An mIDH1 small molecule inhibitor, preparation method thereof and anti-glioma application thereof

Abstract

The application belongs to the field of pharmaceutical chemistry and medicine, and discloses a kind of mIDH1 Small molecule inhibitor and its preparation method and application in preparation of anti-glioma drug.The structure general formula of the mIDH1 Small molecule inhibitor is shown in the description, wherein R is selected from the description.The application simultaneously discloses the preparation method of the mIDH1 Small molecule inhibitor and its application in preparation of anti-glioma drug, and the compound has mIDH1 R132H The mutant enzyme has strong inhibitory effect, has significant proliferation inhibitory activity to mIDH1 mutant glioma cell (U87-R132H) and maintains good mutation selectivity, and has great application potential in preparation of anti-glioma drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a method for preparing a mIDH1 small molecule inhibitor and its application in treating glioma. Background Technology

[0002] Gliomas are the most common and most malignant primary tumors of the central nervous system (CNS). Current treatments are limited by blood-brain barrier (BBB) ​​drug delivery barriers, tumor drug resistance, and an immunosuppressive microenvironment, resulting in extremely poor patient prognosis. Mutations in isocitrate dehydrogenase 1 (mIDH1) are a key driving event in glioma development. Its metabolite, 2-hydroxyglutarate (2-HG), accumulates abnormally in mutant mIDH1 tumor cells, leading to glioma proliferation. Based on a deeper understanding of the mIDH1 mutation mechanism, targeted inhibition of mutant mIDH1 has become an important strategy for precision treatment of gliomas.

[0003] Several small molecule inhibitors targeting mutant mIDH1 have been marketed and have achieved significant clinical efficacy, becoming an important strategy for precision treatment of gliomas. However, current mIDH1 small molecule inhibitors face challenges such as tumor resistance, insufficient blood-brain barrier penetration, and off-target toxicity. Therefore, the development of novel mIDH1 small molecule inhibitors is crucial. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this invention provides a mIDH1 small molecule inhibitor, its preparation method, and its application in treating glioma.

[0005] The technical solution of this invention is as follows: A small molecule inhibitor of mIDH1, the general structural formula of which is shown in Formula 1: (Formula 1); Where R is selected from F1, F2, F3, or F4:

[0006] Furthermore, the mIDH1 small molecule inhibitor is a compound with the structural formulas shown in M10, M11, M12, and M13: .

[0007] Furthermore, the mIDH1 small molecule inhibitor is a compound with the structural formula shown in M11: .

[0008] A second objective of this invention is to provide a method for preparing the aforementioned mIDH1 small molecule inhibitor, comprising the following steps: 1. The amino Boc protection of (S)-4-(1-aminoethyl)phenol hydrobromide yields intermediate T1: ; 2. T1 is coupled with 3-(bromomethyl)-2-methyl-1,1'-biphenyl in the presence of potassium carbonate to give intermediate T2: ; 3. T2 undergoes deBoc removal under hydrochloric acid to yield intermediate T3: ; 4. 2,4-Dichloropyrimidine reacts with oxazolidin-2-ones of different substitutions under alkaline low-temperature conditions to give any one of intermediates F1-F4: ; 5. T3 is coupled with F1 and F4 under alkaline conditions to obtain M10-M11, and with F2 and F3 under palladium and phosphine ligand catalysis to obtain M12-M13.

[0009] Preferably, the synthetic route for compounds M10-M13 is as follows:

[0010] A third objective of this invention is to provide the application of the aforementioned mIDH1 small molecule inhibitor in the preparation of anti-glioma drugs.

[0011] Furthermore, the glioma is composed of wild-type human astrocytoma cell lines U87 or mIDH1. R132H It is caused by the mutant astrocytoma cell line U87-R132H.

[0012] Compared with the prior art, the present invention has the following significant advantages: This invention designs and synthesizes a small molecule inhibitor of mIDH1 that targets and inhibits the R132H mutation site of mIDH1, and exhibits a lower IC50 compared to the positive control drug in in vitro enzyme inhibition activity assays and antitumor activity assays. 50 It has the potential to improve the value of mutant cells and maintain the selectivity of mutant cells, thus showing promise for clinical translation. Attached Figure Description

[0013] Figure 1 For compound M10 in Example 8 1 H NMR spectrum.

[0014] Figure 2 For compound M11 in Example 9 1 H NMR spectrum.

[0015] Figure 3 For compound M12 in Example 10 1 H NMR spectrum.

