Small molecule compound and application thereof in in-vitro amplification of hematopoietic stem cells and / or progenitor cells

Abstract

The invention discloses a small-molecule compound and application thereof in in-vitro amplification of hematopoietic stem cells and / or progenitor cells. The structural general formula of the small-molecule compound is as shown in formula (I). The series of compounds disclosed by the invention can be used in the medical field of related diseases taking hematopoietic stem cell transplantation as a treatment method, including in-vitro amplification culture of hematopoietic stem cells and progenitor cells from umbilical cord blood, bone marrow and mobilized peripheral blood and culture, maintenance and amplification of genetically modified hematopoietic stem and progenitor cells; meanwhile, the invention is suitable for application of downstream derived cell products generated by hematopoietic stem cell differentiation, including red blood cells, blood platelets, T cells, B cells and NK cells, in drugs for treating related diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of bioengineering and biomedicine technology, and in particular to a small molecule compound and its application in in vitro amplification of hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC). Background Art

[0002] Hematopoietic stem cell transplantation is an effective method widely used in the clinic to treat a variety of malignant and non-malignant blood system and other related diseases. It has been used to treat more than 200 diseases. The indications mainly include various types of leukemia, myelodysplastic syndrome, pernicious anemia, etc., as well as some non-hematological diseases including but not limited to AIDS, leukoencephalopathy and metabolic diseases. There are three main sources of hematopoietic stem cells available for clinical transplantation: bone marrow, mobilized peripheral blood and umbilical cord blood. Compared with hematopoietic stem cells from bone marrow and peripheral blood, umbilical cord blood hematopoietic stem cells have many advantages: rapid donor effect, relatively low HLA matching stringency, low incidence of graft-versus-host disease (GvHD), low viral infection rate and low relapse rate in patients with minimal residual disease. The limited number of functional hematopoietic stem cells is the main obstacle to the wider application of hematopoietic stem cell transplantation in the clinical treatment of related diseases. One of the effective ways to solve this key problem is to expand hematopoietic stem cells in vitro.

[0003] In vitro expansion is a potential method to solve the limitation of limited hematopoietic stem cells in a single umbilical cord blood unit. In order to effectively realize the gene editing of hematopoietic stem cells to treat blood system-related diseases, the establishment of an in vitro expansion system of hematopoietic stem cells is also of great significance. In vitro expansion of hematopoietic stem cells can provide enough stem cells for genetic engineering modification for immunotherapy of various cancer patients. Although a series of preliminary progress has been made in the research related to in vitro expansion of hematopoietic stem cells, so far no mature and reliable in vitro expansion method of hematopoietic stem cells has been approved by the FDA for clinical use. One of the possible main reasons is that we have insufficient understanding of the regulatory mechanism of maintaining the stemness of functional hematopoietic stem cells under the stress conditions of in vitro culture expansion, and there is a lack of a reliable screening system for functional hematopoietic stem cells.

[0004] Small molecule compounds are easily absorbed by cells, and the method of treating cells with small molecules can detect many proteins or signaling pathways with biological functions. At present, studies have reported that some small molecule compounds can be used for in vitro expansion of umbilical cord blood hematopoietic stem cells and progenitor cells, including diethylaminobenzaldehyde (DEAB), LG1506 (retinoic acid receptor antagonist), GW9662 (PPARG antagonist), SR1 (AhR antagonist), UM171, LSD inhibitor, BIO (GSK3β inhibitor), NR-101 (c-MPL agonist), HDAC inhibitor (trichostatin A and valproic acid), garcinol (GAR), nicotinamide riboside (NR), CPI-203 (BET inhibitor), of which the main two are SR1 and UM171.

[0005] The effectiveness of small molecule compounds for in vitro expansion of hematopoietic stem cells that are already under development, such as SR1 and UM171, has not yet been widely recognized. Therefore, new small molecule compounds are urgently needed to expand hematopoietic stem cells and hematopoietic progenitor cells to provide effective treatments for patients in need of hematopoietic stem cell transplantation. Summary of the invention

[0006] The object of the present invention is to provide a small molecule compound or a salt thereof that can be used to effectively expand human hematopoietic stem cells and hematopoietic progenitor cells, and the use of the small molecule compound or its salt in the preparation of a drug for expanding hematopoietic stem cells and / or hematopoietic progenitor cells. The compound can also be used for drug treatment of diseases related to HSC and / or HPC.

[0007] In order to achieve the above object, the present invention provides a small molecule compound or a salt thereof, the structural formula of the compound is shown in formula (I):

[0008]

[0009] Where R 1 is phenyl, aromatic heterocycle or substituted phenyl, aromatic heterocycle; R 2 is H, OH, NH2 or substituted amino; R 3 is formate or formamide; X is S, O or N.

[0010] As a preferred embodiment, the compound of formula (I) is further selected from the following formula (II):

[0011]

[0012] Where R 4 is single or multiple H, halogen, alkyl, alkoxy, cyano or aromatic; R 5 is an alkoxy group or a substituted amino group.

[0013] As a preferred embodiment, the compound structural formula is shown in any of the following:

[0014]

[0015]

[0016] The present invention also provides a pharmaceutical composition, comprising the above-mentioned small molecule compound or a salt thereof, and a pharmaceutically acceptable carrier.

[0017] The present invention also provides the use of the above-mentioned small molecule compound or its salt in the preparation of a drug for expanding hematopoietic stem cells and / or hematopoietic progenitor cells.

[0018] The present invention also provides a pharmaceutical composition, comprising a hematopoietic stem cell and / or hematopoietic progenitor cell population expanded by the above-mentioned small molecule compound or a salt thereof, and a pharmaceutically acceptable carrier.

[0019] The present invention also provides the use of hematopoietic stem cells and / or hematopoietic progenitor cells amplified by the above-mentioned small molecule compound or its salt in the preparation of drugs for hematopoietic dysfunction / malignant tumors, autoimmune diseases and / or hereditary immunodeficiency diseases.

[0020] The present invention also provides the use of the above-mentioned small molecule compound or its salt in the preparation of medicines for hematopoietic dysfunction / malignant tumors, autoimmune diseases and / or hereditary immunodeficiency diseases.

[0021] As a preferred embodiment, the hematopoietic dysfunction / malignancy, autoimmune disease and / or inherited immunodeficiency disease include bone marrow failure, lupus, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

[0022] The present invention also provides a kit for amplifying hematopoietic stem cells and / or hematopoietic progenitor cells, the kit comprising the above-mentioned small molecule compound or a salt thereof.

[0023] The compounds of the present invention include compounds that are prepared, purified and / or processed in prodrug form. Thus, the term "prodrug" as used herein refers to a compound that produces a desired active compound when metabolized. Generally, a prodrug is inactive or less active than the desired active compound, but may provide advantageous handling, administration or metabolic properties. Unless otherwise specified, reference to a particular compound also includes its prodrug.

