Improved AST-3424 preparation and purification process

Abstract

The invention provides an improved AST-3424 preparation and purification process, belongs to the field of organic chemical industry, and solves the problems of low yield, long time consumption, high cost and the like of AST-3424 preparation due to the fact that an intermediate and a final product are oily substances and are not easy to purify through conventional pulping, recrystallization and other methods in the AST-3424 preparation process. According to the method, Ag2O is replaced with the NaOH solution, meanwhile, three times of silica gel column chromatography purification processes are reduced into two times of silica gel column chromatography purification processes, the washing process and washing liquid composition of silica gel column chromatography are further optimized, and finally, the new preparation and purification process obtained through optimization is used for solving the problems in AST-3424 preparation.

Description

Technical Field

[0001] The invention relates to a preparation and purification process of AST-3424, and belongs to the field of organic chemical industry. Background Art

[0002] AST-3424 is an AKR1C3 enzyme-activated DNA alkylating agent prodrug that has entered Phase II clinical trials in both China and the United States (China's registered clinical trials are CTR20201915, CTR20201908, CTR20191399, and CTR20191371, and the United States' registered clinical trials are NCT04315324 and NCT03592264). It is specifically activated by the AKR1C3 enzyme that is highly expressed in various tumors to release DNA alkylating agents and thus exert anti-tumor effects.

[0003] This type of compound was first disclosed in patent applications WO2016145092 and WO2017087428, in which the following two synthetic routes of TH2870 (racemic form of AST-3424) were disclosed:

[0004]

[0005] The following preparation process is also disclosed in patent application WO2020172506:

[0006]

[0007] Among them, routes 1, 2, and 3 are basically similar, and their specific operations are recorded in the relevant preparation process of WO2017087428:

[0008] Preparation of compound 5. To POCl 3 Compound 4 (2 g, 10.8 mmol) was added to a solution of 4-bromoethylamine (2 ml, 21.6 mmol) in DCM (20 ml), and then TEA (3.6 ml, 27 mmol) in DCM (10 ml) was added at -40 °C under N2, and the mixture was stirred at -40 °C for 5 hours. 2-Bromoethylamine hydrobromide (17.6 g, 86.8 mmol) was then added, and TEA (12 ml, 86.8 mmol) in DCM (40 ml) was slowly added to the above solution at -40 °C and stirred for 0.5 h. K was added 2 CO 3 (10%, 10.4 g, 100 ml) solution, stirred at room temperature for 5 minutes, extracted with DCM (300 ml x 3), evaporated, purified by silica gel chromatography (EtOAc flushing) to give compound 5 (2.3 g, 43% yield) as a yellow oil.

[0009] Preparation of Compound 6. Compound 5 (4 g, 8.42 mmol) and Ag were stirred at 65°C. 2 A mixture of 4-(4-(2-chloro-4-oxo-1-yl)-4-nitropropene (5.85 g, 25.26 mmol) in THF (40 ml) was added for 3 hours, filtered and concentrated. The residue was purified by silica gel column chromatography (EtOAc flushing) to give Compound 6 (2.3 g, 87% yield) as a yellow oil.

[0010] Preparation of compound TH2870. To a solution of compound 7 (1.81 g, 10.95 mmol) in DMF (10 ml) was added NaH (60%, 438 mg, 1095 mmol) at 0°C, stirred for 10 minutes, then added DMF (10 ml) containing compound 6 (2.3 g, 7.3 mol), and stirred at 0°C for 30 minutes. 2 O quenched, extracted with EtOAc (100 ml × 5), 2 O (150 ml), washed with brine, evaporated, purified by silica gel column chromatography (DCM: MeOH = 40: 1) to give compound TH2870 (2.3 g, 69% yield) as a yellow oil.

[0011] In the preparation processes derived from these synthetic routes, since the intermediates and final products are all oily, they are not easy to be purified by conventional methods such as trituration techniques and recrystallization. Therefore, they can only be purified by silica gel column chromatography, which is inefficient, costly, and time-consuming.

[0012] Therefore, it is necessary to further develop new preparation and purification processes to efficiently and cost-effectively prepare and purify AST-3424 (OBI-3424, TH3424) compounds. Summary of the invention

[0013] The inventors have improved and explored the Ag used in the above-mentioned 1, 2, and 3 synthetic routes. 2 O was replaced by NaOH solution, and the three-time silica gel column chromatography purification process was reduced to a two-time silica gel column chromatography purification process, and the flushing process and flushing liquid composition of the silica gel column chromatography were further optimized. Finally, a new preparation and purification process was obtained through the above optimization.

[0014] The improved AST-3424 preparation and purification process includes the following operations:

[0015]

[0016] Operation 1: Wait for POCl 3 The DCM solution was cooled to -50 to -10 °C and Et 3N, DCM solution of raw material A0, keep the temperature at -40 to -20°C and stir the reaction until the raw material A0 is less than 0.5% and then proceed to the next operation;

[0017] Operation 2: Control the temperature at -50 to -10°C and add 2-bromoethylamine hydrobromide in batches, then add Et 3 After the addition of N in DCM solution, stir the reaction at -20 to -10°C for 2 to 6 hours before proceeding to the next step;

[0018] Operation three, adding water to the reaction solution and mixing, separating and collecting the organic phase, adding a desiccant to remove water, and then evaporating to remove the organic solvent to obtain a crude intermediate A1;

