CN118908913A - Compound containing 2, 3-dihydrothiazole-4-carboxylic acid structure, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound containing a2, 3-dihydrothiazole-4-carboxylic acid structure, a preparation method and application thereof, and discloses a compound with a general formula (I) or pharmaceutically acceptable salt thereof: The compound with the structure shown in the formula I or the pharmaceutically acceptable salt thereof has obvious inhibition effect on PHD, and in-vitro and in-vivo experiments prove that the compound with the structure shown in the formula I has obvious inhibition effect on the activity of human PHD2 protein, can stably promote the accumulation of HIF in human Hep3B cells, promote the generation of EPO and can promote the level of mouse plasma EPO; the compound with the structure shown in the formula I or the pharmaceutically acceptable salt thereof has wide application value in treating and preventing diseases and disorders related to HIF, especially in treating anemia and ischemia caused by insufficient or lack of EPO.

Description

Compound containing 2,3-dihydrothiazole-4-carboxylic acid structure and preparation method and use thereof

Technical Field

The present invention relates to the technical field of pharmaceutical chemistry, and more specifically, to a class of compounds having a 2,3-dihydrothiazole-4-carboxylic acid structure and HIF proline hydroxylase inhibitory activity, and a preparation method and use thereof.

Background Art

Renal anemia is caused by insufficient renal erythropoietin (EPO) production due to various factors or by some toxic substances in the plasma of uremic patients interfering with the production and metabolism of red blood cells. It is one of the most important complications of chronic kidney disease (CKD). It not only causes cognitive impairment and reduced quality of life in patients, but also increases cardiovascular and cerebrovascular risks and mortality, and is an important cause of death in CKD patients. Erythropoiesis-stimulating drugs (ESA) are currently the most important clinical therapeutic drugs. Although traditional ESA pulse supplementation of exogenous EPO drugs improves the physiological condition of patients, it leads to a rapid increase in EPO levels, which is prone to cause cardiovascular and cerebrovascular diseases and increase the risk of death; combined with hypertension complications; the need for combined iron supplementation; some patients have low ESA response and EPO resistance; and the route of administration is limited. The use of small molecule targeted drugs to regulate hypoxia-induced erythropoiesis may solve the above clinical problems.

The synthesis of EPO in the kidney and liver is regulated by hypoxia inducible factor (HIF), and the activity of HIF depends on the activity of prolyl hydroxylase (PHD). PHD promotes the degradation of HIF by hydroxylating HIF, thus affecting the treatment of related diseases mediated by HIF. Therefore, PHD has become one of the important targets for the treatment of renal anemia. Studies have confirmed that compared with traditional drugs, PHD inhibitors are new therapeutic drugs based on the HIF-PHD mechanism. They inhibit HIF degradation by inhibiting PHD and maintain HIF levels in the body. They not only stimulate the remaining kidneys to promote the production of EPO by erythrocytes, but also correct iron metabolism abnormalities by inhibiting hepcidin. Comprehensive treatment of CKD anemia is more in line with the hematopoietic mechanism, so it is safer and more effective. In addition, this type of PHD small molecule inhibitor has the advantages of oral administration, relatively low price, and no need for combined use with iron preparations.

Currently, there are relatively few types of PHD inhibitors reported. Therefore, developing PHD inhibitors with structural diversity is very important for studying structure-activity relationships and developing new safe and effective PHD inhibitors.

Summary of the invention

In view of the deficiencies in the prior art, the present invention discloses a compound containing a 2,3-dihydrothiazole-4-carboxylic acid structure and a preparation method and use thereof. The compound or a pharmaceutically acceptable salt thereof can be used as a PHD inhibitor to safely and efficiently treat and prevent HIF-related diseases and disorders.

In order to achieve the above technical objectives, on the one hand, the present invention provides a compound containing a 2,3-dihydrothiazole-4-carboxylic acid structure or a pharmaceutically acceptable salt thereof:

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from hydrogen, methyl, ethyl, isopropyl, cyclohexyl, halogen, hydroxyl, trifluoromethyl, cyano, methoxy, phenyl or phenoxy.

