Preparation and application of 3-(5-methylisoxazole-3-yl) urea compound

Abstract

The invention relates to 3-(5-methylisoxazole-3-yl) urea compounds. The structural formulas of the compounds are shown as I and II. The invention provides a method for preparing the compound and application of the compound in medicines. The compound provided by the invention can well inhibit the growth of medullary blasts, and the proliferation of the medullary blasts can cause the children brain tumor, so that the compound is expected to be further developed into the medicine for treating the children brain tumor.

Description

Technical Field

[0001] This invention relates to the preparation and use of 3-(5-methylisoxazole-3-yl)urea compounds, belonging to the field of pharmaceutical preparation technology. Background Technology

[0002] Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for approximately 30% of all childhood brain tumors. It is highly aggressive, difficult to treat, and nearly 35% of patients are considered to have no cure. Even if treatment is successful, children may experience severe long-term disability and health problems due to the adverse side effects of radiation and chemotherapy, leaving various sequelae, including cognitive impairment and endocrine disorders.

[0003]

[0004] In 2020, Cheng et al. (Cell Reports 31, 107782) reported through experimental testing that GSK126, UNC1999 and EPZ6438 could significantly inhibit the growth of medulloblasts. However, these compounds have drawbacks, such as poor blood-brain barrier permeability, low bioavailability and significant toxic side effects. For these reasons, it is necessary to further search for new and effective small molecule compounds to overcome these shortcomings. Summary of the Invention

[0005] The purpose of this invention is to provide 3-(5-methylisoxazole-3-yl)urea compounds, their preparation methods, and applications, in order to address the current lack of effective drugs for treating medulloblastoma. The compounds provided by this invention can effectively inhibit the growth of medulloblastoma cells and are expected to be further developed into drugs for treating medulloblastoma-related diseases.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides a class of compounds, the structural formulas of which are shown in I and II.

[0008]

[0009] On the other hand, the present invention also provides a method for preparing compounds with the structures shown in Formulas I and II above, the method comprising:

[0010] (a) Synthesize compound I, with the following reaction formula.

[0011]

[0012]

[0013] (b) Synthesize compound II, with the following reaction formula.

[0014]

[0015] (c) In the synthesis of compound I, the coupling agent may be 3-aminophenylboronic acid ester or 3-aminophenylboronic acid; in the synthesis of compound II, the coupling agent may be 4-aminophenylboronic acid ester or 4-aminophenylboronic acid ester.

[0016] The catalyst used in the coupling reaction can be tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), or tert-butylphosphine, PdCl2(dppf), Pd2(dba)3, and palladium acetate, etc. The base used includes, but is not limited to, sodium carbonate, potassium carbonate, and sodium bicarbonate, etc. The reaction solvent includes, but is not limited to, dioxane-water, DMF-water, ethanol-water, ethylene glycol dimethyl ether-water, and toluene-water, etc.

[0017] Beneficial effects:

[0018] This application provides 1-(3-(4-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methylcarbamoyl)-1-isopropyl-1H-indazole-6-yl)phenyl)-3-(5-methylisoxazol-3-yl)urea (compound structural formula I) and 1-(4-(4-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methylcarbamoyl)-1-isopropyl-1H-indazole-6-yl)phenyl)-3-( 5-Methylisoxazole-3-yl)urea (compound structure II) is a novel compound that, according to biological tests, can effectively inhibit the growth of medulloblasts. Compound structure I shows significantly stronger activity than UNC1999, GSK126, and EPZ6438, while compound structure II shows activity similar to UNC1999 but stronger than GSK126 and EPZ6438. Since medulloblast proliferation can cause brain tumors in children, this compound holds promise for further development into a drug for treating childhood brain tumors. Attached Figure Description

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0020] Figure 1 The image shows the 1H NMR spectrum of compound I.

[0021] Figure 2 This is the 1H NMR spectrum of compound II. Detailed Implementation

[0022] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0023] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0024] The 1,1′-carbonyldiimidazole (CDI) and 5-methylisoxazole-3-amine (compound 1) used in this invention were purchased from Shanghai Bid Pharmaceutical.

