Purification method of double-polyamide benzimidazole STING agonist intermediate

Abstract

The invention discloses a method for synthesizing and purifying a double-polyamide benzimidazole STING (interferon gene stimulant) agonist intermediate, which comprises the following steps: dropwise adding a solution B into a solution A, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine into a reaction system after dropwise adding, and reacting at room temperature; after the reaction is finished, carrying out reduced pressure concentration on the reaction liquid to remove N, N-dimethylformamide so as to obtain a residue, carrying out liquid separation extraction on the residue by adopting dichloromethane and pure water, and washing with water for multiple times to ensure that the N, N-dimethylformamide is removed; and continuously washing residues by using a sodium chloride solution, drying by using anhydrous sodium sulfate, concentrating to obtain a crude product, crystallizing the crude product by using acetonitrile and methyl tert-butyl ether, and carrying out suction filtration and drying to obtain the product. A common chemical method is adopted, no pressure is generated in amplification, the process is simple, operation is convenient, the method can be conveniently used for industrial production, and the product purity reaches up to 95%.

Description

TECHNICAL FIELD

[0001] The present application relates to a purification method of a bis-amidobenzimidazole STING (stimulator of interferon genes) agonist intermediate, belonging to the technical field of organic synthesis. BACKGROUND

[0002] The activation of stimulator of interferon genes (STING) is an important strategy in the field of immunotherapy. The first generation of STING agonists (such as cyclic dinucleotide analogs) failed to achieve success in clinical trials due to their inherent defects. Therefore, the development of small molecule STING agonists other than cyclic dinucleotides has become a research hotspot. Among them, bis-amidobenzimidazole compounds have attracted much attention due to their high efficiency. By optimizing the structure of this skeleton, the STING signaling pathway can be significantly activated.

[0003] Compound 4-[3-({3-[(2E)-4-(8-{bis[(4-methoxyphenyl)methyl]amino}-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl]imidozolidin-3-yl] imidazo [5, 4-g] quinolin-3-yl) but-2-enyl] -6-formamido-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl] imidozolidin-3-yl] -1H-benzo [d] imidazol-4-yl} oxy) propyl] piperazine-1-carboxylic acid-2-methylprop-2-yl ester is an intermediate for synthesizing the second generation of STING agonists. However, this compound is relatively new, and the existing purification method is a laboratory-scale preparation. Although the product can be obtained in small scale, there are significant disadvantages such as expensive equipment, complex operation, and high cost, which makes it difficult to meet the needs of industrialized production. SUMMARY

[0004] To solve the above problems, the purpose of the present application is to provide a purification method of a bis-amidobenzimidazole STING agonist intermediate. The purification method is simple, low-cost, easy to scale up, and suitable for industrial production.

[0005] To achieve the above purpose, the technical solution of the present application is:

[0006] A purification method of a bis-amidobenzimidazole STING agonist intermediate, comprising the following steps:

[0007] (1) Dissolve raw material A in N,N-dimethylformamide (DMF) to obtain solution A.

[0008] The raw material A is 4-[3-({3-[(2E)-4-(8-{bis[(4-methoxyphenyl)methyl]amino}-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl]hydrazono]-1H-imidazo[5,4-g]quinazolin-3-yl)but-2-enyl]-6-formamido-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl]hydrazono]-1H-benzo[d]imidazol-4-yl}oxy)propyl]piperazine-1-carboxylic acid-2-methylpropan-2-yl ester, and its structural formula is:

[0009] The mass ratio of the raw material A to N,N-dimethylformamide is 1:7-8.

[0010] (2) The raw material B is dissolved in N,N-dimethylformamide to obtain solution B;

[0011] The raw material B is 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate, and its structural formula is:

[0012] The mass ratio of the raw material B to N,N-dimethylformamide is 1:3-6.

[0013] (3) The solution B is added dropwise to the solution A at room temperature, and after the dropwise addition is completed, hydrochloride and triethylamine (Et3N) are added to the reaction system, and the reaction is carried out at room temperature.

[0014] The hydrochloride is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI).

[0015] The molar ratio of the raw material A, the raw material B, the hydrochloride and triethylamine is 1:1-3:2-4:6-10.

[0016] (4) After the reaction is completed, the reaction liquid is concentrated under reduced pressure to remove N,N-dimethylformamide to obtain a residue, and the residue is subjected to liquid-liquid extraction with dichloromethane and pure water, and is washed with water for multiple times to ensure the removal of N,N-dimethylformamide.

[0017] The mass ratio of the raw material A, dichloromethane and water is 1:25-27:19-21.

[0018] (5) The residue is washed with a sodium chloride solution, dried with anhydrous sodium sulfate and concentrated to obtain a crude product; the crude product is subjected to crystallization with acetonitrile and methyl tert-butyl ether, and after suction filtration and drying, the product is obtained.