[0024] The "salt" of the compound in the present invention is preferably a pharmaceutically acceptable salt, including acid addition salts and base addition salts.

[0025] In the present invention, "substituted phenyl" refers to a C1-6 alkyl, C1-6 alkoxy, nitro, halogen or hydrogen atom in the ortho, meta or para position of the benzene ring. "Aromatic heterocycle" refers to a thienyl, furanyl, pyrrolyl or pyridyl group, and "substituted aromatic heterocycle" refers to an aromatic heterocycle containing a C1-6 alkyl, C1-6 alkoxy, nitro, halogen or hydrogen atom in the ortho, meta or para position.

[0026] In the present invention, "alkyl" includes branched and straight chain saturated aliphatic hydrocarbon groups with a specific number of carbon atoms, for example, C1-C6 in C1-C6 alkyl is defined as including groups with 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched saturated arrangement. Examples of C1-C6 alkyl defined above include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl and hexyl.

[0027] In the present invention, "halogen" means fluorine, chlorine, bromine or iodine.

[0028] In the present invention, "aromatic group" alone or in combination with another group means a carbocyclic aromatic monocyclic group containing 6 carbon atoms, which may be further fused to a second 5- or 6-membered carbocyclic group which may be aromatic, saturated or unsaturated. Examples of aromatic groups include, but are not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl and tetrahydronaphthyl, etc. The aromatic group may be attached to another group at a suitable position on the cycloalkyl ring or the aromatic ring.

[0029] In the present invention, "aromatic heterocycle" refers to a monocyclic or bicyclic ring system of up to 10 atoms, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from the group consisting of O, N and S. The aromatic heterocycle may be attached via one of the heteroatoms or a ring carbon atom, and examples of aromatic heterocycles include, but are not limited to, thienyl, benzimidazolyl, benzo[b]thienyl, furanyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl , indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, dihydroindolinyl, isoindolinyl, thiazolo[4,5-b]-pyridine, tetrazolyl, oxadiazolyl, thiadiazolyl, thienyl and fluorescein derivatives.

[0030] The "hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC)" in the present invention refer to human hematopoietic stem cells and / or progenitor cells, including hematopoietic stem cells and / or progenitor cells derived from bone marrow, hematopoietic stem cells and / or progenitor cells derived from mobilized peripheral blood, hematopoietic stem cells and / or progenitor cells derived from umbilical cord blood, and hematopoietic stem cells and / or progenitor cells derived from embryonic stem cells or induced pluripotent stem cells.

[0031] The present invention provides a method for increasing HSC and / or HPC, the method comprising contacting a starting cell population in the presence of a compound provided by the present invention. In an embodiment of the present invention, the starting cell population is in vivo, in vitro or ex vivo. In addition, in an embodiment of the present invention, the starting cell population comprises CD34+ cells collected from mobilized peripheral blood (mPB), bone marrow (BM) or umbilical cord blood (UCB). + cell.

[0032] According to one aspect of the present invention, the present invention provides a method for treating hematopoietic dysfunction / malignancy, autoimmune disease and / or inherited immunodeficiency disease in a subject, the method comprising administering to a subject in need of such treatment HSC expanded using a compound disclosed herein.

[0033] In an embodiment of the present invention, the hematopoietic dysfunction / malignancy, autoimmune disease and / or inherited immunodeficiency disease include bone marrow failure, various congenital diseases of global concern (such as sickle cell anemia and thalassemia), lupus, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

[0034] The present invention includes administering HSC and / or HPC amplified using the compounds of the present invention to patients suffering from any of the above-mentioned diseases / malignancies, and also includes a cell population obtained after amplification using the method according to the present invention. Hematopoietic stem cells and hematopoietic progenitor cells can be harvested from adult, cord blood, fetal or embryonic sources, and the method of cell amplification using the present invention can lead to an increase in the number of progenitor cells, which can be used to accelerate the time of implantation of, for example, neutrophils or platelets. Such methods include: contacting a starting population comprising HSC and / or HPC with an agent capable of increasing the number of HSC and / or HPC. The starting population can be enriched in cell surface markers of interest or a combination thereof.

[0035] The present invention relates to a method for expanding HSCs and / or HPCs, the method comprising: (a) providing a starting cell population comprising HSCs and / or HPCs, and (b) contacting the starting cell population ex vivo under conditions suitable for expanding HSCs and / or HPCs, such as contacting the starting cell population ex vivo in the presence of a compound or composition of the present invention.

[0036] The amount of umbilical cord blood from a newborn is often insufficient to treat an adult or older child. An advantage of the expansion method using the compounds or compositions of the present invention is that it is possible to generate sufficient amounts of HSCs and / or HPCs from one umbilical cord blood unit. Thus, in one embodiment, the starting cell population is derived from a CD34-enriched + In a related embodiment, the starting cell population is derived from one or two umbilical cord blood units. In another embodiment, the starting cell population is derived from a CD34-enriched neonatal umbilical cord blood cell population. + In a related embodiment, the starting cell population is derived from human mobilized peripheral blood cells isolated from a patient.

[0037] The starting cell population may preferably contain at least 50% CD34 + cells, in some embodiments, greater than 90% CD34 + Cells. The culture conditions for expansion of the starting cell population will vary depending on the starting cell population, the desired final number of cells, and the desired final ratio of HSCs and / or HPCs.

[0038] In one embodiment, a sample enriched for CD34 + The culture conditions include the use of other cell expansion factors generally known in the art for HSC / HPC expansion, such as cytokines and growth factors.

[0039] The above-mentioned chemical components and biological components can be used not only by adding them to a culture medium, but also by fixing them on the surface of a substrate or support for culture, more specifically, by dissolving the components to be used in a suitable solvent, coating the substrate or support with the resulting solution, and then washing away the excess components. Such components to be used can be added to a substrate or support previously coated with a substance to which the components are bound.

[0040] The expansion of HSC can be carried out in natural culture medium, semi-synthetic culture medium or synthetic culture medium according to the composition, and can be solid culture medium, semi-solid culture medium or liquid culture medium according to the shape, and any nutrient medium for hematopoietic stem cell and / or hematopoietic progenitor cell culture, which can be supplemented with a mixture of the above-mentioned cell expansion factor. Such culture medium generally includes sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acid, vitamin, cytokine, hormone, antibiotic, serum, fatty acid or sugar etc. In cultivation, other chemical components or biological components can be introduced separately or in combination according to the situation. Such components introduced in the culture medium can be fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, fetal bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines or various growth factors etc.

