CN117683066A

CN117683066A - Improved AST-3424 preparation process

Info

Publication number: CN117683066A
Application number: CN202211080291.0A
Authority: CN
Inventors: 蔡晓宏; 段建新; 陈涛; 曲桐; 阮美珍; 卢兆强
Original assignee: Shenzhen Ascentawits Pharmaceutical Technology Co ltd
Current assignee: Shenzhen Ascentawits Pharmaceutical Technology Co ltd
Priority date: 2022-09-05
Filing date: 2022-09-05
Publication date: 2024-03-12

Abstract

The invention relates to an improved AST-3424 preparation process, which is used for preparing AST-3424 with high optical purity, and compound A and compound B are reacted in cesium carbonate as a base and acetone as a solvent, and is characterized in that the ee% value of the prepared AST-3424 with high optical purity is greater than or equal to 96%, and the reaction time is 2-8 hours. Cesium carbonate is used as a base, acetone is used as a solvent for reaction, the reaction time is controlled to be 2-8 hours, and the ee% value of the finally prepared AST-3424 is more than or equal to 96%.

Description

Improved AST-3424 preparation process

Technical Field

The invention relates to a preparation process and a purification method of an anti-cancer candidate drug AST-3424 (OBI-3424) bulk drug.

Background

DNA alkylating cancer therapeutic drug AST-3424 (see patent application: DNA alkylating agent, corresponding to PCT application No. PCT/US2016/021581, publication No. WO2016/145092, corresponding to China application No. 2016800150788, publication No. CN107530556A, disclosed compound TH2870, (R) -1- (3- (3-N, N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl) -1-ethyl-N, N '-bis (ethylenephosphoramidate), composition and method of use and preparation thereof, corresponding to PCT application No. PCT/US2016/062114, publication No. WO2017087428A1, corresponding to PCT application No. 2016800446081, publication No. CN108290911A, chinese name (S) -1- (3- (3-N, N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl) -1-ethyl-N, N' -bis (ethylenephosphoramidate, also referred to as I-24, OB-70) and structure of CAS No. 2097713-2825A), which was developed by I company, is characterized by the following structure:

AST-3424 (OBI-3424) chemical formula

At present, the medicine is subjected to phase II clinical experiments in China and the United states.

The only three patent application documents disclose the preparation of this compound or of the racemate TH-2870 of this compound.

PCT/US2016/021581, publication WO2016/145092 provides the following routes and methods (method I):

the preparation process comprises the following steps:

to a mixture of compound 7 in DMF (60 mL) was added NaH (60%, 0.508g,12.7 mmol) in portions at 0deg.C. The mixture was stirred at 0 ℃ for 0.5h, then compound 6 (2 g,6.35 mmol) was added and then stirred at 0 ℃ for 2.5h. The mixture was diluted with EtOAc (500 mL), washed with brine (50 mL. Times.3), and dried over Na ₂ SO ₄ Dried, filtered, concentrated under reduced pressure and purified by FCC (silica gel, acetone/hexane)To obtain AST-2870 as a yellow oil.

Final purification of TH-2870:

TH2870 as mentioned above is purified by semi-preparative HPLC (C18 column, acetonitrile/water). The combined collections were concentrated under reduced pressure to obtain a pale yellow oil as the final product. Acetonitrile was added to the evaporate as an azeotroping agent to remove water.

Or by a silica gel column (DCM: meoh=40:1) to give compound TH-2870 as a yellow oil.

PCT/US2016/062114, publication number WO2017087428A1 provides the following synthetic routes and methods of preparation (method II):

preparation of AST-3424 by separation of enantiomers of TH2870 by preparative chiral chromatography

983mg of TH2870 was dissolved in 36mL of methanol, 1mL of the above methanol solution was injected onto CHIRALPAK OZ-H4.6X105 mm,5 μm (Daicel) in SFC-80Method Station (Thar, waters) at a column temperature of 35℃to 40℃at a flow rate of 3.0mL/min and a back pressure of 120 bar, and was injected onto a column of methanol at CO ₂ The mixture of methanol (65-60/35-40) was eluted at this flow rate. The enantiomer of formula (Ia) (configuration (R)) was obtained in 86.5% yield and 100% enantiomeric purity. The enantiomer of formula (Ib) (configuration (S)) was obtained in 83.8% yield and 100% enantiomeric purity.

Synthesis of AST-3424 (TH-3424) by Using chiral starting materials

Compound 7 (1.0 g), compound 8 (785 mg), K were stirred at room temperature ₂ CO ₃ (880 mg) in DMF (8 mL) overnight. The mixture was diluted with water, extracted with DCM, and taken up in Na ₂ SO ₄ And (5) drying. Filtration, concentration and then flash chromatography gave compound 9 (1.1 g) as a yellow oil.

These methods use alkaline reagents NaH, K in the final step ₂ CO ₃ And at the rearThe subsequent purification step is carried out by conventional column chromatography or preparative HPLC.

PCT/US2020/019176, publication No. WO2020172506 provides the following technical details (scheme III):

this application provides only the roadmap described above, and does not specifically mention the course of operation, as well as other details.

Disclosure of Invention

In the subsequent industrial mass production process, the skilled person found to use NaH/K ₂ CO ₃ In order to solve this problem, after a series of technological condition searches have been made, a technical proposal is made to prepare high optical purity AST-3424 by reacting cesium carbonate with acetone as a solvent for a reaction time strictly controlled to 2-8 hours, and the ee% value of the finally prepared AST-3424 is 96% or more.

An improved process for preparing AST-3424 with high optical purity by reacting compound A with compound B in cesium carbonate as base and acetone as solvent,

the ee percent value of the prepared AST-3424 with high optical purity is more than or equal to 96 percent,

the reaction time is 2-8 hours.

