TWI905781B - Prolyl hydroxylase inhibitor and use thereof - Google Patents

Abstract

The present invention discloses a prolyl hydroxylase inhibitor, which belongs to the technical field of medicines. The present invention provides a pyridino-triazole compound having a structure represented by the formula below. The pyridino-triazole compound has relatively high prolyl hydroxylase inhibitory activity and EPO induction activity, can effectively treat and prevent HIF-related and/or EPO-related diseases, and provides a new thought for dealing with diseases such as anemia, ischemia, and hypoxia.

Description

Proline hydroxylase inhibitors and their uses

This invention belongs to the field of pharmaceutical technology, specifically relating to a pyridotriazole compound and its pharmaceutical composition that has prolyl hydroxylase inhibitory activity, and further relating to its preparation method and pharmaceutical use.

In cases of anemia, trauma, tissue necrosis, and loss, tissues or cells are often in a state of hypoxia. Hypoxia leads to the expression of a series of transcription factors, which are involved in angiogenesis, iron, glucose metabolism, and cell growth and proliferation. Among them, hypoxia-inducible factor (HIF) is a transcription factor activated in somatic cells under hypoxic conditions, responding to cellular hypoxia by mediating a series of gene regulations. HIF is a heterodimer containing an oxygen-regulated α-subunit (HIFα) and a β-subunit for compositional expression (HIFβ/ARNT). In oxygen-rich (normally oxygenated) cells, HIFα subunits are rapidly degraded via ubiquitination of the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Under hypoxic conditions, HIFα is not degraded, and the active HIFα/β complex accumulates in the cell nucleus, activating the expression of various genes, including glycolytic enzymes, glucose transporters, erythropoietin (EPO), and vascular endothelial growth factor (VEGF).

Erythropoietin (EPO) is a naturally occurring hormone produced in conjunction with HIFα. It stimulates the production of erythrocytes (red blood cells) that carry oxygen throughout the body. EPO is normally secreted by the kidneys, and endogenous EPO increases under conditions of reduced oxygen (hypoxia). All types of anemia are characterized by a reduced capacity of the blood to carry oxygen, and are thus accompanied by similar signs and symptoms, including pale skin and mucous membranes, weakness, dizziness, fatigue, and drowsiness, leading to a decline in quality of life. Anemia is usually associated with a lack of blood in red blood cells or hemoglobin. Common causes of anemia include deficiencies in iron, vitamin B12, and folic acid, and it can also occur as a complication of chronic diseases, such as inflammatory diseases, including those with secondary myelosuppression. Anemia is also associated with kidney dysfunction; most kidney failure patients who undergo dialysis regularly suffer from chronic anemia.

Prolyl hydroxylase (PHD) is a key regulator of HIF. Under normoxic conditions, PHD hydroxylates two key proline residues, Pro402 and Pro564, of HIFα, increasing its affinity for pVHL and accelerating its degradation. Under hypoxic and other pathological conditions, the PHD-catalyzed HIF reaction is inhibited, the rate of protease degradation slows down, and HIFα accumulates intracellularly, leading to a series of adaptive responses in the cell to hypoxia. Inhibiting PHD with PHD inhibitors prolongs the action of HIF, thereby increasing the expression of genes such as EPO, which can effectively treat and prevent HIF-related and/or EPO-related conditions, such as anemia, ischemia, and hypoxia.

For example, US Patent Application US16757333 discloses a crystal form of an alkynylpyridine prolylhydroxylase inhibitor and its preparation method, wherein the structural formula of the alkynylpyridine prolylhydroxylase inhibitor is as follows:

.

Currently, there are prolyl hydroxylase inhibitors on the market, including GlaxoSmithKline's Daprodustat, AstraZeneca's Roxadustat, and Nippon Tobacco Industries Co., Ltd.'s Enarodustat.

Due to the limited development of prolyl hydroxylase inhibitors and the high demand, there is an urgent need to develop such compounds to treat conditions such as anemia, ischemia, and hypoxia.

The purpose of this invention is to provide a pyridotriazole compound and its drug combination as an HIF-PHD inhibitor, which can be used to treat various HIF-related or EPO-related diseases, such as local ischemia and hypoxia.

To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

On the one hand, the present invention provides a compound of formula (I) or a stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvent, pharmaceutically acceptable salt or prodrug thereof;

(I)

R is selected from members of the following atoms or groups:

, , ,or ;

L is _-CH2- or _-CH2O- ; n is an integer between 0 and 2;

^R1 , ^R2 , and ^R3 are each independently selected from substituted or unsubstituted alkyl, cycloalkyl, aryl, or heterocyclic groups.

Preferably, the aryl group includes an aromatic heterocyclic ring.

In some embodiments, L is _-CH2- or _-CH2O- ; the value of n is 0 or 1;

^R1 is a substituted or unsubstituted _{C1-4 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is at least one of _{C1-4 alkyl} , _{C1-4 alkoxy} , _C1-4 halogenated alkyl, halogen, cyano, phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, _or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group;

^R2 is a substituted or unsubstituted _{C1-4 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is at least one of _{C1-4 alkyl} , _{C1-4 alkoxy} , _C1-4 halogenated alkyl, halogen, cyano, phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, _or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group;

^R3 is a substituted or unsubstituted _{C1-10 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is at least one of _{C1-4 alkyl} , _C1-4 alkoxy, _{C1-4 halogenated} alkyl, halogen, cyano, phenyl, or oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, or oxygen- and/or nitrogen _- containing 5-6-membered heterocyclic group.

R ⁴ is selected from at least one of hydrogen, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, aryl, heterocyclic.

In some embodiments, L is _-CH2- or _-CH2O- ; the value of n is 0 or 1;

^R1 is a substituted or unsubstituted phenyl group, wherein the substituent is at least one of _C1-4 alkyl, _C1-4 alkoxy, _C1-4 halogenated alkyl, halogen, cyano, phenyl, or a 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen, or a 5-6 member heterocyclic group containing oxygen and/or nitrogen.

^R2 is a substituted or unsubstituted phenyl group, wherein the substituent is at least one of _C1-4 alkyl, _C1-4 alkoxy, _C1-4 halogenated alkyl, halogen, cyano, phenyl, 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen, and 5-6 member heterocyclic group containing oxygen and/or nitrogen.

^R3 is a substituted or unsubstituted _{C1-10 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is at least one of _{C1-4 alkyl} , _{C1-4 alkoxy} , halogen, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group.

^R4 is at least one of hydrogen, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, aryl, and heterocyclic groups.

Alternatively, ^R4 is hydrogen, a substituted or unsubstituted _{C1-10 alkyl} , _C1-10 alkoxy, phenyl, a 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen, or a 5-6 member heterocyclic group containing oxygen and/or nitrogen, wherein the substituent is at least _one of _{C1-4 alkyl} , _C1-4 alkoxy, _{C1-4 halogenated} alkyl, halogen, cyano, phenyl, or a 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen.

Alternatively, ^R4 is hydrogen, a substituted or unsubstituted _{C1-10 alkyl} , _C1-10 alkoxy, phenyl, _a 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen, or a 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen, wherein the substituent is at least one of _{C1-4 alkyl} , _C1-4 alkoxy, halogen, a 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen, or a 5-6 member aryl heterocyclic group _containing oxygen and/or nitrogen.

In some implementations, the value of n is 0 or 1;

^R1 is a substituted or unsubstituted phenyl group, wherein the substituent is a _C1-4 alkyl, _C1-4 alkoxy, halogen, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group; preferably, the _C1-4 alkyl, _C1-4 alkoxy, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure;

^R2 is a substituted or unsubstituted phenyl group, wherein the substituent is a _C1-4 alkyl, _C1-4 alkoxy, halogen, or a 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen; preferably, the _C1-4 alkyl, _C1-4 alkoxy, and 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen are para-substituted relative to the parent nucleus, and the halogen is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

^R3 is a substituted or unsubstituted _{C1-5 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is a _C1-4 alkyl, _C1-4 alkoxy, halogen, oxygen- and/or nitrogen-containing 5-6 _- membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group; preferably, _{the C1-4} alkyl, _C1-4 alkoxy, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure.

^R4 is _hydrogen , substituted or unsubstituted _{C1-5 alkyl} , _C1-5 alkoxy, phenyl, oxygen- and/or nitrogen-containing 5-6 member aromatic heterocyclic group or oxygen- and _/ or nitrogen-containing 5-6 member heterocyclic group, wherein the substituent is _C1-4 alkyl, _C1-4 alkoxy, halogen, oxygen- and/or nitrogen _- containing 5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group; preferably, the _C1-4 alkyl, _C1-4 alkoxy, oxygen- and/or nitrogen-containing 5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure.

In some implementations, the value of n is 0 or 1;

^R1 is a substituted or unsubstituted phenyl group, wherein the substituent is a _C1-3 alkyl, _C1-3 alkoxy, halogen, oxygen- and/or nitrogen-containing 5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group; preferably, the _C1-3 alkyl, _C1-3 alkoxy, oxygen- and/or nitrogen-containing 5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure;

^R2 is a substituted or unsubstituted phenyl group, wherein the substituent is a _C1-3 alkyl, _C1-3 alkoxy, halogen, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group; preferably, the _C1-3 alkyl, _C1-3 alkoxy, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure;

^R3 is a substituted or unsubstituted _{C1-5 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is a _C1-3 alkyl, _C1-3 alkoxy, halogen, oxygen- and/or nitrogen-containing 5-6 _- membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group; preferably, _{the C1-3} alkyl, _C1-3 alkoxy, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure.

^R4 is hydrogen, substituted or unsubstituted _{C1-5 alkyl} , _C1-5 alkoxy, phenyl, oxygen- and/or nitrogen-containing 5-6 member aryl heterocyclic group, or oxygen- and/or nitrogen _- containing 5-6 member aryl heterocyclic group, wherein the substituent is _C1-3 alkyl, _{C1-3 alkoxy} , halogen, oxygen- and/or nitrogen-containing 5-6 member aryl heterocyclic group, or oxygen- and/or nitrogen-containing 5-6 member aryl heterocyclic group; preferably, the _C1-3 alkyl, _C1-3 alkoxy, oxygen- and/or nitrogen-containing 5-6 member aryl heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member aryl heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure.

In some implementations, the value of n is 0 or 1;

^R1 is a substituted or unsubstituted phenyl group, wherein the substituent is a _C1-2 alkyl, _C1-2 alkoxy, F, Cl, Br, I, or a 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen; preferably, the _C1-2 alkyl, _C1-2 alkoxy, or 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen is para-substituted relative to the parent nucleus, and the F, Cl, Br, I is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

^R2 is a substituted or unsubstituted phenyl group, wherein the substituent is a _C1-2 alkyl, _C1-2 alkoxy, F, Cl, Br, I, or a 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen; preferably, the _C1-2 alkyl, _C1-2 alkoxy, or 5-6 member aryl heterocyclic group containing oxygen and/or nitrogen is para-substituted relative to the parent nucleus, and the F, Cl, Br, I is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

^R3 is a substituted or unsubstituted _{C1-4 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, wherein the substituent is a _C1-2 alkyl, _C1-2 alkoxy, F, Cl, Br, I, oxygen- and/or nitrogen-containing _5-6 -membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group; preferably, _{the C1-2} alkyl, _C1-2 alkoxy, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure.

^R4 is _hydrogen , substituted or unsubstituted _C1-4 alkyl, _C1-4 alkoxy, phenyl, oxygen- and/or nitrogen _- containing 5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group, wherein the substituent is _C1-2 alkyl, _C1-2 alkoxy, F, Cl, Br, I, oxygen- and/or nitrogen-containing 5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group; preferably, _{the C1-2 alkyl} , _C1-2 alkoxy, oxygen- and/or nitrogen-containing _5-6 member aromatic heterocyclic group or oxygen- and/or nitrogen-containing 5-6 member heterocyclic group is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted or para-substituted relative to the parent nucleus structure.

In some implementations, the value of n is 0 or 1;

^R1 is a substituted or unsubstituted phenyl group, wherein the substituent is methyl, methoxy, F, Cl, 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen, or 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen; preferably, the methyl, methoxy, 5-6 member aromatic heterocyclic group containing oxygen and/or nitrogen is para-substituted relative to the parent nucleus, and the F and Cl are ortho-, meta-, or para-substituted relative to the parent nucleus.

^R2 is a substituted or unsubstituted phenyl group, wherein the substituent is methyl, methoxy, F, Cl or a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen; preferably, the methyl, methoxy and the 5-6 member aromatic heterocycle containing oxygen and/or nitrogen are para-substituted relative to the parent nucleus, and the F and Cl are ortho-, meta-, or para-substituted relative to the parent nucleus.

^R3 is a substituted or unsubstituted _{C1-3 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocycle, wherein the substituent of the substituted phenyl is methyl, methoxy, F, Cl, or an oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocycle; preferably, the methyl, methoxy, and oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocycle are para-substituted relative to the parent nucleus, and the halogen is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

^R4 is hydrogen, a substituted or unsubstituted _{C1-3 alkyl} , _C1-3 alkoxy, phenyl, or _a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen, wherein the substituent of the substituted phenyl group is methyl, methoxy, F, Cl, or a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen; preferably, the methyl, methoxy, and 5-6 member aromatic heterocycle containing oxygen and/or nitrogen are para-substituted relative to the parent nucleus, and the halogen is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

In some implementations, the value of n is 0 or 1;

R ¹ is a substituted or unsubstituted phenyl group, wherein the substituent is a methyl group or a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen; preferably, the methyl group, the 5-6 member aromatic heterocycle containing oxygen and/or nitrogen is para-substituted relative to the parent nucleus structure.

