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

Abstract

The present invention discloses a compound containing a 2,3-dihydrothiazole-4-carboxylic acid structure, a preparation method, and uses thereof, and discloses a compound of the general formula (I) or a pharmaceutically acceptable salt thereof: #imgabs0# 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, can stably promote the accumulation of HIF in human Hep3B cells, 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 treating and preventing diseases and disorders related to HIF, especially in treating anemia and ischemia caused by EPO deficiency or lack thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and more specifically, to a class of compounds having a 2,3-dihydrothiazole-4-carboxylic acid structure and HIF proline hydroxylase inhibitory activity, as well as preparation methods and uses thereof. Background Art

[0002] Renal anemia is caused by insufficient renal erythropoietin (EPO) production due to various factors, or by toxic substances in the plasma of uremic patients that interfere with red blood cell production and metabolism. It is one of the most common complications of chronic kidney disease (CKD). It not only causes cognitive impairment and a decreased quality of life in patients, but also increases cardiovascular and cerebrovascular risks and mortality, making it a major cause of death in CKD patients. Erythropoiesis-stimulating drugs (ESAs) are currently the mainstay of clinical treatment. Traditional ESAs, which pulse supplement with exogenous EPO, improve patients' physiological conditions but also lead to rapid increases in EPO levels, which can easily lead to cardiovascular and cerebrovascular disease and increase mortality risk. They are also associated with hypertension complications, require iron supplementation, are hyporesponsive to ESAs and resistant to EPO in some patients, and have limited routes of administration. Using small molecule targeted drugs to regulate hypoxia-induced erythropoiesis has the potential to address these clinical challenges.

[0003] 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 its proline hydroxylase (PHD). PHD promotes its degradation by hydroxylating HIF, thereby affecting the treatment of related diseases mediated by HIF. Therefore, PHD has become one of the important targets for the current 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, maintaining HIF levels in the body, not only stimulating the production of EPO by residual renal erythrocytes, but also correcting abnormal iron metabolism by inhibiting hepcidin. This comprehensive treatment of CKD anemia is more consistent with the hematopoietic mechanism and is therefore safer and more effective. In addition, this type of PHD small molecule inhibitor has the advantages of being orally available, relatively low price, and not requiring co-administration with iron supplements.

[0004] 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 safe and effective new PHD inhibitors. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention discloses a compound containing a 2,3-dihydrothiazole-4-carboxylic acid structure, a preparation method, and uses 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.

[0006] 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:

[0007]

[0008] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each is independently selected from hydrogen, methyl, ethyl, isopropyl, cyclohexyl, halogen, hydroxy, trifluoromethyl, cyano, methoxy, phenyl or phenoxy.

[0009] Furthermore, the compound containing 2,3-dihydrothiazole-4-carboxylic acid structure or a pharmaceutically acceptable salt thereof is selected from the following compounds:

[0010]

[0011] Furthermore, pharmaceutically acceptable salts of the compounds include pharmaceutically acceptable metal salts, which are non-toxic salt forms of the compounds that are therapeutically effective. Furthermore, the present invention prefers cationic salts formed by the compounds with acidic groups (e.g., carboxyl groups), and further salts of alkali metals (e.g., sodium and potassium) and alkaline earth metals (magnesium and calcium) are also preferred. It should be noted that the present invention is not limited to the type of pharmaceutically acceptable salt of a specific component. During the actual preparation process, those skilled in the art may choose one salt over another based on factors such as solubility, stability, and ease of formulation. The determination and optimization of these salts are within the experience of skilled artisans.

[0012] 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:

[0013]

[0014] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6The definition of is the same as that of the above-mentioned compound or its pharmaceutically acceptable salt. The specific preparation process can be as follows: ethyl 2-aminothiazole-4-carboxylate is reacted with a carboxylic acid compound at 30-90°C in a solvent such as DMF or DMSO, using N,N-diisopropylethylamine, triethylamine, or pyridine as an acid-binding agent, catalyzed by a coupling reagent such as HATU, HBTU, HCTU, DCC / DMAP, EDC / DMAP, or PyBop, to produce 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. A hydrolysis reaction is carried out at 0-60°C to obtain the target compound I.

[0015] In addition, the compound I is dissolved or suspended in DMF, acetonitrile, acetone, methanol, ethanol, or diethyl ether and reacted with an inorganic base to form a pharmaceutically acceptable salt. The specific preparation process can be as follows: various compounds are dissolved or suspended in one of diethyl ether, DMF, acetone, methanol, ethanol, isopropanol, ethyl acetate, acetonitrile, or DMSO, and an equimolar aqueous sodium hydroxide solution is added dropwise under an ice-water bath to form the sodium salt of the compound; or various compounds are dissolved in one of diethyl ether, DMF, acetone, methanol, ethanol, isopropanol, ethyl acetate, acetonitrile, or DMSO, 0.5 times the molar amount of calcium carbonate is added, and the mixture is heated and stirred to form the calcium salt thereof, etc.