[0041] In which the starting cell population consists essentially of CD34-rich cells from one or two cord blood units or from mobilized PB cells or from harvested bone marrow + In a specific embodiment of the cell composition, the cells are grown under conditions for HSC and / or HPC expansion, for example, 2 to 21 days and / or until a specified multiple expansion, and a characteristic cell population is obtained. In a specific embodiment, the cells are grown in vitro under conditions for HSC and / or HPC expansion for no more than 21 days, 14 days, 10 days or 7 days. The cell population is then washed to remove the compound or composition of the present invention, and / or other components in the cell culture, and resuspended in a suitable cell suspension medium for short-term use or resuspended in a long-term storage medium, such as a medium suitable for cryopreservation.

[0042] The present invention also provides a cell population with expanded HSC and / or HPC, which can be obtained by the above-mentioned expansion method. In a specific embodiment, such a cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition. The present invention also provides a cell population or a composition thereof with expanded HSC and / or HPC for autologous or allogeneic stem cell transplantation in a mammalian subject.

[0043] The composition is formulated in any conventional manner for use in the methods described herein. In an embodiment, the composition of the present invention comprises a compound described herein. In other embodiments, the composition of the present invention comprises a HSC and / or HPC group amplified using a compound described herein. The composition of the present invention may also comprise a pharmaceutically acceptable carrier. The administration of the composition is by any known effective route by those skilled in the art. For example, the composition is administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, in vitro, intranasally or topically.

[0044] The preferred method of administration is intravenous infusion. The number of cells infused will take into account various factors, such as sex, age, weight, type of disease or condition, stage of condition, percentage of desired cells in the cell population, and number of cells required to produce therapeutic benefit. In one embodiment, the infused cells are all derived from expanded umbilical cord blood cells from a single newborn.

[0045] The expanded HCS and / or HPC can be infused by drip (e.g., in the case of treating leukemia) into a patient who has been pretreated with anticancer drugs, whole body irradiation or immunosuppressive drugs to eradicate cancer cells or promote the implantation of donor cells. As described above, the present invention makes it possible to expand HCS and / or HPC, and to perform transplantation therapy safely and easily in a short time by using the expanded HCS and / or HPC.

[0046] The present invention also provides a kit comprising one or more containers filled with one or more compounds of the present invention, and optionally comprising a solution and a buffer. The kit optionally includes an amplified population of stem cells manufactured by the above method, and may include a container or composition for manufacturing an amplified population of HSC and / or HPC. In particular, the present invention provides a kit for ex vivo expansion of hematopoietic stem cells, comprising a compound of the present invention and instructions for using such a compound in a HSC and / or HPC expansion method, and optional one or more cell expansion factors, or a culture medium for cell growth, particularly a culture medium for HSC and / or HPC growth as described above.

[0047] The advantage of the present invention is that the series of compounds disclosed in the present invention are sufficient to effectively expand human hematopoietic stem cells and hematopoietic progenitor cells, and can be used in the medical field of related diseases using hematopoietic stem cell transplantation as a treatment method, including in vitro expansion and culture of hematopoietic stem cells and progenitor cells derived from umbilical cord blood, bone marrow and mobilized peripheral blood, and the culture, maintenance and expansion of gene-modified hematopoietic stem and progenitor cells; at the same time, they are suitable for the use of downstream derivative cell products generated by hematopoietic stem cell differentiation, including red blood cells, platelets, T cells, B cells and NK cells in drugs for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Flow chart of the preliminary screening experiment for the effect of small molecule compound activators on the expansion of human hematopoietic stem cells.

[0049] Figure 2 CD34 + ADGRG1 + Small molecule compound activator that has cell proliferation effect.

[0050] Figure 3 Effects of different concentrations of compound 1-37 on umbilical cord blood CD34 + Expansion effect of hematopoietic stem and progenitor cells.

[0051] Figure 4 HSCs expanded by compound 3 have hematopoietic activity and self-renewal ability: (A) The number of colony-forming units formed by CD34+ cells after 7 days of culture with compound 3 (n=3); (B) The proportion of human CD45+ cells in the bone marrow of mice transplanted for the first time (n=8 mice / group); (C) The proportion of human CD45+ cells in the bone marrow of mice transplanted for the first time (n=8 mice / group).

[0052] Figure 5 Human CD45 in the bone marrow of secondary transplant recipient mice + Cell ratio: (A) Representative flow cytometry display of the ratio of human CD45+ cells in the bone marrow of secondary transplanted mice (n = 8 mice / group); (B) Representative flow cytometry display of the ratio of human CD45+ cells in the bone marrow of secondary transplanted mice (n = 8 mice / group); + The ratio of cells (n=8 mice / group). DETAILED DESCRIPTION

[0053] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0054] Screening experiments:

[0055] First, we used StemSpan serum-free HSC expansion medium containing 100 ng / mL SCF, TPO, and Flt3L to isolate and purify CD34 + The cells were resuspended and plated in 24-well plates, and then a series of small molecule compounds were added to the cells for in vitro amplification screening experiments. After 7 days of in vitro culture, the cells in the wells were phenotyped by flow cytometry to detect the effects of small molecule compounds on CD34 + ADGR + The expansion effect of the cell population. Screening schemes such as Figure 1As shown in the figure, we screened more than 4,000 compounds, eliminated the fluorescence interference of the compounds themselves, and after repeated verification, we finally found a compound that inhibited CD34 in vitro. + CD133 + ADGRG1 + The cells have a significant proliferation effect ( Figure 2 ), which is the compound "2-(5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)thiazole-4-carboxylic acid ethyl ester" (Compound 3). Subsequently, a series of derivatives were developed and synthesized based on Compound 3 to verify its effectiveness.

[0056] Biological testing:

[0057] Based on the structure of ethyl 2-(5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)thiazole-4-carboxylate (compound 3), we synthesized analogs and tested their in vitro amplification effects. Compound 3 had a significant effect on CD34 proliferation in SFEM medium containing 100 ng / mL SCF, TPO, and Flt3L at either 1 μM or 5 μM concentrations. + The cell populations all had significant in vitro proliferation promoting effects. Compounds 1-37 had different concentrations for the proliferation of umbilical cord blood CD34 + The in vitro expansion of hematopoietic stem and progenitor cells has a certain degree of promoting effect ( Figure 3 When the final concentration of the compound in the culture medium was 1 micromolar, compounds 1, 2, and 9 had no significant effect on CD34 + The in vitro proliferation effect of cells was similar to that of compound 3, about 2 times; while compounds 4, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 had an in vitro proliferation effect on CD34 + The cell population expansion effect was between 1-1.5 times. When the medium compound concentration reached 5 micromolar, compounds 1, 2, 3, 4, 5, 6, 9, 10, 13, 22, 23, 24, 25, 26, 27, 28, 29, 32, 35 had an effect on CD34 + The expansion effect of the population was close to 2-2.5 times; while compounds 8, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 34, 36, and 37 had an effect on CD34 at 5 micromolar. + The expansion effect of the cell population is between 1-1.5 times.