Furthermore, the above-mentioned compound containing 2,3-dihydrothiazole-4-carboxylic acid structure or a pharmaceutically acceptable salt thereof is selected from the following compounds:

Further, the pharmaceutically acceptable salt of the compound includes a pharmaceutically acceptable metal salt, which is a salt form of the compound that is therapeutic and non-toxic; further, the present invention preferably forms a cationic salt of the compound with an acidic group (such as a carboxyl group), and further, an alkali metal (such as sodium and potassium) and an alkaline earth metal (magnesium and calcium) salt may be selected. It should be noted that the present invention is not limited to the type of pharmaceutically acceptable salt of a specific component. In the actual preparation process, a person skilled in the art may choose a certain salt and discard another salt based on factors such as solubility, stability, and ease of preparation; the determination and optimization of these salts are within the experience of skilled technicians.

On the other hand, the present invention provides a method for preparing the compound or a pharmaceutically acceptable salt thereof, wherein the compound is prepared by the following route:

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and R ⁶ are defined as the above compounds or their pharmaceutically acceptable salts. In the specific preparation process, ethyl 2-aminothiazole-4-carboxylate is reacted with carboxylic acid compounds at 30-90°C in a solvent such as DMF or DMSO, with N,N-diisopropylethylamine, triethylamine or pyridine as an acid-binding agent, catalyzed by coupling reagents such as HATU, HBTU, HCTU, DCC/DMAP, EDC/DMAP or PyBop, to obtain intermediate II. Intermediate II is stirred in methanol, ethanol or tetrahydrofuran, and an aqueous solution of sodium hydroxide, potassium hydroxide or lithium hydroxide is added dropwise, and the target compound I is obtained by hydrolysis reaction at 0-60°C.

In addition, the compound I is dissolved or suspended in DMF, acetonitrile, acetone, methanol, ethanol or ether to prepare a pharmaceutically acceptable salt with an inorganic base. The specific preparation process can be selected as follows: various compounds are dissolved or suspended in one of ether, DMF, acetone, methanol, ethanol, isopropanol, ethyl acetate, acetonitrile or DMSO, and an equimolar sodium hydroxide aqueous solution is added dropwise under an ice-water bath to prepare the sodium salt of the compound; or various compounds are dissolved in one of ether, DMF, acetone, methanol, ethanol, isopropanol, ethyl acetate, acetonitrile or DMSO, 0.5 times the mole of calcium carbonate is added, and the calcium salt thereof is obtained by heating and stirring, etc.

The research and development team of the present invention has verified through a large amount of experimental data that the compound containing 2,3-dihydrothiazole-4-carboxylic acid structure or its pharmaceutically acceptable salt proposed in the present invention is effective for treating and/or preventing anemia caused by decreased or insufficient erythropoietin, especially renal anemia. Although the compounds of the present invention can be directly administered without any preparation, the various compounds are preferably used in the form of pharmaceutical preparations, and the administration route can be parenteral route (such as intravenous, intramuscular administration) and oral administration.

On the other hand, the present invention provides a pharmaceutical composition, which comprises the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or a combination thereof.

On the other hand, the present invention provides a pharmaceutical preparation, which comprises the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or a combination thereof.

Furthermore, the pharmaceutical preparation is a tablet, capsule, injection or lyophilized powder injection.

Further, the preparation method of the pharmaceutical composition or pharmaceutical preparation of the compounds of the present invention is as follows: using standard and conventional technology, the compounds of the present invention are combined with solid or liquid carriers acceptable in pharmaceutics, and are arbitrarily combined with adjuvants and excipients acceptable in pharmaceutics to form microparticles or microspheres. Wherein, solid dosage forms can be selected as tablets, dispersed particles, capsules, sustained-release tablets, sustained-release micropills, etc.; solid carriers can be at least one substance, which can serve as diluents, flavoring agents, solubilizers, lubricants, suspending agents, adhesives, disintegrants and wrappers; inert solid carriers include magnesium phosphate, magnesium stearate, slip, lactose, pectin, propylene glycol, polysorbate 80, dextrin, starch, gelatin, cellulose materials such as methylcellulose, microcrystalline cellulose, low melting point paraffin, polyethylene glycol, mannitol, cocoa butter, etc.; and liquid dosage forms include solvents, suspensions such as injections, powders, etc.

