A method for preparing a peptide epoxy ketone compound

Abstract

This invention provides a method for preparing peptide epoxy ketone compounds, specifically including the following steps: Compound 35 undergoes a debenzylation reaction to obtain an oily substance, Compound 36; the oily substance is then separated by pulping and dried to obtain a solid compound 36; the solid compound 36 is condensed with Compound 10 ((R)-5-methyl-1-((S)-2-methylepoxyethylene-2-yl)-1-oxohex-4-en-2-yl)carbamate tert-butyl ester to obtain Compound 37. This invention overcomes the shortcomings of existing technologies and provides a simple and feasible conversion method, transforming the originally oily intermediate Compound 36 into a solid, achieving easy purification, storage, and weighing of the product, suitable for large-scale production and downstream reaction applications, and possessing the advantages of high yield and high purity of peptide epoxy ketone compound 36.

Description

Technical Field

[0001] This invention relates to a method for preparing peptide epoxy ketone compounds, and more particularly to a simple and controllable method for preparing peptide epoxy ketone compounds, belonging to the pharmaceutical field. Background Technology

[0002] Autoimmune diseases (AIDs) are a group of chronic diseases caused by the immune system losing its tolerance to self-antigens, further inducing tissue damage and inflammatory responses, and ultimately leading to target organ damage. Common autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus. These diseases affect approximately 10% of the global population. Their exact causes and pathogenesis remain unclear, and there is currently no cure. They significantly impact patients' work capacity and quality of life, even threatening their lives, and consuming substantial healthcare resources, thus becoming a significant public health problem affecting human health.

[0003] Currently, immunomodulatory drugs used to treat autoimmune diseases often cause side effects such as infections and malignant transformations due to their broad-spectrum and non-disease-specific nature. Therefore, patients urgently need targeted therapies to alleviate these issues. Immunoplasminosomal cells (IPCs) are constitutive proteasomes generated under the induction of cytokines such as TNF-α and interferon-gamma, participating in cellular immunity and subsequently leading to diseases such as autoimmune disorders. IPC inhibitors can block the expression of the IPC subunit LMP2 / 7 by inhibiting the production of related cytokine-inducing factors and the activity of autoreactive T cells, thereby achieving therapeutic goals. Compound 37's maleate salt exerts its therapeutic mechanism by blocking the expression of IPC subunits. As a selective IPC inhibitor, it will provide a safer and more effective treatment option for patients with autoimmune diseases.

[0004] Compound 36 is a key intermediate for the synthesis of maleate of compound 37. It plays a crucial role in the overall synthesis process and is an essential and irreplaceable intermediate for the synthesis of the target product. It cannot be bypassed by other routes and has a strong process dependence.

[0005] The synthetic process of peptide epoxy ketone compound 36 disclosed in WO2026 / 041079 A1 uses compound 35 as a raw material, adds palladium on carbon, and prepares compound 36 by stirring under a hydrogen atmosphere. Compound 36 is condensed to obtain compound 37. However, this route yields compound 37 with low purity and difficult post-processing. Byproduct impurities are difficult to remove and require silica gel column purification, which is costly and not conducive to industrial production. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide a simple and feasible conversion method to transform the originally oily intermediate of compound 36 into a solid, achieving easy purification, storage, and weighing of the product, suitable for large-scale production and downstream reaction applications, and yielding a high yield of peptide-epoxyketone compound 36. To achieve the above objectives, this invention provides a method for preparing peptide-epoxyketone compounds, specifically including the following steps:

[0007] ;

[0008] (a) Compound 35 undergoes debenzylation to give compound 36 as an oily substance;

[0009] (b) The oily substance of compound 36 was separated by pulping and drying to obtain solid compound 36;

[0010] (c) Compound 37 was obtained by condensation of solid compound 36 with compound 10 ((R)-5-methyl-1-((S)-2-methylepoxyethylene-2-yl)-1-oxohex-4-en-2-yl)carbamate tert-butyl ester;

[0011] In step (b), the solvent used for pulping is acetonitrile, or a mixture of acetonitrile and C. 1-4 Alkyl alcohols or C 1-4 A binary mixed solvent composed of any one of the fatty ketones.

[0012] Preferably, the C 1-4 The alkyl alcohol is selected from ethanol, propanol, isopropanol, or tert-butanol, wherein C 1-4 The fatty ketones are selected from acetone or isobutyl ketone.

[0013] More preferably, in step (b), the solvent used for pulping is selected from acetonitrile and acetone, and the weight ratio of acetonitrile to acetone is 200~1000:1.

