Method for synthesizing 2-amino-3-hydroxy-4-oxobutyric acid derivative through enzyme catalysis

Abstract

According to the method for synthesizing the 2-amino-3-hydroxy-4-oxobutyric acid derivative through enzyme catalysis, threonine aldolase LTA and / or threonine transaldolase LTTA are / is adopted for catalysis, and the 2-amino-3-hydroxy-4-oxobutyric acid derivative with double chiral centers can be efficiently and selectively synthesized.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and specifically relates to a method for synthesizing 2-amino-3-hydroxy-4-oxobutyric acid derivatives by enzyme catalysis. Background Art

[0002] In most mammalian tissue cells, tryptophan ingested by the diet is almost entirely metabolized by the kynurenine pathway (KP), which breaks down tryptophan into a variety of neuroactive substances, such as kynurenine (KYN), kynurenic acid (KYNA), and 3-hydroxykynurenine (3-HK), and is ultimately metabolized into nicotinamide adenine dinucleotide (NAD + ) (Nat Med, 2016, 22(2): 202-209.) The dysregulation or disorder of this metabolic pathway is closely related to inflammatory neurological diseases such as Alzheimer's disease, autoimmune diseases, cerebral ischemia and tumor proliferation, and the homeostasis of metabolic intermediates is closely related to the expression level and activity of enzymes in this pathway.

[0003] Kynurenine-3-monooxygenase (KMO) is a flavoprotein monooxygenase that uses NADPH as a cosubstrate and catalyzes L -Kynurenic acid is hydroxylated to 3-hydroxykynurenine, which is a key branch of the kynurenine metabolic pathway. KMO is highly expressed in the liver, kidney tissues and monocytes of the human body, and is also expressed in small amounts in microglia and macrophages in the brain. 3-HK and xanthurenic acid produced by kynurenine metabolism have been shown to have significant neurotoxicity; in contrast, KYNA, which is metabolized by kynurenine aminotransferase (KAT), has a neuroprotective effect. Therefore, inhibiting KMO hydroxylation activity, thereby reducing the levels of 3-HK and quinolinic acid in the body and increasing the level of KYNA, can play a therapeutic role in neurological diseases including Alzheimer's disease, Huntington's disease and Parkinson's disease (Nat commun, 2017, 8(1): 15812-15827.).

[0004] The literature (J Med Chem, 2017, 60(8): 3383-3404) summarizes several KMO inhibitors based on the natural substrate KYN structure, and their specific structures are shown in the following formula. The reported KMO competitive inhibitors are based on the aromatic acyl alanine structure, such as m-NBA and FCE 28833A. On this basis, further structural modification has been carried out to eliminate the enantioselective configuration restriction, such as UPF-648 and CHEMBL3407866.

[0005]

[0006] The currently reported aromatic acyl alanine inhibitors are mainly synthesized by chemical methods through more than five steps of reactions. The existing methods generally have problems such as poor stereoselectivity, cumbersome steps, and low yield, which are not conducive to industrial scale-up production. There is no suitable method to prepare such inhibitors efficiently and selectively. Summary of the invention

[0007] The purpose of the present invention is to provide a method with convenient operation and high stereoselectivity for preparing a KMO inhibitor with a chiral center.

[0008] In the first aspect of the present invention, a method for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided.

[0009]

[0010] Wherein, ring A is selected from the following groups: C3-C8 saturated or partially unsaturated carbocyclic group, 3-12 membered saturated or partially unsaturated heterocyclic group, C6-C 10 Aryl, 5-12 membered heteroaryl;

[0011] Each R a Each is independently selected from the following group: a deuterium atom, a halogen, a nitro group, a hydroxyl group, an amino group, a thiol group, a carboxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, -C1-C6 alkyl-NH2, -NH-C1-C6 alkyl group, -C1-C6 alkyl-NH-C1-C6 alkyl group, a C1-C6 alkyl group substituted with a thiol group, a C2-C6 acyl group, a C3-C8 saturated or partially unsaturated carbocyclic group, a 3-12-membered saturated or partially unsaturated heterocyclic group, a C6-C 10 Aryl, 5-12 membered heteroaryl;

[0012] *Carbon atoms can be in R or S configuration;

[0013] Wherein, the heterocyclic group and heteroaryl group each independently and optionally include 1-4 ring atoms selected from the following group: N, O, S;

[0014] The method comprises the steps of:

[0015] (1) providing a reaction system, wherein the reaction system comprises a cofactor;

[0016] (2) In the reaction system, compound a reacts with glycine or threonine in the presence of a catalyst to obtain a compound of formula (I);

[0017]

[0018] in,

[0019] The catalyst is selected from the group consisting of threonine aldolase LTA, threonine transaldolase LTTA, a strain expressing threonine aldolase, a strain expressing threonine transaldolase, or a combination thereof.