[0058] Compound 3 expands HSCs with hematopoietic activity and self-renewal ability

[0059] Next, to determine whether the HSC population expanded by compound 3 in vitro has corresponding activity in vivo, we transplanted freshly isolated cells and cells from the Vehicle group and compound 3 group that had been cultured in vitro for 7 days into sublethally irradiated immunodeficient recipient mice. + The proportion of cells ( Figure 4 ). The results showed that compared with freshly isolated CD34 + CD34 cells and vehicles treated + Compared with the cell group, compound 3 expanded the cultured umbilical cord blood CD34 + CD45 + The cell chimeras were significantly higher than those in the other two control groups. This result indicated that CD34 + Compound 3 can promote the engraftment of hematopoietic stem and progenitor cells in mice after expansion.

[0060] In order to further evaluate the long-term engraftment and self-renewal ability of the expanded cells, 1×10 7 The secondary transplantation experiment was completed by transplanting new sublethally irradiated NSG mice. The results showed that at 16 weeks after transplantation, the hCD45 in the bone marrow of the mice in the compound 3 group was + The proportion of cells in the vehicle group increased significantly compared with that in the vehicle group. This result indicates that compound 3 has an + The in vitro expansion culture of cells not only has significant advantages in a single transplant, but also has long-term implantation and self-renewal capabilities. The above results show that the HSC in vitro expansion activator compound 3 obtained by screening using ADGRG1 as a marker can significantly promote the in vitro expansion of functional HSCs from umbilical cord blood.

[0061] Synthesis methods of compounds

[0062] The present invention also provides a method for preparing the compound shown in Formula I (including Formula II), but is not limited to the method described below. All raw materials are prepared or directly purchased according to the group characteristics of the target molecule that conforms to the general formula, and by the schemes in these routes, methods well known to those of ordinary skill in the field of organic chemistry. The following method can be combined with a known synthesis method in the field of synthetic organic chemistry or a related modification method recognized by those skilled in the art to synthesize the compounds of the present invention. It is known to those skilled in the art that, according to a specific target structure, one or more of the following schemes can be optionally used to combine, or any steps in one or more of the schemes can be combined to obtain a synthesis scheme.

[0063] The synthetic route of some preferred compounds of the general formula I is as follows: acetylthiosemicarbazide (I-1) and ethyl 3-bromopyruvate (I-2) are heated under reflux for 5 hours in a 1:1 molar ratio in ethanol as a solvent to obtain yellow solid ethyl 2-hydrazinothiazole-4-carboxylate bromate (I-3). Intermediate I-3 and formylacetonitrile (I-4) containing different substituents are heated under reflux for 5 hours in a 1:1 molar ratio in ethanol / acetic acid mixed solvent (2:1), and the target compound Ia is obtained by cooling and precipitation or silica gel column chromatography.

[0064]

[0065] Specific Example 1: Preparation of ethyl 2-(5-amino-3-phenyl-1H-pyrazol-1-yl)thiazole-4-carboxylate (Compound 1)

[0066]

[0067] Step 1: Preparation of ethyl 2-hydrazinothiazole-4-carboxylate bromide (intermediate I-3)

[0068]

[0069] Acetylthiosemicarbazide (raw material I-1, 6.65 g, 50 mmol) and solvent ethanol (80 mL) were added to a round-bottom flask, and then ethyl 3-bromopyruvate (raw material I-2, 9.75 g, 50 mmol) was added. The mixture was heated under reflux for 5 hours, cooled to room temperature, concentrated under reduced pressure, ethyl acetate was added for pulping, and filtered to obtain 13.10 g of yellow solid 2-hydrazinothiazole-4-carboxylic acid ethyl ester bromide (intermediate I-3), with a yield of 97.8%. 1 HNMR(400MHz, DMSO-d6)δ8.01(s,1H),4.28(q,J=7.1Hz,2H),1.29(t,J=7.0Hz,3H).ESI-MS(m / z):188,[M+H] + .

[0070] Step 2: Preparation of ethyl 2-(5-amino-3-phenyl-1H-pyrazol-1-yl)thiazole-4-carboxylate (Compound 1)

[0071]

[0072] 2-Hydrazinothiazole-4-carboxylic acid ethyl ester bromide (intermediate I-3, 268 mg, 1 mmol) and benzoyl acetonitrile (raw material I-4a, 145 mg, 1 mmol) were added to a round-bottom flask, and ethanol (4 mL) and acetic acid (2 mL) were added. The mixture was heated under reflux for 5 hours and cooled to room temperature to precipitate 180 mg of white solid 2-(5-amino-3-phenyl-1H-pyrazol-1-yl)thiazole-4-carboxylic acid ethyl ester (Compound 1), with a yield of 57.3%. 1 H NMR(400MHz,DMSO-d6)δ8.26(s,1H),7.86–7.77(m,2H),7.50–7.37(m,3H),6.90(s,2H) ),5.94(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):315,[M+H] + .

[0073] Specific Example 2: Preparation of ethyl 2-[5-amino-3-(4-methylphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 2)

[0074]

[0075] The benzoyl acetonitrile was replaced by 4-methylbenzoyl acetonitrile, and the compound 2 was obtained as a light yellow solid with a yield of 88.3% by referring to the synthesis method of Specific Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.24(s,1H),7.71(d,J=7.8Hz,2H),7.25(d,J=7.8Hz,2H),6.88(s,2H), 5.91(s,1H),4.33(q,J=7.1Hz,2H),2.34(s,3H),1.33(t,J=7.0Hz,3H).ESI-MS(m / z):329,[M+H] + .

[0076] Specific Example 3: Preparation of ethyl 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 3)

[0077]

[0078] The benzoyl acetonitrile was replaced by 4-methoxybenzoyl acetonitrile, and the compound 3 was obtained as a white solid with a yield of 56.6% by referring to the synthesis method of specific example 1. 1H NMR (400MHz, DMSO-d6) δ8.23(s,1H),7.75(d,J=8.6Hz,2H),7.00(d,J=8.7Hz,2H),6.92(d,J=43.8Hz, 2H),5.87(s,1H),4.33(q,J=7.1Hz,2H),3.80(s,3H),1.33(t,J=7.0Hz,3H).ESI-MS(m / z):345,[M+H] + .

[0079] Specific Example 4: Preparation of ethyl 2-[5-amino-3-(4-fluorophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 4)

[0080]

[0081] The benzoyl acetonitrile was replaced by 4-fluorobenzoyl acetonitrile, and the compound 4 was obtained as a white solid with a yield of 33.1% by referring to the synthesis method of Specific Example 1. 1 H NMR(400MHz,DMSO-d6)δ8.25(s,1H),7.91–7.82(m,2H),7.33–7.23(m,2H),6.91(s,2H) ),5.94(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):333,[M+H] + .