[0016] The research and development team of the present invention has verified through a large amount of experimental data that the compounds containing the 2,3-dihydrothiazole-4-carboxylic acid structure or pharmaceutically acceptable salts thereof proposed in the present invention are 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 administered directly without any preparation, the various compounds are preferably used in the form of pharmaceutical preparations, and the routes of administration can be parenteral (such as intravenous, intramuscular) and oral.

[0017] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0018] On the other hand, the present invention provides a pharmaceutical preparation comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0019] Furthermore, the pharmaceutical preparation is a tablet, capsule, injection or lyophilized powder injection.

[0020] Furthermore, the preparation method of the pharmaceutical composition or pharmaceutical preparation of the compound of the present invention is as follows: using standard and conventional techniques, the compound of the present invention is combined with a solid or liquid carrier acceptable in pharmaceutics, and arbitrarily combined with an adjuvant and excipient acceptable in pharmaceutics to form microparticles or microspheres. Wherein, the solid dosage form can be optionally tablets, dispersed granules, capsules, sustained-release tablets, sustained-release pellets, etc.; the solid carrier can be at least one substance, which can serve as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, adhesive, disintegrant, and encapsulating agent; inert solid carriers include magnesium phosphate, magnesium stearate, sucrose, 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.

[0021] 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 wide range. Generally, the amount of the active compound can be selected in the range of 0.5% to 90% (by weight) of the composition; a further optional range is 0.5%-70%.

[0022] On the other hand, the present invention provides the use of the above-mentioned compound or its pharmaceutically acceptable salt, pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for preventing and / or treating ischemic diseases, in particular, in the prevention and / or treatment of anemia and ischemic diseases caused by EPO deficiency.

[0023] 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.

[0024] 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 treating and preventing HIF-related diseases and disorders, especially in treating anemia and ischemia caused by EPO deficiency or deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 The experimental results of Example 5 showing that the compound having the structure of Formula I of the present invention promotes EPO levels in ICR mice. DETAILED DESCRIPTION

[0027] To facilitate understanding of the present invention, the present invention will be described in more detail below, with preferred embodiments of the present invention provided. However, it should be understood that these embodiments are merely for the purpose of further explanation and are not to be construed as limiting the present invention in any form, i.e., they are not intended to limit the scope of protection of the present invention.

[0028] 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). Subsequent analysis can be performed using methods such as infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 The structure was further confirmed by C NMR, mass spectrometry (MS), etc. The experimental methods described above are all conventional methods unless otherwise specified.

[0029] Unless otherwise defined, all technical and scientific terms herein have the same meanings as commonly understood by those skilled in the art to which the subject matter of the claims pertains. Unless otherwise indicated, all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

[0030] 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 limit the subject matter of the present invention in any way. 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 forms used in this specification and claims include the plural forms of the things referred to. It should also be noted that unless otherwise stated, the use of "or" and "or" means "and / or". In addition, the use of the term "including" and other forms, such as "comprising", "including" and "containing" are not restrictive.

[0031] In the present invention, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0032] 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 can 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.

[0033] In the present invention, a "pharmaceutical composition" refers to a formulation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0034] As used herein, the term "pharmaceutically acceptable" 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 an adverse biological response or interacting in an adverse manner with any components contained in the composition.

[0035] In the present invention, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant government regulatory authorities as acceptable for use by humans or livestock.

[0036] As used herein, the terms "prophylactic," "prevention," and "preventing" include reducing the likelihood of a disease or condition occurring or worsening in a patient.

[0037] As used herein, the terms "treat," "treat," "treat," "treat," and similar synonyms include the following: (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 disease or condition to regress; or (iv) alleviating the symptoms caused by the disease or condition.

[0038] Example 1

[0039] A method for preparing a compound containing a 2,3-dihydrothiazole-4-carboxylic acid structure, using the following preparation route:

[0040]

[0041] The method comprises step 1, preparing intermediate II; step 2, preparing the compound from intermediate II; specifically:

[0042] Step 1

[0043] Taking the preparation of intermediate II-1 as an example, its preparation route is:

[0044]

[0045] 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 flask equipped with a thermometer and a stirring device in sequence, 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). Solid precipitated and was 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%. 1 HNMR(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.