The reaction of A and B is nucleophilic substitution reaction of phenolic hydroxyl and halohydrocarbon, and needs participation of alkali:

the first step: the phenolic hydroxyl in B generates alkoxy anions under the condition of alkali;

and a second step of: alkoxy anions as nucleophiles attack A, while halide anions F ^- Leaving, i.e. attaching the alkoxy anion to the halogen F ^- Ion(s)Leaving to finally produce the product AST-3424.

The key step is that the first step is to generate alkoxy anions under the action of alkali, and the alkali cannot be too weak:

in method I, a strong base NaH is used, and compound B is added after a period of reaction (30 minutes);

the weaker K is used in method II and route III ₂ CO ₃ Or Cs ₂ CO ₃ And the compound A, the compound B and the alkali are put into a mixer and stirred for reaction.

Applicant found in subsequent large scale (above 10 g) process validation experiments and GMP batch (100 g and Kg scale) reactions that the use of too strong a base may lead to racemisation of the compound, resulting in a final product AST-3424 with reduced chiral purity compared to reactant a, i.e. too strong a base may reduce the chiral purity of the product.

Further studies have found that even if a weaker base (K ₂ CO ₃ Or Cs ₂ CO ₃ ) The chiral purity is also reduced even in the case of an excessively long reaction time, and it is finally confirmed that the optical (chiral) purity of the AST-3424 product obtained by using cesium carbonate as a base, acetone as a solvent and the reaction time of 2 to 8 hours is high (up to 98%) based on the experimental search of the applicant.

Optical (chiral) purity may be expressed in a variety of ways, such as chiral HPLC, where the optical (chiral) purity referred to herein may be expressed as an HPLC purity of AST-3424 of greater than or equal to 98%, such as an ee% of greater than or equal to 96% expressed as an ee% value.

The synthetic preparation method of the compound A and the compound B can be referred to PCT/US2016/021581, and the publication number WO2016/145092; PCT/US2016/062114, publication number WO2017087428A 1.

Preferably, the ratio of the amounts of substances of compound a to cesium carbonate is 1:2-2.4; the ratio of the amounts of the substances of compound a to compound B is 1:1-1.80.

Theoretically, the ratio of the amounts of substances of compound a to cesium carbonate is 1:1 is the theoretical reaction ratio, but in practice cesium carbonate is added in excess: on the one hand, high conversion of the valuable starting compounds A can be provided, and on the other hand, the reaction rate can be increased to some extent. However, too much base is likely to destroy compound a, product AST-3424, and therefore cannot be excessive too much, and experiments show that the ratio of the amounts of substances of compound a to cesium carbonate is 1:2-2.4, which combines the two aspects.

Preferably, the solvent acetone is added in a 1g ratio to compound A corresponding to 18-24ml of acetone.

Since acetone is used as the solvent in the reaction, the amount of solvent should be appropriate: too low results in incomplete dissolution of the reaction system, too high solvent amounts dilute the reactant concentration and result in too slow reaction rate, too much solvent increases post-treatment workload and effluent discharge, and many experiments find that the ratio of 1g of compound A to 18-24ml of acetone is suitable.

Preferably, the reaction temperature is 15-35 ℃.

The reaction temperature is directly related to the reaction rate and whether or not the reaction has side reactions. The nucleophilic substitution reaction of the compound A and the compound B produces a product AST-3424. In the presence of a base, an elimination reaction occurs to produce an olefin along with a nucleophilic substitution reaction. Generally, the high temperature promotes the elimination reaction to produce byproducts, and the reaction should be controlled to be carried out at a relatively low temperature in order to suppress the occurrence of the elimination reaction as much as possible; lower reaction temperatures will reduce the reaction rate and ultimately extend the reaction time, which has been shown experimentally to result in racemization of the final product AST-3424.

The proper reaction temperature is necessary, so that the elimination reaction can be carried out as little as possible in the reaction, and the reaction can be completed in a short time as possible, so that the optical purity of the generated AST-3424 is high, and the experiment shows that the reaction temperature is proper at 15-35 ℃.

Preferably, the specific feeding steps of the process of the present application are as follows:

mixing and stirring the compound A with a solvent acetone, dissolving, and then adding the compound B and cesium carbonate;

stirring and reacting after the material feeding is finished.

Preferably, the post-treatment step after completion of the reaction is as follows:

after the reaction is finished, filtering the reaction liquid, washing a filter cake by using acetone, and collecting filtrate;

concentrating the filtrate, and purifying by column chromatography to obtain AST-3424.

The invention also provides a purification method of AST-3424 prepared by the process.

In the prior art: method I was purified using semi-preparative HPLC (C18 column, acetonitrile/water), or ordinary column chromatography; method II chiral separation using Supercritical Fluid Chromatography (SFC) using CO in supercritical fluid state as mobile phase ₂ Adding methanol.

The preparation type HPLC uses acetonitrile as a mobile phase, so that the preparation type HPLC has high cost, cannot be used in industrial production (hundred gram scale and even kilogram scale) on a large scale, and has extremely long separation time due to the small production capacity of the HPLC, and large solvent amount is used, so that the preparation type HPLC is uneconomical, and the mass production cannot be used for separation production by using the HPLC.

Supercritical fluid chromatography is high in efficiency and speed, but is only used in laboratories at present, a large number of separation and purification technologies are not mature, and the method is difficult to apply to industrial production and has high cost.

The common column chromatography can improve the primary separation amount and separation effect by using a chromatographic column with larger diameter and adjusting the proportion of mobile phase and even using gradient elution, but the separation effect and the yield difference between batches are larger due to the fact that the chromatographic columns of different batches are difficult to have stable separation effect, which is unfavorable for quality control, so that the common column chromatography is not suitable for large-scale kilogram-scale production.

The applicant carries out separation by using a dynamic axial compression column system through experimental optimization and selection, and selects proper silica gel with specific particle size (particle size distribution is 10-50 um), a specific loading amount is used during operation, and an ethanol-ethyl acetate mixed solvent with the volume percent of 4-10% of ethanol is used for carrying out isocratic elution during elution, so that the requirement of industrial mass production can be met: hundred gram-grade large-scale separation and good separation effect.