[0019] The mass ratio of the raw material A, acetonitrile and methyl tert-butyl ether is 1:2-3:4-5.

[0020] The reaction route is as follows:

[0021] The present application has the following advantages:

[0022] 1. The present application first provides a scalable purification process for the key intermediate of the STING agonist. By using conventional extraction, washing, crystallization and other unit operations, the whole process is simple, stable, low in equipment requirements and suitable for large-scale industrial production.

[0023] 2. By using specific purification step combination, especially using acetonitrile / methyl tert-butyl ether mixed solvent system for crystallization, the present application can effectively remove by-products and impurities, and the final product has a purity of more than 95%. BRIEF DESCRIPTION OF DRAWINGS

[0024] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0025] Figure 1 The HPLC detection chromatogram of the product obtained in Example 1 is shown in the following figure.

[0026] Figure 2 The HPLC detection chromatogram of the product obtained in Example 2 is shown in the following figure.

[0027] Figure 3 The HPLC detection chromatogram of the product obtained in Example 3 is shown in the following figure.

[0028] Figure 4 The LCMS detection chromatogram of the product obtained in Example 1 is shown in the following figure.

[0029] Figure 5 The nuclear magnetic resonance spectrum of the product obtained in Example 1 is shown in the following figure. DETAILED DESCRIPTION

[0030] The starting material A in the following example is 4-[3-({3-[(2E)-4-(8-{bis[(4- methoxyphenyl)methyl]amino}-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl]hydrazono]- 1H-imidazo[5,4-g]quinazolin-3-yl)but-2-enyl]-6-formamido-2-[(E)-[(2-ethyl-5- methylpyrazol-3-yl)carbonyl]hydrazono]-1H-benzo[d]imidazol-4-yl}oxy)propyl]piperazin-1- carboxylate-2-methylpropan-2-yl ester; the starting material B is 1-ethyl-3-methyl-1H-pyrazole-5- carbonyl isothiocyanate (CAS No.: 1489546-98-6); the hydrochloride salt is 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS No.: 7084-11-9); the N,N- dimethylformamide (CAS No.: 68-12-2); the triethylamine (CAS No.: 121-44-8); the acetonitrile (CAS No.: 75-05-8); the methyl tert-butyl ether (CAS No.: 1634-04-4).

[0031] Example 1

[0032] (1) In a 10 L four-necked flask, the starting material A (773 g, 0.89 mol) was dissolved in N,N-dimethylformamide (6.18 L) under nitrogen protection to obtain solution A.

[0033] (2) The starting material B (348 g, 1.78 mol) was dissolved in N,N-dimethylformamide (1.55 L) to obtain solution B.

[0034] (3) Solution B was added dropwise to solution A at room temperature, and after the dropwise addition was completed, the hydrochloride salt (516 g, 2.70 mol) and triethylamine (546 g, 5.396 mol) were added to the reaction flask. After reaction at room temperature for 2 h, the sample was detected to be less than 6% of the intermediate, otherwise the reaction was continued.

[0035] (4) After the reaction was completed, the reaction liquid was concentrated under reduced pressure to remove N,N-dimethylformamide to obtain a residue; the residue was added to dichloromethane (15.46 L) and pure water (15.46 L) for liquid-liquid extraction, and washed with the same amount of water for multiple times until it was ensured that there was no residual N,N-dimethylformamide.

[0036] (5) Then the residue was further washed with a 10% sodium chloride solution (7.7 L), and after drying over anhydrous sodium sulfate, it was concentrated to obtain a crude product, which was added to acetonitrile (2.32 L) and methyl tert-butyl ether (4.64 L) for crystallization. After suction filtration and drying, the product (664 g, 0.56 mol) was obtained, and its purity was 94.75% by HPLC analysis (its HPLC detection chromatogram is shown in FIG. 1). Figure 1), which was verified by LCMS (its LCMS detection chromatogram is shown in Figure 4 ), and was also verified by NMR (its NMR chart is shown in Figure 5 )

[0037] The product yield was 62.5%.

[0038] Example 2

[0039] (1) In a 10L four-necked flask, under nitrogen protection, the raw material A (447g, 0.55mol) was dissolved in N,N-dimethylformamide (3.6L) to obtain solution A.

[0040] (2) The raw material B (162.24g, 0.83mol) was dissolved in N,N-dimethylformamide (0.9L) to obtain solution B.

[0041] (3) At room temperature, solution B was added dropwise to solution A, after the dropwise addition was completed, hydrochloride (210.65g, 1.1mol) and triethylamine (546g, 5.396mol) were added to the reaction flask, and after 2h of reaction at room temperature, the sample was detected to be less than 6% of the intermediate, otherwise continue to react.