[0025] 6-Bromo-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (compound 3) was synthesized according to WO2011140325A1.

[0026] Example 1:

[0027]

[0028]

[0029] (1) Synthesis of N-(5-methylisoxazol-3-yl)-1H-imidazol-1-carboxamide (i.e., compound 2).

[0030] 1,1′-carbonyldiimidazole (CDI) (1.25 g, 7.7 mmol) was added to dichloroethane (8 mL), heated to 50 °C, and 5-methylisoxazole-3-amine (compound 1, 0.69 g, 7.0 mmol) was slowly added. The mixture was stirred at 50 °C for 3 hours under nitrogen protection. After cooling, a solid precipitated out. The solid was filtered and dried to give an off-white solid N-(5-methylisoxazole-3-yl)-1H-imidazolium-1-carboxamide, designated as compound 2, 0.81 g, 4.2 mmol, with a yield of 60.2%, which was used directly in the next reaction.

[0031] (2) Synthesis of 6-(3-aminophenyl)-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (i.e., compound 4).

[0032] 6-Bromo-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (compound 3, 200 mg, 0.48 mmol), 3-aminophenylboronic acid ester (157 mg, 0.72 mmol), Pd(PPh3)4 (55.5 mg, 0.048 mmol), sodium carbonate (152.6 mg, 1.44 mmol), and dioxane-water (10 mL - 3 mL) were refluxed under nitrogen protection for 7 hours, cooled, concentrated to dryness, and water (50 mL) was added. The mixture was shaken, filtered, and the solid was purified by silica gel column chromatography (V).二氯甲烷 V 甲醇 =20:1 elution), concentrated to give a grayish-white solid 6-(3-aminophenyl)-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide, designated as compound 4, 130 mg, 0.30 mmol, yield 62.5%.

[0033] 1 H NMR (DMSO-d6, 400MHz): δ (ppm) 1.53 (d, J = 8.0Hz, 6H), 2.15 (s, 3H), 2.24 (s, 3H), 4.42 (d, J = 4.0Hz, 2H), 5.18-5.13 (m, 3H), 5.91 (s, 1H), 6 .64(d,J=8.0Hz,1H),7.00-6.97(m,2H),7.18-7.14(t,J=8.0Hz,1H),7.80(s,1H),7.97(s,1H),8.37(s,1H),8.61(s,1H),11.53(s,1H).

[0034] MS(ESI): m / z 430.5 [M+H] +

[0035] (3) Synthesis of 1-(3-(4-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methylcarbamoyl)-1-isopropyl-1H-indazole-6-yl)phenyl)-3-(5-methylisoxazole-3-yl)urea (i.e., compound I)

[0036] N-(5-methylisoxazol-3-yl)-1H-imidazol-1-carboxamide (compound 2, 64.5 mg, 0.336 mmol, prepared in step 1) and 6-(3-aminophenyl)-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (compound 4, 72 mg, 0.168 mmol, prepared in step 2) were suspended in chloroform (5 mL) and heated to 50 °C under nitrogen protection. The solid dissolved, and the reaction was continued at this temperature with stirring for 4 hours. The reaction solution was cooled, extracted with dichloromethane (100 mL), washed with water (15 mL), washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel rapid column chromatography (V) with mixing. 二氯甲烷 V 甲醇=20:1 elution), concentrated, to give a white solid 1-(3-(4-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methylcarbamoyl)-1-isopropyl-1H-indazole-6-yl)phenyl)-3-(5-methylisoxazole-3-yl)urea, designated as compound I, 38 mg, 0.069 mmol, yield 41.1%.

[0037] 1 H NMR (DMSO-d6, 400MHz): δ (ppm) 1.54 (d, J = 4.0Hz, 6H), 2.15 (s, 3H), 2.25 (s, 3H), 2.40 (s, 3H), 4.43 (d, J = 4.0Hz, 2H), 5.20-5.17 (m, 1H), 5.91 (s, 1H) ),6.59(s,1H),7.57-7.44(m,3H),7.80(s,1H),7.85(s,1H),8.08(s,1H) ,8.41(s,1H),8.64-8.62(m,1H),8.98(s,1H),9.52(s,1H),11.53(s,1H).