The method for purifying AST-3424 produced by the preparation process by column chromatography is characterized by comprising the following steps of:

column chromatography was performed using a dynamic axial compression column system,

the filler is silica gel, the particle size distribution is 10-50 μm, and the pore diameter is

The sample loading amount is 2-5% of the mass of the silica gel, namely, the net content of AST-3424 corresponding to 1kg of silica gel is 20-50g,

the elution process comprises

And (3) performing isocratic elution by using an ethanol-ethyl acetate mixed solvent with the mobile phase of 4-10% of ethanol by volume percent, and collecting eluent.

Experimental exploration determines that the volume of the chromatographic column packing subjected to isocratic elution by using an ethanol-ethyl acetate mixed solvent with the volume percent of 4-10% is more than 13 times, preferably 13-55 times.

Preferably, the flow rate of the mobile phase is 500-600ml per minute: the flow rate of a 15cm diameter, i.e. other specification column, is 500-600ml per minute by passing through a column with a 15cm internal diameter.

In column chromatography, the speed of flow rate determines the separation efficiency, and in general, the low speed is beneficial to separation, namely, the low-speed mobile phase washes the silica gel loaded with the compound, so that the separation effect of the compound is better; however, too low a rate may result in a compound to silica gel combination time that is too long to allow reactive groups on the silica gel to react with the compound (AST-3424 has multiple reactive groups that may react), so the flow rates should be suitable, and it has been found experimentally that a 15cm internal diameter column will have a flow rate of 500 to 600ml per minute, such as a 7.5cm internal diameter column phase flow rate of 125 to 150ml per minute, and a 30cm internal diameter column phase flow rate of 2000 to 2400ml per minute.

The usual methods for column chromatography loading (loading, i.e. loading the sample to be purified onto silica gel) are wet loading and dry loading. Wet loading is the direct injection of a solution of the compound to be separated into a chromatographic column filled with silica gel. The dry loading is complicated by adding a proper amount of silica gel to the sample solution to be purified, removing the solvent to obtain dry silica gel loaded with the sample compound, and loading the dry silica gel on a dry silica gel column. The mobile phase is flushed after passing through.

The dry loading and the wet loading can be used, but the AST-3424 has larger polarity, the wet loading can be completely adsorbed in the silica gel, and the dry loading is not needed to strengthen the adsorption, so the wet loading is suggested, namely, the ethyl acetate solution containing the AST-3424 to be purified is injected into the chromatographic column after the column loading is finished.

The elution process also comprises the step of using ethyl acetate for flushing, wherein the ethyl acetate flushing is carried out before the ethanol-ethyl acetate mixed solvent with the volume percent of 4-10% of ethanol is subjected to isocratic elution;

preferably, the amount of ethyl acetate rinse used in the elution is more than 2.8 times the column packing volume, preferably 2.8-8.2 times.

After the elution process is finished, washing the chromatographic column by using an ethanol-ethyl acetate mixed solvent with the ethanol volume ratio of 85-95%;

preferably, the volume of the ethanol-ethyl acetate mixed solvent with the ethanol volume ratio of 85-95% of the washing preparation column is 2.8-3.2 times of the volume of the chromatographic column packing.

The chromatographic column can be reused for 3-5 times, and further, the chromatographic column is preferably reused for 3-4 times, and after the chromatographic column is reused for 4 times, the chromatographic column can be reused after the chromatographic column is dried and reloaded after washing.

And collecting 40-90% of the volume of the eluent eluted by the ethanol-ethyl acetate mixed solvent, namely collecting from 40% of the total volume of the eluent to 90% of the total volume of the eluent, and concentrating the solvent to obtain AST-3424. The eluate thus collected has high purity of AST-3424. The eluent at the head or the eluent at the tail contains other impurities.

Drawings

FIG. 1 is a first sample injection purification elution profile of a first set of experimental chromatographic columns, beginning with 1 for AST-3424 collection and ending at 7 for AST-3424 collection;

FIG. 2 is a first sample injection purification elution profile of a third set of experimental chromatographic columns, beginning with 1 for AST-3424 collection and ending at 9 for AST-3424 collection;

FIG. 3 is a second sample injection purification elution profile of a third set of experimental chromatographic columns, beginning with 1 for AST-3424 collection and ending at 9 for AST-3424 collection;

FIG. 4 is a third sample injection purification elution profile of a third set of experimental chromatographic columns, beginning with 1 for AST-3424 collection and ending at 10 for AST-3424 collection;

FIG. 5 is a fourth sample injection purification elution profile of a third set of experimental chromatographic columns, beginning with collection of AST-3424 at 10 and ending with collection of AST-3424 at 2;

FIG. 6 is a fifth sample injection purification elution profile of the third set of experimental chromatographic columns, beginning with collection of AST-3424 at reference numeral 2 and ending with collection of AST-3424 at reference numeral 10.

Detailed Description

Description of the experiment

All experimental reagents were analytically pure unless otherwise specified.

HPLC testing uses an instrument that is an agilent 1200 liquid chromatograph.

HPLC purity testing of AST-3424 uses a C8 chromatographic column, acetonitrile/water system mobile phase (see CN108290911A, WOWO 2017/087428)

Chiral HPLC purity test of AST-3424 uses chiral chromatography columns (packing is polysaccharide derivative, such as the macrocellulol Daicel CHIRALPAK series) using n-hexane/isopropanol system mobile phase.

Dynamic axial compression column system equipment models DAC150 and DAC50.

Compound A

Compound A, B was prepared by the applicant according to the methods disclosed in PCT/US2016/062114, publication No. WO2017087428A1 or PCT/US2020/019176, publication No. WO 2020/172506.

HPLC, high performance liquid chromatography.

TLC, thin layer chromatography.

DMF, N-dimethylformamide.

DMSO, dimethyl sulfoxide.

THF, tetrahydrofuran.

ee%, percent enantiomeric excess.

eq, molar equivalent.