^R2 is a substituted or unsubstituted phenyl group, wherein the substituent is a methyl group or a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen; preferably, the methyl group or the 5-6 member aromatic heterocycle containing oxygen and/or nitrogen is para-substituted relative to the parent nucleus structure.

^R3 is a substituted or unsubstituted _{C1-3 alkyl} , phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocycle, wherein the substituent is methyl, methoxy, F, Cl, or oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocycle; preferably, the methyl, methoxy, and oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocycle are para-substituted relative to the parent nucleus, and the halogen is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

^R4 is a hydrogen, substituted or unsubstituted _{C1-3 alkyl} , _C1-3 alkoxy, phenyl, or _a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen, wherein the substituent is methyl, methoxy, F, Cl, or a 5-6 member aromatic heterocycle containing oxygen and/or nitrogen; preferably, the methyl, methoxy, and 5-6 member aromatic heterocycle containing oxygen and/or nitrogen are para-substituted relative to the parent nucleus, and the halogen is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus.

In some embodiments, n is 0 or 1; ^R1 is phenyl; ^R2 is phenyl; ^R3 is _a substituted or unsubstituted _C1-3 alkyl, phenyl, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic compound, wherein the substituent is methyl, methoxy, halogen, or oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic compound; preferably, the methyl, methoxy, oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic compound or oxygen- and/or nitrogen-containing 5-6-membered heterocyclic compound is para-substituted relative to the parent nucleus structure, and the halogen is ortho-substituted, meta-substituted, or para-substituted relative to the parent nucleus structure; ^R4 is hydrogen.

The term "parent nucleus structure" refers to the main structure of the compound shown in formula (I), excluding the R groups.

In some embodiments, the compound is selected from the following structures:

.

In some embodiments, the compound is selected from the following structures:

.

In some embodiments, the compound is selected from the following structures:

.

In some embodiments, the compound is selected from the following structures:

.

In some embodiments, the compound is selected from the following structures:

.

In some embodiments, the present invention provides a compound of formula (II) or a stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvent, pharmaceutically acceptable salt or prodrug thereof;

(II)

Where n is an integer between 0 and 3; _R1 is selected from mono- or poly-substituted hydrogen, halogen, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, aryl, or heterocyclic groups on the aromatic ring.

In some embodiments, the aryl group of a compound of formula (II) or a stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvent, pharmaceutically acceptable salt, or prodrug includes an aromatic heterocycle.

In some embodiments, the value of n in the compound of formula (II) or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvents, pharmaceutically acceptable salts or prodrugs is an integer between 0 and 2; _R1 is selected from hydrogen, halogens, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, aryl, heterocyclic groups.

In some embodiments, the value of n for the compound of formula (II) or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvents, pharmaceutically acceptable salts or prodrugs is 0 or 1; _R1 is selected from hydrogen, halogens, substituted or unsubstituted alkyl, cycloalkyl, alkoxy, aryl, heterocyclic groups.

In some embodiments, the value of n in the compound of formula (II) or its stereoisomers, geometric isomers, tautomers, oxynitride hydrates, solvents, pharmaceutically acceptable salts or prodrugs is 0 or 1; _R1 is hydrogen, a substituted or unsubstituted _C1-10 alkyl, _C1-10 alkoxy, phenyl, an oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, an oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group, and the substituent is at least one of _C1-4 alkyl, _C1-4 alkoxy, _C1-4 halogenated alkyl, halogen, cyano, phenyl, or an oxygen- and/or nitrogen-containing 5-6-membered aromatic heterocyclic group, an oxygen- and/or nitrogen-containing 5-6-membered heterocyclic group.

In some embodiments, the value of n is 1 for the compound of formula (II) or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvents, pharmaceutically acceptable salts or prodrugs; _R1 is hydrogen.

In some embodiments, the compound of formula (II) or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvents, pharmaceutically acceptable salts, or prodrugs are selected from the following structures:

.

The enantiomers of the two compounds exhibited excellent pharmacological effects in continuous chronic administration, with significant data showing that they significantly increased hemoglobin and EPO levels in SD rats, and the overall effect was significantly better than that of Enarodustat.

In some embodiments, the preparation of compounds of formula (II) uses the following synthetic route:

SFC stands for supercritical fluid chromatography.

This application also discloses the acquisition of chiral isomer A of [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridin-8-carbonyl)amino]acetic acid and [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)- A high-performance liquid chromatography (HPLC) method was used to analyze the chiral isomer B of [1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid, with the following chromatographic conditions: chromatographic column: chiral column (S,S)Whelk-O1; mobile phase A: carbon dioxide; mobile phase B: a mixed solution of propionitrile and isopropanol; elution was performed using an isocratic elution program; preferably, the volume ratio of propionitrile to isopropanol was 1:0.5-1.5, more preferably 1:1.

Furthermore, this application discloses a crystal of a compound, the structure of which is as follows:

.

The crystal belongs to the monoclinic crystal system, space group P2 ₁ , with cell parameters a = 13.2287(2) [Å], b = 5.10690(10) [Å], c = 13.7391(2) [Å], α = 90°, β = 104.3640(10)°, γ = 90°, cell volume V = 899.17(3) [Å], and minimum asymmetric unit number Z = 2. The crystal of the compound has an XRPD pattern as shown in Figure 11 or essentially as shown in Figure 11, where "essentially" means that the difference in the number of peaks is within 85%.

This application also discloses a method for preparing crystals of chiral isomer A of [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid, wherein the compound is prepared by a programmed cooling method using acetonitrile as a solvent; preferably, the compound described in claim 10 is added to acetonitrile while stirring at 50°C until the solution is clear, and then the temperature is programmed down to 30°C at a rate of 0.01°C/min, and then maintained at 30°C for 2 to 3 days.

This application also discloses crystals of another compound, the structure of which is as follows:

,

The crystal belongs to the monoclinic crystal system, space group P2 ₁ , with cell parameters a = 13.2296(2) [Å], b = 5.10250(10) [Å], c = 13.7423(2) [Å], α = 90°, β = 104.357(2)°, γ = 90°, cell volume V = 898.69(3) [Å], and minimum number of asymmetric members in the cell Z = 2. The crystal of the compound has an XRPD pattern as shown in Figure 5 or essentially as shown in Figure 5, where essentially means that the difference in the number of peaks is within 85%.

This application further discloses a method for preparing crystals of chiral isomer B of [(7-hydroxy-5-(1,2,3,4-tetrahydronaphth-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid, by using dimethyl sulfoxide as a solvent and slowly evaporating the compound at room temperature; preferably, the compound described in claim 13 is added to dimethyl sulfoxide while stirring at room temperature until the solution is clear, filtered, and the filtrate is placed in a container, capped with a perforated lid, and allowed to evaporate at room temperature.

In some embodiments, a pharmaceutical composition comprises a compound of formula (II) or a stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvent, pharmaceutically acceptable salt or prodrug, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

In some embodiments, the use of the compound represented by formula (II) or a stereoisomer, geometric isomer, tautomer, nitride, hydrate, solvent, pharmaceutically acceptable salt or prodrug, or the above-described drug composition, in the preparation of a drug for treating prolylhydroxylase-mediated diseases by inhibiting prolylhydroxylase.

In some embodiments, the diseases mediated by the inhibition of prolylhydroxylase are anemia, local ischemia, and hypoxia.

In some embodiments, the disease mediated by the inhibition of prolylhydroxylase is renal anemia.

In this invention, FG-4592 is roxadustat.

[(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid showed excellent pharmacological effects in continuous chronic administration, with significant data showing that it could significantly increase hemoglobin and EPO levels in SD rats, and the overall effect was significantly better than that of the positive drug Enarodustat.

Preferably, the intermediates prepared in each step of the present invention have a purity of over 95%.

Unless otherwise stated, the term "alkyl" as used herein includes branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms, including all isomers. Common abbreviations for alkyl include, for example, methyl can be represented by "Me" or CH ₃ , ethyl by "Et" or CH ₂ CH ₃ , propyl by "Pr" or CH ₂ CH ₂ CH ₃ , butyl by "Bu" or CH ₂ CH ₂ CH ₂ CH ₃ , etc. For example, "C _1-4 alkyl" (or "C ₁ -C ₄ alkyl") refers to straight-chain or branched alkyl groups having a specific number of carbon atoms, including all isomers. _{C 1-4} alkyl includes n-, iso-, secondary and tert-butyl, n- and isopropyl, ethyl and methyl. The terms "C _1-10 alkyl" etc. have similar meanings.

The term "alkoxy" refers to a straight-chain and branched alkyl group with a specified number of carbon atoms connected by an oxygen bridge.

The term "halogen" (or "halogenated") refers to fluorine, chlorine, bromine, and iodine (or fluorinated (F), chlorinated (Cl), brominated (Br), and iodinated (I)).

The term "aryl" refers to aromatic mono- and polycyclic ring systems, in which the carbon rings are fused together or linked together by single bonds. Common aryl groups include phenyl, naphthyl, and biphenyl.

The term "heterocyclic" refers to a ring structure composed of carbon atoms and non-carbon atoms. Examples of non-carbon atoms in the ring include nitrogen, oxygen, and sulfur. Common heterocyclic groups include pyridine, quinoline, tropane, phenothiazine, benzodiazepine, furan, pyrazolone, and pyrimidine.

The term "aromatic heterocycle" refers to a 5- or 6-membered monocyclic aromatic ring or a 7-12-membered bicyclic ring, composed of a carbon atom and one or more heteroatoms selected from N, O, and S. Examples of aromatic heterocycles include pyridinyl, pyrroloyl, pyrazinyl, pyrimidinyl, darazinyl, thiophene (or thiophenyl), thiazolyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, azole, isozolyl, diazolyl, thiazolyl, isothiazolyl and thiadiazolyl, benzotriazolyl, indole, isoyindolyl, indole, and dihydrogen. Indole, isodihydroindole, quinoxalinyl, quinazolinyl, serotonyl, chromium, isochoryl, tetrahydroquinolinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo-1,4-dienyl, imidazo(2,1-b)(1,3)thiazole and benzo-1,3-m-dioxanecyclopentenyl.

The aryl group in the term "substituted aryl" is defined as previously. When the substituent of the substituted aryl group is not specified, the substituent may be selected from the following groups, including but not limited to: halogen, _C1 - _{C20 alkyl} , _CF3 , NH2, N( _C1 - _C6 alkyl) ₂ , _NO2 , oxo, CN, _N3 , -OH, -O( _C1 - _C6 alkyl), _C3 - _C10 cycloalkyl, _C2 - _C6 alkenyl, C2- _C6 ynyl, ( _{C0 - C6} alkyl)S(O) _0-2- , aryl-S(O) _0-2- , (C0-C6 alkyl)S(O) _0-2 ( _C0 - _C6 alkyl)-, ( _C0 - _C6 alkyl)C(O)NH-, _H2NC (NH)-, -O( _C1 - _C20 alkyl)-, and -O( _C1 -C20 alkyl)-. (C_₆alkyl)CF _₃ , (C_₀-C_₆alkyl)C(O)-, (C_₀-C_₆alkyl) _OC (O)-, (C _₀-C_₆alkyl)_₂NC(O)-(C_₀-C_₆alkyl)O(C_₁-C_₆alkyl)-, (C _₀-C_₆alkyl)C(O)_₁-2(C₀-C₆alkyl)-, (C₀-C_₆alkyl)OC(O)NH-, aryl, aralkyl, heteroaryl, heterocyclic alkyl, halogen-aryl, halogen-aralkyl, halogen-heterocyclic, halogen-heterocyclic alkyl, cyano-aryl, cyano-aralkyl, cyano-heterocyclic and cyano-heterocyclic alkyl. The term "substituted phenyl" has a similar definition.

Unless otherwise stated, all ranges listed in this article are inclusive. For example, "n is an integer between 0 and 2" means that n can be 0, 1, or 2.

The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic base or acid. When the compounds of the present invention are acidic, their corresponding salts can be readily prepared from inorganic or organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (copper and copper ore), iron, ferrous iron, lithium, magnesium, manganese (manganese and manganese ore), potassium, sodium, zinc, etc. Preferred salts are ammonium, calcium, magnesium, potassium, and sodium. Salts prepared from organic bases include primary, secondary, and tertiary amines derived from natural and synthetic sources. Pharmaceutically acceptable, non-toxic organic alkalis that can form salts include arginine, betaine, caffeine, choline, N,N'-dibenzyl ethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylguanidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, dicyclohexylamine, lysine, methylglucosamine, morpholine, guanidine, guanidine, polyamine resins, procaine, purines, cocoa base, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When the compounds of the present invention are basic, their corresponding salts can be readily prepared from inorganic or organic acids. Such acids include, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, hydroxyethylsulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucilage, nitric acid, succinic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc.

The term "solvent" refers to a complex of variable stoichiometry formed by a solute (i.e., a compound of formula (I)) or a pharmaceutically acceptable salt thereof and a solvent that does not impede the biological activity of the solute. Examples of solvents include, but are not limited to, water, ethanol, and acetic acid. When the solvent is water, the solvent is called a hydrate. Hydrates include, but are not limited to, hemihydrates, monohydrates, sesquihydrates, dihydrates, and trihydrates.

The term "prodrug" refers to a functional derivative of the compound of this invention, which is readily converted into the desired compound in the body.

On the other hand, the present invention provides methods for preparing the above-mentioned compounds, including the following four synthetic routes:

Route 1:

Route 2:

Route 3:

Route 4:

Among them, ^R1 , ^R2 , ^R3 , and ^R4 have the same definitions as those mentioned above.