[0014] More preferably, in step (b), the solvent used for pulping is selected from acetonitrile and acetone, and the weight ratio of acetonitrile to acetone is 200~400:1.

[0015] More preferably, in step (b), the weight ratio of the solvent acetonitrile used in pulping to the oily substance of compound 36 is 1 to 100:1, or even more preferably, the weight ratio of the solvent acetonitrile used in pulping to the oily substance of compound 36 is 5 to 30:1.

[0016] Preferably, step (b) pulping is carried out at a temperature of -10°C to 30°C, more preferably, the pulping temperature is 0°C to 25°C, and even more preferably, the pulping temperature is 0°C to 10°C, resulting in a higher purity of the solid compound 36.

[0017] Preferably, in step (c), the condensation is carried out in the presence of a condensing agent, an activator, and an acid-binding agent, wherein the condensing agent is a carbodiimide or an onium salt.

[0018] Further preferably, in step (c), the condensing agent is selected from EDCI, CDI, or BOP, the activator is selected from HOBT or Oxyma, and the acid-binding agent is selected from DIPEA, TEA, or sodium carbonate. Even more preferably, the condensing agent, activator, and acid-binding agent are selected from combinations of EDCI, HOBT, and DIPEA, respectively, and the condensation reaction is carried out in the presence of the combination of EDCI, HOBT, and DIPEA.

[0019] Preferably, in step (c), the molar ratio of condensing agent, activator, acid-binding agent and compound 36 is 1~2:1~2:1~2:1. More preferably, the molar ratio of EDCI, HOBT, DIPEA and compound 36 is 1~2:1~2:1~2:1. Even more preferably, the molar ratio of EDCI, HOBT, DIPEA and compound 36 is 1.2:1.2:2.0:1.

[0020] Preferably, in step (c), the solvent used is dichloromethane, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, etc. More preferably, dichloromethane is used as the reaction solvent, and the weight ratio of compound 36 to dichloromethane is 1:10~50. Even more preferably, the weight ratio of compound 36 to dichloromethane is 1:13.3.

[0021] In step (c), when dichloromethane is used as the reaction solvent, the reaction substrate has high reactivity, the yield of product compound 37 is greater than 80%, and the purity is above 85%. When acetonitrile, tetrahydrofuran, and N,N-dimethylformamide are used as solvents, the yield and purity are not as good as those of dichloromethane. Therefore, dichloromethane is preferred as the reaction solvent in step (c).

[0022] Preferably, in step (a), Pd / C is used as a catalyst with ammonium formate, formic acid, sodium formate, cyclohexene, and hydrazine hydrate. More preferably, Pd / C is used as a catalyst with ammonium formate, the weight ratio of Pd / C to compound 35 is 0.01~0.2:1, and the molar ratio of ammonium formate to compound 35 is 3~10:1. Even more preferably, the weight ratio of Pd / C to compound 35 is 0.03:1, and the molar ratio of ammonium formate to compound 35 is 5:1.

[0023] Beneficial effects: Overcoming the shortcomings of existing technologies, a simple and feasible conversion method is provided. The inventors unexpectedly discovered that by using a specific solvent system for pulping and recrystallization, compound 36 can be converted from an oily state to a solid state, solving the problems of low yield and difficult post-processing of compound 37. It also overcomes the problems of the important intermediate of compound 36 prepared by the current synthesis process being an oily substance that cannot be routinely recrystallized, having a high impurity content in the product, and having poor fluidity, easy moisture absorption and decomposition of the oily substance, making precise quantitative feeding difficult. The yield of compound 36 reaches 70%-80%, and the HPLC purity reaches over 95%. Attached Figure Description

[0024] Figure 1 The HPLC purity chromatogram of compound 36 prepared in Example 1 is shown.

[0025] Figure 2 The MS spectrum of compound 36 prepared in Example 1;

[0026] Figure 3 The HNMR spectrum of compound 36 prepared in Example 1;

[0027] Figure 4 The CNMR spectrum of compound 36 prepared in Example 1;

[0028] Figure 5 The HPLC purity chromatogram of compound 36 prepared in Example 2 is shown.

[0029] Figure 6 The HPLC purity chromatogram of compound 36 prepared in Example 3 is shown.

[0030] Figure 7 The HPLC purity chromatogram of compound 37 prepared in Example 4 is shown.

[0031] Figure 8 The HPLC purity chromatogram of compound 36 prepared in Comparative Example 1 is shown.