Furthermore, the amount of the active ingredient (the compound containing 2,3-dihydrothiazole-4-carboxylic acid structure or a pharmaceutically acceptable salt thereof) contained in the above-mentioned pharmaceutical composition and pharmaceutical dosage form can be specifically used according to the patient's condition and the doctor's diagnosis. The amount or concentration of the compound used can be adjusted within a wider range. Generally, the amount of the active compound can be selected in the range of 0.5% to 90% (weight) of the composition; a further optional range is 0.5%-70%.

On the other hand, the present invention proposes the use of the above-mentioned compound or its pharmaceutically acceptable salt, pharmaceutical composition or pharmaceutical preparation in the preparation of drugs for preventing and/or treating ischemic diseases, especially in the prevention and/or treatment of anemia and ischemic diseases caused by EPO deficiency.

Furthermore, the anemia and ischemic diseases include renal anemia, ischemia, vascular disease, stroke, angina pectoris and apoplexy caused by anemia and/or ischemia; further, the ischemic diseases include anemia and ischemia caused by EPO deficiency or lack.

Compared with the prior art, the compound having the structure of formula I of the present invention or a pharmaceutically acceptable salt thereof has a significant inhibitory effect on PHD. In vitro and in vivo experiments have confirmed that the compound having the structure of formula I has a relatively significant inhibitory effect on the activity of human PHD2 protein, and can stably promote the accumulation of HIF in human Hep3B cells, as well as promote EPO production, and can increase the level of EPO in mouse plasma; the compound having the structure of formula I of the present invention or a pharmaceutically acceptable salt thereof has broad application value in the treatment and prevention of HIF-related diseases and disorders, especially in the treatment of anemia and ischemia caused by insufficient or deficient EPO.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

FIG1 shows the experimental results of Example 5 in which the compound having the structure of Formula I of the present invention promotes the EPO level in ICR mice.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, it should be understood that these embodiments are only used for more detailed description and should not be understood as limiting the present invention in any form, i.e., not intended to limit the scope of protection of the present invention.

Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the compounds are detected by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Subsequently, infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy ( ¹ H NMR, ¹³ C NMR), mass spectrometry (MS) and the like can be used to further confirm their structures. The experimental methods, unless otherwise specified, are conventional methods.

Unless otherwise defined, all technical terms herein have the same meaning as commonly understood by those skilled in the art to which the subject matter of the claims belongs. Unless otherwise indicated, all patents, patent applications, and public materials cited in the entirety of this article are incorporated herein by reference in their entirety.

It should be understood that the above brief description and the detailed description below are exemplary and are only used for explanation, and do not impose any restrictions on the subject matter of the present invention. In the present invention, unless otherwise specifically stated, the use of the singular also includes the plural. It must be noted that unless otherwise clearly stated in the text, the singular form used in this specification and claims includes the plural form of the referred thing. It should also be noted that unless otherwise stated, the "or" and "or" used mean "and/or". In addition, the term "including" and other forms, such as "including", "containing" and "containing" are not restrictive.

In the present invention, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

The compounds of the present invention or their pharmaceutically acceptable salts may contain one or more chiral carbon atoms and may therefore produce enantiomers, diastereomers and other stereoisomeric forms. Each chiral carbon atom may be defined as (R) or (S) based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the present invention may be prepared using racemates, diastereomers or enantiomers as starting materials or intermediates. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

In the present invention, "pharmaceutical composition" refers to a preparation of the compound of the present invention and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of the organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

The term "pharmaceutically acceptable" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, that is, the substance can be administered to a subject without causing adverse biological reactions or interacting in an adverse manner with any components contained in the composition.