[0020] In some embodiments, the cofactor is selected from the group consisting of pyridoxal phosphate (PLP).

[0021] In some embodiments, the cofactor concentration is 0.01 mM to 1 mM, preferably 0.02 to 0.5 mM, and more preferably 0.05 to 0.2 mM.

[0022] In some embodiments, the catalyst concentration is 0.05-5 mg / mL, preferably 0.1-4 mg / mL, and more preferably 0.2-3 mg / mL.

[0023] In some embodiments, the threonine aldolase LTA has an amino acid sequence as shown in SEQ ID NO:1.

[0024] In another preferred embodiment, the threonine aldolase LTA has an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 while maintaining the enzyme activity.

[0025] In some embodiments, the threonine transaldolase LTTA has an amino acid sequence as shown in SEQ ID NO:3.

[0026] In another preferred embodiment, the threonine transaldolase LTTA has an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3 while maintaining the enzyme activity.

[0027] In some embodiments, the strain expressing threonine aldolase is a strain expressing a protein having an amino acid sequence as shown in SEQ ID NO: 1;

[0028] The strain expressing threonine aldolase is a strain expressing a protein having an amino acid sequence as shown in SEQ ID NO:3.

[0029] In another preferred embodiment, the strain expressing threonine aldolase is a strain having a DNA with a nucleotide sequence as shown in SEQ ID NO: 2;

[0030] The strain expressing threonine transaldolase is a strain having a DNA with a nucleotide sequence as shown in SEQ ID NO:3.

[0031] In another preferred embodiment, the strain may be a genetically engineered strain carrying the above gene.

[0032] In some embodiments, the reaction system further comprises metal ions, and the metal ions are selected from the following group: Mg 2+ , Ca 2+ , or a combination thereof.

[0033] In another preferred embodiment, the metal ion concentration is 0.01 to 5 M, preferably 0.05 to 1 M, and more preferably 0.1 to 0.5 M.

[0034] In some embodiments, the reaction is carried out at 10-50°C, preferably at 15-40°C, and more preferably at 20-30°C.

[0035] In some embodiments, the method further comprises the steps of:

[0036] (3) isolating the compound of formula (I) or a pharmaceutically acceptable salt thereof from the reaction system.

[0037] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The structures of pET-28a and pET-22b vectors carrying SEQ ID NO: 2 or SEQ ID NO: 4 and SEQ ID NO: 6 are shown.

[0039] Figure 2 The results of preliminary screening of KMO inhibitors are shown.

[0040] Figure 3 The dose-effect curve fitting of the compounds with high inhibitory activity obtained from the preliminary screening and the IC of each compound under the experimental conditions of the present invention are shown. 50 . DETAILED DESCRIPTION

[0041] After extensive and in-depth research, the inventor unexpectedly found that an enzyme with a specific structure can catalyze the preparation of 2-amino-3-hydroxy-4-oxobutyric acid derivatives with high catalytic efficiency and high chiral selectivity. On this basis, the present invention was completed.

[0042] the term

[0043] Unless otherwise specified, the terms used in the present invention have the following definitions:

[0044] The term "halogen" as used herein means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I); preferably fluorine or chlorine.

[0045] The term "hydrocarbon group" used in the present invention refers to a straight-chain or branched saturated or unsaturated hydrocarbon group, that is, the group remaining after the corresponding hydrocarbon loses a hydrogen atom (H), including alkyl, alkenyl, and alkynyl.

[0046] The term "alkyl" as used in the present invention refers to a straight-chain or branched saturated hydrocarbon group, such as C1-C 10 Alkyl refers to a straight or branched saturated hydrocarbon group containing 1 to 10 carbon atoms, and includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-ethylpropyl, isopentyl, neopentyl, n-hexyl or isohexyl, etc., preferably methyl, ethyl, n-propyl or isopropyl; it can be monosubstituted (such as -CH2F) or polysubstituted (such as -CF3).

[0047] The term "alkenyl" as used herein refers to a straight or branched hydrocarbon group containing one or more carbon-carbon double bonds, which may be located at any position of the hydrocarbon group. 10 Alkenyl, preferably C2-C4 alkenyl, includes but is not limited to ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl or octenyl.

[0048] The term "alkynyl" as used herein refers to a straight or branched hydrocarbon group containing one or more carbon-carbon triple bonds, which may be located at any position of the hydrocarbon group. 10 Alkynyl, preferably C2-C4 alkynyl, includes but is not limited to ethynyl, 1-propynyl, 1-butynyl, heptynyl or octynyl.