On the other hand, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvents, pharmaceutically acceptable salts or prodrugs, and one or more pharmaceutically acceptable carriers, diluents, and excipients.

In pharmaceutical compositions, the term "composition" includes products comprising an active ingredient and an inert component (pharmaceutical-acceptable excipient) constituting a carrier, as well as any product obtained directly or indirectly from the combination, complexation, or aggregation of two or more components, the decomposition of one or more components, or other types of reactions or interactions of one or more components. Therefore, pharmaceutical compositions of the present invention include any composition prepared by mixing compounds of formula (I) and/or formula (II), other active ingredients, and pharmaceutically acceptable excipients.

The pharmaceutical compositions of the present invention comprise a compound of formula (I) and/or formula (II) (or a pharmaceutically acceptable salt or solvent thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optional other therapeutic components or adjuvants. The pharmaceutical compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route of administration in any given case depends on the specific subject, the nature and severity of the condition to which the active ingredient is administered. The pharmaceutical compositions can be prepared by any method known in the field of pharmaceutics.

The active ingredient can be administered orally in solid or liquid dosage forms, such as capsules, tablets, tablets, sugar tablets, granules, and powders, and such as elixirs, syrups, emulsions, dispersions, and suspensions. The active ingredient can also be administered parenterally in sterile liquid dosage forms such as dispersions, suspensions, or solutions. Other dosage forms that can be used to administer active ingredients include ointments, creams, drops, transdermal patches, or powders for topical administration; ophthalmic solutions or suspensions (eye drops) for ophthalmic administration; sprays or powder formulations for inhalation or nasal administration; and creams, ointments, sprays, or suppositories for rectal or vaginal administration. Gelatin capsules contain the active ingredient and a powdered carrier, such as lactose, starch, cellulose derivatives, magnesium stearate, or stearic acid. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products to provide sustained drug release over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from air exposure, or they can be enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colorants and flavoring agents to increase patient acceptability. Generally, water, suitable oils, saline solutions, dextrose (glucose) solutions, and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are suitable carriers for parenteral solutions. Preferred parenteral solutions contain water-soluble salts of the active ingredient, suitable stabilizers, and buffers as needed. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts, as well as sodium EDTA, can also be used. Furthermore, the parenteral solution may contain preservatives such as benzoyl chloride, methylparaben, or propylparaben and chlorobutanol. For inhalation administration, the compounds of the invention can be conveniently delivered as a spray from a pressurized package or a nebulizer. The compounds can also be delivered in the form of a formulated powder, which can be inhaled according to the instructions of a powder inhaler device. Preferred delivery systems for inhalation are metered-dose inhalation (MDI) aerosols, which can be formulated as suspensions or solutions of compounds of formula (I) and/or formula (II) in suitable propellants, such as fluorocarbons or hydrocarbons. For ophthalmic administration, ophthalmic preparations can be formulated as solutions or suspensions of compounds of formula (I) and/or formula (II) in suitable ophthalmic carriers at appropriate weight percentages, thereby maintaining sufficient contact between the compound and the ocular surface for adequate penetration into the cornea and inner regions of the eye.

Useful pharmaceutical dosage forms for administering the compounds of the present invention include, but are not limited to, hard and soft gelatin capsules, tablets, parenteral injections, and oral suspensions.

When the compounds of the present invention are administered stepwise or in combination with other therapeutic agents, the same dosage form as described above may be used. When the drugs are administered in physical combination, the dosage form and route of administration should be selected based on the compatibility of the combined drugs. The compounds of the present invention may be administered as the sole active ingredient or in combination with a second active ingredient, the second active ingredient including those known to be effective in increasing erythropoietin levels in patients.

On the other hand, the present invention provides the use of compounds of formula (I) and formula (II) or their stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvents, pharmaceutically acceptable salts or prodrugs, or the above-described drug compositions in the preparation of drugs for treating prolylhydroxylase-mediated diseases by inhibiting prolylhydroxylase.

In some embodiments, the diseases mediated by the inhibition of prolylhydroxylase are anemia, local ischemia, and hypoxia.

In some embodiments, the anemia is renal anemia.

The beneficial effects of this invention are:

A novel compound is proposed as a prolyl hydroxylase inhibitor with high prolyl hydroxylase inhibitory activity and EPO induction activity. It can effectively treat and prevent HIF-related and/or EPO-related diseases, providing new insights for treating anemia, local ischemia, and hypoxia.

The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application. Those skilled in the art can make various changes and modifications to the invention based on the disclosed content, and these modifications should also fall within the scope of protection claimed in this application.

The invention will be further described below with specific examples. Unless otherwise specified, all chemical reagents used in the embodiments of the invention are obtained through conventional commercial means.

Example 1: Preparation of (7-hydroxy-5-(phenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid.

Step 1: Preparation of tert-butyl 5-(phenoxy)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

1.0 g of 5-iodo-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester, phenol (310 mg), 2-pyridinecarboxylic acid (55 mg), copper iodide (42 mg), potassium phosphate (942 mg), and dimethyl sulfoxide (5.0 mL) were added sequentially to a reaction flask, and the mixture was purged with nitrogen three times. The temperature was raised to 65 ^° C, and the reaction was carried out overnight until the reactants were completely reacted as monitored by TLC. After cooling to room temperature, the mixture was extracted with ethyl acetate, washed with saturated brine, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and precipitated by column chromatography to obtain 450 mg of a white solid.

MS m/z (ESI)[M+H] ⁺ : 418.30

Step 2: Preparation of 5-(phenoxy)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

450 mg of 5-(phenoxy)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was dissolved in 10 mL of a 1:1 mixture of toluene and tetrahydrofuran. Then, 0.5 mL of methanesulfonic acid was added and the mixture was stirred, maintaining the reaction temperature between 60 and 70 ^° C. The reaction was stopped when TLC monitoring showed complete reaction of the starting material. After cooling to room temperature, a small amount of ethyl acetate was added, and the mixture was filtered to obtain 440 mg of a white solid.

MS m/z (ESI) [M+H] ⁺ : 362.22

Step 3: Preparation of methyl [(7-(benzyloxy)-5-(phenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate:

5-(phenoxy)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid (440 mg), EDCI (280 mg), HoBt (197 mg), N,N-dimethylformamide (5.0 mL), and triethylamine (247 mg) were added sequentially to a reaction flask and stirred at room temperature for 10 minutes. Then, glycine methyl hydrochloride (306 mg) was added to the system, and the temperature was raised to 60 ^° C for 1.5 hours. The reaction was stopped, cooled to room temperature, and quenched with a saturated sodium bicarbonate aqueous solution. The mixture was filtered to obtain 300 mg of a yellow solid.

MS m/z (ESI) [M+H] ⁺ : 433.31

Step 4: Preparation of methyl [(7-hydroxy-5-(phenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate:

Methyl [(7-(benzyloxy)-5-(phenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate (300 mg) and palladium carbon (30 mg) were dissolved in a 1:1 mixture of methanol and tetrahydrofuran (4.0 mL). The mixture was stirred at room temperature under hydrogen atmosphere until the reaction was complete as monitored by TLC, at which point the reaction was stopped. The solvent was removed by reduced pressure concentration, and the product was separated by column chromatography to give 170 mg of product.

MS m/z (ESI) [M+H] ⁺ : 343.22

Step 5: Preparation of [(7-hydroxy-5-(phenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid:

[(7-hydroxy-5-(phenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]methyl acetate (170 mg) was dissolved in methanol (2.0 mL), followed by the addition of 30% sodium hydroxide aqueous solution (1.0 mL). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The pH was adjusted to between 1 and 2 with dilute hydrochloric acid, and the mixture was filtered to obtain 150 mg of product.

¹ H NMR (400 MHz, d-DMSO) δ: 14.52 (s, 1H), 12.95 (brs, 1H), 9.71 (s, 1H), 8.59 (s, 1H), 7.59–7.53 (m, 2H), 7.44–7.41 (m, 3H), 5.83 (s, 1H), 4.20 (d, J = 5.6 Hz, 2H)

MS m/z (ESI): 329.09

Example 2 Preparation of (5-(2-chlorophenoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with 428.1 mg of 2-chlorophenol to obtain 108.9 mg of product.

¹ H NMR (400 MHz, d-DMSO) δ: 14.58 (s, 1H), 12.97 (brs, 1H), 9.71 (t, J = 5.6 Hz, 1H), 8.63 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.47 (t, J = 7.2 Hz, 1H), 5.89 (s, 1H), 4.21 (d, J = 5.6 Hz, 2H)

MS m/z (ESI) [M+H] ⁺ : 363.17

Example 3 Preparation of (5-(3-chlorophenoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with 428.1 mg of 3-chlorophenol to obtain 201.6 mg of product.

¹ H NMR (400 MHz, d-DMSO)14.54 (s, 1H), 12.97 (brs, 1H), 9.72 (s, 1H), 8.58 (s, 1H), 7.62 (s, 1H), 7.57 (t, J = 8.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 6.10 (s, 1H), 4.21 (d, J = 5.6 Hz, 2H)

MS m/z (ESI) [M+H]+: 363.14

Example 4: Preparation of (5-(4-chlorophenoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with 428.1 mg of 4-chlorophenol to obtain 182.1 mg of product.

¹ H NMR (400 MHz, d-DMSO) δ: 14.54 (s, 1H), 12.96 (s, 1H), 9.72 (t, J = 5.6 Hz, 1H), 8.60 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.02 (s, 1H), 4.21 (d, J = 5.6 Hz, 2H)

MS m/z (ESI) [M+H] ⁺ : 363.14

Example 5: Preparation of (5-(p-tolyloxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with 356.9 mg of 4-methylphenol to obtain 87.7 mg of product.

¹ H NMR (400 MHz, d-DMSO) δ: 14.51 (s, 1H), 12.95 (s, 1H), 9.69 (t, J = 5.6 Hz, 1H), 8.59 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 5.77 (s, 1H), 4.20 (d, J = 5.6 Hz, 2H), 2.37 (s, 3H)

MS m/z (ESI) [M+H] ⁺ : 343.16

Example 6: Preparation of (5-(p-methoxyphenoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with (409.2 mg) 4-methoxyphenol to obtain 223.7 mg of product.

¹ H NMR (400 MHz, d-DMSO) δ: 14.51 (s, 1H), 12.96 (s, 1H), 9.70 (t, J = 5.6 Hz, 1H), 8.60 (s, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.11(d, J = 8.8 Hz, 2H), 5.72 (s, 1H), 4.21 (d, J = 5.6 Hz, 2H), 3.82 (s, 3H)

MS m/z (ESI) [M+H] ⁺ :359.22

Example 7 Preparation of (5-(3-morpholinophenoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with (590 mg) 3-morpholinophenol to obtain a product of 110.7 mg.

¹ H NMR (400 MHz, d-DMSO) δ: 14.51 (s, 1H), 12.95 (brs, 1H), 9.70 (t, J = 5.2 Hz, 1H), 8.59 (s, 1H), 7.39 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 12.0 Hz, 2H), 6.80 (d, J = 7.6 Hz, 2H), 5.82 (s, 1H), 4.21 (d, J = 5.6 Hz, 2H), 3.73 (t, J = 4.8 Hz, 4H), 3.18 (t, J = 4.8 Hz, 4H)

MS m/z (ESI) [M+H] ⁺ : 414.23

Example 8: Preparation of (5-(4-fluorophenoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with (369.6 mg) 4-fluorophenol to obtain a product of 239.3 mg.

¹ H NMR (400 MHz, d-DMSO) 14.53 (s, 1H), 12.98 (brs, 1H), 9.71 (s, 1H), 8.60 (s, 1H), 7.53–7.49 (m, 2H), 7.43–7.38 (m, 2H), 5.86 (s, 1H), 4.20 (d, J = 5.6 Hz, 2H)

MS m/z (ESI) [M+H] ⁺ :347.14

Example 9 Preparation of (5-(propoxy)-7-hydroxy-[1,2,4]triazolyl[1,5-a]pyridine-8-carbonyl)glycine.

Following the method of Example 1, phenol was replaced with (2 ml) n-propanol to obtain 20.1 mg of product.

¹ H NMR (400 MHz, d-DMSO) 14.51 (s, 1H), 12.92 (brs, 1H), 9.65 (t, J = 5.6 Hz, 1H), 8.47 (s, 1H), 6.44 (s, 1H), 4.39 (t, J = 6.4 Hz, 2H), 4.18 (d, J = 5.6 Hz, 2H), 1.91–1.82 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H)

MS m/z (ESI) [M+H] ⁺ : 295.12

Example 10: Preparation of [(7-hydroxy-5-(phenylethynyl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid.

Step 1: Preparation of tert-butyl 7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

In a reaction flask, 15 g of 7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was dissolved in a mixed solution of methanol and tetrahydrofuran (25 mL/50 mL), and stirred at room temperature under hydrogen atmosphere until the reaction was complete as monitored by TLC. The reaction was then stopped. Palladium carbon in the solution was removed by filtration, and the filtrate was concentrated under reduced pressure to give 11.7 g of crude product. MS m/z (ESI): [M+H] ⁺ : 236.24

Step 2: Preparation of 7-(tert-butyl pentapentyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

10.7 g of 7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was placed in a reaction flask and 65 mL of tetrahydrofuran was added, followed by 8.0 mL of triethylamine and 9.1 g of tert-pentachlor chloride. The reaction flask was purged with nitrogen three times and then sealed. The reaction mixture was heated to 60 ^° C and stirred overnight. The reaction was stopped when the reaction was confirmed to be complete by TLC. After the reaction solution was cooled to room temperature, it was extracted with ethyl acetate, washed successively with saturated sodium bicarbonate aqueous solution, saturated ammonium chloride aqueous solution, and saturated brine, and then concentrated under reduced pressure to obtain 12.0 g of a yellow solid.