[0032] Figure 9 The HPLC purity chromatogram of compound 36 prepared in Comparative Example 2 is shown.

[0033] Figure 10 The HPLC purity chromatogram of compound 36 (before column chromatography) prepared in Comparative Example 14 is shown.

[0034] Figure 11 The HPLC purity chromatogram of compound 36 (after column chromatography) prepared in Comparative Example 14 is shown.

[0035] Figure 12The HPLC purity chromatogram of compound 36 prepared in Comparative Example 15 is shown. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the embodiments.

[0037] Starting material compound 35: Compound 35 was prepared according to the preparation method disclosed in patent WO2026 / 041079 A1 and used in the following examples; all reagents used in the examples are commercially available.

[0038] The HPLC analysis method for compound 36 is as follows:

[0039] The chromatographic column was Thermo BDS-3, Hypersil™ C18, 4.6 × 250 mm, 5 μm, SN: 20466887;

[0040] Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: 0.1% TFA acetonitrile solution;

[0041] High-performance liquid chromatograph: Vanquish Core / Ultimate 3000;

[0042] Flow rate: 1 ml / min, wavelength: 210 nm, column temperature: 30 ℃, injection volume: 10 μL, injection plate temperature: 5 ℃, detector: UV;

[0043] Gradient procedure:

[0044]

[0045] The HPLC analysis method for compound 37 is as follows:

[0046] Column: Inertsil™ ODS-3, 4.6×150mm, 3μm, SN: 24D0488944; or a column with equivalent energy efficiency;

[0047] Mobile phase A: 35 mmol / L potassium dihydrogen phosphate solution (phosphate pH 3.0);

[0048] Mobile phase B: Acetonitrile: Methanol = 90:10;

[0049] High-performance liquid chromatograph: Vanquish Core / Ultimate 3000;

[0050] Flow rate: 0.8 ml / min, wavelength: 210 nm, column temperature: 50 °C, injection volume: 10 μL, injection plate temperature: 50 °C, detector: UV;

[0051] Gradient procedure:

[0052]

[0053] Example 1: Preparation of Compound 36

[0054] Step (a): Compound 35 (8.5 g, 20.04 mmol) was dissolved in ethanol (50 ml, 39.45 g), and 3% Pd / C (300 mg) and ammonium formate (6.3 g, 100 mmol) were added. The mixture was refluxed at 60 °C and stirred for 2 h. After the reaction was complete, the mixture was filtered, and the filtrate was evaporated to dryness to obtain compound 36 as an oil.

[0055] Step (b): The oily compound 36 (7 g) was placed in a reaction flask, and acetonitrile (80 ml, 62.88 g) and acetone (0.2 ml, 0.16 g) were added. After stirring at 0-10 °C for 4 h, the mixture was filtered and dried to obtain a white solid compound 36 (4.9 g, yield 70%, HPLC: 97.78%).

[0056] 1 H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.0Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 8.5 Hz, 2H), 5.07 (d, J = 3.0Hz, 1H), 4.44 – 4.36 (m, 2H), 3.71 (s, 3H), 3.54 (t, J = 4.3 Hz, 5H), 2.89 (dd, J = 51.5, 15.5 Hz, 2H), 2.42 – 2.30 (m, 4H), 1.17 (d, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6): δ 172.47, 172.12, 168.76, 158.70, 134.75, 127.99,113.45, 72.10, 66.58, 61.77, 58.68, 55.39, 53.62, 47.87, 19.22. MS (ESI) m / z410.06 [M+H] + .

[0057] Example 2 Preparation of Compound 36

[0058] Step (a) is the same as in Example 1, except for step (b): 10.50 g of the oily compound 36 was placed in a reaction flask, and acetonitrile (80 ml, 62.88 g) was added. After stirring at 0-10°C for 4 h, the mixture was filtered and dried to obtain a white solid compound 36 (7.95 g, yield 76%, HPLC: 88.74%).

[0059] Example 3 Preparation of Compound 36

[0060] Step (a) is the same as in Example 1, except for step (b): 7.67 g of the oily compound 36 was placed in a reaction flask, and acetonitrile (80 ml, 62.88 g) and ethanol (0.2 ml, 0.16 g) were added. After stirring at 0-10°C for 4 h, the mixture was filtered and dried to obtain a white solid compound 36 (5.5 g, yield 72%, HPLC: 93.94%).