[0016] Figure 4For compound M13 in Example 11 1 H NMR spectrum. Detailed Implementation

[0017] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0018] Example 1: Synthesis of intermediate T1 (S)-4-(1-aminoethyl)phenol hydrobromide (1 g, 4.58 mmol), (Boc)₂O (1.1 g, 5.04 mmol), and triethylamine (973.68 mg, 9.62 mmol) were dissolved in 30 mL of dichloromethane (DCM) at room temperature with stirring. The mixture was then analyzed by TLC. After the reaction was complete, the solution was concentrated under reduced pressure, dissolved in ethyl acetate (EA), washed with 1 M hydrochloric acid, water, and saturated brine, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. Separation was performed by silica gel column chromatography (MeOH / DCM = 1 / 20) to give a white solid T1 (984.6 mg, 4.58 mmol), with a yield of 90.56%. The NMR spectrum of T1 is as follows: 1 H NMR (400MHz, Chloroform-d) δ 7.11 (d, J = 8.0 Hz, 2H), 6.77–6.70 (m, 2H), 4.71 (s, 2H), 1.41 (d, J = 9.1 Hz, 12H). Example 2: Synthesis of intermediate T2 At room temperature, T1 (1 g, 3.83 mmol) was dissolved in 15 mL of N,N-dimethylformamide (DMF) with 3-(bromomethyl)-2-methyl-1,1-biphenyl (837.07 mg, 4.59 mmol) and K2CO3 (1.06 mg, 7.66 mmol). The mixture was stirred at 60 °C for 5 h, and the reaction was monitored by TLC (EA / PE = 1 / 3). After the reaction was complete, 150 mL of water was added, and the mixture was stirred at room temperature for 2 h. The mixture was then filtered, and the filter cake was separated by silica gel column chromatography (EA / PE = 1 / 10 - 1 / 5) to give a white solid T2 (1.21 g, 3.34 mmol), with a yield of 81.48%. The NMR spectrum data of T2 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 7.45 – 7.26 (m, 8H), 7.24 (d, J=5.2 Hz, 2H), 7.02 – 6.95 (m, 2H), 5.08 (s, 2H), 4.75 (s, 1H), 2.25 (s, 3H), 1.44 (d, J = 10.9 Hz, 12H). Example 3: Synthesis of intermediate T3 Compound T2 (500 mg, 1.2 mmol) was dissolved in 10 mL MeOH at room temperature, and 3 mL of 12M hydrochloric acid was added with stirring. After the reaction was complete, the solution was alkalized to pH 9-10 with potassium carbonate solution, concentrated under reduced pressure, dissolved in DCM, washed with water and saturated brine respectively, dried over anhydrous Na2SO4, and concentrated under reduced pressure. Separation was performed by silica gel column chromatography (MeOH / DCM = 1 / 10) to give a white solid T3 (246 mg, 774.9 μmol), with a yield of 64.7%. The NMR spectrum data of T3 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.63 – 8.58 (m, 2H), 7.45 – 7.33 (m, 6H), 7.30 – 7.26 (m, 2H), 7.22 –7.18 (m, 2H), 7.04 – 6.96 (m, 2H), 5.04 (s, 2H), 4.36 – 4.28 (m, 1H), 2.20 (s, 3H), 1.64 (d, J = 6.7 Hz, 3H). Example 4: Synthesis of intermediate F1 2,4-Dichloropyrimidine (1.49 g, 10 mmol) and (S)-4-isopropyl-2-oxazolidinone (1.29 g, 10 mmol) were dissolved in 20 mL of anhydrous DMF at -10 to 0 °C. A DMF suspension prepared with sodium hydride (NaH) (288 mg, 60% mineral oil dispersion, 12 mmol) was slowly added dropwise, and the mixture was stirred for 1 h. After the reaction was complete, 100 mL of ice water was added to quench the reaction, and the mixture was extracted with EA. The organic phases were combined and washed with saturated sodium bicarbonate and brine, respectively. The mixture was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Separation was performed by silica gel column chromatography (EA / PE = 1 / 10) to give a white solid F1 (1.623 g, 6.72 mmol), with a yield of 67.2%. The NMR spectra of F1 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.47 (d, J = 5.8 Hz, 1H), 8.18 (d, J= 5.8 Hz, 1H), 4.79 (dt, J = 8.1, 3.5 Hz, 1H), 4.45 – 4.31 (m, 2H), 2.60 (dtq, J = 10.2, 7.0,3.5 Hz, 1H), 0.99 (d, J = 7.0 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H). Example 5: Synthesis of intermediate F2 The method was the same as in Example 4, except that (S)-4-isopropyl-2-oxazolidinone was used instead of (S)-4-phenyl-2-oxazolidinone as the starting material, yielding a white solid F2 in 40.5% yield. The NMR spectrum data for F2 are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.41 (d, J = 5.8 Hz, 1H), 8.14 (d, J = 5.8 Hz, 1H), 7.41 – 7.30(m, 5H), 5.79 (dd, J = 8.7, 3.5 Hz, 1H), 4.83 (t, J = 8.8 Hz, 1H), 4.45 (dd, J =8.9, 3.6 Hz, 1H). Example 6: Synthesis of intermediate F3 The method is the same as in Example 4, except that (S)-4-isopropyl-2-oxazolidinone was used instead of (S)-4-benzyl-2-oxazolidinone as the starting material, yielding a white solid F3 in 54.7% yield. The NMR spectrum data for F3 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.51 (d, J = 5.8 Hz, 1H), 8.17 (d, J = 5.8 Hz, 1H), 7.39 – 7.23(m, 5H), 5.01 (ddt, J = 9.9, 6.7, 3.4 Hz, 1H), 4.37 – 4.31 (m, 2H), 3.46 (dd, J =13.4, 3.2 Hz, 1H), 2.85 (dd, J = 13.3, 9.4 Hz, 1H). Example 7: Synthesis of intermediate F4 The method was the same as in Example 4, except that (R)-4-isopropyl-2-oxazolidinone was used instead of (S)-4-isopropyl-2-oxazolidinone as the starting material, yielding a white solid F4 in 43.5% yield. The NMR spectrum data for F4 are as follows: 1 H NMR (400MHz, Chloroform- d ) δ 8.47 (d, J = 5.8 Hz, 1H), 8.18 (dd, J = 5.8, 0.9 Hz, 1H), 5.39 – 5.20 (m, 1H), 4.78 (dddd, J = 26.5, 9.1, 3.5, 1.3 Hz, 1H), 4.62 (dd, J =9.0, 3.4 Hz, 1H), 4.48 (td, J = 9.0, 1.3 Hz, 1H), 1.41 (dd, J = 23.2, 6.6 Hz, 3H). Example 8: Synthesis of compound M10 Compound T3 (270 mg, 850.58 μmol), compound F1 (205.57 mg, 850.58 μmol), and N,N-diisopropylethylamine (DIPEA) (550 mg, 4.26 mmol) were dissolved in 10 mL DMSO. The reaction was carried out under argon protection at 110 °C for 16 h, and detected by TLC. After the reaction, the mixture was cooled to room temperature, diluted with 100 mL of water, extracted with EA, and the organic phases were combined and washed with water and saturated brine, respectively. The mixture was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Separation was performed by silica gel column chromatography (EA / PE = 1 / 6 - 1 / 1) to give a white solid M10 (231 mg, 441.98 μmol), with a yield of 51.96%. The NMR spectrum data of M10 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.16 (d, J = 5.8 Hz, 1H), 7.47 – 7.39 (m, 4H), 7.39 – 7.34 (m,1H), 7.34 – 7.26 (m, 3H), 7.25 (d, J = 1.9 Hz, 3H), 7.00 – 6.92 (m, 2H), 5.06 (s, 2H), 4.99 (s, 1H), 4.60 (dd, J= 7.9, 3.9 Hz, 1H), 4.28 (t, J = 8.7 Hz, 1H), 4.21 (dd, J = 9.0, 3.2 Hz, 1H), 2.24 (s, 3H), 1.54 (d, J = 6.9 Hz, 3H), 0.81 –0.57 (m, 6H). Example 9: Synthesis of compound M11 The method was the same as in Example 8, except that compound F4 was used instead of F1 as the raw material, yielding a white solid M11 with a yield of 66.31%. The NMR spectrum data for M11 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.20 (d, J = 5.8 Hz, 1H), 7.48 (d, J = 5.8 Hz, 1H), 7.44 – 7.40 (m, 3H), 7.38 – 7.26 (m, 4H), 7.25 –7.21 (m, 3H), 7.02 – 6.93 (m, 