Example 1 (Small Scale experiment)

Operating procedure

1. Compound a (1.0 eq) and solvent acetone (19 ml acetone/1 g compound a) were charged into a four-necked flask under nitrogen atmosphere.

2. Stirring was started and the compounds B (1.3 eq) and K were added ₂ CO ₃ (2.0 eq) powder

The reaction temperature was adjusted to 25 ℃ ± 5 ℃ for stirring reaction, and the progress of the reaction was detected by HPLC: and judging the end of the reaction when the content of the compound A in the reaction liquid is not detected.

Post-treatment

3. The reaction solution was filtered and the filter cake was washed with acetone (the washing amount was about 3-10 times the volume of the filter cake), and the filtrate was collected and evaporated under reduced pressure to give a crude solution.

The crude solution was separated using a common chromatographic column with the mobile phase methanol: the volume ratio of the ethyl acetate is 7:93, and 0.5% by volume of triethylamine was added, and the chiral purity of the final product was tested after separation using chiral HPLC.

9 sets of experiments were performed, respectively, with the final results shown in Table 1 below.

Table 1: results of pilot experiments after changing conditions on the basis of typical 1, 2, 3 operating methods

Experimental batch	Reaction variation (on the basis of the above-described typical methods of operation 1, 2, 3)	Amount of compound A to be fed	Duration of the reaction	ee％
					1	Typical 1, 2, 3 step operation methods described above	1g	72～96hours	96.6％
2	2.0eq of compound B and 4.0eq of K are added ₂ CO ₃	1.1g	24hours	95.24％
					3	DMSO was used as a reaction solvent	1g	1hour	98.3％
4	THF was used as the reaction solvent	1g	72hours	97.02％
					5	DMSO was used: acetone volumeThe ratio is 1:2, a mixed solvent agent	1g	24hours	94.26％
6	DMF was used as the reaction solvent	1g	24hours	96.1％
					7	Cesium carbonate is used as base	2g	48hours	96.18％
8	Cesium carbonate is used as base	2g	24hours	97.58％
					9	Cesium carbonate is used as base	25g	36hours	96.96％
10	2.4eq Cs was used ₂ CO ₃ And 1.56eq of Compound B	20g	24hours	96.96％

The reaction variation was a variation based on the typical 1, 2, 3 operation methods described above.

As is clear from the above experiments, the solvent used for the reaction, the base used for the reaction (K ₂ CO ₃ Or Cs ₂ CO ₃ ) The final chiral purity (ee%) of the reaction is affected by the reaction time, etc.

Based on K ₂ CO ₃ DMF, naH/DMF and Cs ₂ CO ₃ Comparison of experimental data of acetone, and representation of Cs on the data ₂ CO ₃ Advantages of acetone, K is used ₂ CO ₃ The ee value is inferior to that of cesium carbonate, the reaction time is longer, and the reaction speed of cesium carbonate as alkali is faster; on the other hand, the use of DMF as a solvent for post-treatment can generate more wastewater, the post-treatment is complex, naH can generate a large amount of hydrogen in the reaction process and the post-treatment, and especially the post-treatment has larger potential safety hazard and is unfavorable for the safety production.

DMSO, although it also gives a product of high chiral purity and a relatively short time, is hardly removed from the product and is not suitable as a reaction solvent; and after being mixed with acetone, the chiral purity of the obtained product is too low, so that DMSO is excluded as a reaction solvent for process production.

For this purpose, a single-factor experimental search was performed.

Example 2

Experiments were performed at different reaction times to examine the optical purity of the final product (chiral HPLC purity, i.e. the relative percentage of AST-3424 to its enantiomer in chiral HPLC tests, as measured by area normalization method as 92.61% for chiral HPLC purity of AST-3424 of a certain batch, the relative percentage of its enantiomer was 7.39%, in this case the ee% of AST-3424 was 85.22%) and the results are given in table 2 below.

Table 2: experimental results were searched for reaction time

The results show that an increase in the reaction time leads to a decrease in the optical purity, and it is preferable to compare the case of not more than 8 hours, and a reaction time of 2 to 8 hours is preferable in view of the fact that too short a time will not allow the compound A to react completely. The reaction needs to be immediately filtered after 2 to 8 hours, so that the chiral purity is prevented from being reduced due to long-time alkaline environment.

Example 3

Experiments were performed with varying amounts of cesium carbonate to examine the optical purity of the final product, and the results are given in table 3 below.

Table 3: experimental results were found using cesium carbonate

The results show that too much cesium carbonate would lead to a decrease in chiral purity, and that too much would not be possible.

Example 4

Experiments with different solvents were performed to examine the optical purity of the final product, and the results are shown in table 4 below.

Table 4: experimental results were searched for different reaction solvents

From a pilot comparison, the ee value of acetone was close to 100% and the ee value of other solvents was lower than that of acetone.

The use of DMSO and DMF can be quenched with a large amount of water, resulting in more waste water, and in addition, requires extraction of the product with an organic solvent, which is less suitable than acetone, and the process is more complicated.

Example 5

Examination of the ee% of the reaction mixture shows that at lower temperatures the ee% decreases slowly with increasing reaction time; when the reaction temperature is raised to 45-50 ℃, the racemization reaction is obviously accelerated, and the ee% is reduced rapidly. The results are shown in Table 5 below.

Table 5: experimental results of different reaction temperatures

Example 6

Use of Cs after new improvement ₂ CO ₃ The acetone is reacted, and the specific process operation is as follows:

feeding material

15.76kg of an acetone solution of Compound A (0.65 kg of Compound A after conversion) was charged into an 80L glass reactor. Stirring is started. The temperature was controlled and compound B (0.50 kg) was added to the 80L tank at 15-25 ℃. Cesium carbonate (1.52 kg) was added to the 80L kettle at 15-25℃and the kettle wall was rinsed with acetone (0.26 kg) to clean the wall of the kettle.

Reaction

The temperature of the system is regulated to 10-20 ℃ for heat preservation reaction, sampling is started after 1-2 h, sampling is performed every 1-4 h, and the content of the compound is detected to be less than or equal to 1% by HPLC tracking. The reaction is completed after about 2 to 8 hours.