[0019] Operation 4, dissolve the crude intermediate A1 in THF, add NaOH aqueous solution after cooling to -10 to 10°C, stir the reaction after the addition is complete until A1 is less than 0.5% and then stop the reaction;

[0020] Operation 5, post-treating the reaction solution to obtain a crude product A2;

[0021] Operation six, the crude product A2 is added to a silica gel column for separation and purification, and the sample is washed and dissolved with DCM, firstly washed with EA:DCM in a volume ratio of 1:1 to remove impurities, then washed with a ternary mixed solvent of EA, DCM, and EtOH and the washing liquid is collected, and the organic solvent is evaporated and concentrated to obtain the purified product A2;

[0022] Operation 7: After the temperature of the DMF solution of the purified product A2 and 3-hydroxy-N,N-dimethylbenzamide has dropped to -10 to 10°C, add Cs 2 CO 3 After the addition is completed, stir the reaction until A2 is less than 0.5% and then stop the reaction;

[0023] Operation eight, post-treating the reaction solution to obtain a crude product of AST-3424;

[0024] Operation nine: the crude AST-3424 is added to a silica gel column for column chromatography separation and purification.

[0025] Operation 3: Adding water is to quench the reaction and remove the unreacted POCl 3 , 2-bromoethylamine hydrobromide, Et 3 N is decomposed and dissolved under the action of water, thereby generating inorganic salts soluble in water; the decomposition and dissolution process can be accelerated by stirring, and after stirring, the mixture is allowed to stand for stratification. Depending on the amount of water added, the density of the final aqueous phase is different. The DCM organic phase may be in the upper layer or in the lower layer, and most of the final product A1 will be dissolved in the DCM organic phase, but some inorganic salts or Et 3N, there will be a small amount of product in the aqueous phase, so the organic phase obtained after separation can be washed 2-4 times with clean water. Before heating and evaporating the organic phase, a neutral desiccant, such as anhydrous CaCl 2 、Anhydrous MgSO 4 、Anhydrous Na 2 SO 4 Remove moisture to prevent product A1 from reacting with water and decomposing during the subsequent heating evaporation process; further, heating evaporation concentration should be carried out by reduced pressure evaporation concentration, and the temperature should not exceed 35°C. Based on the cost and convenience of industrial large-scale operation, anhydrous Na 2 SO 4 Drying the DCM organic phase is suitable and meets the requirements, and the drying time should be controlled to be more than 1 hour, preferably 1.5-2.5 hours. After drying, filter, wash the filter cake with DCM, combine the filtrate and the washing liquid, and finally concentrate the organic solution under reduced pressure.

[0026] In operation five, NaCl or a similar salt can be added to the reaction solution and allowed to stand for stratification, the organic phase can be collected, EA or other solvents can be added to the aqueous phase for extraction, the organic phases can be combined and washed with a saturated NaCl or similar salt solution, a desiccant can be added to remove water, and then the organic solvent can be evaporated to obtain a crude intermediate A2.

[0027] The purpose of using NaCl or similar salts is to promote the separation of the organic phase and the aqueous phase after the stirring is completed. Since THF is used as a solvent in the reaction and an aqueous NaOH solution is added at the same time, the mixed system formed after the stirring is completed will be emulsified to a certain extent due to the solubility of THF and water. At this time, it is difficult to separate the phases. The addition of NaCl or similar salts can change the density difference, change the surface tension, etc. to promote separation. Similar salts include LiCl, NaBr, etc.

[0028] The desiccant water removal operation in operation five is similar to that in operation three.

[0029] Operation six is ​​column chromatography purification operation. The filler of column chromatography is silica gel particles, and neutral silica gel of 200-500 mesh can be used, preferably neutral silica gel of 300-400 mesh. After the column is filled, dry loading or wet loading is used.

[0030] For dry loading, a low boiling point solvent (such as DCM) is usually used to dissolve the crude A2, and 1-2 times the amount of silica gel as the crude A2 is added to mix the sample. The silica gel adsorbed with the crude A2 is evaporated under reduced pressure to obtain the silica gel, and then the silica gel adsorbed with the crude A2 is evenly spread over the silica gel chromatography column.

[0031] For wet loading, EA or DCM was directly used to completely dissolve the crude A2 to obtain a solution, and then the solution was evenly added dropwise along the inner wall of the column into the silica gel chromatography column soaked with EA.

[0032] Dry loading is recommended.

[0033] After loading, DCM was used to rinse and dissolve the sample to ensure that the crude A2 product was adsorbed on the silica gel.

[0034] First, the less polar impurities were removed by washing with EA:DCM in a volume ratio of 1:1.

[0035] Then, gradient washing is performed using EA:DCM:EtOH in a volume ratio of 100:100:2 to 100:100:8. Generally, the first washing liquid and the last washing liquid (which may contain some impurities) are discarded, and the washing liquid is collected and concentrated by reduced pressure evaporation to obtain product A2.

[0036] The solvent type, ratio and gradient of the above-mentioned washing liquid can remove the substances generated by the NaOH reaction of the impurities produced in the A0-A1 step to the greatest extent. Since the impurities are quite different from A2 after the NaOH reaction, it is easier to remove them at this stage using the above-mentioned silica gel column chromatography process, and the removal rate is high.

[0037] The post-processing process of operation eight is similar to that of operation none.

[0038] The silica gel column chromatography process of operation nine is similar to that of operation six.