[0082] Specific Example 5: Preparation of ethyl 2-[5-amino-3-(4-chlorophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 5)

[0083]

[0084] The benzoyl acetonitrile was replaced by 4-fluorobenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 5 as a white solid with a yield of 45.5%. 1 H NMR(400MHz,DMSO-d6)δ8.26(s,1H),7.87–7.81(m,2H),7.52–7.47(m,2H),6.93(s,2H) ),5.96(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):349,[M+H] + .

[0085] Specific Example 6: Preparation of ethyl 2-[5-amino-3-(4-bromophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 6)

[0086]

[0087] The benzoyl acetonitrile was replaced by 4-bromobenzoyl acetonitrile, and the compound 6 was obtained as a white solid with a yield of 68.7% by referring to the synthesis method of Specific Example 1. 1 H NMR(400MHz,DMSO-d6)δ8.26(s,1H),7.81–7.75(m,2H),7.66–7.60(m,2H),6.93(s,2H) ),5.96(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):393,[M+H] + .

[0088] Specific Example 7: Preparation of ethyl 2-[5-amino-3-(4-trifluoromethoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 7)

[0089]

[0090] The benzoyl acetonitrile was replaced by 4-trifluoromethoxybenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 7 as a light yellow solid with a yield of 62.8%. 1 H NMR(400MHz,DMSO-d6)δ8.27(s,1H),8.00–7.89(m,2H),7.50–7.38(m,2H),6.94(s,2H) ),5.98(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):399,[M+H] + .

[0091] Specific Example 8: Preparation of ethyl 2-[5-amino-3-(4-cyanophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 8)

[0092]

[0093] The benzoyl acetonitrile was replaced by 4-cyanobenzoyl acetonitrile, and the compound 8 was obtained as a light yellow solid with a yield of 46.7% by referring to the synthesis method of Specific Example 1. 1H NMR(400MHz,DMSO-d6)δ8.26(s,1H),8.02–7.96(m,2H),7.92–7.84(m,2H),6.97(s,2H) ),6.05(s,1H),4.32(q,J=7.1Hz,2H),1.32(t,J=7.1Hz,3H).ESI-MS(m / z):340,[M+H] + .

[0094] Specific Example 9: Preparation of ethyl 2-[5-amino-3-(2-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 9)

[0095]

[0096] The benzoyl acetonitrile was replaced by 2-methoxybenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 9 as a white solid with a yield of 61.0%. 1 H NMR (400MHz, DMSO-d6) δ8.28(s,1H),7.90(d,J=7.6Hz,1H),7.42(t,J=7.7Hz,1H),7.17(d,J=8.4Hz,1H),7.06(t,J=7. 4Hz,1H),6.81(s,2H),6.03(s,1H),4.37(q,J=7.0Hz,2H),3.91(s,3H),1.37(t,J=7.0Hz,3H).ESI-MS(m / z):345,[M+H] + .

[0097] Specific Example 10: Preparation of ethyl 2-[5-amino-3-(2-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 10)

[0098]

[0099] The benzoyl acetonitrile was replaced by 3-methoxybenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 10 as a slightly yellow solid with a yield of 40.7%. 1 H NMR (400MHz, DMSO-d6) δ8.25 (s, 1H), 7.44–7.31 (m, 3H), 6.97 (ddd, J = 7.8, 2.7, 1.3Hz, 1H), 6.89 (s, 2 H),5.95(s,1H),4.32(q,J=7.1Hz,2H),3.82(s,3H),1.32(t,J=7.0Hz,3H).ESI-MS(m / z):345,[M+H] + .

[0100] Specific Example 11: Preparation of ethyl 2-[5-amino-3-(2-fluorophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 11)

[0101]

[0102] The benzoyl acetonitrile was replaced by 2-fluorobenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 11 as a white solid with a yield of 48.1%. 1 H NMR(400MHz, DMSO-d6)δ8.28(s,1H),7.95(td,J=7.8,1.7Hz,1H),7.46(tdd,J=7.0,6.1,1.8Hz,1H),7.35–7 .28(m,3H),6.90(s,2H),5.89(s,1H),4.34(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):333,[M+H] + Specific Example 12: Preparation of ethyl 2-[5-amino-3-(2-chlorophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 12)

[0103]

[0104] The benzoyl acetonitrile was replaced by 2-chlorobenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 12 as an off-white solid with a yield of 51.7%. 1 H NMR(400MHz, DMSO-d6)δ8.27(s,1H),7.78(dd,J=6.0,3.5Hz,1H),7.60–7.52(m,1H),7.44(dd,J=5.9,3.4 Hz,3H),6.89(s,2H),5.94(s,1H),4.34(q,J=7.1Hz,2H),1.34(t,J=7.1Hz,3H).ESI-MS(m / z):349,[M+H] + .

[0105] Specific Example 13: Preparation of ethyl 2-[5-amino-3-(3-bromophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 13)

[0106]

[0107] The benzoyl acetonitrile was replaced by 3-bromobenzoyl acetonitrile, and the compound 13 was obtained as a yellow solid with a yield of 39.7% by referring to the synthesis method of Specific Example 1. 1H NMR (400MHz, DMSO-d6) δ8.28(s,1H),7.99(t,J=1.8Hz,1H),7.83(dt,J=7.8,1.3Hz,1H),7.59(ddd,J=8.0,2.1,1.0Hz,1H ),7.41(t,J=7.9Hz,1H),6.95(s,2H),6.02(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):393,[M+H] + .

[0108] Specific Example 14: Preparation of ethyl 2-[5-amino-3-(3-biphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 14)

[0109]

[0110] The benzoyl acetonitrile was replaced by 3-bibenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 14 as a yellow solid with a yield of 54.5%. 1 H NMR(400MHz, DMSO-d6)δ8.27(s,1H),8.05(t,J=1.8Hz,1H),7.84(dt,J=7.8,1.4Hz,1H),7.77–7.71(m,2H),7.68(dt,J=7.8,1.4Hz,1H),7.53 (dt,J=15.0,7.8Hz,3H),7.45–7.38(m,1H),6.93(s,2H),6.08(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):391,[M+H] + Specific Example 15: Preparation of ethyl 2-[5-amino-3-(3,5-dichlorophenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 15)

[0111]

[0112] The benzoyl acetonitrile was replaced with 3,5-dichlorobenzoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain Compound 15 as a yellow solid with a yield of 83.6%. 1H NMR (400MHz, DMSO-d6) δ8.28(s,1H),7.83(d,J=1.9Hz,2H),7.61(t,J=1.9Hz,1H),6.99(s ,2H),6.09(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):383,[M+H] + .

[0113] Specific Example 16: Preparation of ethyl 2-[5-amino-3-(2-pyridyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 16)

[0114]

[0115] The benzoylacetonitrile was replaced by 2-pyridinecarbonylacetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 16 as a brown solid with a yield of 53.8%. 1 H NMR (400MHz, DMSO-d6) δ8.78–8.70(m,1H),8.34(s,1H),8.10–7.92(m,2H),7.55(ddd,J=7.4,4.9 ,1.4Hz,1H),6.22(s,1H),4.34(q,J=7.1Hz,2H),1.34(t,J=7.1Hz,3H).ESI-MS(m / z):316,[M+H] + .

