A pyrimidine carboxamide compound and a preparation method and application thereof

Abstract

The application belongs to the field of medicine, and relates to a pyrimidine carboxamide compound and a preparation method and application thereof.The pyrimidine carboxamide compound has the following structure: the compound is verified by experiments to have significant anti-inflammatory activity. ELISA experiments and qPCRs prove that the compound can significantly inhibit the expression of IFN-beta and other inflammatory factors, and promote the release of inflammatory factors. The compound can be used for preparing a medicine for treating inflammation, in particular, for treating inflammatory bowel disease, celiac disease, colitis, Crohn's disease and the like, and has potential medical value and important medical prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals and relates to a pyrimidine formamide compound, its preparation method, and its application. Background Technology

[0002] Inflammation is the symptom basis of human diseases, interacting with many disease lesions and serving as a key link in the pathological process of disease. However, current anti-inflammatory drugs, such as corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs), still have many problems in clinical application, such as easily causing gastrointestinal discomfort and bleeding, and increasing the risk of adverse reactions such as heart disease or systemic coagulation disorders. Therefore, finding safer and more effective anti-inflammatory drugs remains an important task in current anti-inflammatory drug research and development.

[0003] Existing technologies have been used to isolate and identify various natural products with novel structures and significant activities from natural medicines. For example, modern chemical and pharmacological studies have shown that jackfruit is rich in isopentenylphenolic components, which possess a wide range of pharmacological activities. In preliminary activity screening, the chloroform extract of the 95% ethanol extract of jackfruit root showed strong inhibitory activity against respiratory burst in rat PMNs. However, many problems still exist in the clinical application of these substances. Summary of the Invention

[0004] The purpose of this invention is to provide a novel pyrimidine formamide compound, its preparation method, and its anti-inflammatory application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A pyrimidine formamide compound has the following structure: .

[0006] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows: Based on the same inventive concept, this invention also claims protection for a method for preparing the pyrimidine formamide compound, comprising the following steps: S1, raw material 1a and pinacol isopropenyl borate are reacted to obtain intermediate I-1; phenylsilane and NaHCO3 are added to intermediate I-1 and reacted sequentially to obtain intermediate I-2; S2, raw material 1b reacts with N,N-diisopropylethylamine and (S)-2-amino-2-phenylethanol to give intermediate I-3; intermediate I-3 reacts with intermediate I-2 to give intermediate I-4; S3, intermediate I-4, and base react to give intermediate I-5; intermediate I-5 is condensed with 3-aminopyridine and N,N-diisopropylethylamine to give the pyrimidine formamide compound. The structure of raw material 1a is as follows: ; The structure of intermediate I-1 is as follows: ; The structure of intermediate I-2 is as follows: ; The structure of raw material 1b is as follows: ; The structure of intermediate I-3 is as follows: ; The structure of intermediate I-4 is as follows: ; The structure of intermediate I-5 is as follows: .

[0007] In one preferred embodiment, a noble metal catalyst is added during the reaction of raw material 1a and pinacol isopropenylboronic acid ester.

[0008] In one preferred embodiment, the catalyst is Pd(dppf)Cl. 2， Full Chinese name: Dichloro-1,1'-bis(diphenylphosphino)ferrocene-palladium(II).

[0009] In one preferred embodiment, the reaction temperature of raw material 1a and pinacol isopropenyl borate is 75°C-85°C.

[0010] In one preferred embodiment, a solvent is added during the reaction of raw material 1a and pinacol isopropenylborate, said solvent being a mixture of 1,4-dioxane and water.

[0011] In one preferred embodiment, the volume ratio of 1,4-dioxane to water in the solvent is 60-67:10-13.

[0012] In one preferred embodiment, the catalyst for the reaction of intermediate I-1 and phenylsilane is Mn(dpm)3, also known as tris(dipentanoylmethane)manganese(III).

[0013] In one preferred embodiment, a solvent, which is a mixture of isopropanol and dichloromethane, is added during the reaction of intermediate I-1 and phenylsilane.

[0014] In one preferred embodiment, the volume ratio of isopropanol to dichloromethane in the solvent is 70-77:2-5.

[0015] In one preferred embodiment, the crude product of intermediate I-1 and phenylsilane is dissolved in a mixed solvent, and then NaHCO3 is added to react.