[0039] Preferably, in operation 1, raw materials A0, POCl 3 and DCM solution of POCl 3 The concentration is not more than 0.06g / ml, Et 3 The concentration of triethylamine in the DCM solution of N is not more than 0.25 g / ml, the concentration of raw material A0 is not more than 0.25 g / ml, POCl 3 With Et 3 The molar ratio of N is 1:1.25-1.30, and the raw material A0 and POCl 3 The amount of substance ratio is 1:1.35-1.45.

[0040] Preferably, in operation 2, 2-bromoethylamine hydrobromide, Et 3 N Et in DCM 3 N concentration is not more than 0.53g / ml, 2-bromoethylamine hydrobromide and Et 3 The molar ratio of N is 1:1.50-1.80.

[0042] Preferably, the raw materials A0 and POCl in operation 1 and 2 are 3 The molar ratio of the three substances, including 2-bromoethylamine hydrobromide, is 1:1.25-1.30:2.75-2.95.

[0043] Preferably, in operation 4, the concentration of crude A1 in the solution obtained after crude A1 is dissolved in THF is not greater than 0.11 g / ml, the concentration of the aqueous NaOH solution is 0.95-1.05 mol / L, and the molar ratio of crude A1 to NaOH is 1:2.0-2.3.

[0044] Preferably, in operation six, the crude product A2 is added to the silica gel column, and the mass ratio of the crude product A2 to the silica gel is 1:10-15, the silica gel specification is 300-400 mesh neutral silica gel, the volume of DCM flushing is greater than 2 times the volume of the silica gel column, and the volume of the flushing liquid with a volume ratio of EA:DCM of 1:1 is greater than 4 times the volume of the silica gel column.

[0045] The volume ratio of EA:DCM:EtOH was 100:100:2 to 100:100:8, using gradient washes or fixed volume ratio solvent washes.

[0046] Preferably, in operation six, a gradient washing solution with a volume ratio of EA:DCM:EtOH of 100:100:2 to 100:100:8 is used for washing, and the volume of the washing solution is about 3 times (3±0.3) the volume of the silica gel chromatography column.

[0047] Preferably, in operation seven, the concentration of A2 purified product and 3-hydroxy-N,N-dimethylbenzamide in the DMF solution is not more than 0.12 g / ml, the concentration of 3-hydroxy-N,N-dimethylbenzamide is not more than 0.13 g / ml, and the purified product A2, 3-hydroxy-N,N-dimethylbenzamide and Cs 2 CO 3 The molar ratio of substances is 1:1.28-1.33:1.35-1.45.

[0048] Preferably, in operation nine, the crude AST-3424 is added to the silica gel column, and the mass ratio of the crude AST-3424 to the silica gel is required to be 1:14-20, and the silica gel specification is 300-400 mesh neutral silica gel;

[0049] The solvents used in column chromatography included those with or without the addition of Et 3 N's EA, EtOH mixed solvent, DCM, EA mixed solvent.

[0050] In certain embodiments, in operation three, the HPLC purity of the crude intermediate A1 is not less than 92%, in operation six, the HPLC purity of the purified product A2 is not less than 97%, and in operation nine, the HPLC purity of the purified product AST-3424 is not less than 99%.

[0051] Beneficial Effects

[0052] In the prior art synthesis process A1-A2 reaction, Ag is used 2 When O is used for the reaction, its usage is usually greater than 100% (more than 1 times the mass of reactant A2), but due to Ag 2 O has a high density and will gradually sink to the bottom during the stirring reaction and cannot be stirred to participate in the reaction. Therefore, Ag is often needed to be added in the middle of the reaction. 2 O, resulting in increased dosage.

[0053] Use Ag 2 Although the reaction can be carried out, it has the following shortcomings in the subsequent large-scale production process:

[0054] In terms of economic cost, on the one hand, due to Ag 2 O is expensive, and the AgCl or AgBr generated by the reaction is easy to attach to Ag. 2 O surface, causing Ag 2 O inactivation further increases Ag 2 The amount of O used makes the reaction material cost high. On the other hand, the Ag used 2 O dosage is large, the final product needs to control silver ions, but silver ions are not easy to remove, and the process of removing silver ions is complex, difficult and costly;

[0055] In terms of process operation, on the one hand, the AgCl or AgBr generated by the reaction is easy to adhere to Ag 2 O surface, causing Ag 2 O inactivation requires mid-course replacement and addition of new Ag 2 O, the operation is cumbersome; in particular, Ag 2 O powder is heavy and easily sinks to the bottom of the reactor, and cannot fully contact with the reaction materials, thus affecting the reaction rate. On the other hand, silver oxide ring closure generally requires heating to reflux the solvent, and the reaction time is relatively long.

[0056] The new solution uses NaOH solution instead of Ag2O. On the one hand, it reduces the cost by using low-value materials; at the same time, it uses the solution form, completely avoiding the problem of adding materials and cleaning during the process, and reducing the reaction time; more importantly, it avoids Ag 2 O, the final product does not need to be controlled and monitored for Ag residues.

[0057] The new scheme further changes the column chromatography separation and purification operation of the intermediate, separates the intermediate A2, and reduces the time and solvent consumption of the column chromatography purification of the final unit product, further reducing the operating cost of the new process. DETAILED DESCRIPTION

[0058] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0059] The experimental methods in the following examples are all conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used are all commercially available products unless otherwise specified.

[0060] The preparation and purification process is specifically described below through examples.

[0061]

[0062] The raw material A0 was synthesized by the method described in WO2017087428. The HPLC method was a conventional C18 column and a UV238nm detector.