MS m/z (ESI): [M+H] ⁺ :320.36

Step 3: Preparation of 5-iodo-7-(tert-pentayloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester

10.0 g of 7-(pentafenoxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was dissolved in tetrahydrofuran (50 mL), followed by the addition of elemental iodine (8.75 g), and the reaction temperature was maintained below -60 ^° C. After 5 minutes, hexamethyldisiloxane (62.7 mL, 1.0 M) was added in portions, and the mixture was stirred until the reaction was complete as monitored by TLC. The reaction was then quenched with ethyl acetate hydrochloride solution, extracted with ethyl acetate, and washed with sodium sulfite aqueous solution. The organic phase was separated and washed sequentially with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine. The organic phase was separated and concentrated under reduced pressure. The crude product was then subjected to column chromatography to obtain 5.9 g of product.

¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.36 (d, J = 5.2 Hz, 1H), 7.45 (d, J = 5.2 Hz, 1H), 2.31 (s, 6H)

MS m/z (ESI): [M+H] ⁺ : 446.26

Step 4: Preparation of tert-butyl 5-(phenylethynyl)-7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid

5-Iodo-7-(tert-butyl iodide)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester (5.9 g), copper iodide (70 mg), Pd(dppf) _Cl₂ (285 mg), tetrahydrofuran (30 mL), triethylamine (6.6 g), and phenylacetylene (2.1 g) were added sequentially to a reaction flask. The atmosphere was replaced with nitrogen three times, and the reaction was heated to 50 ^° C until the reactants were completely reacted as monitored by TLC. The reaction was then quenched by adding ammonia. The organic phase was separated by extraction with ethyl acetate. The ammonia layer was washed with hydrochloric acid, extracted with ethyl acetate, and the organic phases were then combined, concentrated under reduced pressure, and purified by column chromatography to give 1.9 g of a red solid.

MS m/z (ESI): [M+H] ⁺ : 336.36

Step 5: Preparation of 5-(phenylethynyl)-7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid

1.8 g of 5-(phenylethynyl)-7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was dissolved in 24 mL of a 2:1 mixture of toluene and ethyl acetate. Then, 2.1 g of methanesulfonic acid was added and the mixture was stirred, maintaining the reaction temperature between 60 and 70 ^° C. The reaction was stopped when TLC monitoring showed complete reaction of the starting material. After the reaction solution cooled to room temperature, 10 mL of ethyl acetate was added, precipitating a large amount of solid. Filtration yielded a green solid. The solid was then dissolved in a small amount of N,N-dimethylacetamide, and ethyl acetate was added to the system, precipitating the solid again. Filtration yielded 1.0 g of crude product.

MS m/z (ESI): [M+H] ⁺ : 280.26

Step 6: Preparation of methyl [(7-hydroxy-5-(phenylethynyl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate

In reaction flask A, 0.8 g of 5-(phenylethynyl)-7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid was dissolved in 20 mL of dichloromethane, and 0.76 g of oxaloyl chloride was added dropwise, stirring at room temperature. In reaction flask B, 0.68 g of glycine methyl hydrochloride, 20 mL of dichloromethane, and 3.0 mL of triethylamine were added sequentially, and stirring was carried out at 0 ^° C. TLC monitoring in reaction flask A showed complete reaction of the raw materials. Stirring in reaction flask A was stopped, and the material from reaction flask A was added to reaction flask B. Stirring was carried out at 0 ^° C until TLC monitoring showed complete reaction of the raw materials, and the reaction was quenched with water. Extracted with dichloromethane, the organic phase was washed sequentially with saturated ammonium chloride aqueous solution and saturated sodium bicarbonate aqueous solution. The organic phases were separated and combined, concentrated under reduced pressure, and then separated by column chromatography to obtain 0.8 g of yellow solid.

MS m/z (ESI): [M+H] ⁺ : 351.33

Step 7: Preparation of [(7-hydroxy-5-(phenylethynyl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid

Methyl [(7-hydroxy-5-phenylethynyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate (0.8 g) was dissolved in ethanol (10 mL), followed by the addition of sodium hydroxide aqueous solution (18 mL, 2.0 M). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The pH was adjusted to 4-5 with dilute hydrochloric acid, and the mixture was filtered to obtain 0.42 g of a pale yellow solid.

¹ H NMR (400 MHz, d-DMSO) δ: 14.33 (s, 1H), 13.04 (s, 1H), 9.90 (s, 1H), 8.64 (s, 1H), 7.74–7.72 (m, 2H), 7.61–7.53 (m, 3H), 7.37 (s, 1H), 4.24 (d, J = 5.6 Hz, 2H)

MS m/z (ESI): [M+H] ⁺ : 337.31

Example 11 Preparation of [(7-hydroxy-5-(styryl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid.

Step 1: Preparation of tert-butyl 5-(styryl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

500 mg of 5-iodo-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester, _70.2 mg of Pd( _PPh3 ) _2Cl2 , 360 mg of sodium carbonate, and 200 mg of styrene-boronic acid were dissolved in a 1:1 mixture of ethylene glycol dimethyl ether and degassed water (30 mL), and the mixture was purged with nitrogen three times. The mixture was then stirred at 80 ^° C until the reaction was complete as monitored by TLC, at which point heating was stopped. After the reaction solution cooled to room temperature, it was extracted with ethyl acetate and washed with saturated brine. The organic phase was separated, concentrated under reduced pressure, and separated by column chromatography to give 340 mg of crude product.

Step 2: Preparation of 5-(styryl)-7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

290 mg of 5-(styryl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was dissolved in 10 mL of dichloromethane and cooled to -65 ^° C. Then, 3.4 mL of boron tribromide in dichloromethane (1.0 M) was added to the reaction system and stirred until the reactants were completely reacted as monitored by TLC. The reaction was stopped, and the solution was concentrated under reduced pressure. The residue was then added to a mixture of water (10 mL) and methanol (3.0 mL) and stirred at room temperature for 1 hour. The mixture was then filtered to obtain 250 mg of solid.

MS m/z (ESI): [M+H] ⁺ : 282.04

Step 3: Preparation of methyl [(7-hydroxy-5-(styryl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate:

5-(styryl)-7-hydroxy-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid (143 mg), N,N-dimethylformamide (10 mL), DIPEA (329 mg), PyBOP (395 mg), and methyl glycine hydrochloride (178 mg) were added sequentially to a reaction flask, stirred overnight at room temperature, and the solid was filtered off to obtain 50 mg of crude product.

MS m/z (ESI): [M+H] ⁺ : 353.28

Step 4: Preparation of [(7-hydroxy-5-(styryl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid:

Methyl [(7-hydroxy-5-(styryl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate (50 mg) was dissolved in methanol (2.0 mL), followed by the addition of 30% sodium hydroxide aqueous solution (1.0 mL). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The pH was adjusted to between 1 and 2 with dilute hydrochloric acid, and the mixture was filtered to obtain 10 mg of the product.

¹ H NMR (400 MHz, d-DMSO) δ: 14.26 (s, 1H), 12.98 (s, 1H), 9.90 (t, J = 16.4 Hz, 1H), 8.63 (s, 1H), 8.22 (d, J = 16.4 Hz, 1H), 7.76–7.71 (m, 3H), 7.50–7.40 (m, 4H), 4.23 (d, J = 5.6 Hz, 2H)

MS m/z (ESI): [M+H] ⁺ : 339.21

Example 12 Preparation of [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid.

Step 1: Preparation of tert-butyl 5-(3,4-dihydronaphthyl-2-yl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

5-Iodo-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester (5.40 g), Pd( _PPh3 ) _2Cl2 (800 mg), potassium carbonate (4.20 g), _and 3,4-dihydronaphthyl-2-boronic acid pinacol ester (3.10 g) were dissolved in a 2:1 mixture of ethylene glycol dimethyl ether and degassed water (30 mL), and the mixture was purged with nitrogen three times. The mixture was then stirred at 80 ^° C until the reaction was complete as monitored by TLC, at which point heating was stopped. After the reaction solution cooled to room temperature, it was extracted with ethyl acetate, and the organic phase was washed with saturated brine. The organic phase was separated, concentrated under reduced pressure, and separated by column chromatography to obtain 3.30 g of crude product.

Step 2: Preparation of 5-(3,4-dihydronaphth-2-yl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid:

3.30 g of 5-(3,4-dihydronaphthyl-2-yl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester was dissolved in a 3:1 mixture of toluene and ethyl acetate (28 mL). Then, 2.80 g of methanesulfonic acid was added to the reaction system and stirred until the reaction was complete as monitored by TLC. Ethyl acetate (20.0 mL) was added to the reaction system and stirred to induce crystallization. The solid was then filtered and collected. Subsequently, the mixture was slurried with DMF/ _H₂O (20.0 mL/40.0 mL), stirred to induce crystallization, filtered, and collected to obtain 2.70 g of a pale blue solid.

Step 3: Preparation of [5-(3,4-dihydronaphth-2-yl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]methyl acetate:

5-(3,4-dihydronaphthyl-2-yl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carboxylic acid tert-butyl ester (2.70 g), N,N-dimethylformamide (20.0 mL), HOBt (1.10 g), EDCI (1.40 g), glycine methyl ester hydrochloride (0.90 g), and triethylamine (1.50 g) were added sequentially to a reaction flask and stirred at room temperature until the reaction was complete as monitored by TLC. Water (30.0 mL) was added to the reaction system and stirred to induce crystallization. The crystals were filtered, collected, and dried to obtain an off-white solid (2.2 g).

Step 4: Preparation of methyl [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate:

[5-(3,4-dihydronaphthyl-2-yl)-7-(benzyloxy)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid (1.0 g) was dissolved in tetrahydrofuran solution (8.0 mL), followed by the addition of palladium carbon (300 mg), the hydrogen gas was replaced, and the mixture was stirred at room temperature until the reaction of the raw materials was complete as monitored by TLC. The mixture was filtered through diatomaceous earth, concentrated under reduced pressure, and then slurried with isopropyl ether/n-hexane (1.5 mL/1.5 mL), filtered, and approximately 500 mg of a white solid was obtained.

Step 5: Preparation of [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid

Methyl acetate [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate (0.20 g) was dissolved in ethanol solution (2.0 mL), followed by the addition of sodium hydroxide aqueous solution (2.0 mL, 4.0 M). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The pH was adjusted to between 6 and 7 with dilute hydrochloric acid, and the mixture was filtered to obtain 200 mg of a grayish-white solid.

¹ H NMR (400 MHz, d-DMSO) δ: 14.37 (brs, 1H), 9.93 (s, 1H), 8.56 (s, 1H), 7.13 (s, 4H), 6.78 (s, 1H), 4.12 (d, J = 4.0 Hz, 2H), 3.81–3.76 (m, 1H), 3.26–2.86 (m, 4H), 2.26–2.05 (m, 2H),

MS m/z (ESI): [M+H] ⁺ : 367.32

Implementation Examples 13 [(7- Fiber -5-(1,2,3,4- Tetrahydronaphthalene -2- base )- [1,2,4] Triazolidinedione [1,5-a] Pyridine -8- carbonyl ) amino ] Chiral isomers of acetic acid A Preparation.

Step 1: Prepare methyl acetate [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino] according to steps 1-4 of Example 12;

Step 2: The product obtained in step (1) was subjected to chiral separation, yielding 5g of the separated product. Separation conditions: (Chiral column: (S,S)Whelk-O1, 5*25 cm, 10 μm; mobile phase A: _CO2 , mobile phase B: ACN:IPA=1:1; flow rate: 200 mL/min; gradient: 50%-50%; B flow for 12 min; wavelength: 220 nm; elution time: 8.50 min; sample solution: methanol: dichloromethane=1:1; injection volume: 2 mL; number of injection needles: 50). The product was concentrated under reduced pressure to obtain chiral isomer A, a white solid (2.060 g, yield 41.2%).

Step 3: Preparation of chiral isomer A of [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid:

0.20 g of chiral isomer A of methyl [(7-hydroxy-5-(1,2,3,4-tetrahydronaphth-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetate was dissolved in ethanol solution (2.0 mL), followed by the addition of sodium hydroxide aqueous solution (1.0 mL, 4.0 M). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The pH was adjusted to between 1 and 2 with dilute hydrochloric acid, and the mixture was filtered to obtain 200 mg of a grayish-white solid.

1H NMR (400 MHz, d-DMSO) δ: 14.37 (brs, 1H), 9.93 (s, 1H), 8.56 (s, 1H), 7.13 (s, 4H), 6.78 (s, 1H), 4.12 (d, J = 4.0 Hz, 2H), 3.81–3.76 (m, 1H), 3.26–2.86 (m, 4H), 2.26–2.05 (m, 2H),

MS m/z (ESI): [M+H] ⁺ : 367.32

[α]D ²⁰ = +37.384 (c 1.01, DMF).

Implementation Examples 14 [(7- Fiber -5-(1,2,3,4- Tetrahydronaphthalene -2- base )- [1,2,4] Triazolidinedione [1,5-a] Pyridine -8- carbonyl ) amino ] Chiral isomers of acetic acid B Preparation

Following the preparation method of Example 13, the synthesized [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]methyl acetate was subjected to chiral resolution under the same conditions. The chiral isomer B had a peak elution time of 10.09 min, yielding [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]methyl acetate chiral isomer B (methyl acetate chiral isomer B): white solid (1.997 g, yield 39.94%).