[0016] In one preferred embodiment, the volume ratio of DMF to water in the mixed solvent is 1-2:1-2.

[0017] In one preferred embodiment, a solvent is added during the reaction of raw material 1b with N,N-diisopropylethylamine and (S)-2-amino-2-phenylethanol; the solvent is acetonitrile.

[0018] In one preferred embodiment, intermediates I-3 and I-2 are reacted at a temperature of 70°C-80°C overnight.

[0019] In one preferred embodiment, a solvent is added during the reaction of intermediates I-3 and I-2; the solvent is N-methylpyrrolidone.

[0020] In one preferred embodiment, an acid is added during the reaction of intermediates I-3 and I-2; the acid is concentrated hydrochloric acid or concentrated nitric acid.

[0021] In one preferred embodiment, intermediate I-4 is dissolved in THF and then reacted with a base.

[0022] In one preferred embodiment, the base in step S3 is an inorganic base.

[0023] In one preferred embodiment, the alkali is one or more of NaOH, KOH, and Ba(OH)2.

[0024] In one preferred embodiment, a Carter condensing agent is added during the condensation of intermediate I-5 and 3-aminopyridine.

[0025] In one preferred embodiment, the synthetic route of the pyrimidine formamide compound is as follows:

[0026] Based on the same inventive concept, this invention also claims the use of pyrimidine formamide compounds in the preparation of anti-inflammatory reagents. The structure of the pyrimidine formamide compounds is shown below: .

[0027] In one preferred embodiment, the inflammation is inflammatory bowel disease, ciliated enterocolitis, colitis, or Crohn's disease, etc.

[0028] Based on the same inventive concept, the present invention also claims protection for an anti-inflammatory drug, wherein the active ingredient of the anti-inflammatory drug includes the pyrimidine carboxamide compound.

[0029] In one preferred embodiment, the inflammation is inflammatory bowel disease, ciliated enterocolitis, colitis, or Crohn's disease, etc.

[0030] The inflammatory bowel disease, celiac disease, or Crohn's disease mentioned above are inflammatory diseases whose treatment mechanisms and methods are almost identical or very similar to those for treating colitis. The present invention has verified through experimental data that the compounds of the present invention can treat colitis. Therefore, according to common medical knowledge, the compounds of the present invention can also treat inflammatory bowel disease, celiac disease, or Crohn's disease.

[0031] Compared with the prior art, the beneficial effects of the present invention are: This invention experimentally verifies that the compounds of this invention possess significant anti-inflammatory activity. ELISA and qPCR experiments demonstrate that the compounds of this invention can significantly inhibit the expression of IFN-β and other inflammatory factors. Their use in the preparation of drugs for treating inflammation, particularly inflammatory bowel disease, ciliated colitis, colitis, or Crohn's disease, has potential medical value and significant medical prospects. Attached Figure Description

[0032] Figure 1 The IC50 of compounds of formulas 1-3 on STING-Reporter-HEK293 cells 50 Result image.

[0033] Figure 2 This is a bar graph showing the effect of compounds of formula 1-2 on the expression of inflammatory factors in L929 cells; where, Figure 2 A is a bar graph showing the effect of compounds of formula 1-2 on IFN-β expression in L929 cells. Figure 2 B is a bar graph showing the effect of compounds of formula 1-2 on IL-6 expression in L929 cells. Figure 2 C is a bar graph showing the effect of compounds of formula 1-2 on CXCL10 expression in L929 cells. Figure 2 D is a bar graph showing the effect of compounds of formula 1-2 on TNF-α expression in L929 cells. Figure 2 E is a bar graph showing the effect of compounds of formula 1-2 on ISG15 expression in L929 cells.

[0034] Figure 3 This is a bar graph of the results from a compound-based ELISA experiment.

[0035] Figure 4 This is an HE staining image showing the effects of compound 1 on mice with colitis; where, Figure 4 A shows the HE staining image of the colon of control mice. Figure 4 B shows the HE staining image of the colon of mice in the DSS model group. Figure 4 HE staining image of the effect of compound of formula 1 (C = 20 mg / kg) on ​​mice with colitis. Figure 4 HE staining image of the effect of compound of formula 1 (D = 40 mg / kg) on ​​mice with colitis.