[0049] The term "alkyloxy" used in the present invention refers to a group formed by a straight chain or branched chain alkyl group and an oxygen atom. 10 The "alkyloxy" may include C1-C 10 Alkyloxy, C2-C 10 Alkenyloxy, C2-C 10 Alkynyloxy, wherein "alkyl", "alkenyl" and "alkynyl" are as defined above. In some embodiments, preferably C1-C 10 Alkoxy, more preferably C1-C4 alkoxy, includes but is not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy or isobutoxy.

[0050] The term "alkylamino" used in the present invention refers to a group formed by connecting a straight chain or branched chain hydrocarbon group and an amine group, "C1-C 10The "alkylamino group" may include C1-C 10 Alkylamine, C2-C 10 Alkenylamine, C2-C 10 Alkynylamino, wherein "alkyl", "alkenyl" and "alkynyl" are as defined above. In some embodiments, preferably C1-C 10 The alkylamino group, more preferably a C1-C4 alkylamino group, includes but is not limited to a methylamino group, an ethylamino group, a n-propylamino group, an isopropylamino group, a n-butylamino group or an isobutylamino group.

[0051] The term "alkylthiol" as used in the present invention refers to a group formed by connecting a straight chain or branched chain hydrocarbon group with a sulfur atom, "C1-C 10 The "alkylmercapto" may include C1-C 10 Alkylthio, C2-C 10 Alkenylthiol, C2-C 10 Alkynylthiol, wherein "alkyl", "alkenyl" and "alkynyl" are as defined above. In some embodiments, preferably C1-C 10 Alkylmercapto, more preferably C1-C4 alkylmercapto, includes but is not limited to methylmercapto, ethylmercapto, n-propylmercapto, isopropylmercapto, n-butylmercapto or isobutylmercapto.

[0052] The term "acyl" used in the present invention refers to a group formed by connecting a straight-chain or branched saturated or unsaturated hydrocarbon group and a carbonyl group, preferably an alkyl acyl group, including but not limited to formyl, acetyl or propionyl.

[0053] The term "alicyclic hydrocarbon group" used in the present invention refers to saturated alicyclic hydrocarbons and unsaturated alicyclic hydrocarbons (alicyclic hydrocarbons containing one or more carbon-carbon double bonds in the molecule), including monocyclic and polycyclic alicyclic hydrocarbons. 10 The "alicyclic hydrocarbon group" may include C3-C 10 Cycloalkyl, C3-C 10 Cycloalkenyl or C3-C 10 Cycloalkynyl, preferably C3-C 10 Cycloalkyl. "C3-C 10 "Cycloalkyl" refers to a saturated cyclic hydrocarbon group containing 3-10 carbon atoms, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. "C3-C 10 "Cycloalkenyl" refers to a cyclic hydrocarbon group containing 3-10 carbon atoms and one or more carbon-carbon double bonds, including but not limited to cyclopropenyl, cyclobutenyl, cyclopentenyl or cyclohexenyl. "C3-C 10 "Cycloalkynyl" refers to a cyclic hydrocarbon group containing 3 to 10 carbon atoms and containing one or more carbon-carbon triple bonds.

[0054] The term "alicyclic hydrocarbon group" used in the present invention refers to a group formed by connecting a saturated alicyclic hydrocarbon or an unsaturated alicyclic hydrocarbon with an oxygen atom, "C3-C 10The "alicyclic hydrocarbon group" may include C3-C 10 Cycloalkyloxy, C3-C 10 Cycloalkenyloxy, C3-C 10 The cycloalkynyloxy group is preferably a C3-C7 cycloalkoxy group, and more preferably a C3-C6 cycloalkoxy group or a C5-C6 cycloalkoxy group.

[0055] The term "alicyclic hydrocarbon amino group" used in the present invention refers to a group formed by connecting a saturated alicyclic hydrocarbon or an unsaturated alicyclic hydrocarbon with an amino group, "C3-C 10 The "alicyclic hydrocarbon amino group" may include C3-C 10 Cycloalkylamine, C3-C 10 Cycloalkenylamine, C3-C 10 The cycloalkynylamino group is preferably a C3-C7 cycloalkylamino group, and more preferably a C3-C6 cycloalkylamino group or a C5-C6 cycloalkylamino group.

