A 2-aminopyrimidine compound and its preparation method and application

Abstract

The invention discloses a 2-aminopyrimidine compound, the structural formula of which is as follows: the compound can significantly inhibit the proliferation and migration of glioblastoma cells, induce late cell apoptosis, promote tumor cell necrosis, and block the cell cycle at the G2 / M phase, and can target CDK4 / 6 to inhibit the phosphorylation of Rb, thereby inhibiting the activity of CDK4 and CDK6, and has potential application prospects in the preparation of glioblastoma therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to novel compounds and applications, and in particular to a 2-aminopyrimidine compound and a preparation method and application thereof. Background Art

[0002] Glioblastoma (GBM) is the most common and deadliest malignant brain tumor, with high molecular heterogeneity and poor overall prognosis, with a 10-year survival rate of less than 1%. The case fatality rate is close to 80%, and even if surgical resection is performed quickly, it will recur because it is difficult to completely resect.

[0003] Current clinical studies have shown that glioblastoma chemotherapy drugs such as temozolomide, nitrosoureas, and cisplatin have shown therapeutic effects, but there are problems with drug resistance or side effects. For example, temozolomide is an alkylating agent that is used as a standardized treatment for newly diagnosed glioblastoma patients. It has been shown to cause cells to arrest in the G2 / M phase and mediate DNA damage and apoptosis. Although oral temozolomide increases the overall survival of glioblastoma patients, the drug resistance induced by cancer cells limits its further treatment. Therefore, the study of new glioblastoma drugs remains an important challenge in current medicine.

[0004] In order to solve the problem of chemotherapy drug resistance, CDK4 / 6 inhibitors, as a rising anti-cancer "magic drug" in recent years, are rapidly changing the treatment pattern of hormone receptor (HR)-positive human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer, effectively overcoming or delaying the emergence of endocrine resistance, and gaining more survival time for advanced patients. The CDK inhibitors approved by the FDA are Pfizer's Ibrance (palbociclib), Novartis' Kisqali (Ribociclib), and Eli Lilly's Verzenio (abemaciclib).

[0005] Glioblastoma is a fatal brain tumor characterized by abnormal signal transduction and abnormal cell cycle regulation. Molecular analysis has confirmed that the CDK4 / 6-Rb axis is dysregulated in approximately 80% of GBM patients. Therefore, it is necessary to use CDK inhibitors that block specific cell cycle regulation in combination with chemotherapy drugs to treat advanced or metastatic cancer. Recently, Chinese patent CN202111306309.X disclosed that the combination of a small molecule compound ADAMTS5 inhibitor and temozolomide (TMZ) showed good anti-glioblastoma activity, which is expected to provide a new target for anti-tumor drugs and reduce the drug resistance of tumor cells.

[0006] Therefore, it is necessary to develop a new drug that can block the cell cycle regulation of glioblastoma. Summary of the invention

[0007] In view of this, the present invention provides a 2-aminopyrimidine compound, the structural formula of which is as follows:

[0008]

[0009] On the other hand, the present invention also provides a method for preparing 2-aminopyrimidine compounds, comprising the following steps: weighing 77 mg of 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine, 59 mg of 4-morpholinopyridine-2-amine, 14 mg of Pd 2 (dba) 3 , 12 mg of Brettphos catalyst and 195 mg of cesium carbonate were added with 2 mL of dioxane for dissolution, and the mixture was reacted at 90 ° C for 2 h under argon protection. The mixture was filtered with diatomaceous earth and extracted with ethyl acetate / water. The organic phase solution was collected and concentrated to obtain a crude product, which was separated and purified by silica gel column chromatography to obtain 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(4-morpholinopyridin-2-yl)pyrimidin-2-amine.

[0010] The present invention also provides the use of 2-aminopyrimidine compounds in preparing drugs for treating human brain glioblastoma.

[0011] The 2-aminopyrimidine compound LH20 provided by the present invention can significantly inhibit the proliferation and migration of glioblastoma cells, induce late cell apoptosis, promote tumor cell necrosis, and block the cell cycle at the G2 / M phase. It can target CDK4 / 6 to inhibit the phosphorylation of Rb, thereby inhibiting the activity of CDK4 and CDK6, and has potential application prospects in the preparation of glioblastoma therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The H NMR spectrum and C NMR spectrum of the novel compound LH20 of the present invention, wherein (A) represents 1 H NMR; (B) represents) 13 C NMR.