[0063] Embodiment 1

[0064] Synthesis of A1

[0065] Add 300 ml of DCM (dichloromethane) to the reaction bottle, cool to about -30°C under nitrogen protection, and then add POCl 3 (17.41 g, 0.114 mol), then slowly dropwise add A0 (15.00 g, 0.081 mol) dissolved in (14.74 g, 0.146 mol) Et 3 N (triethylamine) and 60ml DCM solution, add it, raise the temperature to about -22℃, react for 15h, take samples for monitoring (A0<0.5%), cool it to about -30℃, add 2-bromoethylamine hydrobromide (56.48g, 0.276mol) in batches, and then slowly add Et 3 N (47.50 g, 0.470 mol) was dissolved in 90 ml of DCM solution, and the temperature was raised to about -20 °C, and the reaction was continued for 4 h, and the reaction was stopped. The reaction solution was washed with water three times, 400 ml each time, and the organic phase was collected and 30 g of anhydrous Na 2 SO 4 Dry for 2 h, filter, wash the filter residue with a small amount of DCM, combine the filtrate and the washing liquid, and concentrate to dryness under reduced pressure to obtain 50.15 g of a yellowish yellow compound with a purity of 92.69% and a yield of 130.21%, which is directly used in the next step.

[0066] Synthesis of A2

[0067] The crude product of A1 prepared in the previous step (45.04 g, equivalent to 0.094 mol according to the purity of 100%) and 450 ml of THF (tetrahydrofuran) were added to the reaction flask. Under nitrogen protection, the temperature was lowered to about 0°C, and then 1 mol / L NaOH solution (213 ml) was slowly added dropwise. After the addition was completed, the reaction was allowed to proceed for 4 h. The reaction was monitored by HPLC (high performance liquid chromatography) (A1 < 0.5%). The reaction was stopped, 100 g of NaCl was added, the mixture was allowed to stand for 5 min, and the liquid was separated. The organic phase was retained. The aqueous phase was extracted with 300 ml of EA (ethyl acetate), all organic phases were combined, and the organic phase was washed twice with saturated brine, 600 ml each time. The organic phase was collected, and 40 g of anhydrous Na 2 SO 4 Dry for 1.5 h, filter, wash the filter residue with a small amount of EA, combine the filtrate and the washing liquid, concentrate to dryness under reduced pressure, and add the residue to silica gel column chromatography for separation and purification.

[0068] Use dry loading method, dissolve 27.50g of crude A2 in 300ml of DCM, add 28g of 300-400 mesh neutral silica gel to mix the sample, evaporate under reduced pressure to obtain silica gel adsorbing crude A2, and then evenly spread the silica gel adsorbing crude A2 on the silica gel chromatography column.

[0069] The column of the silica gel chromatography column is loaded with 280g of 300-400 mesh neutral silica gel (about 800cm 3 , 10.18 times the mass of the crude product), the final column height after EA flushing and balancing the silica gel chromatography column was 20 cm, the inner diameter of the column was 7.4 cm, and the column volume was about 860 cm 3 .

[0070] First, pure DCM was used to press the column, and the sample was washed and dissolved and adsorbed on the silica gel. The amount used was about 2000 ml (about 2.325 times the volume of the chromatography column).

[0071] Then, the impurities were removed by washing with EA:DCM in a volume ratio of 1:1, and the amount used was about 3500 ml (about 4.069 times the volume of the chromatography column).

[0072] Finally, the column was gradiently washed with EA:DCM:EtOH (ethanol) in a volume ratio of 100:100:2 to 100:100:8, and the washing liquid was collected. The amount used was about 2500 ml (about 2.906 times the volume of the chromatography column).

[0073] During the flushing process, the flow rate of the flushing liquid should be about 2 ml per second. The above flushing process takes about 2 hours and consumes a total of 8 L of solvent.

[0074] The initial and final rinsing liquids which may contain some impurities were discarded, and the rinsing liquids were collected and combined (the rinsing liquids with HPLC purity higher than 97% were collected and combined, and those with purity lower than this were collected separately and then purified by repeating the above-mentioned silica gel column chromatography process), and the organic solvent was removed by evaporation and concentrated to obtain 15.73 g of A2 purified product in the form of a yellow buttery compound with a purity of 97.95% and a two-step reaction yield of 68.57%.

[0075] Synthesis of AST-3424

[0076] Add A2 (7.03 g, 0.022 mol), 3-hydroxy-N,N-dimethylbenzamide (7.79 g, 0.029 mol) and 60 ml DMF to the reaction flask. Under nitrogen protection, cool to about 0 ° C, then add (10.18 g, 0.031 mol) in batches, react for 16 hours, monitor by HPLC (A2 < 0.5%), and stop the reaction. Add 180 ml ice water, 54 g NaCl and 180 ml EA, stir for 2-5 minutes, separate the liquids, collect the EA phase, extract the aqueous phase with 180 ml EA again, combine all EA phases, add 30 g anhydrous Na 2 SO 4 Dry for 1.5 h, filter, and concentrate to dryness under reduced pressure. The crude product is separated and purified by silica gel column chromatography.

[0077] Use dry loading method, dissolve 10.50g crude AST-3424 in 200ml EA, add 10g 300-400 mesh neutral silica gel to mix the sample, evaporate under reduced pressure to obtain silica gel adsorbing crude AST-3424, and then evenly spread the silica gel adsorbing crude AST-3424 on the silica gel chromatography column.

