 1:1 to 10:1, preferably about 2:1 to 5:

1.

30. A process for preparing a compound of formula X or a salt thereof, It is characterized in that The method comprises the following steps: xi) providing a solution of a compound of formula A in a solvent; x-ii) At elevated temperature, with acid R 3 -COOH acylates the compound of formula A to provide the compound of formula X; and x-iii) optionally, isolating and purifying the compound of formula X; Where R 3 is H, or substituted or unsubstituted C 1-4 Alkyl, preferably H or methyl, more preferably H.

31. The method according to claim 30, characterized in that R 3 is H; and / or In step xi), the volume ratio of the solvent to the compound of formula A is about 5:1 to 30:1, preferably about 5:1 to 10:1; and / or In step xi), the solvent is N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran and / or 1,4-dioxane, preferably DMF; and / or The elevated temperature in step x-ii) is about 55 to 75°C, preferably about 70°C; and / or In step x-ii), the acid R 3 The volume ratio of -COOH to the compound of formula A is about 0.5:1 to 10:1, preferably about 1:1 to 5:1; and / or In step x-ii), the reaction time is about 1 to 20 h, preferably about 1 to 5 h; and / or The step x-iii) comprises: 1) concentrating the reaction mixture in step xi) until there is no fraction, and then adding ethyl acetate dropwise to the residue to crystallize the compound of formula X, wherein preferably, the volume ratio of ethyl acetate to the compound of formula A is about 15:1 to 90:1, preferably about 15:1 to 30:1; and 2) dissolving the isolated compound of formula X in the solvent used in step xi), then adding ethyl acetate dropwise to crystallize the compound of formula X, then filtering, and drying in vacuo at elevated temperature, wherein preferably, the volume ratio of the solvent to the compound of formula A is about 0.5:1 to 5:1, preferably about 1:1 to 2:1; and / or the volume ratio of ethyl acetate to the compound of formula A is about 1:1 to 10:1, preferably about 2:1 to 4:1; 3) repeating the recrystallization of steps 1) and 2); and 4) Slurrying the dried compound of formula X obtained in step 3) with ethyl acetate, filtering, and vacuum drying at elevated temperature for more than 16 hours, wherein preferably, the volume ratio of ethyl acetate to the compound of formula A is about 1:1 to 10:1, preferably about 2:1 to 5:

1.

32. A process for preparing a compound of formula XI or a salt thereof, It is characterized in that The method comprises the following steps: xi-i) hydrolyzing a compound of formula A in water in the presence of a base at elevated temperature to provide the compound of formula XI or a salt thereof; as well as xi-ii) Optionally, isolating and purifying the compound of formula XI or a salt thereof.

33. The method according to claim 32, characterized in that In step xi-i), the volume ratio of water to the compound of formula A is about 5:1 to 30:1, preferably about 5:1 to 10:1; and / or In step xi-i), the molar ratio of the base to the compound of formula A is about 1:1 to 5:1, preferably about 2:1 to 5:1; and / or The base is an inorganic base, preferably potassium hydroxide, sodium hydroxide and a mixture thereof, more preferably hydroxide. Potassium chloride; and / or In step xi-i), the elevated temperature is about 50 to 100°C, preferably about 80 to 90°C; and / or In step xi-i), the hydrolysis reaction is carried out for about 5 to 48 hours, preferably about 10 to 20 hours; and / or The step xi-ii) comprises: 1) adding silica gel to the reaction mixture in step xi-ii) to concentrate the reaction mixture and prepare silica gel powder, and subjecting the silica gel powder to silica gel column chromatography to obtain a solid crude product of the compound of formula XI or a salt thereof, Preferably, the mass ratio of the added silica gel to the compound of formula A is about 0.5:1 to 2:1, preferably about 2:1; and / or the silica gel chromatography column is gradient eluted with EA / MeOH (1% TFA) as the eluent, wherein the volume ratio of EA:MeOH (1% TFA) is about 50:1 to 1:1, preferably about 30:1 to 10:1; 2) slurrying the crude solid obtained from step xii-1) with methanol, filtering, and then vacuum drying at elevated temperature for more than 16 hours to obtain a purified compound of formula XI or a salt thereof, Preferably, the volume ratio of the methanol to the compound of formula A is about 1:1 to 10:1, preferably about 2:1 to 5:

1.