[0056] The term "alicyclic hydrocarbon thiol" used in the present invention refers to a group formed by connecting a saturated alicyclic hydrocarbon or an unsaturated alicyclic hydrocarbon with a thiol group, "C3-C 10 The "alicyclic hydrocarbon thiol" may include C3-C 10 Cycloalkylthiol, C3-C 10 Cycloalkenylthiol, C3-C 10 The cycloalkynylmercapto group is preferably a C3-C7 cycloalkanemercapto group, and more preferably a C3-C6 cycloalkanemercapto group or a C5-C6 cycloalkanemercapto group.

[0057] The term "C6-C 12 The term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group containing 6 to 12 carbon atoms and no heteroatoms, such as phenyl and naphthyl.

[0058] The term "5-12 membered heteroaryl" as used in the present invention refers to a 5-12 membered aromatic hydrocarbon ring containing 1-4 atoms selected from nitrogen, oxygen or sulfur atoms, and the remaining atoms are carbon atoms, including monocyclic, bicyclic or condensed rings. Examples of heteroaryl include, but are not limited to, pyrrolyl, triazolyl, thiadiazolyl, tetrazolyl, imidazolyl, pyrazolyl, isothiazolyl, thiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, furazanyl, furanyl, thienyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl and triazinyl.

[0059] The term "C6-C 12 Aryl C1-C 10 "Hydrocarbon" refers to C6-C 12 Aryl and C1-C 10 Hydrocarbon-linked groups, including C6-C 12 Aryl C1-C 10 Alkyl, C6-C 12 Aryl C2-C 10 Alkenyl, C6-C12 Aryl C2-C 10 Alkynyl, preferably C6-C 12 Aryl C1-C 10 Alkyl, more preferably C6-C 12 Aryl C1-C4 alkyl, such as benzyl, phenethyl or phenylpropyl.

[0060] The term "5-12 membered heteroaryl C1-C 10 "Hydrocarbon" refers to a 5-12 membered heteroaryl group with a C1-C 10 Hydrocarbon-linked groups, including 5-12 membered heteroaryl C1-C 10 Alkyl, 5-12 membered heteroaryl C2-C 10 Alkenyl, 5-12 membered heteroaryl C2-C 10 Alkynyl, preferably 5-12 membered heteroaryl C1-C 10 The hydrocarbon alkyl group is more preferably a 5-12 membered heteroaryl C1-C4 alkyl group, such as pyrrolylmethyl, furanylmethyl or thienylethyl.

[0061] The term "oxo" used in the present invention refers to "=O", that is, an oxygen atom is connected to other atoms through a double bond.

[0062] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, used in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0063] The term "pharmaceutically acceptable salt" as used herein refers to the form of salts corresponding to the compounds of the present invention, which are prepared by reacting the compounds with specific substituents obtainable by the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic groups, base addition salts can be obtained by adding a sufficient amount of base to the solution to contact the neutral form of such compounds. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amines or magnesium salts and the like. When the compounds of the present invention contain relatively basic groups, acid addition salts can be obtained by adding a sufficient amount of acid to the solution to contact the neutral form of such compounds. Pharmaceutically acceptable acid addition salts include inorganic acid salts and organic acid salts, the inorganic acid includes hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, etc.; the organic acid includes acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, lactic acid, mandelic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. The compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any acid or base addition salts.

[0064] Main reagents and consumables

[0065] The gene sequences of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 were commissioned to be synthesized by GENEWIZ and cloned into the commonly used plasmid pET28a or pET22b of Escherichia coli. The competent expression strain E. coli BL21 (DE3) was purchased from Sangon Biotech.

[0066] The antibiotics (kanamycin, ampicillin), culture medium components Tryptone, Yeastextract and NaCl, agar and the inducer isopropyl β-D-thiogalactoside (IPTG) required for protein expression used in the present invention were purchased from Sangon Biotech. The oxoacetaldehyde substrates required for the synthesis of the target compound were synthesized according to references or purchased from Bidex Pharmaceuticals. L-threonine and glycine were purchased from Bidex Pharmaceuticals, and the cofactor pyridoxal phosphate (PLP) was purchased from J&K SCIENTIFIC LTD; the PfKMO reaction substrate L-kynurenine was purchased from ACMEC, and the cofactors reduced nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD) and magnesium formate were purchased from Bidex Pharmaceuticals. Deuterated reagents were purchased from J&K SCIENTIFIC LTD. Ni NTA unionrose 6FF used for protein purification was purchased from Union Biotech.

[0067] Synthesis method

[0068] The present invention provides an enzyme-catalyzed method for synthesizing a two-handed center 2-amino-3-hydroxy-4-oxobutyric acid derivative, which is achieved by the following route:

[0069]

[0070] Wherein, Ra and n are defined as above;

[0071] (1) providing a reaction system, wherein the reaction system comprises a cofactor;

[0072] (2) In the reaction system, compound a reacts with glycine or threonine in the presence of a catalyst to obtain a compound of formula (I).