[0078] The column of the silica gel chromatography column is loaded with 150g of 300-400 mesh neutral silica gel (about 430cm 3 , 14.28 times the mass of the crude product), the silica gel chromatography column was balanced by washing with EA, and the final column height was 11 cm, the inner diameter of the column was 7.4 cm, and the column volume was about 473 cm 3 .

[0079] First, 0.5% by mass of Et 3 The chromatography column was flushed with N EA to dissolve the sample and adsorb it on the silica gel. The amount used was about 1000 ml (about 2.114 times the volume of the chromatography column).

[0080] Then, the impurities were removed by gradient washing with EA:EtOH in a volume ratio of 100:1 to 100:2, with a usage of about 5000 ml (about 10.570 times the volume of the chromatography column).

[0081] Finally, the column was rinsed with EA:EtOH in a volume ratio of 100:6 and the rinse solution was collected. The amount used was about 5500 ml (about 11.627 times the volume of the chromatography column).

[0082] During the flushing process, the flow rate of the flushing liquid was ensured to be 4 ml per second. The above flushing process took 0.8 hours.

[0083] The initial and final rinsing liquids which may contain some impurities were discarded, and the rinsing liquids were collected and combined (the rinsing liquids with HPLC purity higher than 98% were collected and combined, and those with purity lower than this were collected separately and then purified by repeating the above-mentioned silica gel column chromatography process), and the organic solvent was removed by evaporation and concentrated to obtain 7.92 g of AST-3424 product as a yellow oily compound with a purity of 99.74% and a yield of 77.15%.

[0084] Calculation of solvent consumption and time spent:

[0085] During the final purification of 7.92 g of AST-3424 product, the column chromatography took 0.8 h and consumed a total of 11.5 L of solvent;

[0086] A2 corresponding to 7.92g AST-3424 used 7.03g of the total preparation amount of 15.73g, i.e. 44.69%. The chromatography column time spent on this part of A2 product is 2×44.69%=0.89 hours, and the solvent consumption is 8L×44.69%=3.57L.

[0087] The new process was used to prepare 7.92 g of AST-3424 product with a purity of 99.74%. The column chromatography time was 1.69 hours and the solvent consumption was equivalent to 15.07 L. After conversion, the average column chromatography time per gram was 0.21 hours and the solvent consumption was 1.90 L.

[0088] Comparative Example 1

[0089] The following is a comparative example, that is, in the A1-A2 reaction, a NaOH solution is used for the reaction, and the A1 crude product and the AST-3424 crude product are separated by silica gel column chromatography.

[0090] Synthesis of A1

[0091] A0 (160 g, 0.86 mol) and 2000 ml DCM were added to the reaction bottle. The temperature was lowered to about -30 °C under nitrogen protection, and POCl was added. 3 (185.2 g, 1.21 mol), and then slowly add Et 3N (279.40 g, 2.77 mol) was dissolved in 400 ml DCM solution, and the temperature was raised to about -20 ° C, reacted for 15 h, and sampled for HPLC monitoring (A0 < 1%). Then the temperature was lowered to about -30 ° C, 2-bromoethylamine hydrobromide (571.40 g, 2.80 mol) was added in batches, and Et 3 N (376.28 g, 3.73 mol) was dissolved in 200 ml of DCM solution, and the temperature was raised to about -15 °C. The reaction was continued for 4 h, and the reaction was stopped. The reaction solution was washed with water three times, 1200 ml each time, and the organic phase was collected. 120 g of anhydrous Na 2 SO 4 Dry for 2 h, filter, wash the filter residue with a small amount of DCM, combine the filtrate and the washing liquid, and concentrate under reduced pressure to obtain 521.72 g of crude product A1.

[0092] Use dry loading method, dissolve 521.72g of crude A1 in 3000ml of DCM, add 350g of 300-400 mesh neutral silica gel to mix the sample, evaporate under reduced pressure to obtain silica gel adsorbing crude A1, and then evenly spread the silica gel adsorbing crude A1 on the silica gel chromatography column.

[0093] Use 5kg 300-400 mesh neutral silica gel (about 14333cm 3 , 8.75 times the mass of the crude product) was loaded into the column. The silica gel chromatography column was flushed with DCM to a final loading height of 30 cm. The inner diameter of the column was 25 cm, and the column volume was approximately 14718 cm 3 .

[0094] Gradient flushing was performed. The flow rate was set at 2.8 ml / s, and the mixture was flushed with a mixed solvent of DCM:EA=4:1 (amount 15 L), a mixed solvent of DCM:EA=2:1 (amount 10.5 L), a mixed solvent of DCM:EA=3:2 (amount 16.8 L), a mixed solvent of DCM:EA=1:1 (amount 50 L), and a mixed solvent of DCM:EA=2:1 containing 2% EtOH (50 L). The flushing liquid was collected and concentrated by evaporation under reduced pressure to obtain 220 g of an oily substance with a purity of 97% and a yield of 53.55%.

[0095] The amount of solvent used was large, a total of 142.3 L, and the total flushing time was 14 h.