0.20 g of chiral isomer B of methyl acetate was dissolved in 2.0 mL of ethanol solution, followed by the addition of 1.0 mL of sodium hydroxide aqueous solution (4.0 M). The mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The pH was adjusted to 1-2 with dilute hydrochloric acid, and the mixture was filtered to obtain 200 mg of a grayish-white solid.

¹ H NMR (400 MHz, d-DMSO) δ: 14.37 (brs, 1H), 9.93 (s, 1H), 8.56 (s, 1H), 7.13 (s, 4H), 6.78 (s, 1H), 4.12 (d, J = 4.0 Hz, 2H), 3.81–3.76 (m, 1H), 3.26–2.86 (m, 4H), 2.26–2.05 (m, 2H),

MS m/z (ESI): [M+H] ⁺ : 367.32

[α]D ²⁰ = –36.601 (c 1.01, DMF).

Implementation Examples 15 Biology test

1 , PHD2 Enzyme activity inhibition detection

1.1 Reagents and Consumables

PHD2 enzyme: purchased from Active Motif; Sodium α-ketoglutarate: purchased from Sigma;

FITC-HIF1α: Purchased from GL; Nunc ^™ 384 perforated plate: Purchased from Thermo Scientific.

1.2 Experimental Methods

(1) Prepare 1×Assay buffer.

(2) Preparation of compound concentration gradients: The test concentration of the test compound was started at 10 μM, diluted 3 times, and tested at 10 concentrations in duplicate wells; the solution was diluted to a final concentration of 100 times in a 384-well plate, and then 100 nL was transferred to a 384-well plate using a non-contact ultrasonic pipetting system Echo550 for later use. 100 nL of 100% DMSO was added to the negative control well and the positive control well, respectively.

(3) Prepare an enzyme solution with a final concentration of 2 times using 1×Assay buffer;

(4) Add 5 μL of enzyme solution at twice the final concentration to the compound well and the positive control well, respectively;

(5) Add 5 μL of 1×Assay buffer to the negative control well;

(6) Centrifuge at 1000 rpm for 30 seconds, shake to mix, and incubate for 15 min;

(7) Prepare a tracer solution with a final concentration of 2 using 1×Assay buffer, add 5.0 μL of the tracer solution with a final concentration of 2 to initiate the reaction;

(8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix for 60 min, and read the mP value from the Envision Multimode Plate Reader (Perkin Elmer). Export the data and process it to obtain the inhibition rate of the test compound. Compound numbers 1-12 correspond to the compounds prepared in Examples 1-12, respectively. The results are shown in the table below:

Table 1 Compound number Compound structural formula IC ₅₀ (nM) FG-4592 89.2 Enarodustat 21.9 Example 1 compound 19.8 Example 2 compound 34.8 Example 3 compound 30.2 Example 4 compound 33.3 Example 5 compound 44.1 Example 6 Compound 47.6 Example 7 Compound 34.1 Example 8 Compound 35.4 Example 9 Compound 34.0 Example 10 Compound 46.7 Example 11 Compound 31.3 Example 12 Compound 34.3

As can be seen from the table above, the compounds of the present invention have good HIF-prolyl hydroxylase inhibitory activity, and it is evident that compounds 1-12 have good inhibitory activity.

2 The in vitro erythropoietin of the compound of this invention (EPO) Induced activity experiment

The in vitro erythropoietin (EPO) induction activity of the compounds of Examples 10-12 of this invention was evaluated using the Hep3B (ATCC) cell line derived from human liver cancer. Hep3B cells were cultured in Eagle's Minimum Essential Medium at 37 °C in the presence of 10% fetal bovine serum (FBS). The experimental procedures are as follows:

(1) Cell preparation: Inoculate cells into 96-well plates at a rate of 2*10^4 cells per well and 100 μL per well.

(2) Preparation of compound concentration gradients: 100 μM, 20 μM, 2 replicates. A 200-fold final concentration solution was prepared in a 96-well plate. The compound was then diluted 200/3 times using cell culture medium, and 50 μL was administered to the cells. 50 μL of DMSO-containing medium was added to the negative control wells to achieve a final concentration of 5‰ DMSO. 50 μL of the highest concentration of the positive control compound was added to the positive control wells. The plates were incubated at 37 °C for 24 hours.

(3) Wash the reaction plate twice with approximately 400 μL of 1X Wash Buffer per well;

(4) Add 100 μL of diluted standard (including standard blank control) to the appropriate well.

(5) Add 50 μL of sample and 50 μL of Sample Diluent to the sample well;

(6) Add 50 μL of 1X Biotin Conjugated Antibody to all wells and incubate at room temperature for 1 hour;

(7) Wash the reaction plate 6 times with approximately 400 μL of 1X Wash Buffer per well;

(8) Add 100 μL of 1X Streptavidin-HRP to each well. Incubate at room temperature for 15 minutes;

(9) Wash the reaction plate 6 times with approximately 400 μL of 1X Wash Buffer per well;

(10) Add 100 μL of TMB Substrate Solution to each well. Incubate at room temperature for 10 minutes;

(11) Add 100 μL of Stop Solution to each well;

(12) Read OD450 using EnSight.

The EPO-induced activity of each compound is expressed as the half-maximal effect concentration (EC50). The experimental results are shown in the table below:

Table 2 Number EC ₅₀ (μM) FG-4592 13.79 Enarodustat 15.06 Example 10 Compound 6.46 Example 11 Compound 1.79 Example 12 Compound 6.79 Example 13 Compound 5.05 Example 14 Compound 4.16

As can be seen from the table above, the compounds of the present invention have high EPO induction activity, indicating that the compounds obtained in Examples 10-14 have good induction activity.

Implementation Examples 16 Pharmacokinetic evaluation of compounds

Experimental Objective

After intravenous or gavage administration of the compounds to SD rats, the concentration of the relevant compounds in plasma was detected by LC-MS/MS to preliminarily evaluate the pharmacokinetics and absolute bioavailability of the compounds prepared in Examples 13 and 14 in rats.

Strain: Sprague Dawley rat, sex: male, weight: 200-250 g

Source: Zhejiang Vitonliwa Experimental Animal Technology Co., Ltd.

Experimental Operation

The pharmacokinetic characteristics of the compounds administered intravenously and orally in rodents were tested using a standard protocol. In the experiment, candidate compounds were prepared into clear solutions and administered to rats via single intravenous injection and gavage. The intravenous solvent was a mixture of N,N-dimethylacetamide (DMA) and 10% Solutol HS15 solution, while the oral solvent was a carboxymethyl cellulose sodium (CMC) suspension. Blood samples were collected from the animals at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h after administration into K2EDTA anticoagulant tubes and stored on ice until centrifugation.

Experimental results: Test sample Half-life T _{_1/2} (h) Concentration integral AUC (hr*ng/mL) Bioavailability F (%) Enarodustat 1.12 11440 30.1 Example 13 Compound 2.02 19130 29.4 Example 14 Compound 2.43 15740 31.3

As can be seen from the table above, the compounds prepared in Examples 13 and 14 of this invention have good PK properties. It is evident that the compounds prepared in Examples 13 and 14 have significantly longer half-lives and higher in vivo exposure levels.

Implementation Examples 17 Pharmacological evaluation of compounds

Experimental objective: The objective of this experiment is to evaluate the effect of continuous administration of the compounds prepared in Examples 12, 13, and 14 on EPO (erythropoietin) in SD rats.

Animal: Species: Rat; Strain: SD (Sprague Dawley);

Age and weight: 8 weeks, weight 200-220g; sex: male; source: Beijing Vital River Laboratory Animal Technology Co., Ltd.

Information on test compounds and positive control drugs

Enarodustat (a male enhancement drug); Description: A commercially available drug approved for marketing in Japan.

Specifications and content: 2mg JTZ-951 (Enarodustat), 140 tablets/box

Experimental plan:

After one week of acclimatization, SD rats were randomly divided into three groups of six each: a blank control group, a positive drug group (Enarodustat), and the compound group from Example 12. Enarodustat tablets were crushed in a mortar and pestle, and 0.5% methylcellulose was added quantitatively, followed by magnetic stirring to mix thoroughly. The compound prepared in Example 12 was weighed, and 0.5% methylcellulose was added quantitatively, followed by magnetic stirring to mix thoroughly. The final concentration of all compounds was 0.3 mg/mL, and the administration volume was 10 mL/kg. Administered once daily for 14 consecutive days. Blood samples were collected 4 hours after administration on day 14 to measure EPO levels.

Results and Analysis

The results showed that 14 days after administration, the serum EPO level of rats in the positive drug Enarodustat and the compounds prepared in Examples 12, 13, and 14 was significantly increased, and the plasma EPO level of the compounds prepared in Examples 12, 13, and 14 was significantly higher than that of the positive drug Enarodustat.

Table 3. EPO levels (pg/mL) 14 days after drug administration Vehicle Enarodustat Example 12 Compound Example 13 Compound Example 14 Compound 1 36.7 452.8 2649.3 2904.6 2279.1 2 30.9 396.3 2904.6 2697.4 2095.6 3 31.8 483.2 2882.6 2904.6 1982.7 4 45.4 404.5 2904.6 2904.6 1943.6 5 49.6 418.4 2904.6 2658.5 2038.9 6 38.1 442.3 2904.6 2772.1 2137.6 average value 38.8 432.9 2858.4 2807.0 2079.6

Note: 2904.6 pg/mL is the upper limit of measurement.

In conclusion, the compounds prepared in Examples 12, 13, and 14 significantly increased EPO levels in SD rats, and their overall effect was significantly better than that of the positive drug Enarodustat, demonstrating excellent pharmacological effects.

Implementation Examples 18 Implementation Examples 13 Chiral isomers of methyl acetate A Determination of chiral configuration of compounds

1. Compound preparation

Referring to Example 13, the method for preparing chiral isomer A of methyl acetate [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]methyl acetate (hereinafter referred to as chiral isomer A) was obtained by chiral resolution, which is the preceding compound of the final product in Example 13.

2. Single crystal culture

Weigh approximately 3-7 mg of methyl acetate chiral isomer A into a vial. While stirring, gradually add acetonitrile at 50 ^° C until the solution is just clear or nearly clear. Filter while hot, transfer the filtrate to a clean vial, and perform a step-by-step cooling process using a Crystal 16 instrument. First, reduce the temperature from 50 ^° C to 30 ^° C at a rate of 0.01 ^° C/min, then maintain the temperature at 30 ^° C for approximately 2 days.

Needle-shaped single crystals were obtained from acetonitrile by cooling crystallization, as shown in Figure 1, and were used for X-ray single crystal diffraction analysis.

3. Instruments and Parameters

Single-crystal data for the chiral isomer A of methyl acetate were obtained using a Rigaku XtaLAB Synergy DW diffractometer at 180 K with Cu Kα radiation (λ = 1.54184 Å). Diffraction data were reduced and corrected for absorption using the CrysAlisPro program, and the structure was resolved using the SHELXT program via a dual-space algorithm. All non-hydrogen atoms were directly located from the difference Fourier plot, with hydrogen atoms filling the parent atom. The final structure was refined using the SHELXL program based on the F2 full-matrix least squares method.

4. X-ray diffraction analysis

The single-crystal structure of methyl acetate chiral isomer A belongs to the monoclinic crystal system, space group P21, _and the molecular formula of the single crystal is _{C20H20N4O4 .} Each _asymmetric unit contains one molecule of methyl acetate chiral isomer A, and each unit cell contains two asymmetric units. The absolute configuration of the chiral carbon is "R".

The refined single-crystal structure is shown in Figures 2 , 3, and 4. The crystal structure parameters are summarized in Table 4, and detailed data on atomic coordinates, anisotropic displacement parameters, twist angles, hydrogen bonds, bond lengths, and bond angles are provided in Tables 5 to 11. The XRPD plot calculated based on the single-crystal structure is basically consistent with the test results of the single-crystal sample, but the peak positions are slightly shifted. This may be because the single-crystal data was collected at 180 K, while the experimentally measured XRPD spectrum was obtained at room temperature (Figure 5).