[0116] Specific Example 17: Preparation of ethyl 2-[5-amino-3-(3-pyridyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 17)

[0117]

[0118] The benzoylacetonitrile was replaced by 3-pyridinecarbonylacetonitrile, and the synthesis method of Example 1 was referred to to obtain compound 17 as a yellow solid with a yield of 81.6%. 1 H NMR (400MHz, DMSO-d6) δ9.25(d,J=2.0Hz,1H),8.83(ddd,J=10.9,5.3,1.5Hz,1H),8.73(dt,J=8.1,1.8Hz,1H),8.32(s, 1H),8.01–7.90(m,1H),7.14(s,2H),6.20(s,1H),4.34(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):316,[M+H] + .

[0119] Specific Example 18: Preparation of ethyl 2-[5-amino-3-(4-pyridyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 18)

[0120]

[0121] The benzoylacetonitrile was replaced by 4-pyridinecarbonylacetonitrile, and the synthesis method of Example 1 was referred to to obtain Compound 18 as a yellow solid with a yield of 68.0%. 1 H NMR(400MHz,DMSO-d6)δ8.99–8.92(m,2H),8.39–8.33(m,3H),7.18(s,2H),6.3 5(s,1H),4.34(q,J=7.1Hz,2H),1.34(t,J=7.1Hz,3H).ESI-MS(m / z):316,[M+H] + .

[0122] Specific Example 19: Preparation of ethyl 2-[5-amino-3-(2-thienyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 19)

[0123]

[0124] The benzoylacetonitrile was replaced by 2-thenoylacetonitrile, and the compound 19 was obtained as a yellow solid with a yield of 62.5% by referring to the synthesis method of Specific Example 1. 1 H NMR (400MHz, DMSO-d6) δ8.24(s,1H),7.59(dd,J=5.0,1.2Hz,1H),7.53(dd,J=3.6,1.2Hz,1H),7.13(dd ,J=5.1,3.6Hz,1H),5.87(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):321,[M+H] + .

[0125] Specific Example 20: Preparation of ethyl 2-[5-amino-3-(3-thienyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 20)

[0126]

[0127] The benzoylacetonitrile was replaced by 3-thenoylacetonitrile, and the compound 20 was obtained as a yellow solid with a yield of 57.2% by referring to the synthesis method of Specific Example 1. 1H NMR (400MHz, DMSO-d6) δ8.23(s,1H),7.94(dd,J=2.9,1.3Hz,1H),7.63(dd,J=5.0,2.9Hz,1H),7.50(dd ,J=5.0,1.2Hz,1H),5.87(s,1H),4.33(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):321,[M+H] + .

[0128] Specific Example 21: Preparation of ethyl 2-[5-amino-3-(2-furyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 21)

[0129]

[0130] The benzoyl acetonitrile was replaced by 2-furoyl acetonitrile, and the synthesis method of Example 1 was referred to to obtain Compound 21 as a light yellow solid with a yield of 23.5%. 1 H NMR (400MHz, DMSO-d6) δ8.23 (s, 1H), 7.86–7.74 (m, 1H), 6.89 (d, J = 3.4Hz, 1H), 6.68–6.57 ( m,1H),5.76(s,1H),4.32(q,J=7.1Hz,2H),1.31(t,J=7.0Hz,3H).ESI-MS(m / z):305,[M+H] + .

[0131] Specific Example 22: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid methyl ester (Compound 22)

[0132]

[0133] Step 1: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid (Intermediate 3a)

[0134]

[0135] A round-bottom flask was added with ethyl 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (compound 3, 344 mg, 1 mmol) and a mixed solvent of tetrahydrofuran / methanol / water (4 mL / 2 mL / 2 mL), and then lithium hydroxide (168 mg, 4 mmol) was added and stirred at room temperature for 2 hours. 5 mL of water and 5 mL of ethyl acetate were added for washing. The aqueous layer was adjusted to acidity with hydrochloric acid to precipitate 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid (intermediate 3a) as a white solid (yield 88.6%). 1 H NMR(400MHz,DMSO-d6)δ13.13(s,1H),8.18(s,1H),7.79–7.68(m,2H),7.03– 6.97(m,2H),6.91(s,2H),5.85(s,1H),3.80(s,3H).ESI-MS(m / z):315,[MH] - .

[0136] Step 2: Preparation of methyl 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 22)

[0137]

[0138] 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid (intermediate 3a, 63 mg, 0.2 mmol), methanol (19 mg, 0.6 mmol) and carbonyldiimidazole (CDI, 41 mg, 0.25 mmol) were dissolved in 2 mL of DMF, and DBU (36 mg, 0.24 mmol) was added dropwise, and stirred at room temperature for 6 hours. 4 mL of water was added, extracted with ethyl acetate, and purified by silica gel column chromatography to obtain 35 mg of methyl 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 22) as a white solid, with a yield of 43.0%. 1 H NMR(400MHz, DMSO-d6)δ8.18(s,1H),7.77–7.71(m,2H),7.01–6.96(m,2H),5.85(s,1H),3.83(s,3H),3.80(s,3H).ESI-MS(m / z):331,[M+H] + .

[0139] Specific Example 23: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid isopropyl ester (Compound 23)

[0140]

[0141] Methanol was replaced with isopropanol, and the synthesis method of specific example 22 was referred to to obtain compound 23 as a white solid with a yield of 48.0%. 1 H NMR(400MHz,DMSO-d6)δ8.20(s,1H),7.78–7.73(m,2H),7.02–6.97(m,2H),6.86(s,2H),5.8 6(s,1H),5.12(p,J=6.2Hz,1H),3.80(s,3H),1.33(d,J=6.3Hz,6H).ESI-MS(m / z):359,[M+H] + .

[0142] Specific Example 24: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid cyclobutyl ester (Compound 24)

[0143]

[0144] Methanol was replaced with cyclobutanol, and the synthesis method of specific example 22 was referred to to obtain compound 24 as a light yellow solid with a yield of 46.0%. 1 H NMR(400MHz,DMSO-d6)δ8.22(s,1H),7.77–7.72(m,2H),7.03–6.98(m,2H),6.85(s,2H),5 .87(s,1H),5.15(m,1H),3.80(s,3H),1.43(m,4H),1.31(m,2H).ESI-MS(m / z):371,[M+H] + .