Post-treatment

The reaction liquid is filtered when the temperature of the white steel filter pressing tank is controlled between 10 and 20 ℃. The filtrate was transferred to a vessel. The filter cake was rinsed with acetone and the leacheate was detected by thin layer chromatography until no product AST-3424 was detected, and the leacheate was collected and combined into a container.

Concentrating

Transferring the filtrate and the leaching solution into a jacketed reaction kettle, controlling the temperature of the jacket T to be less than or equal to 35 ℃, adding ethyl acetate into the system, and concentrating to the residual 1.95-2.60L under the pressure of P to be less than or equal to-0.08 MPa.

Cooling to obtain crude product

The system is cooled to 15-25 ℃, stirred for 10-30 min, and finally 2.59kg of reddish brown liquid is obtained, wherein the mass percent of AST-3424 is 39.04% by quantitative detection, namely, the mass of the converted pure AST-3424 is 1.01kg, and the HPLC purity is 80.50%.

Example 7

And (3) carrying out column chromatography purification process experiments by a dynamic axial compression column system.

The optimal process conditions finally confirmed by the inventor through fumbling experiments are as follows:

column name: dynamic axial compression column DAC-150.

Filling type: spherical silica gel (sphericity more than 90%) 10-50um

Chromatographic column length: 320mm, column inner diameter: 150mm

Filler amount: 3kg of

Sample loading amount: 1/30 filler amount, standard value: 0.1kg, actual: 0.288kg (ethyl acetate solution, content: 39.04%)

Homogenizing solvent: acetic acid ethyl ester

The homogenization proportion is as follows: 1:2.5-3 (kg/L)

The homogenizing and stirring time is more than or equal to 30min

Column packing pressure (required column pressure): 5MPa of

Dwell time: for more than 1h

Detection wavelength: 230nm detection and 285nm monitoring

Eluent flow rate: 550mL/min

Mobile phase ratio: divided into A, B and C, a is 100% ethyl acetate, B is ethyl acetate/absolute 93:7 (mass ratio), C is ethyl acetate/absolute ethyl alcohol 11:89 (mass ratio)

The number of times of the repeated use of the spherical silica gel: 3 times, there is an operation of washing the silica gel column.

The elution procedure is as follows in table 7.

Table 7: elution procedure

Purification operation flow:

1. the mobile phase was formulated.

According to the proportion of mobile phase

A is 100% of ethyl acetate, and the ethyl acetate is taken as the main ingredient,

b is ethyl acetate/absolute ethanol 93:7 (mass ratio),

c is ethyl acetate/absolute ethyl alcohol 11:89 (mass ratio)

Three mobile phases A, B, C were prepared and stored for later use.

2. Mixing with silica gel, and loading into column.

An appropriate amount of ethyl acetate was added to the homogenization tank by peristaltic pump. Adding the calculated amount of spherical silica gel into a homogenizing tank, leaching the homogenizing tank with a proper amount of ethyl acetate, and stirring the system in the homogenizing tank for more than or equal to 0.5h for later use. The final homogenate ratio, i.e. the ratio of silica gel to ethyl acetate, is 1kg silica gel to 2.5-3L ethyl acetate.

The column was tested for tightness by pressurizing with an appropriate amount of ethyl acetate. After the test is passed, the filler slurry which is uniformly stirred in the homogenizing tank is pressed into a preparation column, after the pressing is finished, a proper amount of ethyl acetate is used for leaching the homogenizing tank, leaching liquid is transferred into the preparation column through a pipeline, and the pressure of the preparation column is maintained for more than or equal to 0.5h after the pressure of the preparation column is increased to 5MPa, so that the preparation column is ready for use.

3. Connecting the pipeline with a flow pump, injecting sample and configuring an elution program to perform column chromatography purification operation.

Confirm that pump a was properly connected to the preparative chromatograph: mobile phase A-pump A-preparation chromatograph

Confirm that pump B was properly connected to the preparative chromatograph: mobile phase B-pump B-preparative chromatograph

According to

Detection wavelength: 230nm detection and 285nm monitoring

Eluent flow rate: 550mL/min

An elution procedure was performed.

The a pump was started for 15 minutes to equilibrate.

And after balancing, sampling and eluting.

The sample injection amount of each needle is 0.288Kg (ethyl acetate solution, the content is 39.04%, the AST-3424 is 0.1 Kg), the sample is required to be filtered, 1-2 needles are predicted to be pre-experiments, 5-10 parts of components are collected in the pre-experiments, each part is collected for 1-30 min, the collection plan is determined by single detection HPLC according to the pre-experiment result, every 2-3 needles are combined into a batch, and the corresponding components are combined and then are detected by HPLC respectively.

And during sample injection, a sample injection pump is used for sample injection at a flow rate of 50-100 mL/min.

After the sample injection is finished, the pump A is continuously started to elute for 30 minutes, and then the pump A is closed, and a pipeline of the pump A is connected with the mobile phase C.

Confirm that pump a was properly connected to the preparative chromatograph: mobile phase c→pump a→preparative chromatograph.

The pump B is started to continue elution. The pump B was started for elution from 30.1 minutes to 170 minutes.

The elution procedure is ended and pump B is turned off.

Pump a was started to pump mobile phase C for column flushing. The pump A was started for column flushing from 170.1 minutes to 200 minutes, and the volume of the mobile phase C of the flushing column was about 2.8-3.2 times the volume of the packing of the chromatographic column.

And (3) carrying out next sample injection purification after the column flushing is finished, and directly repeating all the operations of 3 without repeating the operations of mixing silica gel and loading the column of 2.

And (3) after the 3-needle is prepared, the column is required to be reloaded, the operations of mixing silica gel and loading the column in the steps of 2 and 3 are repeated, a pipeline and a flow pump are connected, and the column chromatography purification operation is carried out by injecting sample and configuring an elution program.