[0013] Figure 2 The figures are effect diagrams of the compound LH20 of the present invention on inhibiting the proliferation of GBM cells, wherein (A) represents the changes in morphology and cell density of U87MG cells after being treated with different concentrations of LH20 for 24 h (upper) and 48 h (lower); (B) represents the changes in morphology and cell density of U251 cells after being treated with different concentrations of LH20 for 24 h (upper) and 48 h (lower); (C) represents the effect of different concentrations of LH20 on the proliferation of U87MG cells (24 h and 48 h); (D) represents the effect of LH20 on the proliferation of U251 cells (24 h and 48 h).

[0014] Figure 3The figures show the results of the inhibition of U87MG and U251 cell migration by the compound LH20 of the present invention, respectively, wherein (A) represents the number of U87MG cells that migrated to the gaps counted under an optical microscope after being cultured for 48 hours in the presence of different concentrations of LH20 (4 μM, 8 μM and 10 μM); (B) represents the number of U251 cells that migrated to the gaps counted under an optical microscope after being cultured for 48 hours in the presence of different concentrations of LH20 (4 μM, 8 μM and 10 μM); (C) represents the migration analysis of U87MG after being treated with LH20 for 24 hours; (D) represents the migration analysis of U251 after being treated with LH20 for 24 hours and 48 hours.

[0015] Figure 4 The compound LH20 of the present invention promotes the apoptosis of U87MG and U251 cells, respectively, wherein (A) represents the flow cytometry graph of U87MG cells, (B) and (C) represent the changes in early apoptosis, late apoptosis and necrosis rates of U87MG cells treated with LH20 for 24 h and 48 h, respectively; (D) represents the flow cytometry graph of U251 cells; (E) and (F) represent the changes in early apoptosis, late apoptosis and necrosis rates of U251 cells treated with LH20 for 24 h and 48 h, respectively.

[0016] Figure 5 The figure shows the results of LH20-induced mitochondrial damage in U87-MG and U251 cells.

[0017] Figure 6 Figures 2 and 3 show the blocking effect of LH20 on GBM cells, where (A) represents the flow cytometry graph of U87MG cells; (B) and (C) represent the G0 / G1, S and G2 / M phase rate analysis graphs of U87MG cells treated with LH20 for 24h and 48h, respectively; (D) represents the flow cytometry graph of U251 cells; (E) and (F) represent the G0 / G1, S and G2 / M phase rate analysis graphs of U251 cells treated with LH20 for 24h and 48h. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0019] The raw materials involved in the embodiments of the present invention were purchased from regular manufacturers. The primary GBM cell lines U87MG and U251 cells were purchased from Procell (Wuhan, China), cultured in a high-glucose DMEM medium (DMEM; Gibco, California, USA) containing 10% fetal bovine serum (CLARK Bioscience, Virginia, USA) and 1% antibiotics (streptomycin / penicillin, Biosharp, Anhui, China), and cultured in a humidified incubator with stable CO2 (5%) and temperature (37°C).

[0020] Example 1: The preparation method of 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine is as follows:

[0021] Weigh 2,4-dichloro-5-fluoropyrimidine (5.01 g, 30.00 mmol), 4-fluoro-2-methoxyphenylboronic acid (5.17 g, 30.42 mmol) and Pd (pph 3 ) 4 (1.74 g, 1.5 mmol) was placed in a 100 mL flask and Na 2 CO 3 (6.15 g, 58.57 mmol) was dissolved in an appropriate amount of water and transferred to a flask. 50 mL of solvent (the volume ratio of water to dioxane was 1:4) was added, sealed with a rubber stopper, degassed and protected with argon, and reacted at 95 °C for 2 h. Celite was used to filter to remove the catalyst, and dichloromethane / water was extracted 3 times. The organic phase solution was collected and combined, and anhydrous Na 2 CO 3 The mixture was dehydrated, filtered under reduced pressure, and concentrated using a vacuum rotary evaporator to obtain a crude product. The mixture was separated and purified by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate was 15:1) to obtain 5.00 g of a white solid with a yield of 65%. The NMR characterization confirmed that the solid was 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine, and its structural formula was:

[0022]

[0023] Example 2: The preparation method of 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(4-morpholinopyridin-2-yl)pyrimidin-2-amine (LH20) is as follows:

[0024] Weigh 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine (77 mg, 0.30 mmol) prepared in Example 1, 4-morpholinopyridin-2-amine (59 mg, 0.33 mmol), Pd 2 (dba) 3(14 mg, 0.015 mmol), Brettphos (12 mg, 0.021 mmol) and cesium carbonate (195 mg, 0.60 mmol), dissolved in 2 mL of dioxane, sealed with a rubber stopper, degassed and protected with argon, reacted at 90 ° C for 2 h, filtered with diatomaceous earth to remove the catalyst, extracted with ethyl acetate / water 3 times, collected and combined the organic phase solution, and concentrated with a reduced pressure rotary evaporator to obtain a crude product. After separation and purification by silica gel column chromatography (dichloromethane / methanol, 60:1, v / v), 66 mg of white powder was obtained with a yield of 55%, which was confirmed by nuclear magnetic resonance characterization to be 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(4-morpholinopyridin-2-yl)pyrimidin-2-amine, as shown in Formula 2.

[0025] like Figure 1 The results show that 1H NMR (400MHz, CF 3 COOD)δ=9.0(d,J=2.5,1H),8.1(d,J=7.4,1H),7.9-7.8(m,1H),7.1(ddt,J=15.3, 7.8,2.5,3H),6.8(s,1H),4.2(d,J=4.9,4H),4.2-4.1(m,3H),3.9(d,J=5.2,4H).

[0026] like Figure 2 As shown in the results, 13C NMR (100 MHz, CF3COOD) δ 160.75, 160.64, 158.49, 149.44, 146.46, 136.17, 133.69, 113.54, 109.36, 109.15, 104.97, 101.05, 100.79, 93.98, 65.73, 55.93, 45.86.

[0027]

[0028] Example 3: Preparation of 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(4-methylpyridin-2-yl)pyrimidin-2-amine (MD11)

[0029] Weigh 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine (77 mg, 0.30 mmol), 4-methylpyridin-2-amine (36 mg, 0.33 mmol), Pd 2 (dba) 3(14 mg, 0.015 mmol), Brettphos (12 mg, 0.021 mmol) and cesium carbonate (195 mg, 0.60 mmol), add 2 mL of anhydrous dioxane to dissolve, seal with a rubber stopper, use argon for degassing and protection, and react at 90 ° C for 2 h. Celite is used to filter to remove the catalyst, and ethyl acetate / water is extracted 3 times. The organic phase solution is collected and combined, and the crude product is concentrated by a reduced pressure rotary evaporator. After separation and purification by silica gel column chromatography (dichloromethane / methanol=300:1, v / v), 16 mg of white powder is obtained, and the yield is 17%. It is confirmed by nuclear magnetic characterization that it is 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(4-methylpyridin-2-yl)pyrimidin-2-amine, and the structural formula is as shown in Formula 3.

[0030]

[0031] Example 4: Preparation of 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(5-morpholinopyridin-2-yl)pyrimidin-2-amine (LH18)

[0032] Weigh 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine (77 mg, 0.30 mmol), 5-morpholinopyridin-2-amine (59 mg, 0.33 mmol), Pd 2 (dba) 3 (14 mg, 0.015 mmol), Brettphos (12 mg, 0.021 mmol), cesium carbonate (195 mg, 0.60 mmol), dissolved in 2 ml of anhydrous Dioxane, the feed port was sealed with a rubber stopper, degassed and protected with argon, and reacted at 90 °C for 2 h. Celite assisted filtration to remove the catalyst, EA / H 2 O was extracted three times, and the organic phase solution was collected and combined, and concentrated by a reduced pressure rotary evaporator to obtain a crude product. After separation and purification by silica gel column chromatography (DCM / MeOH, 80:1, v / v), 62 mg of light yellow solid was obtained, with a yield of 52%; nuclear magnetic resonance characterization confirmed that it was 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(5-morpholinopyridin-2-yl)pyrimidin-2-amine, with a structural formula as shown in Formula 4.