[0145] Specific Example 25: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxamide (Compound 25)

[0146]

[0147] 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid (intermediate 3a, 63 mg, 0.2 mmol), ethylamine (11 mg, 0.24 mmol) and HATU (114 mg, 0.3 mmol) were dissolved in 2 mL of DMF, and DIEA (36 mg, 0.6 mmol) was added dropwise, and stirred at room temperature for 6 hours. 4 mL of water was added, extracted with ethyl acetate, and purified by silica gel column chromatography to obtain 42 mg of yellow solid 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxamide (compound 25), with a yield of 61.1%. 1 HNMR(400MHz,DMSO-d6)δ8.84(t,J=6.0Hz,1H),7.91(s,1H),7.79–7.69(m,2H),7.03–6.98(m,2H),6.96 (s,2H),5.79(s,1H),3.80(s,3H),3.30(q,J=6.8Hz,2H),1.14(t,J=7.2Hz,3H).ESI-MS(m / z):344,[M+H] + .

[0148] Specific Example 26: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxamide (Compound 26)

[0149]

[0150] Ethylamine was replaced with methylethylamine, and the synthesis method of specific example 25 was referred to to obtain compound 26 as an off-white solid with a yield of 56.0%. 1 H NMR(400MHz,DMSO-d6)δ7.95(s,1H),7.76–7.73(m,2H),7.05–6.94(m,2H),6.73(s,2H),5.85(s,1H), 3.80(s,3H),3.49(d,J=8.5Hz,2H),2.89(s,3H),1.17(q,J=6.8,6.2Hz,3H).ESI-MS(m / z):358,[M+H] + .

[0151] Specific Example 27: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid cyclopropylamine (Compound 27)

[0152]

[0153] Ethylamine was replaced with cyclopropylamine, and the synthesis method of specific example 25 was referred to to obtain compound 27 as a yellow solid with a yield of 61.9%. 1 H NMR (400MHz, DMSO-d6) δ8.74(d,J=3.8Hz,1H),7.93(s,1H),7.76–7.69(m,2H),7.03–6.97(m,2H),6.94(s,2H),5.7 8(s,1H),3.80(s,3H),2.79(tt,J=7.6,3.8Hz,1H),0.77–0.71(m,2H),0.66–0.60(m,2H).ESI-MS(m / z):356,[M+H] + .

[0154] Specific Example 28: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid cyclobutylamine (Compound 28)

[0155]

[0156] Ethylamine was replaced with cyclobutylamine, and the synthesis method of specific example 25 was referred to to obtain compound 28 as a white solid with a yield of 56.8%. 1 H NMR (400MHz, DMSO-d6) δ8.83(d,J=8.1Hz,1H),7.92(s,1H),7.78–7.70(m,2H),7.02–6.99(m,2H),6.98(s,2H), 5.81(s,1H),4.46(h,J=8.4Hz,1H),3.80(s,3H),2.25–2.15(m,4H),1.74–1.63(m,2H).ESI-MS(m / z):370,[M+H] + .

[0157] Specific Example 29: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid cyclopentylamine (Compound 29)

[0158]

[0159] Ethylamine was replaced with cyclopentylamine, and the synthesis method of specific example 25 was referred to to obtain compound 29 as a white solid with a yield of 78.2%. 1H NMR (400MHz, DMSO-d6) δ8.51(d,J=7.9Hz,1H),7.92(s,1H),7.77–7.70(m,2H),7.03–6.96(m,2H),6.94(s,2H),5.8 0(s,1H),4.25(q,J=7.6Hz,1H),3.80(s,3H),1.91(m,2H),1.72(m,2H),1.63–1.52(m,4H).ESI-MS(m / z):384,[M+H] + .

[0160] Specific Example 30: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid cyclohexylamine (Compound 30)

[0161]

[0162] Ethylamine was replaced with cyclohexylamine, and the synthesis method of specific example 25 was referred to to obtain compound 30 as a white solid with a yield of 62.9%. 1 H NMR(400MHz, DMSO-d6)δ8.45(d,J=8.6Hz,1H),7.92(s,1H),7.76–7.70(m,2H),7.02–6.97(m,2H),6.94(s, 2H),5.79(s,1H),3.80(s,3H),1.86–1.71(m,4H),1.64(m,1H),1.47–1.10(m,6H).ESI-MS(m / z):398,[M+H] + .

[0163] Specific Example 31: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid morpholine (Compound 31)

[0164]

[0165] Ethylamine was replaced with morpholine, and the synthesis method of specific example 25 was referred to to obtain compound 31 as a light yellow solid with a yield of 62.3%. 1 H NMR(400MHz,DMSO-d6)δ7.79(s,1H),7.77–7.72(m,2H),7.06–6.94(m,2H),6 .72(s,2H),5.85(s,1H),3.80(s,3H),3.66(s,8H).ESI-MS(m / z):386,[M+H] + .

[0166] Specific Example 32: Preparation of 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxanilide (Compound 32)

[0167]

[0168] Ethylamine was replaced with aniline, and the synthesis method of specific example 25 was referred to to obtain compound 32 as a white solid with a yield of 43.5%. 1 H NMR (400MHz, DMSO-d6) δ10.34(s,1H),8.13(s,1H),7.79–7.72(m,4H),7.40(t,J=7.9Hz,2H),7.20 –7.12(m,1H),7.05(s,2H),7.03–6.97(m,2H),5.82(s,1H),3.80(s,3H).ESI-MS(m / z):392,[M+H] + .

[0169] Specific Example 33: Preparation of ethyl 2-[5-acetylamino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 33)

[0170]

[0171] 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylic acid ethyl ester (Compound 3, 69 mg, 0.2 mmol), acetic anhydride (204 mg, 2 mmol) and pyridine (16 mg, 0.2 mmol) were added to a round-bottom flask and stirred at 100°C for 2 hours. After cooling to room temperature, a white solid 2-[5-acetylamino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxanilide (Compound 33) was precipitated.

[0172] 75 mg, yield 97.0%. 1 H NMR (400MHz, DMSO-d6) δ10.93(s,1H),8.35(s,1H),7.84(d,J=8.2Hz,2H),7.08(s,1H),7.03(d,J=8.3Hz, 2H),4.34(q,J=7.3,6.9Hz,2H),3.81(s,4H),2.21(s,3H),1.34(t,J=7.2Hz,3H).ESI-MS(m / z):387,[M+H] + .

[0173] Specific Example 34: Preparation of ethyl 2-[5-hydroxy-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 34)

[0174]

[0175] 2-Hydrazothiazole-4-carboxylic acid ethyl ester bromide (intermediate I-3, 268 mg, 1 mmol) and ethyl 4-methoxybenzoyl acetate (222 mg, 1 mmol) were added to a round-bottom flask, and ethanol (4 mL) and acetic acid (2 mL) were added. The mixture was heated to reflux for 5 hours and cooled to room temperature to precipitate 200 mg of ethyl 2-[5-hydroxy-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (compound 34) as a white solid with a yield of 57.3%. 1 H NMR(400MHz,DMSO-d6)δ8.25(s,1H),7.86–7.82(m,2H),7.09–7.06(m,2H),6.02(d,J=1.0H z,1H),4.34(q,J=7.1Hz,2H),3.84(s,3H),1.33(t,J=7.1Hz,2H).ESI-MS(m / z):346,[M+H] + .