[0073] The catalyst is selected from the group consisting of threonine aldolase LTA, threonine transaldolase LTTA, or a combination thereof;

[0074] The threonine may be D-threonine or L-threonine.

[0075] The catalyst may have a sequence selected from the group consisting of:

[0076] (a) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0077] (b) An amino acid sequence obtained by replacing, deleting or inserting one or more amino acids based on SEQ ID NO: 1 or SEQ ID NO: 3 while maintaining the enzyme activity.

[0078] The coding gene sequence of the enzyme can be selected from the following group:

[0079] (a) the nucleotide sequence described in SEQ ID NO: 2 or SEQ ID NO: 4;

[0080] (b) a polynucleotide complementary to the sequence defined in (a); or

[0081] (c) any polynucleotide or complementary sequence having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity with the sequence defined in (a).

[0082] The sources of LTA or LTTA enzyme in the present invention include but are not limited to Pseudomonas, Escherichia coli, Candida, nematodes, etc.; preferably, the LTA or LTTA enzyme is a coding sequence derived from Pseudomonas.

[0083] The present invention also provides a recombinant vector carrying the above-mentioned gene, wherein the coding gene of LTA or LTTA enzyme is introduced into an expression vector through molecular cloning technology, and Escherichia coli or yeast is used as a host for heterologous expression of the protein. The expression vector includes but is not limited to Escherichia coli expression vectors such as pETDuet, pACYCDuet, pRSFDuet, pET28a or pET22b.

[0084] In the present invention, the recombinant plasmid containing the LTA or LTTA gene sequence is transformed into competent E. coli cells, and 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) is used to induce protein expression at 18°C ​​for 20 hours. The pure LTA and LTTA enzymes and bacterial cells have catalytic activity at 20-50°C, preferably 25-30°C.

[0085] The reaction has catalytic activity at pH 5.0-10.0, preferably pH 8.0.

[0086] Metal ion Mg 2+ , Ca 2+ It has a promoting effect on the catalytic activity of threonine transaldolase LTTA, preferably with the same concentration of Mg as the substrate. 2+ ;

[0087] The reaction time may be 1 to 12 hours. Preferably, the reaction time of LTA is 1 to 2 hours, and the reaction time of LTTA is 4 to 6 hours.

[0088] In the reaction system, the molar ratio of oxoacetaldehyde compounds to glycine is 1:10-10000, preferably 1:50, the initial concentration of the oxoacetaldehyde substrate is 0.01-1000mM, preferably 10mM, and the PLP concentration is 0.001-10mM, preferably 0.1mM; in the LTTA-catalyzed reaction system, the molar ratio of oxoacetaldehyde compounds to L-threonine is 1:1-500, preferably 1:10, the initial concentration of the oxoacetaldehyde substrate is 0.01-1000mM, preferably 5mM; the PLP concentration is 0.001-10mM, and the PLP concentration is preferably 0.2mM.

[0089] The concentration of LTA enzyme can be 0.01 to 10 mg / mL, preferably 0.5 mg / mL; the concentration of LTTA enzyme can be 0.01 to 10 mg / mL, preferably 1 mg / mL.

[0090] The reaction system may use a buffer including but not limited to the following: phosphate buffer, Tris-HCl buffer, ammonium acetate buffer and HEPES-NaOH buffer.

[0091] The reaction system may contain a cosolvent, which may be selected from organic solvents such as dimethyl sulfoxide, methanol, ethanol, isopropanol, etc. Preferably, the cosolvent is dimethyl sulfoxide to enhance the solubility of the substrate. The cosolvent accounts for 5-25% of the total reaction volume.

[0092] The main advantages of the present invention include:

[0093] 1. The method of the present invention has high reaction efficiency, mild reaction conditions and is easy to operate.

[0094] 2. When LTA is used as a catalyst in the method of the present invention, the catalytic efficiency and yield are high.

[0095] 3. When the method of the present invention uses LTTA as a catalyst, the selectivity is high and a high-purity single-configuration product can be obtained.

[0096] The present invention is described in detail below by way of examples, and it should be noted that the following examples do not limit the present invention in any way. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments exemplified below, and other biocatalytic or chemical synthesis methods, and those skilled in the art can replace them by equivalent means well known to themselves.