34. A method for quality control of a bulk drug or a formulation of 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine, comprising determining the content of a compound of formula I as described in any one of claims 1 to 7, preferably a compound of formula I-1 as described in claim 10, a compound of formula I-2 as described in claim 11, a compound of formula I-3 as described in claim 12, a compound of formula VI as described in claim 21, a compound of formula X as described in claim 30, and / or a compound of formula XI as described in claim 32 in the bulk drug or formulation, and optionally comparing with a specified content of each of the compounds; Preferably, for the compound of formula I, the specified content is: based on the total weight of the drug substance, not more than about 0.9%, preferably not more than about 0.6%, more preferably not more than about 0.45%; For the compound of formula I-1, the specified content is: based on the total weight of the raw material, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15%; or, based on the total weight of the preparation, no more than about 0.40%, preferably no more than about 0.3%, more preferably Not more than about 0.20%; For the compound of formula I-2, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15%; For the compound of formula I-3, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15%; For the compound of formula VI, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15%; For the compound of formula X, the specified content is: based on the total weight of the drug substance, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15%; and / or For the compound of formula XI, the specified content is: based on the total weight of the raw material, no more than about 0.2%, preferably no more than about 0.15%, more preferably no more than about 0.10%; or, based on the total weight of the formulation, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20%.

35. A compound of formula I as described in any one of claims 1 to 7, preferably a compound of formula I-1 as described in claim 10, a compound of formula I-2 as described in claim 11, a compound of formula I-3 as described in claim 12, a compound of formula VI as described in claim 21, a compound of formula X as described in claim 30, and / or a compound of formula XI as described in claim 32 as a standard or reference substance for quality control of a bulk drug or a preparation of 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine.

36. A 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine API having a low content of related compounds, characterized in that: The related compound is a compound of formula I as described in any one of claims 1 to 7, preferably a compound of formula I-1 as described in claim 10, a compound of formula I-2 as described in claim 11, a compound of formula I-3 as described in claim 12, a compound of formula VI as described in claim 21, a compound of formula X as described in claim 30, and / or a compound of formula XI as described in claim 32; and The drug substance has one or more of the following contents: Based on the total weight of the bulk drug, no more than about 0.9%, preferably no more than about 0.6%, more preferably no more than about 0.45% of the compound of formula I, Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula I-1, Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula I-2, Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula I-3, Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula VI, Based on the total weight of the bulk drug, no more than about 0.3%, preferably no more than about 0.25%, more preferably no more than about 0.15% of the compound of formula X, Based on the total weight of the bulk drug, no more than about 0.2%, preferably no more than about 0.15%, more preferably no more than about 0.10% of the compound of formula XI.

37. A 3,6-diamino-2,5-bis{N-[(1R)-1-carboxylic acid-2-hydroxyethyl]carbamoyl}pyrazine preparation having a low content of related compounds, characterized in that The related compound is selected from the compound of formula I-1 as claimed in claim 10 and the compound of formula XI as claimed in claim 32; and The preparation has one or both of the following contents: Based on the total weight of the formulation, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20% of the compound of formula I-1; Based on the total weight of the formulation, no more than about 0.40%, preferably no more than about 0.3%, more preferably no more than about 0.20% of the compound of formula XI.

38. The method according to claim 34, the use according to claim 35, the bulk drug according to claim 36, the preparation according to claim 37, wherein the preparation is a solution for injection or a lyophilized powder for injection, preferably a solution for injection.