[0036] Figure 5 This is an HE staining image showing the effects of compound 2 on mice with colitis; where, Figure 5 A shows the HE staining image of the colon of control mice. Figure 5 B shows the HE staining image of the colon of mice in the DSS model group. Figure 5 HE staining image of the effect of compound of formula 2 (C = 20 mg / kg) on ​​mice with colitis. Figure 5 HE staining image of the effect of compound of formula 2 (D = 40 mg / kg) on ​​mice with colitis. Detailed Implementation

[0037] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0038] The compound shown in Formula 1 was purchased from Bid Pharmaceutical Co., Ltd. (Catalog No.: BD01306085).

[0039] Example 1

[0040] ( S Preparation of 2-((3,3-dimethyl-1-oxo-1,3-dihydroisobenzofuran-5-yl)amino)-4-((2-hydroxy-1-phenylethyl)amino)-N-(pyridin-3-yl)pyrimidine-5-carboxamide (compound of formula 2) The synthesis route is as follows:

[0041] Specifically, the following steps are included: Synthesis of Intermediate I-1 Under argon protection, starting material 1a (2.67 g, 13.5 mmol), pinacol isopropenylborate (2.73 g, 16.24 mmol), K₂CO₃ (4.67 g, 33.84 mmol), and Pd(dppf)Cl₂ (97.6 mg, 0.133 mmol) were dissolved in a mixed solvent of 1,4-dioxane (67 mL) / water (13 mL). The reaction mixture was stirred overnight at 80 °C. After the reaction was complete, the mixture was cooled to room temperature, diluted with water, and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed successively with saturated NaCl solution, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give intermediate I-1 (1.85 g, 87%).

[0042] Synthesis of Intermediate I-2 Under an oxygen atmosphere, intermediate I-1 (1.83 g, 11.6 mmol) and Mn(dpm)3 (0.70 g, 1.16 mmol) were dissolved in a mixed solvent of isopropanol (73 mL) and dichloromethane (3.3 mL). The reaction system was cooled to 0 °C, and phenylsilane (2.51 g, 23.18 mmol) was slowly added, with stirring continued for 4 h. After the reaction was complete, the reaction was quenched with 20% Na2S2O3 aqueous solution, and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude 4-amino-2-(2-hydroxypropyl-2-yl)benzyl nitrile. The crude product was dissolved in DMF (23 mL) / water (23 mL), and NaHCO3 (3.89 g, 46.3 mmol) was added in portions, with stirring at 80 °C for 24 h. After the reaction was complete and cooled to room temperature, the mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrates were combined and extracted with ethyl acetate (10 mL × 3). The organic layer was washed with saturated NaCl, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography to give intermediate I-2 (1.57 g, 77%).

[0043] Synthesis of Intermediate I-3 Starting material 1b (2.00 g, 9.05 mmol) was dissolved in acetonitrile (23 mL), followed by the addition of DIPEA (3.0 mL, 18.1 mmol) and (S)-2-amino-2-phenylethanol (1.36 g, 9.9 mmol). The mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give intermediate I-3 (2.32 g, 79%).

[0044] Synthesis of Intermediate I-4 Intermediate I-2 (0.98 g, 5.55 mmol) and intermediate I-3 (1.78 g, 5.55 mmol) were dissolved in N-methylpyrrolidone (14 mL). Concentrated hydrochloric acid (0.33 mL) was slowly added dropwise to the reaction system, and the mixture was stirred overnight at 80 °C. After the reaction was complete, the mixture was cooled to room temperature, and triethylamine was added to neutralize the reaction system. The mixture was then extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated NaCl, dried over anhydrous Na₂SO₄, filtered, and concentrated to give intermediate I-4 (2.17 g, 84%).

[0045] Synthesis of Intermediate I-5 Intermediate I-4 (2.00 g, 4.30 mmol) was dissolved in 17 mL of THF, and 5 mL of 2 M NaOH aqueous solution and 1.7 mL of methanol were added. The mixture was stirred overnight at room temperature. After the reaction was complete, the pH of the reaction system was adjusted to ≈4 with 2 M HCl, and the mixture was filtered to obtain a white solid intermediate I-5 (1.37 g, 73%).