[0061] Example 4 Preparation of Compound 37

[0062] Compound 10 (3.14 g, 11.10 mmol) was dissolved in DCM (30.14 mL, 40 g), and TFA (8.00 mL, 11.9 g) was added in an ice bath and reacted for 30 min. The solvent was removed by rotary evaporation to obtain the trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one.

[0063] The solid (2-morpholinylacetyl)-L-alanine compound 36 (5 g, 12.22 mmol) obtained in Example 1 was added to a reaction flask. A mixed solution of dichloromethane (50.00 ml, 66.5 g) and N,N-diisopropylethylamine (DIPEA) (3.20 g, 24.81 mmol) was added at 5 °C and stirred. After 10 min, 1-hydroxybenzotriazole (HOBT) (1.95 g, 14.44 mmol) was added and stirring continued. After 10 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2.80 g, 14.44 mmol) was added and activated for 30 min. Finally, the trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one was added, and the reaction was then moved to room temperature (25 °C) for reaction. After the reaction was complete, the solution was poured into a 6% sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 37, a pale yellow solid (5.34 g, yield 84.49%, HPLC: 88.95%). MS (ESI) m / z 575.28 [M+H] + .

[0064] Comparative Example 1: Preparation of Compound 36

[0065] Step (a) is the same as in Example 1. Step (b) is as follows: 500 mg of compound 36 oil was placed in a reaction flask, and dichloromethane (5 ml, 7.4 g) and ethyl acetate (1 ml, 0.9 g) were added. The mixture was stirred at 0-10°C for 4 h, filtered, and dried to obtain yellow viscous compound 36 (315 mg, yield 63%, purity 48.86%).

[0066] Preparation of Compound 36 (Comparative Example 2)

[0067] Step (a) is the same as in Example 1. Step (b) is as follows: 2 g of the oily compound 36 is placed in a reaction flask, and dichloromethane (5 ml, 6.7 g) and tetrahydrofuran (1 ml, 0.9 g) are added. Ethyl acetate is added in portions (9 ml, 8.1 g in total). After stirring at 0-10°C for 4 h, the mixture is filtered and dried to obtain a yellow foamy solid compound 36 (0.66 g, yield 33%, purity 79.83%).

[0068] Preparation of Compound 36 (Comparative Example 3-13)

[0069] Solid compound 36 was obtained according to Comparative Example 1, wherein the slurry solvent was replaced with other solvents shown in Table 1, and the reaction results are shown in Table 3. The purity and impurity status of Examples 1-3 and Comparative Examples 1-2 are summarized in Table 4.

[0070]

[0071]

[0072] Preparation of Compound 37 (Comparative Example 14)

[0073] Compound 10 (10.10 g, 35.69 mmol) was dissolved in DCM (101.00 mL, 133.8 g), and TFA (12.22 mL, 18.2 g) was added in an ice bath and reacted for 30 min. The solvent was removed by rotary evaporation to obtain the trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one.

[0074] The oily compound (2-morpholinylacetyl)-L-alanine compound 36 (7.64 g, 18.68 mmol) prepared in step (a) of Example 1 was added to a reaction flask. A mixture of dichloromethane (115.00 ml, 152.4 g) and N,N-diisopropylethylamine (DIPEA) (9.12 g, 70.70 mmol) was added at 5°C and stirred. After 10 min, 1-hydroxybenzotriazole (HOBT) (7.00 g, 51.85 mmol) was added and stirring continued. After 10 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (10.10 g, 51.85 mmol) was added and activated for 30 min. Finally, trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one was added, and the mixture was then transferred to room temperature (25°C). The reaction was carried out at ℃. After the reaction was complete, the mixture was poured into a 6% sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, the solvent was evaporated, and the product was purified by silica gel column chromatography to give compound 37 as a pale yellow solid (4.33 g, yield 44.87%, HPLC: 91.15%, HPLC before silica gel column purification: 58.09%).

[0075] Preparation of Compound 37 (Comparative Example 15)

[0076] Compound 10 (62 mg, 0.22 mmol) was dissolved in DCM (1.00 mL, 1.3 g), and TFA (80 mg) was added in an ice bath and reacted for 30 min. The solvent was removed by rotary evaporation to obtain the trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one.

[0077] The solid (2-morpholinylacetyl)-L-alanine compound 36 (100 mg, 0.24 mmol) prepared in step (b) of Example 1 was added to a reaction flask. A mixed solution of dichloromethane (5.00 ml) and triethylamine (TEA) (49 mg, 0.39 mmol) was added at 5 °C and stirred. After 10 min, 1-hydroxybenzotriazole (HOBT) (56 mg, 0.28 mmol) was added and stirring continued. After 10 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (80 mg, 0.28 mmol) was added and activated for 30 min. Finally, the trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one was added and the reaction was then moved to room temperature (25 °C). After the reaction was complete, the solution was poured into a 6% sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 37, a pale yellow foamy solid (68 mg, yield 53.84%, HPLC: 78.23%).