Table 4. Crystal structure parameters of chiral isomer A of methyl acetate Molecular formula C ₂₀ H ₂₀ N ₄ O ₄ molecular weight 380.40 Temperature/K 180.00(10) Crystal system Monoclinic crystal system Space Group P 2 ₁ a /Å 13.2296(2) b /Å 5.10250(10) c /Å 13.7423(2) α /° 90 β /° 104.357(2) γ /° 90 Unit cell volume / Å ³ 898.69(3) Z 2 ρ _calc g/cm ³ 1.406 μ /mm ^-1 0.828 F (000) 400.0 Crystal size / mm ³ 0.48 × 0.04 × 0.02 Diffraction source Cu Kα ( λ = 1.54184 Å) Data collection 2θ range / ° 6.64 to 145.488 Diffraction index range -16 ≤ h ≤ 15, -6 ≤ k ≤ 6, -16 ≤ l ≤ 16 Number of diffraction points collected 15082 Independent diffraction points 3389 [ R _int = 0.0370, R _sigma = 0.0282] Data/Limitations/Parameters 3389/3/260 Fitting accuracy ^based on F2 1.063 Final R value [ I ＞=2 σ ( I )] R _₁ = 0.0389, wR_₂ = 0.1045 Final R- value [All Data] R _₁ = 0.0407, wR_₂ = 0.1067 Peak/valley/e ^Å⁻³ of maximum residual electron density 0.53/-0.21 Flack parameters 0.04(9)

Table 5. Atomic coordinates (× ^10⁴ ) and equivalent isotropic shift parameters ( ^Ų × ^10³ ) of chiral isomer A of methyl acetate atom x y z U(eq) O1 3838.8(15) 12688(4) -49.0(13) 46.2(5) O2 3791.7(17) 9024(6) 837.1(17) 65.1(7) O3 7203.8(14) 12162(4) 2559.3(12) 40.2(4) O4 8666.9(13) 10360(4) 3967.4(13) 39.3(4) N1 5615.5(16) 10253(5) 2171.4(14) 36.6(4) N2 5282.0(14) 6067(4) 3404.3(14) 35.3(4) N3 5897.1(15) 3302(4) 4724.3(14) 35.4(4) N4 6661.4(14) 5018(4) 4612.7(14) 30.5(4) C1 2834(2) 12039(7) -710(2) 49.8(7) C2 4200.2(19) 11068(6) 711.2(17) 39.1(5) C3 5227(2) 12043(5) 1345.5(17) 39.9(5) C4 6593.7(18) 10453(5) 2731.0(16) 32.7(5) C5 6942.0(17) 8563(5) 3556.8(16) 30.8(4) C6 6278.8(17) 6657(5) 3811.2(16) 31.0(5) C7 5107.2(17) 4063(5) 3986.1(17) 36.7(5) C8 7674.1(17) 5054(5) 5193.2(16) 30.9(4) C9 8322.9(17) 6845(5) 4945.1(16) 32.4(5) C10 7971.4(17) 8634(5) 4133.5(16) 31.7(5) C11 7945.5(16) 3092(4) 6037.9(16) 30.5(5) C12 7426.2(17) 3712(5) 6898.2(17) 35.4(5) C13 7585.6(18) 1429(6) 7632.6(18) 39.6(5) C14 8675.6(18) 307(5) 7883.5(16) 34.4(5) C15 8965(2) -1499(6) 8668.7(18) 43.1(6) C16 9942(2) -2652(6) 8904.0(19) 47.7(6) C17 10655(2) -2009(6) 8356(2) 46.1(6) C18 10377.8(19) -210(5) 7578.0(19) 40.6(5) C19 9391.3(17) 938(5) 7326.8(16) 32.4(5) C20 9122.4(17) 2870(5) 6468.3(17) 34.1(5)

Table 6. Anisotropic shift parameters of chiral isomer A of methyl acetate (Å ² × 10 ³ ) atom U ₁₁ U ₂₂ U ₃₃ U ₂₃ U ₁₃ U ₁₂ O1 49.2(10) 48.8(11) 33.1(8) 8.3(8) -4.2(7) 1.8(8) O2 51.3(11) 77.9(16) 56.9(12) 25.9(12) -4.4(9) -19.0(11) O3 42.9(9) 43.1(9) 32.3(8) 6.1(7) 5.0(7) -2.9(8) O4 34.1(8) 43.9(10) 37.8(8) 6.8(8) 5.0(7) -4.3(7) N1 39.9(10) 41.0(11) 25.9(9) 6.4(8) 2.2(8) -0.6(9) N2 29.6(9) 46.1(11) 28.3(9) 1.7(8) 3.4(7) 1.2(8) N3 31.5(9) 41.9(11) 31.2(9) 2.3(8) 4.9(7) -3.8(8) N4 29.5(9) 34.4(9) 26.2(8) 1.0(7) 4.5(7) 1.6(7) C1 39.1(12) 69.3(18) 35.4(12) 6.1(13) -1.4(10) 9.9(13) C2 37.1(11) 51.1(14) 28.8(10) 7.9(10) 7.5(9) 2.3(11) C3 45.6(13) 40.6(13) 29.1(10) 5.8(10) 0.9(9) -0.2(10) C4 38.6(11) 34.5(11) 25.3(9) -0.5(9) 8.4(8) 1.5(9) C5 33.0(10) 33.8(11) 25.1(9) -1.6(8) 5.9(8) 4.0(9) C6 31.1(10) 37.0(12) 24.1(9) -0.3(9) 5.3(8) 5.6(8) C7 30.0(10) 46.0(13) 32.0(10) 1.3(10) 3.9(9) -3.5(10) C8 29.4(10) 34.3(11) 28.0(10) -2.7(9) 5.1(8) 3.3(9) C9 29.1(10) 36.9(11) 28.9(10) -1.7(9) 2.7(8) 3.2(9) C10 31.8(10) 34.4(11) 29.8(10) -1.0(9) 9.2(8) 1.1(9) C11 29.1(10) 32.7(11) 27.8(10) -0.5(8) 3.4(8) 2.7(8) C12 32.8(10) 42.5(12) 29.5(10) -1.0(9) 5.0(8) 6.6(10) C13 33.1(11) 54.0(14) 32.0(11) 4.8(10) 8.3(9) 4.7(10) C14 35.1(11) 40.1(12) 24.2(10) -2.1(9) 0.1(8) 0.5(9) C15 43.4(12) 54.1(15) 28.9(11) 2.3(10) 3.2(9) -3.8(11) C16 51.6(14) 49.7(15) 32.9(11) 7.9(11) -6.1(10) 4.4(12) C17 38.4(12) 49.8(14) 43.0(13) 6.4(11) -3.3(10) 10.4(11) C18 34.5(11) 46.1(13) 37.9(12) 1.6(10) 2.4(9) 3.7(10) C19 31.0(10) 34.3(11) 28.3(10) -2.4(8) 0.4(8) 0.1(9) C20 29.3(10) 38.6(12) 32.5(11) 3.8(9) 4.0(8) 0.7(9)

Table 7. Bond lengths of chiral isomer A of methyl acetate atom atom Key length / Å atom atom Key length / Å O1 C1 1.450(3) C5 C10 1.396(3) O1 C2 1.325(3) C8 C9 1.354(3) O2 C2 1.206(4) C8 C11 1.507(3) O3 C4 1.250(3) C9 C10 1.427(3) O4 C10 1.334(3) C11 C12 1.540(3) N1 C3 1.448(3) C11 C20 1.526(3) N1 C4 1.335(3) C12 C13 1.521(3) N2 C6 1.333(3) C13 C14 1.510(3) N2 C7 1.353(3) C14 C15 1.398(4) N3 N4 1.375(3) C14 C19 1.395(3) N3 C7 1.321(3) C15 C16 1.383(4) N4 C6 1.376(3) C16 C17 1.385(4) N4 C8 1.379(3) C17 C18 1.387(4) C2 C3 1.505(3) C18 C19 1.394(3) C4 C5 1.473(3) C19 C20 1.511(3) C5 C6 1.411(3)

Table 8. Bond angles of chiral isomer A of methyl acetate atom atom atom Keyboard corner /˚ atom atom atom Keyboard corner /˚ C2 O1 C1 116.5(2) C9 C8 N4 116.7(2) C4 N1 C3 120.6(2) C9 C8 C11 126.69(19) C6 N2 C7 102.66(19) C8 C9 C10 121.63(19) C7 N3 N4 101.00(19) O4 C10 C5 122.5(2) N3 N4 C6 109.98(18) O4 C10 C9 116.8(2) N3 N4 C8 125.45(19) C5 C10 C9 120.7(2) C6 N4 C8 124.54(19) C8 C11 C12 112.85(18) O1 C2 C3 110.4(2) C8 C11 C20 111.90(19) O2 C2 O1 124.4(2) C20 C11 C12 108.91(17) O2 C2 C3 125.1(2) C13 C12 C11 109.70(19) N1 C3 C2 109.9(2) C14 C13 C12 114.16(19) O3 C4 N1 121.6(2) C15 C14 C13 119.3(2) O3 C4 C5 120.6(2) C19 C14 C13 121.8(2) N1 C4 C5 117.8(2) C19 C14 C15 118.8(2) C6 C5 C4 123.2(2) C16 C15 C14 121.5(2) C10 C5 C4 119.7(2) C15 C16 C17 119.7(2) C10 C5 C6 117.1(2) C16 C17 C18 119.3(2) N2 C6 N4 109.1(2) C17 C18 C19 121.4(2) N2 C6 C5 131.5(2) C14 C19 C20 121.2(2) N4 C6 C5 119.3(2) C18 C19 C14 119.2(2) N3 C7 N2 117.2(2) C18 C19 C20 119.5(2) N4 C8 C11 116.64(19) C19 C20 C11 111.78(19)

Table 9. Twist angles of chiral isomer A of methyl acetate A B C D Twist angle /˚ A B C D Twist angle /˚ O1 C2 C3 N1 -179.8(2) C7 N3 N4 C8 -178.7(2) O2 C2 C3 N1 -4.0(4) C8 N4 C6 N2 178.6(2) O3 C4 C5 C6 177.5(2) C8 N4 C6 C5 -1.3(3) O3 C4 C5 C10 -1.9(3) C8 C9 C10 O4 178.4(2) N1 C4 C5 C6 -3.3(3) C8 C9 C10 C5 -0.9(3) N1 C4 C5 C10 177.3(2) C8 C11 C12 C13 170.60(19) N3 N4 C6 N2 0.6(2) C8 C11 C20 C19 178.99(18) N3 N4 C6 C5 -179.36(19) C9 C8 C11 C12 110.1(2) N3 N4 C8 C9 177.9(2) C9 C8 C11 C20 -13.2(3) N3 N4 C8 C11 -2.0(3) C10 C5 C6 N2 -178.6(2) N4 N3 C7 N2 0.6(3) C10 C5 C6 N4 1.3(3) N4 C8 C9 C10 1.0(3) C11 C8 C9 C10 -179.1(2) N4 C8 C11 C12 -70.0(3) C11 C12 C13 C14 43.5(3) N4 C8 C11 C20 166.74(19) C12 C11 C20 C19 53.5(3) C1 O1 C2 O2 6.0(4) C12 C13 C14 C15 169.6(2) C1 O1 C2 C3 -178.1(2) C12 C13 C14 C19 -13.1(3) C3 N1 C4 O3 -0.5(4) C13 C14 C15 C16 177.7(3) C3 N1 C4 C5 -179.7(2) C13 C14 C19 C18 -178.4(2) C4 N1 C3 C2 168.2(2) C13 C14 C19 C20 2.5(3) C4 C5 C6 N2 2.0(4) C14 C15 C16 C17 0.2(4) C4 C5 C6 N4 -178.12(19) C14 C19 C20 C11 -23.4(3) C4 C5 C10 O4 -0.1(3) C15 C14 C19 C18 -1.1(3) C4 C5 C10 C9 179.18(19) C15 C14 C19 C20 179.9(2) C6 N2 C7 N3 -0.3(3) C15 C16 C17 C18 0.1(4) C6 N4 C8 C9 0.1(3) C16 C17 C18 C19 -0.9(4) C6 N4 C8 C11 -179.77(19) C17 C18 C19 C14 1.4(4) C6 C5 C10 O4 -179.6(2) C17 C18 C19 C20 -179.5(2) C6 C5 C10 C9 -0.2(3) C18 C19 C20 C11 157.6(2) C7 N2 C6 N4 -0.2(2) C19 C14 C15 C16 0.3(4) C7 N2 C6 C5 179.7(2) C20 C11 C12 C13 -64.5(2) C7 N3 N4 C6 -0.7(2)

Table 10. Hydrogen bonds in chiral isomer A of methyl acetate D H A d(DH)/Å d(HA)/Å d(DA)/Å DHA/° O4 H4 O3 0.81(2) 1.83(3) 2.545(2) 146(3) N1 H1 N2 0.88(2) 2.09(3) 2.828(3) 141(3)

Table 11. Atomic coordinates (× ^10⁴ ) and isotropic shift parameters ( ^Ų × ^10³ ) of chiral isomer A of methyl acetate atom x y z U(eq) H4 8390(20) 11350(60) 3510(20) 47 H1 5220(20) 9070(60) 2360(20) 44 H1A 2928 10715 -1197 75 H1B 2522 13618 -1067 75 H1C 2373 11346 -312 75 H3A 5738 12183 930 48 H3B 5133 13806 1611 48 H7 4444 3237 3869 44 H9 9030 6913 5321 39 H11 7686 1340 5757 37 H12A 7736 5322 7252 42 H12B 6671 4021 6621 42 H13A 7421 2023 8262 48 H13B 7086 twenty one 7344 48 H15 8480 -1944 9049 52 H16 10123 -3879 9439 57 H17 11328 -2792 8511 55 H18 10871 248 7209 49 H20A 9453 2305 5931 41 H20B 9407 4613 6708 41

5 Analytical methods

5.1 Polarizing Microscope (PLM)

PLM analysis was performed using an ECLIPSE LV100POL (Nikon, JPN) polarizing microscope. A small amount of sample was spread evenly on a glass slide, cedarwood oil was added to disperse the sample, and a coverslip was placed on top. The sample was then observed under a 10x objective lens.

5.2 X-ray Powder Diffraction (XRPD)

The solid sample was examined using an X-ray diffractometer. The sample was laid flat on a zero-background single-crystal silicon sample tray, and after being gently pressed flat, it was analyzed according to the parameters in Table 12.

Table 12. XRPD Test Parameters equipment Brook, D8 Advance light source Cu Kα (λ = 1.5418 Å) Detector LynxEye XE-T Scanning angle 3-40° (2θ) Scan step size 0.02° (2θ) Scanning speed 0.1 s/step Phototube voltage/current 40 kV/40 mA Diverging Narrow Seam 0.6 mm Rotation 30 rpm Sample tray Zero-background sample tray

As can be seen from the single-crystal structure analysis above, the chiral configuration of the methyl acetate chiral isomer A is the "R" configuration, as shown in Figure 6. Therefore, in Example 13, when the methyl ester is removed using a sodium hydroxide aqueous solution to obtain the carboxylic acid product, the already generated chiral configuration is not changed. Thus, it can be inferred that the chiral configuration of the product [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid chiral isomer A in Example 13 is the "R" configuration.