[0046] Synthesis of final product 1 (compound of formula 2) Intermediate I-5 (30 mg, 0.070 mmol) and 3-aminopyridine (9.7 mg, 0.104 mmol) were dissolved in DMF (0.67 mL), and Carter's condensing agent (46 mg, 0.104 mmol) was added, followed by the slow addition of DIPEA (18 mg, 0.14 mmol). The mixture was stirred at room temperature for 6 h. After the reaction was complete, water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic layers were combined and washed successively with water (10 mL × 3) and saturated NaCl solution. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give target compound 1 (compound 2) (32 mg, 90%).

[0047] After characterization, the structural formula of compound 2 is as follows: .

[0048] The characterization data are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 10.32 (s, 1H), 10.26 (s, 1H), 9.47 (d, J =7.9 Hz, 1H), 8.85 (d, J = 2.6 Hz, 1H), 8.84 (s, 1H), 8.32 (dd, J= 4.7, 1.5Hz, 1H), 8.13 (ddd, J = 8.4, 2.6, 1.5 Hz, 1H), 8.00 (s, 1H), 7.69 (dd, J =8.5, 1.7 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.42-7.35 (m, 5H), 7.28-7.24 (m,1H), 5.34 (dd, J = 8.3, 4.6 Hz, 1H), 5.20 (t, J = 5.0 Hz, 1H), 3.86 (dt, J =9.5, 4.7 Hz, 1H), 3.80-3.76 (m, 1H), 1.58 (s, 3H), 1.52 (s, 3H). Example 2

[0049] The synthesis of compound 2 and compound 3 is carried out as follows: In the synthesis of intermediate I-3, starting material 1b (2.00 g, 9.05 mmol) was replaced with an equimolar amount of 2,4-dichloro-5-trifluoromethylpyrimidine, dissolved in acetonitrile (23 mL), otherwise the same as in Example 1. The resulting compound (white solid, 60 mg, 67%) was obtained, with the following structure: .

[0050] The structural characterization data of compound formula 3 are as follows: 1 H NMR (600 MHz, DMSO- d 6) δ 10.20 (s, 1H), 8.34 (s, 1H), 7.87 (s, 1H), 7.69 (dd, J = 8.4, 1.8 Hz, 1H), 7.63 (d, J = 8.4Hz, 1H), 7.39 (d, J = 7.4 Hz, 2H), 7.36-7.31 (m, 2H), 7.25-7.21 (m, 1H), 7.08(d, J = 7.7 Hz, 1H), 5.42 (q, J = 6.4 Hz, 1H), 5.17 (t, J = 5.2 Hz, 1H), 3.85 (hept,J = 6.1 Hz, 2H), 1.56 (s, 3H), 1.52 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6)δ 168.62, 160.50, 158.11, 156.20, 155.11 (q, J = 4.4 Hz), 145.74, 140.48,128.33, 127.04, 126.65, 125.68, 124.85 (q, J = 269.9 Hz), 119.98, 117.21,110.11, 98.44 (q, J = 31.7 Hz), 84.50, 64.13, 56.23, 27.14, 27.11. Example 3

[0051] To investigate the effects of compounds on the expression of inflammatory factors in in vitro experiments.

[0052] 1. IC50 of compounds 1-3 50 test 20 µM of compounds (compounds 1, 2, and 3, and H-151) were simultaneously added to STING-Reporter-HEK293 cells (purchased from Shanghai Jiman Biotechnology Co., Ltd., product number: GM-C33256) and cultured for 24 h. After lysis, an equal volume of Luciferase Assay Buffer was added to the well plate and Luciferase was detected.

[0053] The structure of compound 1 is as follows: .

[0054] The structure of the H-151 is as follows: .

[0055] The IC50 testing process is as follows: 1) 16-24 hours before the experiment, remove the cells from the culture flask, digest and centrifuge to collect the cell pellet, resuspend the cells in an appropriate amount of complete culture medium, detect cell viability and count the cells, and then adjust the cell concentration to 2 × 10⁻⁶ cells / year using complete culture medium. 4 Cells / mL were collected, and MSA-2 agonist was added to the resuspension to a working concentration of 20 µM. 90 μL of cells / well was then added to a 96-well plate using a pipette. 100 µL of PBS was added to the surrounding wells. The plate was capped and incubated in an incubator. Once cell confluence reached 90%, the drug was added for further incubation.