[0046] Taking the preparation of intermediate II-5 as an example, its preparation route is:

[0047]

[0048] 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 flask equipped with a thermometer and a stirring device in sequence, 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). Solid precipitated and was 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%. 1 H 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.

[0049] By referring to the synthetic methods of Intermediate II-1 and Intermediate II-5, the following Intermediate II shown in Table 1 can be obtained.

[0050] Table 1

[0051]

[0052]

[0053] Step 2

[0054] 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:

[0055]

[0056] Specifically, intermediate II-1 (3.25 g, 10 mmol) and ethanol (50 mL) were added sequentially to a reaction flask equipped with a thermometer and a stirring device, and stirred. A potassium hydroxide (1.40 g, 25 mmol) aqueous solution was added dropwise in an ice-water bath. The reaction was allowed to proceed overnight at room temperature. TLC showed that the reaction was complete. The pH was adjusted to 6 with acetic acid, and the reaction was filtered. The filter cake was washed with water (30 mL × 3) and dried to obtain compound I-1 as a white solid (2.77 g, yield 93.5%).1 H NMR (600MHz, 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.

[0057] 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.

[0058] Table 2

[0059]

[0060]

[0061] It should be noted that the biological activity of the compounds of the present invention can be evaluated 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 provided merely as examples and are not intended to limit the scope of protection of the present invention.

[0062] Example 2

[0063] In this example, the cytotoxicity of compounds containing 2,3-dihydrothiazole-4-carboxylic acid structure was detected:

[0064] Specifically, the test compound was dissolved in DMSO to prepare a 30 mmol / L stock solution, which was then diluted with culture medium to various concentrations (final DMSO concentration <0.1%) for later use. Human Hep3B cells were seeded into 96-well plates (4-5×10³, 100 μL per well). After 24 hours of cell culture, the test compound was added. The cells were incubated at 37°C, 5% CO₂ for 72 hours. MTT (10 μL, 5 mg / mL in phosphate-buffered saline) was added to each well. After a further 4 hours of incubation, the culture medium was aspirated and DMSO (150 μL) was added to each well. OD values ​​were read at 490 nm using a microplate reader, and the inhibition rate was calculated. The results are shown in Table 3. FG-4592 (Roxadustat), also known as roxadustat, is an orally bioavailable HIF-PHD inhibitor.

[0065] Table 3

[0066]

[0067] 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.

[0068] Example 3

[0069] Detection of the PHD2 inhibitory activity of the compounds of the present invention:

[0070] Experimental materials: human PHD2 recombinant protein, Fitc-HIF-1α (556-574), hydroxyethylpiperazineethionine (Hepes), 2-OG, MnCl2, NaCl, Tween-20.

[0071] Experimental Methods: Prepare buffer (10mM Hepes, 150mM NaCl, 100μM MnCl2, 10μM 2-OG, 0.05% Tween-20), pH 7.4, and set aside. Add 1.2μM PHD2 (181-426) and the test compound to a 96-well microtiter plate, incubate at 37°C for 30 minutes, and then add 60nM Fitc-HIF1α556-574. The final reaction volume is 60μL and the DMSO content is less than 1%. Incubate the 96-well plate at 37°C for 2 hours. Fluorescence intensity (λ ex =485nM±25nM, λ em =535nM±25nM), and calculate the IC of each compound against PHD2 50 The results are shown in Table 4.

[0072] Table 4 IC values ​​of preferred compounds for PHD2 50 Value (μM)

[0073] Group <![CDATA[IC 50 (μM)]]> Group <![CDATA[IC 50 (μM)]]> Group <![CDATA[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

[0074] 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 activities comparable to those of the positive control drug FG-4592. This indicates that the compound of formula I of the present invention has potential PHD2 inhibitory activity.

[0075] Example 4

[0076] Cell-based ELISA assay to detect the ability of compounds to increase EPO levels:

[0077] Experimental materials: human hepatocellular carcinoma 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.

[0078] 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, 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 collected and the EPO concentration in the supernatant was detected using an EPO kit; the experimental results are shown in Table 5.

[0079] Table 5 Relative EPO-promoting ability of the tested compounds

[0080] 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

[0081] aRelative EPO-promoting ability = EPO concentration of the test compound in promoting cell production / ctrl

[0082] The experimental results in Table 5 confirmed that compared with the control group, compounds I-1 to I-11 were able to promote the production of EPO in Hep3B cells, among which compounds I-1, I-4, I-8 and I-11 had an EPO-promoting ability 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.