Implementation Examples 19 Implementation Examples 14 Chiral isomers of methyl acetate B Determination of chiral configuration of compounds

1. Compound preparation

Referring to Example 14, the method for preparing chiral isomer B of methyl acetate [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino] methyl acetate was obtained by chiral resolution. This isomer is the preceding compound to the final product in Example 14.

2. Single crystal culture

Weigh an appropriate amount of methyl acetate chiral isomer B sample into a vial. While stirring, gradually add dimethyl sulfoxide at room temperature until the solution is just clear or nearly clear. Filter the solution, place the filtrate in a clean vial, cap it with a perforated cap, and allow it to evaporate slowly at room temperature.

Needle-shaped single crystals were obtained from dimethyl monoxide by a slow evaporation method, as shown in Figure 7, and were used for X-ray single crystal diffraction analysis.

3. Instruments and Parameters

The selection of instruments and parameters is the same as that shown in Embodiment 18.

4. X-ray diffraction analysis

The single-crystal structure of methyl acetate chiral isomer B belongs to the monoclinic crystal system, space group P21, _and the molecular formula of the single crystal is _{C20H20N4O4 .} Each _asymmetric unit contains one molecule of methyl acetate chiral isomer B, and each unit cell contains two asymmetric units. The absolute configuration of the chiral carbon is "S".

The refined single-crystal structures are shown in Figures 8, 9, and 10. Crystal structure parameters are summarized in Table 13, while atomic coordinates, anisotropic displacement parameters, torsion angles, hydrogen bonds, bond lengths, and bond angles are summarized in Tables 14 to 20. The experimentally measured XRPD pattern of the single-crystal sample has more peaks than the calculated XRPD spectrum; this may be because the solvent was nearly evaporated after the single-crystal test, resulting in the precipitation of other crystal forms during solvent evaporation (Figure 11).

Table 13. Crystal structure parameters of chiral isomer B of methyl acetate Molecular formula C ₂₀ H ₂₀ N ₄ O ₄ molecular weight 380.40 Temperature/K 179.99(10) Crystal system Monoclinic Space Group P 2 ₁ a /Å 13.2287(2) b /Å 5.10690(10) c /Å 13.7391(2) α /° 90 β /° 104.3640(10) γ /° 90 Unit cell volume/Å ³ 899.17(3) Z 2 ρ _calc g/cm ³ 1.405 μ /mm ^-1 0.827 F (000) 400.0 Crystal size / mm ³ 0.29 × 0.03 × 0.02 Diffraction source Cu Kα ( λ = 1.54184 Å) Data collection 2 θ range / ° 6.64 to 145.034 Diffraction index range -16 ≤ h ≤ 16, -6 ≤ k ≤ 6, -16 ≤ l ≤ 16 Number of diffraction points collected 15381 Independent diffraction points 3395 [ R _int = 0.0243, R _sigma = 0.0168] Data/Limitations/Parameters 3395/1/255 Fitting accuracy ^based on F2 1.067 Final R value [ I ＞=2 σ ( I )] R _₁ = 0.0320, wR_₂ = 0.0881 Final R- value [All Data] R _₁ = 0.0326, wR_₂ = 0.0889 Peak/valley/e ^Å⁻³ of maximum residual electron density 0.47/-0.22 Flack parameters -0.02(5)

Table 14. Atomic coordinates (× ^10⁴ ) and equivalent isotropic shift parameters ( ^Ų × ^10³ ) of chiral isomer B of methyl acetate. atom x y z U(eq) O1 3838.3(13) -2686(4) -49.0(12) 45.2(4) O2 3791.2(15) 970(5) 839.5(15) 65.5(6) O3 7202.8(12) -2163(3) 2559.1(11) 39.2(4) O4 8666.7(11) -359(3) 3967.6(11) 38.4(4) N1 5615.2(14) -252(4) 2170.6(12) 35.2(4) N2 5282.3(12) 3932(4) 3404.3(12) 33.9(4) N3 5896.3(12) 6691(4) 4724.6(13) 33.9(4) N4 6661.4(12) 4979(3) 4613.1(12) 29.0(3) C1 2832.0(18) -2032(6) -709.2(17) 48.1(6) C2 4200.6(17) -1071(5) 712.5(15) 37.8(5) C3 5225.7(18) -2041(5) 1345.7(15) 38.4(5) C4 6592.5(16) -444(4) 2732.7(15) 31.8(4) C5 6943.4(15) 1429(4) 3557.1(14) 29.6(4) C6 6280.4(15) 3347(4) 3813.0(14) 29.2(4) C7 5108.6(15) 5935(5) 3986.9(15) 35.2(4) C8 7676.4(15) 4946(4) 5195.7(14) 29.2(4) C9 8322.4(15) 3151(4) 4943.3(15) 31.3(4) C10 7968.9(15) 1369(4) 4133.4(14) 30.8(4) C11 7944.4(14) 6904(4) 6038.2(14) 29.0(4) C12 9122.1(15) 7132(4) 6468.9(15) 32.7(4) C13 9390.5(15) 9063(4) 7327.3(14) 30.8(4) C14 10381.0(16) 10213(5) 7578.5(17) 39.3(5) C15 10653.4(18) 12001(5) 8353.9(18) 45.1(5) C16 9942.0(19) 12652(5) 8904.1(17) 46.5(6) C17 8963.3(18) 11500(5) 8666.0(16) 41.5(5) C18 8675.7(15) 9699(4) 7885.1(14) 32.6(4) C19 7585.8(16) 8562(5) 7634.9(16) 38.2(5) C20 7425.0(15) 6285(4) 6897.7(15) 34.6(4)

Table 15. Anisotropic shift parameters of chiral isomer B of methyl acetate (Å ² × 10 ³ ) atom U ₁₁ U ₂₂ U ₃₃ U ₂₃ U ₁₃ U ₁₂ O1 47.6(9) 45.2(9) 35.6(7) -8.7(7) -3.4(7) -1.2(7) O2 48.8(9) 76.2(14) 62.7(11) -29.4(11) -2.9(8) 18.2(10) O3 41.9(8) 38.5(8) 35.3(7) -6.4(7) 6.0(6) 3.2(7) O4 33.2(7) 40.1(8) 40.4(8) -7.0(7) 6.4(6) 4.9(7) N1 38.0(9) 36.3(9) 28.9(8) -5.3(7) 3.6(7) 1.2(8) N2 27.7(8) 41.3(10) 30.8(8) -1.1(7) 3.7(6) -0.7(7) N3 28.7(8) 37.0(10) 35.0(8) -2.1(7) 5.9(7) 4.4(7) N4 26.7(7) 30.2(8) 29.4(8) -1.2(7) 5.7(6) -1.3(7) C1 37.7(11) 63.6(16) 37.9(11) -6.0(11) -0.3(9) -10.1(11) C2 36.5(10) 45.6(12) 31.5(10) -7.5(9) 8.7(8) -2.2(9) C3 44.5(11) 35.4(11) 31.6(9) -5.4(9) 2.7(8) -0.2(9) C4 37.1(10) 30.8(10) 28.4(9) 1.6(8) 9.8(8) -1.0(8) C5 31.2(9) 29.5(9) 28.1(8) 3.0(8) 7.3(7) -2.7(8) C6 29.0(9) 32.4(10) 25.9(8) 0.7(8) 6.0(7) -4.4(7) C7 26.9(9) 41.6(11) 35.6(10) -1.8(9) 5.0(8) 2.8(8) C8 27.3(9) 29.6(9) 29.6(9) 3.0(8) 4.8(7) -2.8(8) C9 26.7(9) 32.6(10) 32.3(9) 2.0(8) 3.3(7) -2.6(8) C10 31.2(9) 29.2(10) 33.3(9) 2.5(8) 10.6(7) -0.1(8) C11 26.0(9) 28.5(10) 30.7(9) 0.2(8) 3.6(7) -2.8(7) C12 26.1(9) 34.6(10) 35.5(10) -3.6(8) 4.0(7) -1.4(8) C13 28.5(9) 29.7(9) 30.6(9) 3.5(8) 0.6(7) -0.2(8) C14 32.1(10) 41.3(12) 41.3(11) -0.9(9) 2.8(8) -4.1(9) C15 36.3(11) 46.4(13) 45.9(12) -4.9(10) -2.4(9) -9.4(10) C16 49.5(13) 45.6(13) 35.7(10) -9.7(10) -6.0(9) -4.8(11) C17 41.5(11) 49.2(13) 31.3(10) -1.8(9) 4.4(8) 3.7(10) C18 31.6(9) 35.2(10) 27.5(9) 3.3(8) 0.9(7) -0.7(8) C19 30.8(10) 49.7(13) 34.3(10) -5.2(10) 8.6(8) -5.1(9) C20 30.4(9) 38.8(11) 33.1(9) 1.8(9) 5.3(7) -7.1(9)

Table 16. Bond lengths (Å) of chiral isomer B of methyl acetate atom atom Key length / Å atom atom Key length / Å O1 C1 1.452(3) C5 C10 1.391(3) O1 C2 1.325(3) C8 C9 1.356(3) O2 C2 1.207(3) C8 C11 1.504(3) O3 C4 1.255(3) C9 C10 1.424(3) O4 C10 1.337(3) C11 C12 1.528(2) N1 C3 1.447(3) C11 C20 1.539(3) N1 C4 1.335(3) C12 C13 1.510(3) N2 C6 1.334(3) C13 C14 1.399(3) N2 C7 1.353(3) C13 C18 1.395(3) N3 N4 1.375(2) C14 C15 1.381(3) N3 C7 1.318(3) C15 C16 1.386(4) N4 C6 1.372(3) C16 C17 1.386(4) N4 C8 1.382(2) C17 C18 1.393(3) C2 C3 1.502(3) C18 C19 1.513(3) C4 C5 1.467(3) C19 C20 1.522(3) C5 C6 1.416(3)

Table 17. Bond angles of chiral isomer B of methyl acetate atom atom atom Keyboard corner /˚ atom atom atom Keyboard corner /˚ C2 O1 C1 116.49(19) C9 C8 N4 116.31(17) C4 N1 C3 120.83(18) C9 C8 C11 127.11(16) C6 N2 C7 102.46(16) C8 C9 C10 121.77(17) C7 N3 N4 101.04(17) O4 C10 C5 122.32(19) N3 N4 C8 125.41(16) O4 C10 C9 116.64(17) C6 N4 N3 109.96(16) C5 C10 C9 121.04(18) C6 N4 C8 124.60(17) C8 C11 C12 111.84(16) O1 C2 C3 110.47(19) C8 C11 C20 112.89(16) O2 C2 O1 124.4(2) C12 C11 C20 108.96(15) O2 C2 C3 125.0(2) C13 C12 C11 111.83(16) N1 C3 C2 109.90(19) C14 C13 C12 119.42(18) O3 C4 N1 121.17(19) C18 C13 C12 121.38(17) O3 C4 C5 120.50(18) C18 C13 C14 119.19(19) N1 C4 C5 118.33(18) C15 C14 C13 121.1(2) C6 C5 C4 123.08(17) C14 C15 C16 119.8(2) C10 C5 C4 120.14(18) C17 C16 C15 119.4(2) C10 C5 C6 116.78(18) C16 C17 C18 121.5(2) N2 C6 N4 109.27(17) C13 C18 C19 121.57(18) N2 C6 C5 131.26(18) C17 C18 C13 118.95(19) N4 C6 C5 119.47(17) C17 C18 C19 119.43(19) N3 C7 N2 117.27(18) C18 C19 C20 114.36(17) N4 C8 C11 116.58(17) C19 C20 C11 109.77(17)

Table 18. Twist angles of chiral isomer B of methyl acetate A B C D Twist angle /˚ A B C D Twist angle /˚ O1 C2 C3 N1 179.68(18) C7 N3 N4 C8 178.65(18) O2 C2 C3 N1 3.8(3) C8 N4 C6 N2 -178.64(18) O3 C4 C5 C6 -177.55(18) C8 N4 C6 C5 1.5(3) O3 C4 C5 C10 2.2(3) C8 C9 C10 O4 -178.39(19) N1 C4 C5 C6 3.0(3) C8 C9 C10 C5 0.9(3) N1 C4 C5 C10 -177.25(18) C8 C11 C12 C13 -178.98(16) N3 N4 C6 N2 -0.6(2) C8 C11 C20 C19 -170.86(17) N3 N4 C6 C5 179.56(17) C9 C8 C11 C12 13.2(3) N3 N4 C8 C9 -177.93(18) C9 C8 C11 C20 -110.1(2) N3 N4 C8 C11 2.0(3) C10 C5 C6 N2 178.7(2) N4 N3 C7 N2 -0.5(2) C10 C5 C6 N4 -1.5(3) N4 C8 C9 C10 -1.0(3) C11 C8 C9 C10 179.05(18) N4 C8 C11 C12 -166.68(17) C11 C12 C13 C14 -157.64(19) N4 C8 C11 C20 70.0(2) C11 C12 C13 C18 23.2(3) C1 O1 C2 O2 -6.0(3) C12 C11 C20 C19 64.2(2) C1 O1 C2 C3 178.15(19) C12 C13 C14 C15 179.6(2) C3 N1 C4 O3 0.5(3) C12 C13 C18 C17 -179.7(2) C3 N1 C4 C5 179.86(18) C12 C13 C18 C19 -2.2(3) C4 N1 C3 C2 -168.23(19) C13 C14 C15 C16 0.8(4) C4 C5 C6 N2 -1.6(3) C13 C18 C19 C20 12.6(3) C4 C5 C6 N4 178.17(17) C14 C13 C18 C17 1.1(3) C4 C5 C10 O4 0.0(3) C14 C13 C18 C19 178.7(2) C4 C5 C10 C9 -179.34(17) C14 C15 C16 C17 -0.1(4) C6 N2 C7 N3 0.2(2) C15 C16 C17 C18 0.0(4) C6 N4 C8 C9 -0.2(3) C16 C17 C18 C13 -0.5(3) C6 N4 C8 C11 179.72(17) C16 C17 C18 C19 -178.1(2) C6 C5 C10 O4 179.68(18) C17 C18 C19 C20 -169.87(19) C6 C5 C10 C9 0.4(3) C18 C13 C14 C15 -1.3(3) C7 N2 C6 N4 0.3(2) C18 C19 C20 C11 -43.1(2) C7 N2 C6 C5 -179.9(2) C20 C11 C12 C13 -53.5(2) C7 N3 N4 C6 0.6(2)