[0056] 2) The stock solution of each compound was 20 mM. The compound was prepared to 1 mM and then diluted 3 times with blank culture medium to form 10 different concentration gradients. 10 µL of each concentration was added to a 96-well plate and incubated for 24 hours.

[0057] 3) Add an equal volume of the "One-Step" chemiluminescence assay kit (GM One-Step 2.0 Luciferase Reporter GeneAssay, Jiman Biotechnology) for the in vitro detection of firefly luciferase activity to a 96-well plate, pipette the sample five times, wait five minutes, and then pipette an appropriate volume (generally 30-50 μL) into a 384-well plate.

[0058] 4) Use an ELISA reader to detect luciferase, read the values, and calculate the inhibition rate.

[0059] The results are as follows Figure 1 As shown in Table 1.

[0060] Table 1. Half-maximal inhibitory concentration (IC50) of compounds in STING-Reporter-HEK293 cells

[0061] The results show that among the three compounds, compound 1 has the lowest half-maximal inhibitory concentration, indicating that it has the strongest inhibitory activity. Compound 2 has slightly weaker activity than compound 3, and compound 3 has relatively poor activity.

[0062] 2. PCR verification After adding 20µM compounds (compound 1, compound 2, and H-151) to L929 cells and culturing for 1 hour, the cells were treated with the same concentration of MSA and incubated for 5 hours. Cell RNA was then extracted and detected by qPCR.

[0063] The process of extracting RNA from cells is as follows: 1. Cell plating: To extract sufficient RNA, the number of cells plated should be ≥1×10⁻⁶. 6 -5×10 6 .

[0064] 2. Dosing of medication (treatment duration 5 hours): Volume per well: 2 ml.

[0065] Working drug concentrations: [MSA-2] = 20 μM; [Equation 1] = 20 μM; [Equation 2] = 20 μM; [H-151] = 20 μM.

[0066] 3. Extracting cellular RNA 1) Cell culture: Harvest cells, transfer them to a 1.5ml centrifuge tube, add 1ml Trizol, mix well, and let stand at room temperature for 5min.

[0067] 2) Tissue: Take 50-100 ml of tissue (fresh or tissue preserved at -70℃ or in liquid nitrogen) and place it in a 1.5 ml centrifuge tube. Add 1 ml of Trizol and homogenize thoroughly. Let stand at room temperature for 5 min.

[0068] 3) Add 0.2 ml of chloroform, shake for 15 seconds, and let stand for 2 minutes.

[0069] 4) Centrifuge at 4℃ for 12000g for 15min and collect the supernatant.

[0070] 5) Add 0.5 ml of isopropanol, gently mix the liquid in the tube, and let it stand at room temperature for 10 minutes.

[0071] 6) Centrifuge at 4℃, 12000g x 10min, and discard the supernatant.

[0072] 7) Add 1 ml of 75% ethanol and gently wash the precipitate. Incubate at 4℃, 7500 g for 5 min, then discard the supernatant.

[0073] 8) Dry the product and add an appropriate amount of DEPC H2O to dissolve it (promote dissolution at 65℃ for 10-15 minutes).

[0074] 4. qPCR system and procedure Table 2 qPCR system

[0075] Table 3 qPCR primer sequences

[0076] qPCR procedure Real-time quantitative PCR (qPCR) reactions were performed on a real-time PCR instrument. The amplification program was set as follows: First, the template DNA was pre-denatured at 95 °C for 30 s to ensure complete denaturation. Then, 40 cycles of amplification were performed, each cycle consisting of denaturation at 95 °C for 5 s, followed by annealing and extension at 60 °C for 34 s, during which fluorescence signals were acquired. After amplification, the reaction mixture was sequentially incubated at 95 °C for 15 s, at 60 °C for 1 min, and at 95 °C for 15 s to ensure sufficient denaturation of the PCR products and prepare for melting curve analysis.