[0083] Example 5

[0084] Test of the compound of Formula I or its pharmaceutically acceptable salt to increase EPO levels in ICR mice:

[0085] Experimental materials: SPF-grade ICR mice (male, weighing 18-20 g), mouse erythropoietin kit (Nanjing Jiancheng), BIO-TEKUquant multifunctional microplate reader.

[0086] Experimental method: After male ICR mice (18-20g) were adapted to feeding for 7 days, they were randomly divided into blank group, positive drug group and test drug group according to body weight, with 5 mice per 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 20mg / 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 EPO concentration in the plasma was detected using a mouse erythropoietin kit; the experimental results are shown in the figure below. Figure 1 .

[0087] Depend on Figure 1The results of in vitro tests confirmed that when the dosage was 20 mg / kg, compounds I-1 to I-11 could increase the EPO level in mice to varying degrees. This shows that the compound having the structure of formula I has the potential to improve anemia.

[0088] Example 6

[0089] This example illustrates a method for preparing a pharmaceutically acceptable salt of a compound having the 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 complete, stirring was continued under an ice-water bath for about 1 hour, and the solvent was evaporated under reduced pressure to obtain a white solid, namely the sodium salt of Compound I-1.

[0090] To more fully illustrate the pharmaceutical compositions of the present invention containing 2,3-dihydrothiazole-4-carboxylic acid compounds or pharmaceutically acceptable salts thereof, the following pharmaceutical formulation examples 7-10 are provided below. These examples are for illustration only and are not intended to limit the scope of the present invention. The formulations may use any active compound of the present invention and its salt, preferably the compound described in Example 1.

[0091] Example 7

[0092] A pharmaceutical preparation, which is a hard gelatin capsule prepared using the following ingredients:

[0093]

[0094] Preparation process: Dry the raw materials and auxiliary materials in advance, pass them through a 100-mesh sieve and set aside. Mix the above ingredients according to the prescribed amount and fill them into hard gelatin capsules.

[0095] Example 8

[0096] A pharmaceutical preparation, which is a tablet prepared using the following ingredients:

[0097]

[0098] Preparation process: Pre-dry the raw and excipient materials and pass them through a 100-mesh sieve for later use. First, thoroughly mix the prescribed amount of excipients. Add the API to the excipients using an incremental dilution method, mixing thoroughly 2-3 times between additions to ensure thorough mixing of the drug and excipients. Pass through a 20-mesh sieve and dry in a ventilated oven at 55°C for 2 hours. Pass the dried granules through a 16-mesh sieve, determine the intermediate content, mix thoroughly, and compress on a tablet press.

[0099] Example 9

[0100] A pharmaceutical preparation, which is an injection prepared using the following composition:

[0101]

[0102] Preparation: Add the active ingredient to water for injection in which polysorbate and propylene glycol have been dissolved. Add a medicinal acid to adjust the pH to 4-8 to dissolve the active ingredient. Add activated carbon and stir for 30 minutes. Remove the carbon, filter thoroughly, seal, and sterilize.

[0103] Example 10

[0104] A pharmaceutical preparation, which is a lyophilized powder for injection prepared using the following materials:

[0105] Calcium salt of compound I-2 60 mg

[0106] Medicinal base 0.1-7.0%

[0107] Mannitol 55-85%

[0108] Preparation method: Add the active ingredient to water for injection and adjust the pH to 4-8 with a medicinal acid to dissolve it. Then add mannitol and sterilize under high pressure according to the requirements of injection. Add activated carbon and filter through a microporous membrane. The filtrate is packaged and freeze-dried to obtain loose blocks. The blocks are sealed.

[0109] It should be noted that the above content is a further detailed description of the present invention in conjunction with specific implementation methods, and the specific implementation of the present invention cannot be considered to be limited to these descriptions; the dimensional data of this embodiment does not necessarily limit the technical solution, but only illustrates one specific working condition. For those skilled in the art of the present invention, simple improvements and modifications can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: in, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 Each is independently selected from hydrogen, methyl, ethyl, isopropyl, cyclohexyl, halogen, hydroxy, trifluoromethyl, cyano, methoxy, phenyl or phenoxy.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that Selected from the following compounds:

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The pharmaceutically acceptable salts include pharmaceutically acceptable metal salts.

4. A method for preparing the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The compound is prepared by the following route:

5. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The mass content of the compound or its pharmaceutically acceptable salt in the pharmaceutical composition is 0.5% to 90%.

7. A pharmaceutical preparation, characterized in that The invention comprises the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

8. The pharmaceutical preparation according to claim 7, characterized in that The pharmaceutical preparation is a tablet, capsule, injection or freeze-dried 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 medicament 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.

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