In the present invention, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by relevant government regulatory authorities as acceptable for human or livestock use.

As used herein, the terms "prophylactic," "prevention," and "prevention" include reducing the likelihood of a disease or condition occurring or worsening in a patient.

As used herein, the terms "treat," "treat," "treat," and other similar synonyms include the following meanings: (i) preventing a disease or condition from occurring in a mammal, particularly when such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition; (ii) inhibiting the disease or condition, i.e., curbing its development; (iii) alleviating the disease or condition, i.e., causing the state of the disease or condition to regress; or (iv) alleviating the symptoms caused by the disease or condition.

Example 1

A method for preparing a compound containing a 2,3-dihydrothiazole-4-carboxylic acid structure, using the following preparation route:

The method comprises step 1, preparing intermediate II; step 2, preparing the compound from intermediate II; specifically:

Step 1

Taking the preparation of intermediate II-1 as an example, its preparation route is:

Specifically, 3-chlorophenylacetic acid (1.71 g, 10 mmol), DMF (50 ml), N, N-diisopropylethylamine (2.58 g, 20 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBop) (5.20 g, 10 mmol) were added to a reaction bottle equipped with a thermometer and a stirring device, and the mixture was stirred at room temperature for 30 min. Ethyl 2-aminothiazole-4-carboxylate (1.72 g, 10 mmol) was added and heated to 40° C. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into cold water (120 mL). Solids precipitated and were filtered. The filter cake was washed three times with water (30 mL×3). The solid obtained after drying was purified by column chromatography (eluent: v (petroleum ether): v (ethyl acetate) = 4: ¹ ) to obtain intermediate II-1 (2.44 g) as a white solid with a yield of 75.2%. NMR (400MHz, DMSO) δ12.80 (s, 1H), 8.05 (s, 1H), 7.42 (d, J = 1.8Hz, 1H), 7.40-7.32 (m, 2H) ,7.29(dt,J＝7.0,1.7Hz,1H),4.27(q,J＝7.1Hz,2H),3.80(s,2H),1.29(t,J＝7.1Hz,3H). ESI-MS(m/z)[M+H]+:326.0.

Taking the preparation of intermediate II-5 as an example, its preparation route is:

Specifically, phenoxyphenylacetic acid (2.28 g, 10 mmol), DMF (50 ml), triethylamine (2.02 g, 20 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (3.80 g, 10 mmol) were added to a reaction bottle equipped with a thermometer and a stirring device, and the mixture was stirred at room temperature for 30 min. Ethyl 2-aminothiazole-4-carboxylate (1.72 g, 10 mmol) was added and heated to 80° C. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into cold water (120 mL). Solids precipitated and were filtered. The filter cake was washed three times with water (30 mL× ³ ). The solid obtained after drying was purified by column chromatography (eluent: v (petroleum ether): v (ethyl acetate) = 3:1) to obtain intermediate II-5 (2.35 g) as a white solid with a yield of 61.5%. NMR (400MHz, DMSO) δ12.78(s,1H),8.04(s,1H),7.43-7.31(m,4H),7.13(t,J＝6.9Hz,1H) ,6.99(dd,J＝12.9,4.9Hz,4H),4.27(q,J＝7.1Hz,2H),3.75(s,2H),1.29(t,J＝7.1Hz,3H). ESI-MS(m/z)[M+H]+:383.1.

By referring to the synthesis methods of intermediate II-1 and intermediate II-5, the following intermediate II shown in Table 1 can be obtained.