[0176] Specific Example 35: Preparation of ethyl 2-[3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 35)

[0177]

[0178] 3-(4-methoxyphenyl)pyrazole (174 mg, 1 mmol) and ethyl 2-bromothiazole-4-carboxylate (260 mg, 1.1 mmol) were added to a round-bottom flask, and solvent DMSO (3 mL) and potassium carbonate (207 mg, 1.5 mmol) were added, and stirred at 100° C. for 4 hours. 5 mL of water was added, extracted with ethyl acetate, and purified by silica gel column chromatography to obtain 120 mg of slightly yellow solid ethyl 22-[3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-4-carboxylate (Compound 35), with a yield of 36.4%. 1 H NMR(400MHz, DMSO-d6)δ8.58(d,J=2.7Hz,1H),8.34(s,1H),7.91–7.85(m,2H),7.13(d,J=2.7Hz,1H), 7.08–7.01(m,2H),4.33(q,J=7.1Hz,2H),3.82(s,3H),1.33(t,J=7.1Hz,3H).ESI-MS(m / z):330,[M+H]+ .

[0179] Specific Example 36: Preparation of ethyl 2-[3-(4-methoxyphenyl)-1H-pyrazol-1-yl]oxazole-4-carboxylate (Compound 36)

[0180]

[0181] 3-(4-methoxyphenyl)pyrazole (174 mg, 1 mmol) and ethyl 2-chlorooxazole-4-carboxylate (193 mg, 1.1 mmol) were added to a round-bottom flask, and solvent DMSO (3 mL) and potassium carbonate (207 mg, 1.5 mmol) were added, and stirred at 100° C. for 4 hours. 5 mL of water was added, extracted with ethyl acetate, and purified by silica gel column chromatography to obtain 72 mg of yellow solid ethyl 2-[3-(4-methoxyphenyl)-1H-pyrazol-1-yl]oxazole-4-carboxylate (Compound 36), with a yield of 41.5%. 1 H NMR(400MHz,DMSO-d6)δ8.10(d,J=2.5Hz,1H),7.83–7.77(m,2H),7.13(s,1H),7.03–6.99(m,2H),6.8 6(d,J=2.4Hz,1H),4.26(q,J=7.1Hz,2H),3.79(s,3H),1.30(t,J=7.1Hz,3H).ESI-MS(m / z):314,[M+H] + .

[0182] Specific Example 37: Preparation of ethyl 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-5-carboxylate (Compound 37)

[0183]

[0184] Step 1: Preparation of ethyl 2-hydrazinothiazole-5-carboxylate (Intermediate 37-1)

[0185]

[0186] Ethyl 2-chlorooxazole-5-carboxylate (1.91 g, 10 mmol) and tetrahydrofuran (20 mL) were added to a round-bottom flask, followed by hydrazine hydrate (85%, 1.2 mL). The mixture was heated under reflux for 2 hours and cooled to room temperature to precipitate 1.60 g of off-white solid ethyl 2-hydrazinothiazole-5-carboxylate (Intermediate 37-1). The yield was 85.6%. 1H NMR (400MHz, DMSO-d6) δ9.46 (s, 1H), 7.74 (s, 1H), 5.16 (s, 2H), 4.18 (q, J = 7.1Hz, 2H), 1.24 (t, J = 7.1Hz, 3H). ESI-MS (m / z): 188, [M+H] + .

[0187] Step 2: Preparation of ethyl 2-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-5-carboxylate (Compound 37)

[0188]

[0189] 2-Hydrazinothiazole-5-carboxylic acid ethyl ester 5 (Intermediate 37-1, 188 mg, 1 mmol) and 4-methoxyphenylacetonitrile (175 mg, 1 mmol) were added to a round-bottom flask, and ethanol (4 mL) and acetic acid (2 mL) were added. The mixture was heated to reflux for 5 hours and cooled to room temperature to precipitate 155 mg of 22-[5-amino-3-(4-methoxyphenyl)-1H-pyrazol-1-yl]thiazole-5-carboxylic acid ethyl ester (Compound 37) as a white solid with a yield of 45.0%. 1 H NMR(400MHz,DMSO-d6)δ8.24(s,1H),7.81–7.71(m,2H),7.08–6.96(m,2H),6.90(s,2H),5.8 5(s,1H),4.32(q,J=7.1Hz,2H),3.80(s,3H),1.32(t,J=7.1Hz,3H).ESI-MS(m / z):345,[M+H] + .

[0190] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A small molecule compound or a salt thereof, characterized in that: The structural formula of the compound is shown in formula (I): Where R 1 is phenyl, aromatic heterocycle or substituted phenyl, aromatic heterocycle; R 2 is H, OH, NH2 or substituted amino; R 3 is formate or formamide; X is S, O or N.

2. A small molecule compound or a salt thereof according to claim 1, characterized in that: The compound of formula (I) is further selected from the following formula (II): Where R 4 is single or multiple H, halogen, alkyl, alkoxy, cyano or aromatic; R 5 is an alkoxy group or a substituted amino group.

3. A small molecule compound or a salt thereof according to claim 1 or 2, characterized in that: The compound structural formula is shown in any of the following:

4. A pharmaceutical composition comprising the small molecule compound or its salt according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

5. Use of the small molecule compound or its salt according to any one of claims 1 to 3 in the preparation of a drug for expanding hematopoietic stem cells and / or hematopoietic progenitor cells.

6. A pharmaceutical composition comprising a hematopoietic stem cell and / or hematopoietic progenitor cell population expanded by the small molecule compound or a salt thereof according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

7. Use of hematopoietic stem cells and / or hematopoietic progenitor cells expanded using the small molecule compound or a salt thereof according to any one of claims 1 to 3 in the preparation of drugs for hematopoietic dysfunction / malignant tumors, autoimmune diseases and / or hereditary immunodeficiency diseases.

8. Use of the small molecule compound or its salt according to any one of claims 1 to 3 in the preparation of a medicament for hematopoietic dysfunction / malignant tumor, autoimmune disease and / or hereditary immunodeficiency disease.

9. The use according to claim 7 or 8, characterized in that: The hematopoietic disorders / malignancies, autoimmune diseases and / or inherited immunodeficiency diseases include bone marrow failure disorders, lupus, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, myelodysplastic syndrome, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, hemoglobinuria, Fanconi anemia, thalassemia, sickle cell anemia, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

10. A kit for amplifying hematopoietic stem cells and / or hematopoietic progenitor cells, characterized in that: The kit comprises the small molecule compound or its salt according to any one of claims 1 to 3.

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