[0038] MS(ESI): m / z 554.5 [M+H] +

[0039] The 1H NMR spectrum of compound I is shown below. Figure 1 As shown.

[0040] (4) Synthesis of 6-(4-aminophenyl)-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (i.e., compound 5).

[0041] 6-Bromo-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (compound 3, 180 mg, 0.43 mmol), 4-aminophenylboronic acid ester (141 mg, 0.64 mmol), Pd(PPh3)4 (49.7 mg, 0.043 mmol), sodium carbonate (136.7 mg, 1.29 mmol), and dioxane-water (8 mL - 2 mL) were refluxed under nitrogen protection for 7 hours, cooled, concentrated to dryness, and water (50 mL) was added. The mixture was shaken, filtered, and the solid was purified by silica gel column chromatography (V). 二氯甲烷 V 甲醇=20:1 elution), concentrated to give a gray solid 6-(4-aminophenyl)-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide, designated as compound 5, 120 mg, 0.28 mmol, yield 65.1%.

[0042] 1 H NMR (DMSO-d6, 400MHz): δ (ppm) 1.52 (d, J = 8.0Hz, 6H), 2.15 (s, 3H), 2.24 (s, 3H), 4.42 (d, J = 8.0Hz, 2H), 5.15-5.10 (m, 1H), 5.29 (s, 2H) ),5.91(s,1H),6.71(d,J=8.0Hz,2H),7.59(d,J=8.0Hz,2H),7.79(s,1H),7.93(s,1H),8.33(s,1H),8.60-8.57(m,1H),11.53(s,1H).

[0043] MS(ESI): m / z 430.4 [M+H] +

[0044] (5) Synthesis of 1-(4-(4-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methylcarbamoyl)-1-isopropyl-1H-indazole-6-yl)phenyl)-3-(5-methylisoxazole-3-yl)urea (i.e., compound II)

[0045] N-(5-methylisoxazol-3-yl)-1H-imidazol-1-carboxamide (compound 2, 61 mg, 0.32 mmol, prepared in step 1) and 6-(4-aminophenyl)-N-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methyl)-1-isopropyl-1H-indazole-4-carboxamide (compound 5, 70 mg, 0.16 mmol, prepared in step 4) were suspended in chloroform (5 mL). The mixture was heated to 50 °C under nitrogen protection and stirred at this temperature for 4 hours. The reaction solution was then cooled and soluble in dichloroform. Extracted with alkane (100 mL), washed with water (15 mL), washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, concentrated, purified by silica gel column chromatography (V dichloromethane:V methanol = 20:1 elution), concentrated to give a white solid 1-(4-(4-((1,2-dihydro-4,6-dimethyl-2-oxopyridin-3-yl)methylcarbamoyl)-1-isopropyl-1H-indazole-6-yl)phenyl)-3-(5-methylisoxazole-3-yl)urea, designated as compound II, 35 mg, 0.063 mmol, yield 39.4%.

[0046] 1 H NMR (DMSO-d6, 400MHz): δ (ppm) 1.52 (d, J = 8.0Hz, 6H), 2.15 (s, 3H), 2.25 (s, 3H), 2.40 (s, 3H), 4.43 (d, J = 4.0Hz, 2H), 5.20-5.17 (m, 1H), 5.92 ( s,1H),6.60(s,1H),7.62(d,J=8.0Hz,2H),7.84(m,3H),8.10(s,1H),8 .39(s,1H),8.66-8.65(m,1H),9.00(s,1H),9.52(s,1H),11.56(s,1H).

[0047] MS(ESI): m / z 554.4 [M+H] +

[0048] The 1H NMR spectrum of compound II is shown below. Figure 2 As shown.