[0077] The results are as follows Figure 2 As shown, where, Figure 2A is a bar graph showing the effect of compounds of formula 1-2 on IFN-β expression in L929 cells. Figure 2 B is a bar graph showing the effect of compounds of formula 1-2 on IL-6 expression in L929 cells. Figure 2 C is a bar graph showing the effect of compounds of formula 1-2 on CXCL10 expression in L929 cells. Figure 2 D is a bar graph showing the effect of compounds of formula 1-2 on TNF-α expression in L929 cells. Figure 2 E is a bar graph showing the effect of compounds of formula 1-2 on ISG15 expression in L929 cells. Groups are con, MSA, H-151, formula 1, and formula 2. Data are expressed as relative expression levels (normalized to the internal reference gene and standardized with the con group), bars represent the mean, and scatter plots represent independent replicates (n≈3). Comparisons with the MSA group are shown in parentheses; treatment with formula 1 significantly reduced MSA-induced transcription levels of related genes. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0078] qPCR results showed that, compared with H-151, compound 1 significantly and effectively reduced inflammatory factors IFN-β, TNF-α, ISG15, CXCL10, and IL-6, while compound 2 had an effect comparable to H-151.

[0079] 3. ELISA experiment 5 µM of compounds (Formula 1, Formula 2, and H-151) were collected from cell supernatants used for qPCR experiments. The IFN-β levels in the supernatant were detected using the Elabscience (Catalog No. E-EL-M0033) IFN-β assay kit. The ELISA assay procedure is as follows: 1. Add 100 μL of standard working solution or sample to the corresponding well and incubate at 37°C for 90 minutes.

[0080] 2. After discarding the liquid in the plate, immediately add 100 μL of biotinylated antibody working solution and incubate at 37°C for 60 minutes.

[0081] 3. Discard the liquid inside the plate and wash the plate 3 times.

[0082] 4. Add 100 μL of HRP enzyme conjugate working solution to each well and incubate at 37°C for 30 minutes. Discard the liquid in the plate and wash the plate 5 times.

[0083] 5. Add 90 μL of substrate solution to each well and incubate at 37°C for about 15 minutes.

[0084] 6. Add 50 μL of stop solution to each well.

[0085] 7. Immediately take readings at a wavelength of 450nm and process the data.

[0086] The results are as follows Figure 3 As shown. Figure 3 The results of detecting IFN-β release in cell supernatant under different treatment conditions are presented. Groups were con, MSA, H-151 (5 μM), Formula 1 (5 μM), and Formula 2 (5 μM). Bar charts represent the mean, scatter plots represent independent replicates (n=3), and error bars represent the dispersion of the mean. The comparison with the con group is shown in parentheses. MSA treatment significantly increased IFN-β release levels, while the compound in Formula 1 significantly reduced MSA-induced IFN-β levels. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0087] The results showed that, compared with H-151, compound 1 significantly and effectively reduced the expression and release of IFN-β, while compound 2 had an effect comparable to H-151.

[0088] Example 4 Compounds of Formula 1 and Formula 2 can effectively improve colitis in mice.

[0089] To investigate the anti-inflammatory effects of compounds of formulas 1 and 2 in vivo, all animal experiments were conducted in accordance with the Chinese Guidelines and Laws on the Use and Care of Laboratory Animals (GB / T 35892-2018 and GB / T 35823-2018). Female C57BL / 6J mice (18±2 g, 8 to 9 weeks old) were purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd. (Hunan, China) and housed under specific pathogen-free conditions (temperature, 22±℃, relative humidity, 55-60%, and a regular 12 / 12-hour light / dark cycle). All mice were acclimatized to the environment for one week before the formal experiments and then randomly divided into 6 groups (n = 6 per group). The control group (Control) was treated with distilled water; the model group (DSS) was induced with experimental colitis by drinking 3% DSS solution freely for 7 days, after which the 3% DSS solution was replaced with ordinary distilled water. The first intervention group (20 mg / kg Formula 1-DSS) administered 3% DSS solution to the animals via free access to drinking water for 7 days. Seven days after successful model establishment, mice were administered Formula 1 compound via gavage at a dose of 20 mg / kg. The second intervention group (40 mg / kg Formula 1-DSS) administered 3% DSS solution to the animals via free access to drinking water for 7 days. Seven days after successful model establishment, mice were administered Formula 1 compound via gavage at a dose of 40 mg / kg. The third intervention group (20 mg / kg Formula 2-DSS) administered 3% DSS solution to the animals via free access to drinking water for 7 days. Seven days after successful model establishment, mice were administered Formula 2 compound via gavage at a dose of 20 mg / kg. The fourth intervention group (40 mg / kg Formula 2-DSS) administered 3% DSS solution to the animals via free access to drinking water for 7 days. Seven days after successful model establishment, mice were administered Formula 2 compound via gavage at a dose of 40 mg / kg. The intervention group received the medication three times daily, every other day, with body weight recorded daily from the start of the modeling process. Colonic tissue was collected at the end of treatment (day 14) and fixed with paraformaldehyde before sectioning. The tissue sections were then stained with hematoxylin and eosin (HE) and evaluated. Results are as follows: Figures 4-5 As shown. Figure 4 middle, Figure 4 A shows the HE staining image of the colon of control mice. Figure 4 B shows the HE staining image of the colon of mice in the DSS model group. Figure 4 HE staining image of the effect of compound of formula 1 (C = 20 mg / kg) on ​​mice with colitis. Figure 4 HE staining image of the effect of compound of formula 1 (D = 40 mg / kg) on ​​mice with colitis. Figure 5 middle, Figure 5 A shows the HE staining image of the colon of control mice. Figure 5 B shows the HE staining image of the colon of mice in the DSS model group. Figure 5 HE staining image of the effect of compound of formula 2 (C = 20 mg / kg) on ​​mice with colitis. Figure 5 HE staining image of the effect of compound of formula 2 (D = 40 mg / kg) on ​​mice with colitis.