Table 1

Step 2

Taking the preparation of (E)-2-(2-(3-chlorophenyl)acetimidyl)-2,3-dihydrothiazole-4-carboxylic acid (Compound I-1) as an example, the preparation route is:

Specifically, intermediate II-1 (3.25 g, 10 mmol) and ethanol (50 mL) were added to a reaction bottle equipped with a thermometer and a stirring device in sequence, and stirred. A potassium hydroxide (1.40 g, 25 mmol) aqueous solution was added dropwise under ice-water bath conditions. The mixture was reacted overnight at room temperature. TLC showed that the reaction was complete. The pH was adjusted to 6 with acetic acid, filtered, and the filter cake was washed with water (30 mL×3) and dried to obtain compound I-1 as a white solid (2.77 g). The yield was 93.5%. ¹ H NMR (600 MHz, DMSO) δ13.56 (brs, 2H), 7.71 (s, 1H), 7.45 (s, 1H), 7.37-7.31 (m, 3H), 3.96 (s, 2H). ¹³ C NMR (151MHz, DMSO) δ 169.83, 164.28, 157.92, 138.13, 133.31, 130.61, 129.76, 128.63, 127.19, 119.46, 41.47. ESI-MS (m/z) [M+H]+: 298.0.

Referring to the preparation method of compound Ⅰ-1, compounds Ⅰ-2 to Ⅰ-11 can be synthesized. The results are shown in Table 2.

Table 2

It should be noted that the biological activity of the compounds of the present invention can be evaluated by using any conventional known method; suitable analytical methods are well known to those skilled in the art. The biological activity of the compounds of the present invention having the structure of Formula I or their pharmaceutically acceptable salts was tested in the following Examples 2-5. It should be noted that these examples are presented only as examples and are not intended to limit the scope of protection of the present invention.

Example 2

In this example, the cytotoxicity of compounds containing 2,3-dihydrothiazole-4-carboxylic acid structure was detected:

Specifically, the compound to be tested was dissolved in DMSO to prepare a 30 mmol/L stock solution, and diluted with culture medium to various concentrations (final DMSO concentration <0.1%) for later use. Human Hep3B cells were inoculated into 96-well plates (4-5×103, 100 μL per well), and the test compound was added after 24 hours of cell culture. Incubate at 37°C, 5% CO2 for 72 hours, add MTT (10 μL, 5 mg/mL phosphate buffered saline) to each well, continue incubation for 4 hours, then aspirate the culture medium, and add DMSO (150 μL) to each well. The OD value was read at 490 nm by an enzyme reader, and the inhibition rate was calculated. The results are shown in Table 3, where FG-4592 (Roxadustat), also known as Roxadustat, is an orally bioactive HIF-PHD inhibitor.

Table 3

Table 3 shows that the cell survival rate of the compound containing 2,3-dihydrothiazole-4-carboxylic acid structure of the present invention at 50 μM and a higher concentration of 100 μM is higher than that of the positive control drug FG-4592, and the safety at the cellular level is better than that of FG-4592.

Example 3

Detection of the activity of the compounds of the present invention in inhibiting PHD2:

Experimental materials: human PHD2 recombinant protein, Fitc-HIF-1α(556-574), hydroxyethylpiperazineethionine (Hepes), 2-OG, MnCl ₂ , NaCl, Tween-20.

Experimental method: Prepare buffer (10mM Hepes, 150mM NaCl, 100μM MnCl ₂ , 10μM 2-OG, 0.05% Tween-20), pH 7.4, for later use. Add 1.2μM PHD2 (181-426) and the test compound to the 96-well ELISA plate in sequence, incubate at 37°C for 30min, then add 60nM Fitc-HIF1α556-574, the final volume of the reaction solution is 60μL and the DMSO content is less than 1%. Incubate the 96-well plate at 37°C for 2h, use an ELISA reader to detect the fluorescence intensity (λ _ex =485nM±25nM, λ _em =535nM±25nM), and calculate the IC ₅₀ value of each compound against PHD2. The results are shown in Table 4.