The above purification is carried out on the basis that a crude product has been obtained.

Column chromatography purification was performed using a dynamic axial compression column system.

Table 8 below provides four lots of 4 GMP lot production data including yield, HPLC purity, ee% value.

Table 8: GMP product production data for four lots

The data under the condition of 4 batches of GMP (good manufacturing practice) prove that the novel process is completely feasible, and the product quality meets the requirements.

Example 8

The dynamic axial compression column system performs a column chromatography purification process fumbling experiment.

The process parameters used in example 7 were obtained by the following fumbling procedure.

The ethanol and ethyl acetate mixed solvent was experimentally examined for suitable ethanol content as the mobile phase.

First set of experiments (spherical silica gel multiplexing 4 times)

A sample of 5g (ethyl acetate solution) was directly fed thereto, and the amount of the sample was 1.1g according to the content conversion AST-3424.

The filler is spherical silica gel 10-50umAnd total 120g. Using ethyl acetate for homogenization, the ratio is as follows: 1:2.5-3 (kg/L), i.e.1 kg of silica gel using 2.5-3L of ethyl acetate.

The experiment uses a mobile phase: a: acetic acid ethyl ester

B:5% ethanol 95% ethyl acetate (volume ratio)

The flow rate is 60ml/min, the detection wavelength: 230nm detection and 285nm monitoring.

The elution procedure is as follows in table 9:

table 9: elution program table of dynamic axial compression column system for performing column chromatography purification experiment of first group of experiments of small scale

Time	0	30	30.1	220
					Mobile phase B	0	0	100	100
Mobile phase a	100	100	0	0

The elution profile is shown in FIG. 1 (where the upper curve is the 230nm detection curve and the lower curve is the 285nm monitoring curve, as is the remaining figures). The eluents were collected from the spectra labeled 1 and 7 for the period of time to give 7 parts of eluents, labeled 1-7 in the effluent sequence, respectively, with the HPLC test purity results shown in Table 10 below.

Table 10: HPLC purity data of first sample injection purification eluent of first group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5	6	7
								HPLC purity%	97.85	99.56	99.89	99.93	100	100	100

The same elution procedure, same loading and process parameters, were repeated after washing the column, and 6 eluents were collected, labeled 1-6 in the effluent sequence, with the HPLC test purity results as shown in Table 11 below.

Table 11: HPLC (high Performance liquid chromatography) purity data of second sample injection purification eluent of first group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5	6
							HPLC purity%	97.81	99.02	99.73	99.89	99.91	100

The mobile phase was rinsed with ethyl acetate for 30min,5% absolute ethanol: the mixture was washed with 95% ethyl acetate for 190min, and the two needles were repeated, and the result showed that the impurity separation was good. But the preparation time is long and the cost is too high.

The gradient needs to be continuously optimized, the sample injection amount is increased, and the cost is reduced. And simultaneously repeating sample injection and examining the durability of the filler.

Increasing the ethanol content in the mobile phase using the mobile phase: a: acetic acid ethyl ester

B:8% ethanol 92% ethyl acetate (volume ratio)

After washing the column, multiplexing was continued for a third use, the same elution procedure (only time shortened to 200 minutes), the same loading and process parameters, and 5 fractions of eluate were collected, each labeled 1-5 in the effluent sequence, and the HPLC test purity results were as shown in Table 12 below.

Table 12: HPLC purity data of third sample injection purification eluent of first group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5
						HPLC purity%	98.65	99.89	99.97	99.98	99.63
Rrt=1.25 impurities	/	/	/	/	0.37

And/indicates undetected, as follows.

B:8% ethanol 92% ethyl acetate (volume ratio)

And 100% of loading, namely 10g of ethyl acetate solution is added, and the content of the ethyl acetate solution is 2.2g according to the content conversion AST-3424.

After washing the column, multiplexing was continued for the fourth run, the same elution procedure (only total time shortened to 200 minutes), the same loading and process parameters, and 5 fractions of eluate were collected, each labeled 1-5 in the effluent sequence, and the HPLC test purity results were as shown in Table 13 below.

Table 13: HPLC purity data of fourth sample injection purification eluent of first group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5
						HPLC purity%	98.06	99.91	99.72	99.67	98.18
Rrt=1.25 impurities	/	/	/	0.25	1.65

The quality of the sample obtained by separation after four uses of the silica gel chromatographic column packed in the first group of experiments has been reduced: the impurity rrt=1.25 in the 5 th eluate was already as high as 1.65% and was no longer used.

In the subsequent ethanol or ethanol-ethyl acetate mixed solvent washing experiments, the impurities at rrt=1.25 were washed and collected, and the substances were found to be yellow in color, adsorbing the impurities as fillers, which peaked after AST-3424, and appeared in the eluent easily for 3 consecutive needles.

Second set of experiments (spherical silica gel multiplexing 3 times)

Column packing was performed using the parameters of the first set of experiments, with packing of spherical silica gel 20-35um 120gUsing ethyl acetate for homogenization, the ratio is as follows: 1:2.5-3 (kg/L), i.e.1 kg of silica gel using 2.5-3L of ethyl acetate.

The experiment uses a mobile phase: a: acetic acid ethyl ester

B:8% ethanol 92% ethyl acetate (volume ratio)

The sample amount was 11ml (sample amount 10 g), and the content was 2.2g according to the content conversion AST-3424.

The elution procedure is as follows in table 14:

table 14: elution program table for dynamic axial compression column system to perform column chromatography purification experiment of second group of experiments of small test

Time	0	30	30.1	180
					Mobile phase B	0	0	100	100
Mobile phase a	100	100	0	0

The machine was started and the eluents were collected to give 6 parts of eluents, each labeled 1-6 in the effluent sequence, with the HPLC test purity results as shown in Table 15 below.

Table 15: HPLC purity data of first sample injection purification eluent of second group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5	6
							HPLC purity%	98.02	99.79	99.89	99.82	99.05	97.78
Rrt=1.25 impurities	/	/	/	0.091	0.78	2.1

And a 100% loading of 20g of sample (22 ml of ethyl acetate solution) was added thereto, and the amount of the sample was 4.4g according to the content conversion AST-3424.