[0097] Example 1: Gene acquisition and expression purification

[0098] The amino acid sequences of PpLTA from Pseudomonas putida, BuLTTA from Burkholderia diffusa, and PfKMO from Burkholderia diffusa (shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5) were obtained from the PDB database, and GENEWIZ was commissioned to perform codon optimization and gene sequence synthesis, and the optimized nucleotide sequences were cloned into vectors pET28a or pET22b, as shown in the attached figure. Figure 1 The recombinant plasmid was then transformed into the competent cell of E. coli BL21 (DE3), cultured at 37°C for 12 h, and a single colony was picked up with a sterile inoculation loop in a clean bench and inoculated into 100 ml of liquid LB medium containing the corresponding antibiotics (50 μg / ml kanamycin or 100 μg / ml ampicillin). After overnight culture at 37°C and 220 rpm, the inoculation volume was transferred to 1 L of liquid LB medium containing the corresponding antibiotics at a 1% inoculation rate, and cultured at 37°C and 220 rpm until OD 600 When the pH value was 0.8 to 1.0, the temperature was lowered to 18°C ​​and the reaction mixture was kept at 180 rpm. IPTG with a final concentration of 0.1 mM was added to induce expression for 20 h. The bacterial cells were then collected by centrifugation at 9000 rpm for 15 min.

[0099] The collected bacterial cells were resuspended in cell lysis buffer and broken by high-pressure homogenizer to release the target protein. The supernatant was collected by centrifugation at 4°C and 15000rpm for 30min, and the protein was purified by Ni NTA Unionrose 6FF. The sample was repeated 3 to 4 times, and the impurities were washed away with lysis buffer containing 25mM imidazole, and the target protein was eluted with lysis buffer containing 250mM imidazole. The target protein was obtained after dialysis.

[0100] The cell lysis buffer used for PpLTA and BuLTTA was: 50 mM Na2HPO4, 300 mM NaCl, pH 8.0; the dialysate was: 50 mM Na2HPO4, 50 mM NaCl, pH 7.4.

[0101] The cell lysis buffer used for PfKMO was: 50 mM Tris, 150 mM NaCl, pH 7.5; the dialysate was: 50 mM Na2HPO4, 50 mM NaCl, pH 7.5.

[0102] Example 2: Synthesis of 2-amino-3-hydroxy-4-oxobutane derivatives catalyzed by threonine aldolase and threonine transaldolase

[0103] According to the amount of target compound required for subsequent experiments, the reaction volume was determined to be 30 mL. Through research on relevant literature, the reaction system was determined as follows:

[0104] Table 2 Threonine aldolase PpLTA reaction system

[0105]

[0106] The preferred reaction buffer is 50 mM Na2HPO4, 50 mM NaCl, pH 7.4; the reaction is carried out at room temperature on a magnetic stirrer for 1 hour.

[0107] Table 3 Threonine transaldolase BuLTTA reaction system

[0108]

[0109] The preferred reaction buffer is 100 mM HEPES-NaOH, pH 8.0; the reaction is carried out on a magnetic stirrer at room temperature for 4 hours.

[0110] Example 3: Purification of target product, calculation and characterization of dr value and yield

[0111] (1) After the reaction, the reaction solution was centrifuged and the supernatant was taken out using a rapid preparative liquid chromatography column Column (SD-8101-120, 400g), acetonitrile / water (water phase containing 0.1% acid) system gradient elution to purify the product, collect the components containing the product, remove the solvent by rotary evaporator to obtain the product, weigh the product and calculate the yield. Yield calculation formula: product yield = m / M × 100%, where m is the actual product mass; M is the theoretical product mass.

[0112] (2) Take 20 μL of the reaction solution after the reaction is completed, dilute it with 180 μL of 50% methanol, mix it, centrifuge it to obtain the supernatant, and detect it by UPLC-MS. Integrate the peak area of ​​the diastereomers to obtain the dr value of the carbon connected to the β-hydroxyl group of the product. Detection conditions: Detection wavelength: 220 nm; Chromatographic column: ACQUITY BEH C18 (2.1×50mm, 1.7μm); mobile phase: water (0.1% formic acid) / acetonitrile (0.1% formic acid); gradient: 5-95%; acquisition time: 4.5min; flow rate: 0.8mL / min; column temperature: 40℃; injection volume: 2μL.

[0113] (3) Take a small amount of the purified product, add about 600 μL of deuterated water, centrifuge to obtain the supernatant, and collect the compound 1 H NMR, 13 C NMR and fluorinated compounds 19 The structure of the compound was identified by combining F NMR spectrum with high-resolution mass spectrometry. The compound characterization data are shown in Table 6.