Table 19. Hydrogen bonds in chiral isomer B of methyl acetate D H A d(DH)/Å d(HA)/Å d(DA)/Å DHA/° O4 H4 O3 0.84 1.80 2.546(2) 146.7 N1 H1 N2 0.88 2.12 2.829(3) 137.0

Table 20. Hydrogen atom coordinates (× ^10⁴ ) and isotropic shift parameters ( ^Ų × ^10³ ) of chiral isomer B of methyl acetate. atom x y z U(eq) H4 8383 -1329 3483 58 H1 5203 976 2303 42 H1A 2925 -694 -1191 72 H1B 2370 -1358 -309 72 H1C 2522 -3604 -1073 72 H3A 5133 -3802 1612 46 H3B 5736 -2182 929 46 H7 4446 6763 3869 42 H9 9030 3079 5317 38 H11 7683 8652 5757 35 H12A 9408 5392 6709 39 H12B 9452 7699 5931 39 H14 10875 9758 7209 47 H15 11326 12781 8510 54 H16 10124 13877 9440 56 H17 8477 11949 9044 50 H19A 7084 9966 7349 46 H19B 7425 7961 8265 46 H20A 6669 5980 6620 41 H20B 7734 4673 7250 41

5 Analytical methods

5.1 Polarizing Microscope (PLM)

PLM analysis was performed using an ECLIPSE LV100POL (Nikon, JPN) polarizing microscope. A small amount of sample was spread evenly on a glass slide, cedarwood oil was added to disperse the sample, and a coverslip was placed on top. The sample was then observed under the microscope with an objective lens.

5.2 X-ray Powder Diffraction (XRPD)

The solid sample was examined using an X-ray diffractometer. The sample was laid flat on a zero-background single-crystal silicon sample tray, and after being gently pressed flat, it was analyzed according to the parameters in Table 12.

As can be seen from the single-crystal structure analysis above, the chiral configuration of the methyl acetate chiral isomer B is the "S" configuration, as shown in Figure 12. Therefore, in Example 14, when the methyl ester is removed using an aqueous sodium hydroxide solution to obtain the carboxylic acid product, the already generated chiral configuration is not changed. Thus, it can be inferred that the chiral configuration of the product [(7-hydroxy-5-(1,2,3,4-tetrahydronaphthyl-2-yl)-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl)amino]acetic acid chiral isomer B in Example 14 is the "S" configuration.

Implementation Examples 20 Implementation Examples 13 and 14 Subacute toxicity test of compound

1. Experimental materials

1.1 Test sample

surface twenty one Sample Information Compound Name Example 13 Compound Example 14 Compound

1.2 Prescription preparation

Weigh appropriate amounts of compounds 13 and 14 from Example 13 and Example 14 into suitable glass bottles. Then, add DMSO, Solutol HS-15, and 0.9% physiological saline (in a ratio of v:v:v = 5%:10%:85%) to the glass bottles in sequence. Vortex and stir, continuously monitoring the drug dissolution during stirring until completely dissolved/suspended. Finally, prepare a dosing solution of compounds 13 and 14 with a concentration of 0.67 mg/mL, and use immediately after preparation.

2. experimental animals

Animal source and number: Species: rat; strain grade: SD rat, SPF; weight: 160-200g; number of selected animals: 24 (half male and half female).

The animal room is well-ventilated and equipped with air conditioning, maintaining a temperature of 20-25°C and a humidity of 40%-70%, with 12 hours of light and 12 hours of darkness. Animals have free access to food and water. SD rats that have undergone at least 3 days of acclimatization before drug administration and whose physical condition is good, as verified by a veterinarian, are eligible for this experiment.

3. Experimental plan

3.1 Trial cycle and dosage settings

All operations performed on the experimental animals in this experiment complied with the requirements of the "Regulations on the Management of Laboratory Animals" promulgated by the Ministry of Science and Technology of the People's Republic of China. This experiment included a solvent control group, a compound administration group (Example 13), and a compound administration group (Example 14). The dosage for the compound administration groups (Example 13 and Example 14) was 10 mg/kg, administered once daily for 14 consecutive days. The blank control group received an equal volume of blank solvent. The volume of each administration was 15 mL/kg. The toxic reactions and their severity were observed after each administration. Observation continued for 7 days after the last administration. Free access to water was provided throughout the experiment. See Table 22 for details.

surface twenty two Experimental dosage and subgroup design Group Dosage (mg/kg) Concentration (mg/mL) Frequency of drug administration Dosage volume (mL/kg) Number of animals (in animals) ♀ ♂ Blank control group / / Once a day for 14 consecutive days 15 4 4 Example 13 Low-dose group of compounds 10 0.67 Once a day for 14 consecutive days 15 4 4 Example 14: Low-dose group of compounds 10 0.67 Once a day for 14 consecutive days 15 4 4

After weighing, calculate the theoretical drug volume per SD rat using the following formula. The actual drug dosage and sample collection time for each SD rat should be recorded in detail in the corresponding table.

3.2 Experimental observation

During the experiment, researchers and veterinarians must continuously observe the physical characteristics and health status of the experimental animals. Any abnormal behavior in the animals, such as pain, depression, or reduced activity, must be recorded in the original experimental records. If the abnormal behavior of the experimental animals exceeds the requirements of the relevant animal welfare documents of IACUC, the veterinarian may determine whether to terminate the experiment and notify the project leader.

3.3 Observation from the cage

During the experiment, all animals should be observed at least twice daily at the cage edge, and continuously for two hours after drug administration. If any animal exhibits severe toxic side effects or dies during administration, the observation results should be recorded in the appropriate form. Observational indicators include: animal appearance, behavior, secretions, excrement, feeding status, and mortality (number of deaths, time of death, and pre-mortality reactions). If clinical symptoms appear, the frequency of observation should be increased to ensure detailed observation of the animal's poisoning symptoms, including onset time, severity, duration, and reversibility. If any animal is found to be dead or near death, an autopsy should be performed promptly according to regulations.

3.4 weight

Animals must be weighed before each daily administration of medication, and the weight should be recorded in the corresponding table for 14 consecutive days. The food intake of the experimental animals should also be monitored during the trial period.

3.5 Clinical Pathology

Approximately 1.8–2.0 mL of whole blood should be collected before the first administration and 24 hours after the last administration for hematological and blood biochemistry tests. Additionally, a blood sample should be collected 24 hours after the last administration for coagulation tests. If any abnormalities occur in the animal during administration, the hematological, coagulation, and blood biochemistry tests should be performed earlier. The animal must fast for at least 12 hours before blood collection.

Approximately 500 μL of whole blood was placed in an anticoagulant tube containing EDTA-K2 for hematological analysis.

White blood cell count (WBC); Neutrophil count (absolute count, NEUT; percentage, %NEUT); Lymphocyte count (absolute count, LYM; percentage, %LYM); Monocyte count (absolute count, MONO; percentage, %MONO); Eosinophil count (absolute count, EOS; percentage, %EOS); Basophil count (absolute count, BASO; percentage, %BASO); Red blood cells... Cell count (RBC); Hemoglobin (HGB); Hematocrit (HCT); Mean erythrocyte volume (MCV); Mean erythrocyte hemoglobin content (MCH); Mean erythrocyte hemoglobin concentration (MCHC); Erythrocyte variation coefficient (RDW-CV); Standard deviation of erythrocyte distribution width (RDW-SD); Platelet count (PLT); Mean platelet distribution width (MPV); Mean platelet volume (PDW); Plateletcrit (PCT);

Approximately 1.5 mL of whole blood is placed in a separation gel blood collection tube (without anticoagulant) for blood biochemistry analysis.

Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Alkaline phosphatase (ALP); Albumin (ALB); Total cholesterol (TC); Creatinine (CRE); Urea (UREA); Creatine kinase (CK); Triglycerides (TG); Total bilirubin (TBIL); Total protein (TP); Aspartate aminotransferase/alanine aminotransferase (AST/ALT); Globulins (Glo II); Albumin/Globulin (A/G II); Electrolytes (K+/Na+/Cl−/Ca2+).

Conclusion: Under the experimental conditions, compounds 13 and 14 did not cause obvious gastrointestinal necrosis, petechiae, or bloating in the stomach. Their toxicity was lower than that of enarodustat, and they had better safety.

The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

without.

Figure 1 is a micrograph of a single crystal sample of chiral isomer A of methyl acetate. Figure 2 is an asymmetric single-member diagram of the crystal structure of chiral isomer A of methyl acetate. Figure 3 is a unit cell diagram of the crystal structure of chiral isomer A of methyl acetate. Figure 4 is a packing diagram of the crystal structure of chiral isomer A of methyl acetate. Figure 5 is a comparison diagram of the single crystal sample of chiral isomer A of methyl acetate and the calculated XRPD. Figure 6 is a chemical structure diagram of chiral isomer A of methyl acetate. Figure 7 is a micrograph of a single crystal sample of chiral isomer B of methyl acetate. Figure 8 is an asymmetric single-member diagram of the crystal structure of chiral isomer B of methyl acetate. Figure 9 is a unit cell diagram of the crystal structure of chiral isomer B of methyl acetate. Figure 10 is a packing diagram of the crystal structure of chiral isomer B of methyl acetate. Figure 11 is a comparison diagram of XRPD obtained from actual measurement and calculation of single crystal samples of chiral isomer B of methyl acetate. Figure 12 is a chemical structure diagram of chiral isomer B of methyl acetate.

without.

Claims (12)

A compound selected from the following structures or its stereoisomers, geometric isomers, tautomers, hydrates, or pharmaceutically acceptable salts: . A method for preparing the compound as claimed in claim 1, or its stereoisomers, geometric isomers, tautomers, hydrates, or pharmaceutically acceptable salts, wherein the following synthetic route is used: , where n is 1, and _R1 is selected from mono- or poly-substituted hydrogen on an aromatic ring. A high-performance liquid chromatography (HPLC) method for obtaining the compound as described in claim 1 or its stereoisomers, geometric isomers, tautomers, hydrates, or pharmaceutically acceptable salts, wherein the chromatographic conditions are as follows: chromatographic column: chiral column (S,S)Whelk-O1; mobile phase A: carbon dioxide; mobile phase B: a mixed solution of propionitrile and isopropanol; elution using an isocratic elution program. A crystal of a compound, wherein the structure of the compound is as follows: The crystal belongs to the monoclinic system, space group P2 ₁ , with cell parameters a=13.2287(2) [Å], b=5.10690(10) [Å], c=13.7391(2) [Å], α＝90°, β＝104.3640(10)°, γ＝90°, cell volume V=899.17 (3) [Å], and minimum number of asymmetric members in the cell Z＝2. Crystals of the compound as claimed in claim 4, wherein the crystals of the compound have XRPD spectra as shown in Figure 11 or substantially as shown in Figure 11, wherein "substantially" means that the difference in the number of peaks is within 85%. A method for preparing crystals of the compound as described in claim 4 or 5, comprising the following steps: preparing the compound as described in claim 5 by slow volatilization at room temperature using dimethyl monoxide as a solvent. A crystal of a compound, wherein the structure of the compound is as follows: The crystal belongs to the monoclinic system, space group P2 ₁ , with cell parameters a=13.2296(2) [Å], b=5.10250(10) [Å], c=13.7423(2) [Å], α＝90°, β＝104.357(2)°, γ＝90°, cell volume V=898.69(3) [Å], and minimum number of asymmetric members in the cell Z＝2. Crystals of the compound as claimed in claim 7, wherein the crystals of the compound have XRPD spectra as shown in Figure 5 or substantially as shown in Figure 5, wherein "substantially" means that the difference in the number of peaks is within 85%. A method for preparing crystals of the compound as described in claim 7 or 8, wherein the compound as described in claim 8 is prepared by a programmed cooling method using acetonitrile as a solvent. A pharmaceutical composition comprising a compound selected from the following structures or its stereoisomers, geometric isomers, tautomers, hydrates, pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers, diluents, and excipients: . Use of a compound as claimed in claim 1, or any of its stereoisomers, geometric isomers, tautomers, hydrates, pharmaceutically acceptable salts, or crystals of the compounds as claimed in claims 4-5, 7-8, or a pharmaceutical composition as claimed in claim 10, in the preparation of a medicament for treating anemia, local ischemia, and hypoxia by inhibiting prolylhydroxylase. The use as described in claim 11, wherein the use is in the preparation of a medicament for treating renal anemia by inhibiting the prolylhydroxylase.