After washing the column, multiplexing was continued for a second use, and 5 parts of eluent were collected, labeled 1-5 in the effluent sequence, with the following HPLC test purity results, table 16.

Table 16: HPLC purity data of second sample injection purification eluent of second group of experimental chromatographic column

Eluent numbering	1	2	3	4	5
						HPLC purity%	98.26	99.83	99.92	99.48	98.57
Rrt=1.25 impurities	/	/	/	0.52	1.08

The mobile phase was used: a:10% ethanol 90% ethyl acetate

B:5% ethanol 95% ethyl acetate (volume ratio)

The column was washed and reused for a third run with the same elution procedure (except for the total time reduced to 160 minutes) and 6 eluents were collected and labeled 1-6 in each run, with the HPLC test purity results shown in table 17 below.

Table 17: HPLC purity data of third sample injection purification eluent of second group of experimental chromatographic column

Eluent numbering	1	2	3	4	5	6
							HPLC purity%	95.67	99.67	99.75	99.84	99.90	99.86

The failure to detect rrt=1.25 impurity in the third sample-feeding eluent in the 6 th eluent indicates that the impurity is eluted after the elution of AST-3424, and the impurity can be prevented from being mixed in by shortening the elution time or not collecting the tail eluent.

The preparation condition and the analysis result are combined, and the elution adopts 8 percent ethanol and 92 percent ethyl acetate (volume ratio) and can be better separated and purified. The maximum sample injection amount is calculated to be 4.4g according to the feeding amount, the sample injection amount is 20g, and the separation is better.

However, three consecutive injections revealed that other impurity residues, especially rrt=1.25 impurities, were more, and thus the chromatographic column was selected for 3 uses.

The use of pure ethanol to flush the column is not as good as 90% ethanol-10% ethyl acetate (volume ratio).

Third set of experiments (spherical silica gel multiplexing 5 times)

A batch of crude AST-3424 was prepared again and concentrated to give 325ml, totaling 296g, which was tested to contain about 62.92g of AST-3424. The third set of purification experiments used this batch of samples for purification and isolation.

Preparation of the column: spherical silica gel 20-35um300g of the mixture was homogenized with ethyl acetate, the proportions being as follows: 1:2.5-3 (kg/L), i.e. 1kg silica gel with 2.5-3L ethyl acetate, column packing pressure 5mpa.

The experiment uses a mobile phase: a: acetic acid ethyl ester

B:8% ethanol 92% ethyl acetate

The elution procedure is as follows in table 18:

table 18: elution program table of dynamic axial compression column system for performing column chromatography purification experiment of third group of experiments of small scale

Time	0	30	30.1	180
					Mobile phase B	0	0	100	100
Mobile phase a	100	100	0	0

25ml of ethyl acetate solution was introduced for the first time, which was 4.84g in the amount of AST-3424.

The equilibration process was approximately 0.8mpa.

The elution profile is shown in FIG. 2, and 6 fractions of the eluates were collected and each labeled 1-6 in the effluent sequence, and the HPLC test purity results are shown in Table 19 below.

Table 19: HPLC purity data of the first sample introduction purification eluent of the third group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5	6
							HPLC purity%	95.67	99.67	99.75	99.84	99.90	99.86
Rrt=1.25 impurities	/	/	/	/	0.089	0.4

After washing the chromatographic column, multiplexing was performed, 52ml (10 g for AST-3424, 3.33% of the mass of silica gel was used as the corresponding sample), the elution profile was as shown in FIG. 3, 6 eluents were collected and labeled 1-6 as the effluent sequence, and the HPLC test purity results were shown in Table 20 below.

Table 20: HPLC purity data of second sample injection purification eluent of third group of experimental chromatographic column

Eluent numbering	1	2	3	4	5	6
							HPLC purity%	97.55	99.66	99.67	99.72	98.91	98.16
Rrt=1.25 impurities	/	/	/	0.11	0.63	1.23

After washing the column, the column was multiplexed, 39ml (7.6 g, equivalent to AST-3424) was injected for the third time, the elution profile was as shown in FIG. 4, and 6 eluents were collected, each labeled 1-6 in the effluent sequence, and the HPLC test purity results were shown in Table 21 below.

Table 21: third set of experimental chromatographic column third sample injection purification eluent HPLC purity data

Eluent numbering	1	2	3	4	5	6
							HPLC purity%	97.08	99.76	99.88	99.85	98.49	99.15
Rrt=1.25 impurities	/	/	/	0.049	1.33	0.7

The analysis result shows that when the sample injection amount is 52ml (second sample injection in the third group of experiments), the corresponding RRT (relative retention time) =1.10 impurity separation in the HPLC purity detection is poor, and the rrt=1.25 impurity also becomes large: the rrt=1.25 impurities in the 5 th and 6 th portions are 1.33% and 0.7% respectively, so that the loading amount should not be too high, and the loading amount is 2% -5% of the mass of the silica gel.

The packing material is subjected to column injection after being washed by using fourth time, 90% ethanol and 10% ethyl acetate (volume ratio), 25ml of ethyl acetate solution (3.2 g for converting AST-3424) is injected, and the operation pressure is that: 7.2mpa. The elution profile is shown in FIG. 5, and 5 parts of the eluates are collected and labeled 1-5 in the effluent sequence, respectively, and the HPLC test purity results are shown in Table 22 below.

Table 22: HPLC purity data of fourth sample injection purification eluent of third group of experimental chromatographic columns

Eluent numbering	1	2	3	4	5
						HPLC purity%	98.44	99.83	99.99	99.86	99.60

The separation effect developed by the preparation method can be achieved by using DAC50 to prepare the column, wherein the oil pressure of the column is 5.0mpa and the operation pressure is 1.2mpa.

Filtration is recommended to maintain column stability and lifetime due to the high sample impurities and precipitations.