[0033]

[0034] Example 5: Study on the inhibitory activity of LH20 compound, MD11 compound and LH18 compound on the proliferation of U87MG cells

[0035] The cells were seeded in 96-well microplates at a density of 10,000 and treated with LH20 and 24 and 48 hours, respectively. The culture medium was then removed and incubated with fresh culture medium containing 20 μL Cell Counting Kit-8 (CCK8) reagent at 37°C for 30 min, and the cell proliferation capacity was assessed by measuring the absorbance (450 nm) in a microplate reader (Spectra MAX190, Molecular Devices, San Jose, USA).

[0036] Furthermore, the morphological and cell density changes of U87MG and U251 cells after treatment with different concentrations of LH20 for 24h and 48h were shown in Figure 2. Figure 2 As shown in A and 2B, U87MG and U251 cells treated with LH20 showed morphological changes such as cell rounding and disappearance of processes. The results of CCK8 detection showed that after being treated with 4, 8, 10, 20, 100 and 200 μM LH20 for 24 h, the cell survival rates of U87MG (85.85±1.38%~14.86±0.84%) and U251 (89.62±3.55%~48.70±3.75%) were significantly lower than those of the control group (U87MG: 100.00±3.18%, U251: 100.00±3.85%), indicating that after being treated with 4, 8, 10, 20, 100 and 200 μM LH20 for 48 h, its inhibitory ability was enhanced (U87MG: 87.95±3.25%~12.58±0.87% VS 100.00±0.96%).

[0037] U251: (79.83±13.25%~14.21±0.95%VS100.00±4.75%, Figure 2 C and 2D). In particular, 10 μM LH20 administration for 48 h significantly increased the LDH release of U87MG, indicating that prolonged LH20 administration can induce U87MG cell damage.

[0038] According to the above method, under the same conditions, 10uM of MD11 compound and LH18 compound were used to test the inhibitory effect on the activity of U87MG cells. The results are shown in Table 1, indicating that the LH20 compound showed unexpected inhibitory activity on U87MG cells.

[0039] Table 1: Test results of the inhibitory activity of different compounds on U87MG cells

[0040]

[0041] Example 6: Study on the inhibitory effect of LH20 on GBM cell migration

[0042] GBM cells were seeded in 6-well cell culture plates at a cell density of 100,000 cells / well and grown to a density of 90%. The fused monolayer cells were scraped with a pipette tip and each well was rinsed with phosphate buffered saline (PBS) to remove non-adherent cells. The cells were then treated with compound LH20 (4, 8, and 10 μM) and cultured for 24 h or 48 h. The perimeter of the central cell-free zone was determined under an optical microscope. Migration area calculation formula: Migration area (%) = (area [0 day] - area [24 h / 48 h]) / area [0 day] × 100%.

[0043] like Figure 3 As shown in the results of A and 3B, after the cells were cultured in high-glucose DMEM for 24 hours and 48 hours, the scratched cells showed obvious migration. After 24 hours of treatment with 8μM and 10μM LH20, the migration area of ​​U87MG cells (39.84±19.86%) and (26.50±30.78%) was significantly lower than that of the control U87MG cells (100.00±17.02%, Figure 3 C). After 48h of exposure to different concentrations of LH20 (4, 8 and 10 μM), the migration area of ​​U87MG cells was further reduced (51.76±6.26%, 20.22±21.67%, 12.58±16.15% VS 100.00±8.25%, Figure 3 D). U251 cells treated with LH20 also showed similar inhibitory effects (24h: 63.03±14.52%, 64.63±21.40%, 65.52±29.31% VS 123.30±12.57%; 48h: 62.98±31.45%, 53.56±20.31%, 57.36±16.57% VS 113.2±29.24%), indicating that LH20 can significantly inhibit the migration of GBM cells.

[0044] Example 7: Study on the effect of LH20 on GBM cell apoptosis

[0045] Cell apoptosis test method: After treating cells with LH20, trypsin was used to collect cells and centrifuged at 3000g for 5 minutes at 4°C. After centrifugation, 100,000 cells were suspended with 500μL buffer and 5μL staining reagent. After incubation at 37°C in the dark for 5 minutes, the fluorescence intensity of FITC and PI was analyzed by flow cytometry.