[0114] PpLTA has a high catalytic efficiency, but the stereoselectivity of PpLTA for the product β-carbon is very poor. Based on this feature, the present invention can use PpLTA to catalyze the reaction and separate and purify to obtain two products with different β-carbon configurations, thereby expanding the scope of the initial screening. The substrate structure used in the PpLTA catalytic reaction, the obtained product structure and yield are shown in Table 4;

[0115] After preliminary screening of the KMO inhibitory activity of the above compounds (screening scheme see Example 4, the preliminary screening results are shown in the attached Figure 2 As shown), a batch of compounds with good inhibitory activity were obtained. Summarizing the rules, it can be seen that the inhibitory activity of (2S, 3S) type products on KMO is significantly better than that of (2S, 3R) type products. Although the catalytic efficiency of BuLTTA is lower than that of PpLTA, it has strong stereoselectivity for the β-carbon of the product. Based on this feature, threonine transaldolase BuLTTA was used as a catalyst, and several compounds with strong inhibitory activity were selected. According to the reaction system in Example 2-Table 3, (2S, 3S) type products can be synthesized with high selectivity. The substrate structure used in the BuLTTA-catalyzed reaction, the structure of the obtained product, the dr value, and the yield are shown in Table 5;

[0116] The summary of the NMR and high-resolution mass spectra of the 50 compounds obtained above is shown in Table 6:

[0117] Table 4 Threonine aldolase PpLTA reaction yield

[0118]

[0119]

[0120]

[0121] Table 5 dr value and yield of threonine transaldolase BuLTTA reaction

[0122]

[0123]

[0124] Table 6 NMR and HRMS (ESI)

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Example 4: Screening of KMO inhibitors

[0139] After obtaining the above 50 target compounds, these compounds were preliminarily screened for KMO inhibitory activity, that is, L -kynurenine ( L -KYN) hydroxylation reaction system, the above compounds were additionally added to observe their inhibitory effect on the PfKMO reaction. The reaction volume was set to 200 μL, the substrate L -The kynurenine concentration was 2 mM, and the experimental groups to which the above compounds were added were set at two concentrations: 500 μM and 250 μM. The reaction system was shown in Table 7.

[0140] Table 7 Reaction system used for PfKMO inhibitor screening

[0141]

[0142]

[0143] The preferred buffer for the reaction is 25 mM HEPES-NaOH pH 8.0 buffer. The target compound is dissolved in the buffer. Some poorly soluble compounds require the addition of acid or base to promote dissolution.

[0144] According to the above reaction system, add L -KYN components were mixed, PfKMO and the target compound were fully mixed and incubated for 2 minutes, and then added L -KYN. It contains only LThe reaction of -KYN was used as the control group. On this basis, a blank control group without adding PfKMO was set up. The reaction was carried out at 26°C for 1 h, and 800 μL of 50% methanol-water was added to quench the reaction. The peak area of ​​the reaction product 3-OH-KYN in each experimental group was measured by UPLC-MS to determine the percentage of the peak area of ​​the control group. The lower the percentage, the stronger the inhibitory activity.

[0145] UPLC-MS detection conditions: detection wavelength: 220nm; chromatographic column: ACQUITY HSS T3 (2.1×50mm, 1.8μm); mobile phase: water (0.1% formic acid) / acetonitrile (0.1% formic acid); gradient: 0-95%; acquisition time: 4.5min; flow rate: 0.8mL / min; column temperature: 40℃; injection volume: 1μL.

[0146] The inhibitor screening results are shown in the attached Figure 2 Among these compounds, 3b, 6b, 7b, 9b, 10b, 12b, 19a, 19b, 24a, 24b, 25a and 25b showed high inhibitory activity in the initial screening. Among these 12 compounds, the six compounds with the strongest inhibitory activity, namely 6b, 9b, 19b, 24b, 25a and 25b, were finally determined for IC 50 Determination of.

[0147] Example 5: KMO Inhibitor IC 50 Determination of

[0148] In IC 50 In the assay protocol, the reaction volume was set to 200 μL and the substrate L -KYN concentration was 200 μM,

[0149] The concentration of PfKMO was 0.02 mg / mL, and the proportions of the other reaction components were consistent with those in Example 4. Within the concentration range of 0.0001 to 2000 μM, 10 to 15 concentration gradients were set for the above 6 compounds and the currently known commercial KMO inhibitor GSK180. The reaction without additional compound addition was used as the control group, with 3 parallels in each group. The reaction conditions and detection methods were consistent with those in Example 4. The reaction system is shown in Table 8.