After the chromatographic column is washed by using 90% ethanol-10% ethyl acetate (volume ratio), the separation effect is improved.

After washing the column with 90% ethanol-10% ethyl acetate (volume ratio), drying under reduced pressure, homogenizing with ethyl acetate and refilling the column according to the above method, the fifth time the packing was used, the operating pressure: 1.2mpa. The sample was taken in 50ml of ethyl acetate (6.4 g, equivalent to AST-3424), the same elution procedure was followed, the elution profile being shown in FIG. 6, and 5 parts of eluate were collected, each labeled 1-5 in the effluent sequence, and the purity results were determined by HPLC and the fourth approximation (no HPLC purity was detected).

Experimental summary of column chromatography purification process by dynamic axial compression column system

1. The filler is spherical silica gel with particle size distribution of 20-35um and pore diameterOf course, the particle size may be single or mixed; />

2. The sample loading amount is 2-5% of the mass of the silica gel, namely, the net content of AST-3424 corresponding to 1kg of silica gel is 20-50g, and excessive sample loading can deteriorate the purification effect;

3. the elution process comprises the steps of performing isocratic elution by using an ethanol-ethyl acetate mixed solvent with the mobile phase of 4-10% of ethanol by volume percent and collecting eluent;

4. in order to achieve better purification effect and reduce cost as much as possible, the chromatographic column can be reused for 3-5 times, after the elution process before re-sampling is finished in the multiplexing process, the ethanol-ethyl acetate mixed solvent with the ethanol volume ratio of 85-95% is used for flushing the preparation column, impurities in the last sampling are flushed completely through flushing until TLC cannot be detected (flushing solvent with the volume of about 2.8-3.2 times of the volume of the chromatographic column is used);

5. further, the chromatographic column is preferably reused for 3-4 times, and after the chromatographic column is reused for 4 times, if the chromatographic column is required to be reused, the chromatographic column is required to be dried and reloaded after being washed, and the chromatographic column can be reused after being reloaded;

6. further, in order to remove small polar impurities in the sample, an ethyl acetate rinse is also included in the elution process, and the ethyl acetate rinse is performed before the isocratic elution with an ethanol-ethyl acetate mixed solvent having an ethanol volume percentage of 4% -10%.

7. In the elution process, the volume of the chromatographic column packing is more than 2.8 times, preferably 2.8-8.2 times, and the volumes of the chromatographic column packing are 226ml, 226ml and 565ml respectively in the first group, the second group and the third group of experiments, and the washing time is 1800ml (the flow rate is 60 ml/min) corresponding to 30 minutes, so that the corresponding volume of the ethyl acetate washing is 7.96 times, 7.96 times and 3.18 times respectively, and more washing time is better, but 3 times is proper in consideration of efficiency and cost, and the washing is recommended to be performed by using ethyl acetate with the volume of the chromatographic column packing of 2.8-8.2 times;

8. the same volume of the column packing for isocratic elution with ethanol-ethyl acetate mixtures with ethanol volume percentages of 4% -10% is recommended to be 13-fold or more, preferably 13-55-fold. The volume ratios used for the exploration experiment and the GMP batch experiment are listed below in tabular form.

Table 23: exploring volumetric ratio data of ethyl acetate rinse and eluent elution for experimental and GMP batch purification chromatography columns

1-1 represents the first run of the dynamic axial compression column system in a column chromatography purification process fumbling experiment, and so on.

The elution volume of 40-90% of the elution volume, which is about 40% to 90% of the total volume of the elution, was collected by an ethanol-ethyl acetate mixed solvent with an ethanol volume percentage of 4% -10% of AST-3424.

Claims

1. An improved process for preparing AST-3424 with high optical purity by reacting compound A with compound B in cesium carbonate as base and acetone as solvent,

the reaction time is 2-8 hours.

2. The preparation process according to claim 1, wherein,

the ratio of the amounts of the substances of compound a to cesium carbonate is 1:2-2.4;

the ratio of the amounts of the substances of compound a to compound B is 1:1-1.80.

3. The preparation process according to claim 1, wherein the solvent acetone and the compound A are added in a ratio of 1g of compound A to 18-24ml of acetone.

4. The preparation process according to claim 1, wherein the reaction temperature is 10-35 ℃.

5. The preparation process according to claim 1, comprising the following specific feeding steps:

stirring and reacting after the material feeding is finished.

6. The preparation process according to claim 1, wherein the post-treatment step comprises the following steps:

7. A method for purifying AST-3424 produced by the preparation process of claims 1-6 by column chromatography, characterized in that:

the elution process comprises

8. The column chromatography method according to claim 7, wherein:

wherein, the volume of the chromatographic column packing which is subjected to isocratic elution by using an ethanol-ethyl acetate mixed solvent with the volume percent of 4-10% of ethanol is more than 13 times, preferably 13-55 times.

9. The column chromatography method according to claim 7, wherein:

wherein the flow rate of the mobile phase is 500-600ml per minute: the flow rate of a 15cm diameter, i.e. other specification column, is 500-600ml per minute by passing through a column with a 15cm internal diameter.

10. The column chromatography method according to claim 7, wherein:

11. The column chromatography method according to claim 7, wherein:

the chromatographic column is reused for 3-5 times.

12. The column chromatography method according to claim 11, wherein:

preferably, the volume of the ethanol-ethyl acetate mixed solvent with the ethanol volume ratio of 85-95% of the flushing chromatographic column is 2.8-3.2 times of the volume of the chromatographic column packing.

13. The column chromatography method according to claim 7, wherein:

and collecting 40-90% of the volume of the eluent eluted by the ethanol-ethyl acetate mixed solvent, namely collecting from 40% of the total volume of the eluent to 90% of the total volume of the eluent, and concentrating the solvent to obtain AST-3424.

14. The column chromatography method according to claim 7, wherein:

and (3) wet loading, and injecting an ethyl acetate solution containing AST-3424 to be purified into the chromatographic column after the column loading is finished.