[0042] (4) After the reaction was completed, the reaction liquid was concentrated under reduced pressure to remove N,N-dimethylformamide to obtain a residue; the residue was added to dichloromethane (9.5L) and pure water (9.5L) for liquid-liquid extraction, and washed with equal amount of water for multiple times until it was ensured that there was no residual N,N-dimethylformamide;

[0043] (5) Then the residue was washed with a 10% sodium chloride solution (4.8L) and dried with anhydrous sodium sulfate, and then concentrated to obtain a crude product, which was added to acetonitrile (1.4L) and methyl tert-butyl ether (2.86L) for crystallization, and after suction filtration and drying, the product (366.4g, 0.31mol) was obtained, which was analyzed by HPLC to have a purity of 96.75% (its HPLC detection chromatogram is shown in Figure 2 ), and the structure was correctly characterized by NMR.

[0044] The product yield was 56.3%.

[0045] Example 3

[0046] (1) In a 10L four-necked flask, under nitrogen protection, the raw material A (447g, 0.55mol) was dissolved in N,N-dimethylformamide (3.6L) to obtain solution A.

[0047] (2) The raw material B (162.24g, 0.83mol) was dissolved in N,N-dimethylformamide (0.9L) to obtain solution B.

[0048] (3) At room temperature, solution B was added dropwise to solution A, and after the addition was completed, hydrochloride (435.6 g, 2.27 mol) and triethylamine (393.9 g, 3.9 mol) were added to the reaction bottle. After reaction at room temperature for 2 h, the sample was detected to be 0% of intermediate <6%, otherwise the reaction was continued.

[0049] (4) After the reaction was completed, the reaction liquid was concentrated under reduced pressure to remove N,N-dimethylformamide to obtain a residue; the residue was added to dichloromethane (11.2 L) and pure water (11.2 L) for liquid-liquid extraction, and washed with the same amount of water for multiple times until it was ensured that there was no residual N,N-dimethylformamide;

[0050] (5) Then the residue was washed with a 10% sodium chloride solution (5.6 L), and after drying over anhydrous sodium sulfate, it was concentrated to obtain a crude product, which was added to acetonitrile (1.68 L) and methyl tert-butyl ether (3.36 L) for crystallization. After suction filtration and drying, the product (420.76 g, 0.36 mol) was obtained, and its purity was 96.41% by HPLC analysis (its HPLC detection chromatogram is shown in FIG. 1), and the structure was correct by nuclear magnetic resonance characterization. Figure 3

[0051] The product yield was 54.7%.

[0052] The above examples do not limit the present application in any way, and any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present application.​

Claims

1. A method for purifying a polyamide benzimidazole STING agonist intermediate, characterized in that, Includes the following steps: (1) Dissolve raw material A in N,N-dimethylformamide to obtain solution A; Wherein, the raw material A is 4-[3-({3-[(2E)-4-(8-{bis[(4-methoxyphenyl)methyl]amino}-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl]aza-ylidene]-1H-imidazo[5,4-g]quinazolin-3-yl)but-2-enyl]-6-carbamoyl-2-[(E)-[(2-ethyl-5-methylpyrazol-3-yl)carbonyl]aza-ylidene]-1H-benzo[d]imidazo-4-yl}oxy)propyl]piperazine-1-carboxylic acid-2-methylpropyl-2-yl ester; (2) Dissolve raw material B in N,N-dimethylformamide to obtain solution B; Wherein, raw material B is 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate; (3) At room temperature, solution B is added dropwise to solution A. After the addition is complete, hydrochloride and triethylamine are added to the reaction system to react. Wherein, the hydrochloride is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; (4) After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove N,N-dimethylformamide and the residue is obtained. The residue is extracted by separation with dichloromethane and pure water and washed with water multiple times to ensure the removal of N,N-dimethylformamide. (5) The residue was washed with sodium chloride solution, dried with anhydrous sodium sulfate and concentrated to obtain crude product; the crude product was crystallized with acetonitrile and methyl tert-butyl ether, filtered and dried to obtain the product.

2. The purification method for the polyamide benzimidazole STING agonist intermediate according to claim 1, characterized in that, In step (1), the mass ratio of raw material A to N,N-dimethylformamide is 1:7~8.

3. The purification method for the polyamide benzimidazole STING agonist intermediate according to claim 1, characterized in that, In step (2), the mass ratio of raw material B to N,N-dimethylformamide is 1:3~6.

4. The purification method for the polyamide benzimidazole STING agonist intermediate according to claim 1, characterized in that, In step (3), the molar ratio of raw material A, raw material B, hydrochloride and triethylamine is 1:1~3:2~4:6~10.

5. The purification method for the polyamide benzimidazole STING agonist intermediate according to claim 1, characterized in that, In step (4), the mass ratio of raw material A, dichloromethane, and water is 1:25~27:19~21.

6. The purification method for the polyamide benzimidazole STING agonist intermediate according to claim 1, characterized in that, In step (5), the mass ratio of raw material A, acetonitrile, and methyl tert-butyl ether is 1:2~3:4~5.

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