[0096] Synthesis of Compound A2

[0097] A1 (322.70 g, 0.68 mol, A1 intermediate prepared in other batches) and 3000 ml THF were added to the reaction bottle. Under nitrogen protection, the temperature was lowered to about 0°C, and then 1 mol / L NaOH solution (1419 ml) was slowly added dropwise. After the addition was completed, the reaction was continued for 4 h. HPLC monitoring (A1 < 0.5%) was performed to stop the reaction. 100 g NaCl was added, and the mixture was allowed to stand for 5 min. The liquid was separated, and the organic phase was retained. The aqueous phase was extracted with 1500 ml EA, and all organic phases were combined. The organic phase in the previous step was back-extracted twice with 3000 ml saturated brine (a total of 6000 ml saturated brine was used), and 200 g anhydrous Na 2 SO 4 Dry for 2 h; filter again, wash the filter residue with a small amount of EA, combine the filtrate and the washing liquid, and concentrate to dryness under reduced pressure to obtain 202.32 g with a purity of 84.51%.

[0098] Synthesis of AST-3424

[0099] A2 (202.32 g, 0.64 mol), 3-hydroxy-N,N-dimethylbenzamide (128.77 g, 0.78 mol) and 3600 ml of acetone were added to the reaction flask. Under nitrogen protection, the temperature was lowered to about 0°C, and Cs 2 CO 3 (461.44 g, 1.42 mol), react for 21 h, monitor by HPLC (A2 < 2%), stop the reaction. Filter, spin dry the solvent. Dissolve with 1200 ml EA, add 1200 ml saturated sodium bicarbonate twice and stir, wash the EA phase with 800 ml saturated brine three times (a total of 2400 ml saturated brine), add 100 g anhydrous Na 2 SO 4 Dry for 2 h, filter again, wash the filter residue with a small amount of EA, combine the filtrate and the washing liquid, concentrate to dryness under reduced pressure, and separate and purify the residue by silica gel column chromatography.

[0100] Use dry loading method, dissolve 286.08g of crude AST-3424 in 3000ml EA, add 290g 300-400 mesh neutral silica gel to mix the sample, evaporate under reduced pressure to obtain silica gel adsorbing crude AST-3424, and then evenly spread the silica gel adsorbing crude AST-3424 on the silica gel chromatography column.

[0101] Use 3kg 300-400 mesh neutral silica gel (about 8600cm 3 , 10.48 times the mass of the crude product) was loaded into the column. The silica gel chromatography column was flushed with EA and the final loading height was 18 cm. The inner diameter of the column was 25 cm 3 , the column volume is about 8830cm 3 .

[0102] The flow rate was set at 1.8 ml / s, and the volume ratio of EA:DCM:EtOH:Et 3 N = 100:100:1:0.5 mixed solvent (10 L), EA:DCM:EtOH:Et 3 N = 100:100:2:0.5 mixed solvent (58 L), EA:DCM:EtOH:Et 3 The product was rinsed with a mixed solvent of N=100:100:5:0.5 (34 L), and the rinse liquid was collected and concentrated by evaporation under reduced pressure to obtain a batch of 62.52 g, purity 99.71%, yield 21.16%; another batch of 97.78 g, purity 99.02%, yield 33.10%, and a total yield of samples with a purity greater than 99% was 54.26%.

[0103] The amount of solvent used was large, a total of 102 L, and the flushing time was a total of 17 hours.

[0104] Compared with Example 1, the yield and purity are slightly lower.

[0105] Calculation of solvent consumption and time spent:

[0106] During the final purification of 160.3 g of AST-3424 product, column chromatography took 17 hours and consumed a total of 102 L of solvent;

[0107] A1 corresponding to 160.3g AST-3424 used 322.70g of the total preparation amount of 220g, i.e. 146.68%. The chromatography column time for this part of A1 product is 14 hours × 146.68% = 20.53 hours, and the solvent consumption is 142.3L × 146.68% = 208.72L.

[0108] The old process of Comparative Example 1 was used to prepare 160.3 g of AST-3424 product with a purity of 99.728%. The column chromatography time was 37.53 hours and the solvent consumption was 310.72 L. After conversion, the average column chromatography time per gram was 0.23 hours and the solvent consumption was 1.94 L.

[0109] By comparison, it was found that the new process after purification in the second and third steps of the above route can make the product obtained by the silica gel column chromatography process of the final product AST-3424 have high purity and high yield compared with the product obtained by purification in the first and third steps of Comparative Example 1, and the chromatographic separation time is shortened and the amount of solvent is also reduced.

[0110] Comparative Example 2

[0111] The following is a comparative example, that is, in the reaction A1-A2, Ag is used 2 O for reaction, and separation of the crude product of A1 and the crude product of AST-3424 by silica gel column chromatography

[0112] Synthesis of Compound A1

[0113] A0 (9.25 g, 0.05 mol) and 240 ml of DCM were added to the reaction flask. The temperature was lowered to about -25 °C under nitrogen protection, and POCl was added. 3 (12.26 g, 0.0780 mol), and then slowly add Et 3 N (16.03, 0.159 mol), after dropping, raise the temperature to about -20 ° C, react for 7 hours, take samples for HPLC monitoring (A0 <1%). Then lower the temperature to about -25 ° C, add 2-bromoethylamine hydrobromide (38.90 g, 0.190 mol) in batches, and then slowly add Et 3 N (28.66 g, 0.284 mol), after dropping, react for 12 h, stop the reaction, wash the reaction solution with 300 ml of water; then wash the reaction solution with 300 ml of semi-saturated brine; then wash the reaction solution with 300 ml of saturated brine; then add 40 g of anhydrous Na 2 SO 4 Dry for 4 h, filter, wash the filter residue with a small amount of DCM, combine the filtrate and the washing liquid, and concentrate under reduced pressure to obtain 34.68 g of crude product A1 with a purity of 85%.