[0090] HE staining results showed that compounds of formula 1 and formula 2 could significantly alleviate DSS-induced colitis, and the higher dose (i.e., 40 mg / kg group) of compounds of formula 1 and formula 2 had a more significant effect on improving colitis.

[0091] It is evident that compounds of formula 1 and formula 2 have a significant ameliorative effect on DSS-induced colitis in mice at appropriate concentrations.

[0092] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the protection scope of this invention.

Claims

1. A pyrimidine carboxamide compound, characterized in that, Its structure is as follows:

2. The method for preparing pyrimidine formamide compounds according to claim 1, characterized in that, Includes the following steps: S1, raw material 1a and pinacol isopropenyl borate are reacted to obtain intermediate I-1; phenylsilane and NaHCO3 are added to intermediate I-1 and reacted sequentially to obtain intermediate I-2; S2, raw material 1b reacts with N,N-diisopropylethylamine and (S)-2-amino-2-phenylethanol to give intermediate I-3; intermediate I-3 reacts with intermediate I-2 to give intermediate I-4; S3, intermediate I-4, and base react to give intermediate I-5; intermediate I-5 is condensed with 3-aminopyridine and N,N-diisopropylethylamine to give the pyrimidine formamide compound. The structure of raw material 1a is as follows: ; The structure of intermediate I-1 is as follows: ; The structure of intermediate I-2 is as follows: ; The structure of raw material 1b is as follows: ; The structure of intermediate I-3 is as follows: ; The structure of intermediate I-4 is as follows: ; The structure of intermediate I-5 is as follows: .

3. The preparation method according to claim 2, characterized in that, A noble metal catalyst is added during the reaction of raw material 1a and pinacol isopropenylboronic acid ester; preferably, the catalyst is Pd(dppf)Cl2.

4. The preparation method according to claim 2, characterized in that, A solvent is added during the reaction of raw material 1a and pinacol isopropenylboronic acid ester, wherein the solvent is a mixture of 1,4-dioxane and water; preferably, the volume ratio of 1,4-dioxane to water in the solvent is 60-67:10-13.

5. The preparation method according to claim 2, characterized in that, The base is an inorganic base; preferably, the base is one or more of NaOH, KOH, and Ba(OH)2.

6. The preparation method according to claim 2, characterized in that, The synthetic route for the pyrimidine formamide compounds is as follows:

7. The application of a pyrimidine carboxamide compound in the preparation of anti-inflammatory reagents, characterized in that, The structures of the pyrimidine formamide compounds are shown below: 。 8. The application according to claim 7, characterized in that, The inflammation could be inflammatory bowel disease, ciliated enterocolitis, colitis, or Crohn's disease.

9. An anti-inflammatory drug, characterized in that, The active ingredient of the anti-inflammatory drug includes pyrimidine formamide compounds; the structure of the pyrimidine formamide compounds is shown below: 。 10. The anti-inflammatory drug according to claim 9, characterized in that, The inflammation could be inflammatory bowel disease, ciliated enterocolitis, colitis, or Crohn's disease.

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