[0049] Example 2: Effects of compounds I and II on medulloblast proliferation

[0050] 1. Cell Culture

[0051] After primary medulloblastoma cells were dissected and isolated from the brain, they were digested with papain and centrifuged using Percoll to obtain a single-cell suspension. The suspension was then seeded at 2 x 10^5 cells per well on 24-well plates. After two hours of complete cell adhesion, culture medium (B27 medium containing 1 mM sodium pyruvate, 2 mM L-glutamine, and 1% penicillin / streptomycin) was added to a final volume of 500 μL per well, and the plates were incubated at 37°C with 5% CO2 for 48 hours. In 96-well plates, cells were seeded at 100 μL of culture medium at a density of 3 x 10^5 cells per well. After 4-6 hours of complete cell adhesion, the original culture medium was removed, and 100 μL of culture medium containing different concentrations of drugs (prepared as described in "2. Drug Addition Protocol") was added. The plates were then incubated for another 48 hours.

[0052] 2. Dosing regimen

[0053] UNC1999 (reference standard), purchased from Shanghai Bide Pharmaceutical, 98%.

[0054] The test samples (compound I, compound II, and UNC1999) were dissolved in a small amount of DMSO, and then a stock solution with a concentration of 100 μM was prepared using culture medium (B27 medium containing 1 mM sodium pyruvate, 2 mM L-glutamine, and 1% penicillin / streptomycin). The stock solution was then diluted with culture medium to five concentrations: 500 nM, 1 μM, 5 μM, 10 μM, and 20 μM. Three replicates were set up for each concentration.

[0055] Meanwhile, a blank control group was prepared by adding an equal volume of small amount of DMSO and an equal volume of culture medium, and a cell-free group.

[0056] 3. CCK-8 absorbance detection

[0057] After 48 hours of cell culture, 10 μL of CCK-8 was added to each well of a 96-well plate, and the plates were incubated for 3 hours. The absorbance was then measured at 450 nm. The inhibition rate was determined, and the IC50 concentration required to inhibit 50% cell growth was calculated. 50 .

[0058] 4. Data Analysis: All data were analyzed using GraphPad Prism 6.0.

[0059] 5. Test Results

[0060] Table 1. Sample test results for IC50 inhibitory effects on medulloblastocytes. 50

[0061] <![CDATA[IC 50 ]]> Compound I 0.75uM Compound II 2.34uM UNC1999 2.11uM

[0062] Table 2. Cell Reports 2020, 31, 107782 reports on the IC50 inhibitory effects of drugs on medulloblastocytes. 50

[0063] <![CDATA[IC 50 ]]> GSK126 4.64uM EPZ6438 8.74uM UNC1999 2.08uM

[0064] As shown in Tables 1 and 2, the results of determining UNC1999 (reference standard) are basically the same as those reported in the literature, indicating that the test results of compound I and compound II are reliable.

[0065] Compound I, with its structural formula, exhibits significantly stronger inhibitory activity against medulloblastocytes than UNC1999, GSK126, and EPZ6438. Its inhibitory activity is 2.8 times that of UNC1999, 6.2 times that of GSK126, and 11.7 times that of EPZ6438.

[0066] Compound structure II inhibits medulloblast activity similarly to UNC1999, but more strongly than GSK126 and EPZ6438.

[0067] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A class of compounds, characterized in that, The structural formulas of this type of compound are shown in I and II: 。 2. A method for preparing the compound of claim 1, characterized in that, The method includes: (a) Synthesize compound I, with the following reaction formula: ； (b) Synthesize compound II, according to the following reaction formula: ； ； (c) In the synthesis of compound I, the coupling agent may be 3-aminophenylboronic acid ester or 3-aminophenylboronic acid; in the synthesis of compound II, the coupling agent may be 4-aminophenylboronic acid ester or 4-aminophenylboronic acid. The catalyst used in the coupling reaction can be tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), or tert-butylphosphine, PdCl2(dppf), Pd2(dba)3, and palladium acetate, etc. The base used includes, but is not limited to, sodium carbonate, potassium carbonate, sodium bicarbonate, etc. The reaction solvent includes, but is not limited to, dioxane-water, DMF-water, ethanol-water, ethylene glycol dimethyl ether-water, and toluene-water.

3. The use of the compound of claim 1 or a pharmaceutically acceptable salt, solvate or pharmaceutical complex thereof in the preparation of a medicament for treating diseases related to medulloblastoma.

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