[0078] Preparation of Compound 37 (Comparative Example 16)

[0079] Compound 35 (2.12 g, 4.25 mmol) was dissolved in methanol (50 mL), and 10% Pd / C (212 mg) was added. The reaction was carried out under a hydrogen atmosphere at 50 °C for 2 h. After the reaction was completed, the mixture was filtered and the filtrate was evaporated to dryness to obtain compound 36.

[0080] Compound 10 (62 mg, 0.22 mmol) was dissolved in DCM (1 mL, 1.3 g), and TFA (80 mg) was added in an ice bath and reacted for 30 min. The solvent was removed by rotary evaporation to obtain the trifluoroacetate of 2-amino-5-methyl-l-((S)-2-methylepoxyethylene-2-yl)hex-4-en-1-one, which was dissolved in DMF (3 mL, 2.8 g) and stirred in an ice bath for 10 min. Compound 36 (100 mg, 0.24 mmol) and HATU (118 mg, 0.32 mmol) were added to the reaction mixture and stirred for 10 min, followed by the slow addition of DIPEA (50 mg, 0.39 mmol). The mixture was stirred for 30 min and then allowed to react at room temperature. After the reaction was complete, the mixture was poured into water, extracted with ethyl acetate, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and purified by rotary evaporation and silica gel column chromatography to obtain compound 37, a pale yellow foamy solid (80.8 mg, yield 65.20%).

Claims

1. A method for preparing a peptide epoxy ketone compound, characterized in that, Specifically, the steps include the following: ； (a) Compound 35 undergoes debenzylation to give compound 36 as an oily substance; (b) The oily substance of compound 36 was separated by pulping and drying to obtain solid compound 36; (c) Compound 37 was obtained by condensation of solid compound 36 with compound 10 ((R)-5-methyl-1-((S)-2-methylepoxyethylene-2-yl)-1-oxohex-4-en-2-yl)carbamate tert-butyl ester; In step (b), the solvent used for pulping is acetonitrile, or a mixture of acetonitrile and C. 1-4 Alkyl alcohols or C 1-4 A binary mixed solvent composed of any one of the fatty ketones.

2. The method for preparing a peptide epoxy ketone compound according to claim 1, characterized in that, In step (b), the solvent used for pulping is selected from acetonitrile and acetone, with a weight ratio of acetonitrile to acetone of 200~1000:1, and the weight ratio of acetonitrile to the oily substance of compound 36 is 1~100:

1.

3. The method for preparing a peptide epoxy ketone compound according to claim 1, characterized in that, In step (c), the condensation is carried out in the presence of a condensing agent, an activator, and an acid-binding agent, wherein the condensing agent is a carbodiimide or an onium salt.

4. The method for preparing a peptide epoxy ketone compound according to claim 3, characterized in that, In step (c), the condensing agent is EDCI, CDI or BOP, the activator is HOBT or Oxyma, and the acid-binding agent is DIPEA, TEA or sodium carbonate.

5. The method for preparing a peptide epoxy ketone compound according to claim 4, characterized in that, In step (c), the condensing agent, activator, and acid-binding agent are selected from EDCI, HOBT, and DIPEA, respectively.

6. The method for preparing a peptide epoxy ketone compound according to claim 5, characterized in that, In step (c), the molar ratio of condensing agent, activator, acid-binding agent and compound 36 is 1~2:1~2:1~2:

1.

7. The method for preparing a peptide epoxy ketone compound according to claim 1, characterized in that, In step (c), the solvent used for the condensation is dichloromethane, acetonitrile, tetrahydrofuran, or N,N-dimethylformamide.

8. The method for preparing a peptide epoxy ketone compound according to claim 7, characterized in that, In step (c), the solvent used for the condensation is dichloromethane, and the weight ratio of compound 36 to dichloromethane is 1:10~50.

9. The method for preparing a peptide epoxy ketone compound according to claim 1, characterized in that, In step (a), the debenzylation is catalyzed by Pd / C and ammonium formate, with the weight ratio of Pd / C to compound 35 being 0.01~0.2:1 and the molar ratio of ammonium formate to compound 35 being 3~10:1.

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