2H), 5.07 (s, 2H), 4.90 (d, J = 12.3 Hz, 1H), 4.61 (d, J = 8.9 Hz, 1H), 4.54 (d, J = 8.9 Hz, 1H), 4.47 (dd, J = 8.9, 3.4 Hz, 1H), 4.36 (td, J = 9.0, 1.1 Hz, 1H), 2.24 (s, 3H), 1.53 (d, J = 6.9 Hz, 3H), 1.02 (s, 3H). Example 10: Synthesis of compound M12 Compound T3 (150 mg, 472.54 μmol), compound F2 (130.28 mg, 472.54 μmol), tris(dibenzylacetone)palladium (Pd2(dba)3) (8.65 mg, 9.45 μmol), 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) (BINAP) (29.42 mg, 47.25 μmol), and K2CO3 (130.61 mg, 945.08 mmol) were dissolved in toluene (10 mL). The reaction was carried out under argon protection and refluxed at 90 °C for 16 h, followed by TLC analysis. After the reaction was completed, the mixture was cooled to room temperature, and 30 mL of water was added. The mixture was stirred at room temperature for 10 min. The mixture was extracted with EA, and the organic phases were combined and washed with water, saturated sodium bicarbonate, and saturated brine, respectively. The mixture was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Silica gel column chromatography (EA / PE = 1 / 10 - 1 / 1) yielded a white solid M12 (163 mg, 292.81 μmol), with a yield of 61.97%. The NMR spectrum data for M12 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 5.8 Hz, 1H), 7.51– 7.26 (m, 13H), 7.23 – 7.16 (m, 2H), 7.01 (s, 1H), 6.87 (d, J = 8.2 Hz, 2H), 5.74 (dd, J = 8.7, 3.8 Hz, 1H), 5.06 (s, 2H), 4.76 (t, J = 8.7 Hz, 2H), 4.28 (dd, J = 8.7, 3.8 Hz, 1H), 2.25 (s, 3H), 1.49 (d, J = 6.8 Hz, 3H). Example 11: Synthesis of compound M13 The method was the same as in Example 10, except that compound F3 was used instead of F2 as the raw material, yielding a white solid M13. The yield was 59.76%. The NMR spectrum data of M13 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J = 5.8Hz, 1H), 7.50 (d, J= 5.8 Hz, 1H), 7.47 – 7.41 (m, 2H), 7.40 – 7.29 (m, 8H), 7.28 – 7.21 (m, 3H), 7.04 (s, 2H), 6.90 (d, J = 8.2 Hz, 2H), 5.15 (p, J = 6.9 Hz,1H), 5.02 – 4.90 (m, 3H), 4.28 (t, J = 8.4 Hz, 1H), 4.18 (dd, J = 9.0, 2.7 Hz,1H), 3.09 (s, 1H), 2.19 (s, 3H), 1.63 (d, J = 6.8 Hz, 3H). Example 12: Determination of mIDH1 enzyme inhibitory activity of compound M10-13 A two-step experimental protocol was used to determine the enzyme activities of wild-type and mutant mIDH1, respectively: mIDH1 R132H Enzyme inhibitory activity assay: The total volume of the primary reaction system was 10 μL. The reaction buffer composition was: 150 mM NaCl, 20 mM Tris (pH 7.5), 10 mM MgCl2, and 0.05% (w / v) bovine serum albumin. The reaction solution contained 5 nM recombinant mIDH1. R132H Protein (manufacturer: Active Motif, trade name: Recombinant IDH1 (R132H) protein), 1 M m α-ketoglutarate (α-KG), 20 μM NADPH, and analyte compounds M10-M13 at concentrations of 13.3 μM, 4.44 μM, 1.48 μM, 493 nM, 164 nM, 54.9 nM, and 18.3 nM. The reaction system was incubated at 37°C in the dark for 60 min, then 5 μL of secondary reaction initiation buffer (containing 4 U / mL myocardial flavin enzyme and 12 μM resazurine sodium in the above reaction buffer) was added, mixed well, and incubated at 37°C in the dark for another 10 min. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. A maximum signal control group (containing no mIDH1) was also included in the experiment. R132H The fluorescence values ​​of all experimental wells were normalized, and the results were compared with the minimum signal control group (containing all reaction components).