[0150] Table 8 IC of target compounds 50 Reaction system used for determination

[0151]

[0152] The inhibition rate of each group was calculated according to the peak area of ​​the product 3-OH-KYN according to the formula, and the dose-effect curve was fitted by Graphpad to obtain the IC value of each compound under the reaction conditions. 50It should be pointed out that IC 50 It is not the inhibition constant of the compound, but will be affected by the concentration of substrate and enzyme used. Therefore, the IC obtained under different conditions is 50 There will be differences. Inhibition rate (%inhibition) calculation formula:

[0153] IC values ​​of the six compounds of the present invention were determined 50 As shown in Table 9, the corresponding dose-effect curves are shown in the attached Figure 3 .

[0154] Table 9 IC of target compounds 50

[0155]

[0156] The above six compounds obtained by the present invention all showed high KMO inhibitory activity, especially compound 25b. Under the same experimental conditions, IC 50 The concentration of KMO inhibitors is 0.21 μM, which shows an inhibitory activity comparable to that of the commercial inhibitor GSK180. Therefore, this type of compound has a high application value in the research and development of KMO inhibitors and can be applied to KMO-mediated neuropathies or inflammations.

[0157] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for synthesizing a compound of formula (I) or a pharmaceutically acceptable salt thereof, in, Ring A is selected from the following groups: C3-C8 saturated or partially unsaturated carbocyclic group, 3-12 membered saturated or partially unsaturated heterocyclic group, C6-C 10 Aryl, 5-12 membered heteroaryl; Each R a Each is independently selected from the following group: a deuterium atom, a halogen, a nitro group, a hydroxyl group, an amino group, a thiol group, a carboxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group, a C1-C6 haloalkoxy group, -C1-C6 alkyl-NH2, -NH-C1-C6 alkyl group, -C1-C6 alkyl-NH-C1-C6 alkyl group, a C1-C6 alkyl group substituted with a thiol group, a C2-C6 acyl group, a C3-C8 saturated or partially unsaturated carbocyclic group, a 3-12-membered saturated or partially unsaturated heterocyclic group, a C6-C 10 Aryl, 5-12 membered heteroaryl; *Carbon atoms can be in R or S configuration; Wherein, the heterocyclic group and heteroaryl group each independently and optionally include 1-4 ring atoms selected from the following group: N, O, S; The method comprises the steps of: (1) providing a reaction system, wherein the reaction system comprises a cofactor; (2) In the reaction system, compound a reacts with glycine or threonine in the presence of a catalyst to obtain a compound of formula (I); in, The catalyst is selected from the group consisting of threonine aldolase LTA, threonine transaldolase LTTA, a strain expressing threonine aldolase, a strain expressing threonine transaldolase, or a combination thereof.

2. The method according to claim 1, characterized in that The cofactor is selected from the group consisting of pyridoxal phosphate (PLP).

3. The method according to claim 1, characterized in that The cofactor concentration is 0.01mM to 1mM, preferably 0.02 to 0.5mM, and more preferably 0.05 to 0.2mM.

4. The method according to claim 1, characterized in that The catalyst concentration is 0.05-5 mg / mL, preferably 0.1-4 mg / mL, and more preferably 0.2-3 mg / mL.

5. The method according to claim 1, characterized in that The threonine aldolase LTA has an amino acid sequence as shown in SEQ ID NO:

1. In another preferred embodiment, the threonine aldolase LTA has an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 while maintaining the enzyme activity.

6. The method according to claim 1, characterized in that The threonine transaldolase LTTA has the amino acid sequence shown in SEQ ID NO:

3. In another preferred embodiment, the threonine transaldolase LTTA has an amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3 while maintaining the enzyme activity.

7. The method according to claim 1, characterized in that: The strain expressing threonine aldolase is a strain expressing a protein having an amino acid sequence as shown in SEQ ID NO: 1; The strain expressing threonine aldolase is a strain expressing a protein having an amino acid sequence as shown in SEQ ID NO:

3. In another preferred embodiment, the strain expressing threonine aldolase is a strain having a DNA with a nucleotide sequence as shown in SEQ ID NO: 2; The strain expressing threonine transaldolase is a strain having a DNA with a nucleotide sequence as shown in SEQ ID NO:

4.

8. The method according to claim 1, characterized in that The reaction system also includes metal ions, which are selected from the following group: Mg 2+ , Ca 2+ , or a combination thereof. In another preferred embodiment, the metal ion concentration is 0.01-5M, preferably 0.05-1M, and more preferably 0.1-0.5M.

9. The method according to claim 1, characterized in that The reaction is carried out at 10-50°C, preferably at 15-40°C, and more preferably at 20-30°C.

10. The method according to claim 1, characterized in that The method further comprises the steps of: (3) isolating the compound of formula (I) or a pharmaceutically acceptable salt thereof from the reaction system.

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