[0046] To study the effect of LH20 on apoptosis of U87MG and U251 cells, U87MG and U251 cells were treated with 4μM, 8μM and 10μM LH20 for 24h and 48h, respectively, and the effect of LH20 on apoptosis of U87MG and U251 cells was detected by AnnexinⅤ-FITC / PI. Figure 4 A-4C results showed that compared with the control group (4.97±1.71%), the late apoptosis rates of U87MG treated with 8μM LH20 and 10μM LH20 at 24 hours were 10.26±2.19% and 12.13±2.36%, respectively; compared with the control group (3.12±1.63%), the late apoptosis and necrosis rates at 48 hours were 10.61±4.70% and 9.09±1.30%, respectively; compared with the control group (24h: 1.47±0.62%, 48h: 1.37±0.65%), the cell necrosis rates were (24h: 4.14±1.71% and 3.99±1.02% VS1.47±0.62%; 48h: 7.16±1.05% and 9.36±2.61% VS1.37±0.65%). Figure 4 D-4F results showed that LH20 also had a similar apoptotic effect in U251 cells, indicating that LH20 can promote the necrosis of tumor cells.

[0047] Example 8: Study on the effect of LH20 on mitochondrial membrane potential

[0048] Mitochondrial membrane potential (MMP) detection: U87MG and U251 cells were treated with 0, 4, 8 and 10 μM LH20 for 24 h or 48 h, respectively, and then 500 μL JC-1 working solution was added and incubated at 37°C for 20 min. After centrifugation at 3000 g for 5 min, the supernatant was discarded, and the obtained cells were then resuspended in 120 μL JC-1 staining buffer and analyzed by flow cytometry.

[0049] The JC-1 kit was used to detect the mitochondrial membrane potential state of GBM cells. Figure 5 The results confirmed that the red-to-green fluorescence ratio of U87MG and U251 cells treated with LH20 for 24 h and 48 h was lower, indicating that LH20 reduced the mitochondrial membrane potential and induced mitochondrial-dependent apoptosis.

[0050] Example 9: Testing of the effect of LH20 on GBM cell cycle arrest

[0051] Cell cycle analysis was performed on a 24-well plate using a cell cycle staining kit. After the cells were treated with 0, 4, 8, and 10 μM LH 20 for 24 h or 48 h, they were collected with trypsin and washed with PBS buffer solution, followed by the addition of 1 mL of DNA staining solution and 10 μL of membrane permeabilization agent for staining at 37°C for 30 min, and analyzed by flow cytometry and FlowJo software was used to simulate the cell cycle.

[0052] Cell cycle experiment results ( Figure 6AC) showed that when cells were treated with 4, 8 and 10 μM LH20 for 24h and 48h, respectively, it did not increase the proportion of G1 phase cells in U87 MG cells and U251 cells, but promoted the gradual release of G1 phase cells from the G1 / S checkpoint, resulting in an increase in the proportion of G2 / M phase cells, while the proportion of G1 phase cells decreased accordingly. This indicates that LH20 compounds can block the GBM cell cycle in the G2 / M phase and have the function of regulating the GBM cell cycle.

[0053] In summary, the novel pyrimidine diamine compound LH20 provided by the present invention can significantly inhibit the proliferation and migration of U87 MG cells, induce late cell apoptosis, promote tumor cell necrosis, and block the cell cycle in the G2 / M phase. LH20 can inhibit the activity of CDK4 and CDK6 by inhibiting the phosphorylation of Rb. In addition, LH20 may target CDK4 / 6 to inactivate Rb phosphorylation and affect the expression of mTOR, thereby inhibiting the phosphorylation of CDK4 / 6 downstream protein retinoblastoma (Rb). Therefore, the novel pyrimidine diamine compound LH20 has potential prospects in the treatment of human glioblastoma.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A 2-aminopyrimidine compound, characterized in that: The compound structural formula is as follows:

2. The method for preparing the 2-aminopyrimidine compound according to claim 1, characterized in that The method comprises the following steps: weighing 77 mg of 2-chloro-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine, 59 mg of 4-morpholinopyridine-2-amine, 14 mg of Pd2(dba)3, 12 mg of Brettphos catalyst and 195 mg of cesium carbonate, adding 2 mL of dioxane to dissolve, reacting at 90° C. for 2 h under argon protection, filtering with diatomaceous earth, extracting with ethyl acetate / water, collecting and concentrating the organic phase solution to obtain a crude product, and separating and purifying the crude product by silica gel column chromatography to obtain 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-N-(4-morpholinopyridine-2-yl)pyrimidine-2-amine.

3. Use of a 2-aminopyrimidine compound according to claim 1 in the preparation of a drug for treating human glioblastoma.

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