[0114] Use dry loading method, dissolve 34.68g of crude A1 in 500ml DCM, add 35g 300-400mesh neutral silica gel to mix the sample, evaporate under reduced pressure to obtain silica gel adsorbing crude A1, and then evenly spread the silica gel adsorbing crude A1 on the silica gel chromatography column.

[0115] The column was loaded with 400 g of 300-400 mesh neutral silica gel. The silica gel chromatography column was equilibrated with DCM.

[0116] Gradient washing was performed. The mixture was washed with a mixed solvent of DCM:EA=4:1 by volume (2 L), a mixed solvent of DCM:EA=2:1 (3 L), a mixed solvent of DCM:EA=1:1 (4 L), and a mixed solvent of DCM:EA=2:1 containing 2% EtOH (4 L), and the washing liquid was collected and concentrated by evaporation under reduced pressure to obtain 12.11 g of an oily substance with a purity of 95% and a yield of 51.00%.

[0117] 13 L of solvent was consumed and the reaction time was about 1.3 hours.

[0118] Synthesis of Compound A2

[0119] A1 (12.11 g, 0.0255 mol) and 250 ml THF were added to the reaction flask. Under nitrogen protection, the temperature was lowered to about 0°C and Ag was added. 2 O (29.54 g, 0.127 mol) and DIPEA (9.88 g, 0765 mol), then heat to 55°C for reaction, react for 15 h, monitor with HPLC (A1 <1%), stop the reaction, filter on diatomaceous earth, and evaporate the solvent to dryness to obtain 7.89 g with a purity of 92% and a yield of 98.24%.

[0120] Synthesis of compound AST-3424

[0121] A2 (4.23 g, 0.0134 mol), 3-hydroxy-N,N-dimethylbenzamide (2.88 g, 0.0175 mol) and 34 ml DMF were added to the reaction flask. Under nitrogen protection, the temperature was lowered to about 0°C, and Cs 2 CO 3 (6.13g, 0.0188mol), react for 15h, monitor by HPLC (A2<2%), stop the reaction. Add 110ml ice water, 35g NaCl and 110ml EA, stir for 2-5min, separate the liquids, collect the EA phase, extract the aqueous phase with 100ml EA again, combine all the EA phases. Then, at 0℃, wash the EA phase with 250ml 1mol / L NaOH solution for 5-10min twice; wash the EA phase with 200ml saturated brine twice; add 30g anhydrous Na 2 SO 4 The mixture was dried for 3 h, filtered, and concentrated to dryness under reduced pressure to obtain 6.30 g. The crude product was separated and purified by silica gel column chromatography.

[0122] Use dry loading method, dissolve 6.30g crude AST-3424 in 100ml EA, add 9g 300-40 mesh neutral silica gel to mix the sample, evaporate under reduced pressure to obtain silica gel adsorbing crude AST-3424, and then evenly spread the silica gel adsorbing crude AST-3424 on the silica gel chromatography column. Use EA to rinse and balance the silica gel chromatography column.

[0123] First, 0.5% by mass of Et 3 The column was flushed with N EA to dissolve the sample and adsorb it on the silica gel. The amount used was about 1800 ml.

[0124] Then, the impurities were removed by gradient washing with EA:EtOH in a volume ratio of 100:1 to 100:3, with a usage of about 4200 ml.

[0125] Finally, rinse with EA:EtOH in a volume ratio of 100:6 and collect the rinse solution, with a usage of about 3200 ml.

[0126] Discard the initial and final rinses that may contain some impurities, collect and combine the rinses (HPLC purity should be higher than 99% before the rinses are collected and combined, evaporate and remove the organic solvent, and concentrate to obtain AST-3424 product 3.32g yellow oily compound with a purity of 99.60% and a yield of 53.90%

[0127] 9.2 L of solvent was consumed and the reaction time was about 1.53 hours.

[0128] Calculation of solvent consumption and time spent:

[0129] During the final purification of 3.32 g AST-3424 product, the column chromatography took 1.53 hours and consumed a total of 9.2 L of solvent;

[0130] A2 corresponding to 3.32g AST-3424 used 4.23g of the total preparation amount of 7.89g, i.e. 53.61%. The chromatography column time for this part of A1 product is 1.3 hours × 53.61% = 0.70 hours, and the solvent consumption is 13L × 53.61% = 6.97L.

[0131] The old process of Comparative Example 2 was used to prepare 3.32 g of AST-3424 product with a purity of 99.60%. The column chromatography time was 2.23 hours, and the solvent consumption was equivalent to 16.17 L. After conversion, the average column chromatography time per gram was 0.67 hours, and the solvent consumption was 4.87 L.

[0132] By comparison, it is found that the new process after purification in the second and third steps of the above route can make the final product AST-3424 silica gel column chromatography process obtain a product that is purified in the first and third steps and uses Ag as compared to the comparative example 2. 2 The product obtained has high purity and high yield, and the chromatographic separation time is significantly shortened and the amount of solvent is also significantly reduced.

[0133] Other similar embodiments and comparative embodiments of the first embodiment

[0134] The following are the experimental results of different batches, including specific examples 2, 3 and comparative example 3 (A2 without column chromatography) and a brief description of the specific experimental process.

[0135] Brief table of experimental process for different batches.