[0019] mIDH1 WT Enzyme inhibitory activity assay: The total volume of the primary reaction system was 10 μL, and the reaction buffer composition was the same as above. The reaction solution contained 5 nM recombinant mIDH1.WT Protein (manufacturer: Active Motif, trade name: Recombinant IDH1 protein), 1 mM DL isocitrate, 20 μM NADP, and analyte compounds M10-M13 at concentrations of 13.3 μM, 4.44 μM, 1.48 μM, 493 nM, 164 nM, 54.9 nM, and 18.3 nM. After incubation at 37°C in the dark for 60 min, 5 μL of secondary reaction starter solution (containing 4 U / mL myocardial flavin enzyme and 12 μM resazurine sodium in the above reaction buffer) was added, mixed well, and incubated at 37°C in the dark for another 10 min. The microplate reader detection conditions were the same as above. The experiment included a maximum signal control group (containing all reaction components) and a minimum signal control group (excluding mIDH1). WT (protein), and normalized the fluorescence values ​​of all experimental wells.

[0020] Using the approved mMIDH1 inhibitor evanixib (AG-120, manufacturer: McLean) as a positive control, a two-step experimental protocol was employed to determine the effect of compounds M10-M13 on mutant mIDH1. R132H Enzymes and wild-type mIDH1 WT The in vitro inhibitory activity of the enzyme was evaluated to assess the enzyme inhibitory selectivity of the compound, and the results are shown in Table 1.

[0021] Table 1 shows the in vitro inhibitory activity of compounds M10-M13 against mIDH1 enzyme.

[0022] The results show that compounds M10-M13 described in this patent have an effect on mIDH1. R132H All enzymes exhibit inhibitory activity and are effective against mIDH1. WT The enzyme showed no significant inhibition and exhibited good selectivity. Among them, M11 showed the best enzyme inhibitory activity, superior to the positive control drug AG-120, and can be used to prepare novel mIDH1 small molecule inhibitors.

[0023] Example 13: Cytotoxicity evaluation of compounds M10, M11, and M13 Using the approved mIDH1 inhibitors AG-120 and IDH-305 (manufacturer: Bidex Pharmaceuticals) as positive controls, the MTT assay was used to determine the inhibitory activity of compounds M10, M11, and M13 on the proliferation of U87 and U87-R132H cells (all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)). Both U87 and U87-R132H cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% triple antibodies. For U87-R132H cells, an additional 1 μg / mL puromycin was added to the medium. 3000 cells per well were seeded in 96-well plates and incubated for 24 h. Then, gradient concentrations (0.1–10 μM) of the compounds were added, with 6 replicates per group. After 48 h of incubation, MTT (5 mg / mL) was added and incubated for 4 h. Formazan crystals were dissolved in DMSO, and the OD value was measured at 490 nm using a microplate reader. The cell inhibition rate was calculated using the following formula:

[0024] The results are shown in Table 2.

[0025] Table 2 shows the in vitro antitumor activity results of compounds M10, M11, and M13.

[0026] The results showed that compounds M10, M11, and M13 described in this patent all exhibited significant mIDH1-R132H mutant selectivity, and their inhibitory activity against U87-R132H cells was significantly higher than that against U87 wild-type cells, consistent with the results of mIDH1 enzyme activity assays, thus verifying their target specificity and activity consistency. Among them, compound M11 had an IC50 value of [missing information]. 50 It is superior to the positive control drugs AG-120 and IDH-305 and can be used to prepare novel mIDH1 small molecule inhibitors.

[0027] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A small molecule inhibitor of mIDH1, characterized in that, The general structural formula of the mIDH1 small molecule inhibitor is shown in Formula 1: (Formula 1); Where R is selected from F1, F2, F3, or F4: 。 2. The mIDH1 small molecule inhibitor according to claim 1, characterized in that, The mIDH1 small molecule inhibitors are compounds with structural formulas as shown in M10, M11, M12, and M13: 。 3. The mIDH1 small molecule inhibitor according to claim 1, characterized in that, The mIDH1 small molecule inhibitor is a compound with the structural formula shown in M11: .

4. A method for preparing the mIDH1 small molecule inhibitor as described in claim 1, characterized in that, Includes the following steps: 1) The amino-Boc protection of (S)-4-(1-aminoethyl)phenol hydrobromide yields intermediate T1: ; 2) T1 is coupled with 3-(bromomethyl)-2-methyl-1,1'-biphenyl in the presence of potassium carbonate to give intermediate T2: ; 3) T2 undergoes deBoc removal under hydrochloric acid to yield intermediate T3: ; 4) 2,4-Dichloropyrimidine reacts with oxazolidin-2-ones with different substitutions under alkaline low-temperature conditions to give any one of intermediates F1-F4: ; 5) T3 is coupled with F1 and F4 under alkaline conditions to obtain M10-M11, and with F2 and F3 under palladium and phosphine ligand catalysis to obtain M12-M13.

5. The use of the mIDH1 small molecule inhibitor according to any one of claims 1 to 3 in the preparation of an anti-glioma drug.

6. The application according to claim 6, characterized in that, The glioma is composed of wild-type human astrocytoma cell lines U87 and / or mIDH1. R132H It is caused by the mutant astrocytoma cell line U87-R132H.

Images