Table 4 IC ₅₀ values of preferred compounds against PHD2 (μM)

Group IC ₅₀ (μM) Group IC ₅₀ (μM) Group IC ₅₀ (μM) FG-4592 9.1±0.27 Ⅰ-4 9.0±0.58 Ⅰ-8 11.7±0.21 Ⅰ-1 10.5±0.42 Ⅰ-5 12.9±0.24 Ⅰ-9 22.5±0.29 Ⅰ-2 21.4±0.53 Ⅰ-6 63.0±2.11 Ⅰ-10 12.0±0.66 Ⅰ-3 37.0±0.35 Ⅰ-7 14.3±0.55 Ⅰ-11 9.7±0.31

Table 4 shows that compounds Ⅰ-1 to Ⅰ-11 have different degrees of inhibitory effects on PHD2, among which compounds Ⅰ-1, Ⅰ-4 and Ⅰ-11 have comparable activities to the positive control drug FG-4592. This shows that the compound of formula Ⅰ of the present invention has potential PHD2 inhibitory activity.

Example 4

Cell-based ELISA assay to detect the ability of compounds to boost EPO levels:

Experimental materials: human hepatoma cells (Hep3B), culture medium (10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin), 96-well plates, human EPO kit, BIO-TEK Uquant multifunctional microplate reader.

Experimental method: Hep3B cells were suspended in culture medium (10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin) to prepare a suspension, and then inoculated into a 96-well plate and cultured at 37°C, 5% _CO2 . The next day, different concentrations of the test compound were added and cultured for 24 hours. The supernatant was taken and the EPO concentration of the supernatant was detected using an EPO kit; the experimental results are shown in Table 5.

Table 5 Relative EPO-promoting ability of the tested compounds

Group Relative EPO-promoting ^ability Group Relative EPO-promoting ^ability FG-4592 1.27 Ⅰ-6 1.02 Ⅰ-1 1.38 Ⅰ-7 1.18 Ⅰ-2 1.19 Ⅰ-8 1.22 Ⅰ-3 1.17 Ⅰ-9 1.12 Ⅰ-4 1.40 Ⅰ-10 1.07 Ⅰ-5 1.16 Ⅰ-11 1.24

aRelative EPO-promoting ability = EPO concentration of the tested compound in promoting cell production/ctrl

The experimental results in Table 5 confirm that compared with the control group, compounds I-1 to I-11 can promote the production of EPO in Hep3B cells, among which compounds I-1, I-4, I-8 and I-11 have an ability to promote EPO close to that of the positive control drug FG-4592. This shows that this type of compound can promote EPO production at the cellular level and has the potential to treat renal anemia.

Example 5

Test of the compound having the structure of Formula I or its pharmaceutically acceptable salt for increasing the EPO level in ICR mice:

Experimental materials: SPF-grade ICR mice (male, weighing 18-20 g), mouse erythropoietin kit (Nanjing Jiancheng), BIO-TEK Uquant multifunctional microplate reader.

Experimental method: After male ICR mice (18-20g) were adapted to feeding for 7 days, ICR mice were randomly divided into blank group, positive drug group and test drug group according to body weight, with 5 mice/cage in each group. After fasting for 12 hours before the experiment, the mice were gavaged with the corresponding test drug (0.5% CMC suspension) at a dose of 20 mg/kg. After 4 hours of administration, blood was collected from the canthus of each group of animals, anticoagulated with EDTA, and centrifuged at 3000r/min for 20 minutes to collect plasma. The mouse erythropoietin kit was used to detect the EPO concentration in plasma; the experimental results are shown in Figure 1.

The in vitro test results in Figure 1 confirm that when the dosage is 20 mg/kg, compounds Ⅰ-1 to Ⅰ-11 can increase the level of EPO in mice to varying degrees. It can be seen that the compound with the structure of formula Ⅰ has the potential to improve anemia.

Example 6

This example illustrates a method for preparing a pharmaceutically acceptable salt of a compound having a structure of Formula I, taking the sodium salt of Compound I-1 as an example. Specifically, 2.9 g of a white solid product of Compound I-1 was suspended in 12 mL of ethanol, and a 2.0 mol/L aqueous sodium hydroxide solution (5 ml) was added dropwise under an ice-water bath. After the addition was completed, the mixture was stirred under an ice-water bath for about 1 hour, and the solvent was evaporated under reduced pressure to obtain a white solid, i.e., the sodium salt of Compound I-1.