[0136]

[0137]

[0138] By comparing Example 3, it can be seen that even if NaOH solution is used instead of Ag2O for the reaction, if the A2 intermediate is not separated and purified by column chromatography but directly put into the reaction, the purity of the finally prepared AST-3424 after column chromatography separation and purification is still low and cannot reach 99% purity.

Claims

1. The improved AST-3424 preparation and purification process includes the following operations: Operation 1: After the DCM solution of POCl3 is cooled to -50 to -10°C, Et3N and DCM solution of raw material A0 are added, and the temperature is maintained at -40 to -20°C and stirred to react until the raw material A0 is less than 0.5% before proceeding to the next operation; Operation 2: Control the temperature at -50 to -10°C and add 2-bromoethylamine hydrobromide in batches, then add Et3N DCM solution, and after the addition is complete, stir the reaction at -20 to -10°C for 2-6 hours before proceeding to the next operation; Operation three, adding water to the reaction solution and mixing, separating and collecting the organic phase, adding a desiccant to remove water, and then evaporating to remove the organic solvent to obtain a crude intermediate A1; Operation 4, dissolve the crude intermediate A1 in THF, add NaOH aqueous solution after cooling to -10 to 10°C, stir the reaction after the addition is complete until A1 is less than 0.5% and then stop the reaction; Operation 5, post-treating the reaction solution to obtain a crude product A2; Operation six, the crude product A2 is added to a silica gel column for separation and purification, and the sample is washed and dissolved with DCM, firstly washed with EA:DCM in a volume ratio of 1:1 to remove impurities, then washed with a ternary mixed solvent of EA, DCM, and EtOH and the washing liquid is collected, and the organic solvent is evaporated and concentrated to obtain the purified product A2; Operation 7, after the temperature of the DMF solution of the purified product A2 and 3-hydroxy-N,N-dimethylbenzamide has dropped to -10 to 10°C, Cs2CO3 is added, and after the addition is complete, the reaction is stirred until the A2 concentration is less than 0.5% and then the reaction is stopped; Operation eight, post-treating the reaction solution to obtain a crude product of AST-3424; Operation nine: the crude AST-3424 is added to a silica gel column for column chromatography separation and purification.

2. The preparation and purification process according to claim 1, in operation 1, the concentration of POCl3 in the raw material A0, POCl3 and DCM solution is not more than 0.06 g / ml, the concentration of Et3N in the DCM solution of Et3N is not more than 0.25 g / ml, the concentration of raw material A0 is not more than 0.25 g / ml, the molar ratio of POCl3 to Et3N is 1:1.25-1.30, and the molar ratio of raw material A0 to POCl3 is 1:1.35-1.

45.

3. The preparation and purification process according to claim 1, wherein in operation 2, 2-bromoethylamine hydrobromide is added in batches under the condition of controlling the temperature at -30±10°C, the Et3N concentration of the DCM solution of Et3N is not more than 0.53 g / ml, and the molar ratio of 2-bromoethylamine hydrobromide to Et3N is 1:1.50-1.

80.

4. According to the preparation and purification process of claim 1, in operations 1 and 2, the molar ratio of the raw material A0, POCl3 and 2-bromoethylamine hydrobromide is 1:1.25-1.30:2.75-2.

95.

5. The preparation and purification process according to claim 1, wherein in operation 4, the concentration of the crude A1 in the solution obtained after the crude A1 is dissolved in THF is not greater than 0.11 g / ml, the concentration of the NaOH aqueous solution is 0.95-1.05 mol / L, and the molar ratio of the crude A1 to NaOH is 1:2.2-2.

4.

6. The preparation and purification process according to claim 1, in operation 6, the crude product A2 is added to the silica gel column, the mass ratio of the crude product A2 to the silica gel is 1:10-15, the silica gel specification is 300-400 mesh neutral silica gel, the volume of DCM flushing is greater than 2 times the volume of the silica gel column, and the volume of the flushing liquid with a volume ratio of EA:DCM of 1:1 is greater than 4 times the volume of the silica gel column. The volume ratio of EA:DCM:EtOH was 100:100:2 to 100:100:8, using gradient washes or fixed volume ratio solvent washes.

7. The preparation and purification process according to claim 1, wherein in operation 6, 0.5% of the volume of DCM in Et3N is added to the EA:DCM:EtOH mixed solvent.

8. The preparation and purification process according to claim 1, in operation 7, the concentration of the purified product A2 in the DMF solution of 3-hydroxy-N,N-dimethylbenzamide is not more than 0.12 g / ml, the concentration of 3-hydroxy-N,N-dimethylbenzamide is not more than 0.13 g / ml, and the molar ratio of the purified product A2, 3-hydroxy-N,N-dimethylbenzamide and Cs2CO3 is 1:1.28-1.33:1.35-1.

45.

9. The preparation and purification process according to claim 1, in operation 9, the crude AST-3424 is added to the silica gel column, and the mass ratio of the crude AST-3424 to the silica gel is required to be 1:14-20, and the silica gel specification is 300-400 mesh neutral silica gel; The solvents used for column chromatography include a mixed solvent of EA and EtOH with or without the addition of Et3N, and a mixed solvent of DCM and EA.

10. The preparation and purification process according to claim 1, wherein: In operation three, the HPLC purity of the crude intermediate A1 is not less than 92%, in operation six, the HPLC purity of the purified product A2 is not less than 97%, and in operation nine, the HPLC purity of the purified product AST-3424 is not less than 99%.

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