In order to more fully illustrate the pharmaceutical composition containing 2,3-dihydrothiazole-4-carboxylic acid compounds or pharmaceutically acceptable salts thereof of the present invention, the following pharmaceutical preparation examples 7-10 are provided below, which are only for illustration and not for limiting the scope of the present invention. The preparation can use any active compound and salt thereof of the compounds of the present invention, preferably the compound described in Example 1.

Example 7

A pharmaceutical preparation, which is a hard gelatin capsule prepared using the following ingredients:

Preparation process: Dry the raw materials and auxiliary materials in advance, pass them through a 100-mesh sieve for later use. Mix the above ingredients according to the prescribed amount and fill them into hard gelatin capsules.

Example 8

A pharmaceutical preparation, which is a tablet prepared using the following ingredients:

Preparation process: Dry the raw materials and excipients in advance, and pass them through a 100-mesh sieve for later use. First, fully mix the excipients in the prescribed amount. Add the API to the excipients by incremental dilution method, and fully mix 2-3 times each time to ensure that the drug and excipients are fully mixed, pass through a 20-mesh sieve, and dry in a ventilated oven at 55°C for 2 hours. Pass the dry granules through a 16-mesh sieve, determine the intermediate content, mix evenly, and press on a tablet press.

Example 9

A pharmaceutical preparation, which is an injection prepared using the following composition:

Preparation method: Take the active ingredient and add it to the injection water in which polysorbate and propylene glycol have been dissolved, add medicinal acid to adjust the pH value to 4-8 to dissolve it. Add activated carbon, stir and adsorb for 30 minutes, remove carbon, fine filter, potting, and sterilize.

Example 10

A pharmaceutical preparation, which is a lyophilized powder for injection prepared from the following materials:

Calcium salt of compound Ⅰ-2 60mg

Medicinal base 0.1-7.0%

Mannitol 55-85%

Preparation method: Take the active ingredient and add it to water for injection, adjust the pH value to 4-8 with medicinal acid to dissolve it. Then add mannitol, sterilize it under high pressure according to the requirements of injection, add activated carbon, filter it with a microporous filter membrane, package the filtrate, freeze-dry it, obtain loose blocks, seal it, and you are done.

It should be noted that the above contents are further detailed descriptions of the present invention in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions; the dimensional data of this embodiment does not necessarily limit the technical solution, but only shows one of the specific working conditions. For ordinary technicians in the technical field to which the present invention belongs, several simple improvements and modifications can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims (10)

1. A compound of general formula (I) or a pharmaceutically acceptable salt thereof: Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are independently selected from hydrogen, methyl, ethyl, isopropyl, cyclohexyl, halogen, hydroxyl, trifluoromethyl, cyano, methoxy, phenyl or phenoxy. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that it is selected from the following compounds: 3. The compound or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the pharmaceutically acceptable salt comprises a pharmaceutically acceptable metal salt. 4. A method for preparing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that the compound is prepared by the following route: Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , and R ⁶ have the same definitions as in claim 1. 5. A pharmaceutical composition, characterized in that it comprises the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or a combination thereof. 6 . The pharmaceutical composition according to claim 5 , characterized in that the mass content of the compound or the pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.5% to 90%. 7. A pharmaceutical preparation, characterized in that it comprises the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient or a combination thereof. 8. The pharmaceutical preparation according to claim 7, characterized in that the pharmaceutical preparation is a tablet, a capsule, an injection or a lyophilized powder injection. 9. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the pharmaceutical composition according to claim 5 or 6, or the pharmaceutical preparation according to claim 7 or 8 in the preparation of a drug for preventing and/or treating anemia or ischemic diseases. 10. The use according to claim 9, characterized in that the anemia and ischemic diseases include renal anemia, ischemia, vascular disease, stroke, angina pectoris and apoplexy caused by anemia and/or ischemia.