CN118754876A - Purification method and application of a precursor compound targeting FAP - Google Patents

Abstract

The invention relates to the field of compound purification, in particular to a purification method and application of a precursor compound of a target FAP. The purification method mainly adopts three high-pressure preparation liquid phase purification, wherein the first high-pressure preparation liquid phase purification process effectively controls phosphorus impurities in the product, the second high-pressure preparation liquid phase purification process realizes the improvement of the product purity, the third high-pressure preparation liquid phase process realizes the effective control of the TFA content, the product quality is improved, the product stability is improved, the quality and the stability of the medicine are finally improved, the clinical safety risk caused by overhigh impurities is reduced, the medication safety of patients is ensured, and the purification method is suitable for large-scale production and popularization of the medicine.

Description

Purification method and application of a precursor compound targeting FAP

Technical Field

The present invention relates to the field of compound purification, and in particular to a purification method and application of a precursor compound targeting FAP.

Background Art

Fibroblast activation protein (FAP) is a membrane serine peptidase expressed on the surface of activated fibroblasts in tumor stroma. Studies have shown that high expression of FAP is detected on the surface of stromal fibroblasts in more than 90% of epithelial malignancies. Therefore, FAP has become an important target for tumor imaging and treatment.

¹⁸ F-NOTA-FAPI (clinical trial registration number CTR20230410) is a FAP-targeted PET tracer labeled with the positron-emitting radionuclide ¹⁸ F and has been approved for PET clinical studies of FAP solid tumors.

The above gives the necessary preparation process in the synthesis of ¹⁸ F-NOTA-FAPI. In the preparation of its precursor compound NOTA-FAPI, peptide condensation reagents (such as HATU, HBTU, etc.) are inevitably used for the condensation of NOTA-Bis (tBuEster) and compound A, resulting in the introduction of a large amount of phosphorus-containing impurities into the final product, thereby affecting the purity and quality of the final product. In addition, trifluoroacetic acid (TFA) is also introduced into the synthesis of ¹⁸ F-NOTA-FAPI, and TFA is inevitably used as the mobile phase in the subsequent HPLC purification process, resulting in the inability to control the TFA content in the product. Excessive TFA content will cause the final product to be extremely easy to absorb moisture, with low yield and poor product stability.

In order to improve the product quality and stability of ¹⁸ F-NOTA-FAPI, it is necessary to optimize the purification method of its precursor compound NOTA-FAPI.

Summary of the invention

In view of the above problems, the present invention provides a method for purifying a precursor compound NOTA-FAPI of an ¹⁸ F-NOTA-FAPI tracer, which can effectively control the phosphorus impurities and TFA contents in the precursor compound NOTA-FAPI.

Specifically, the present invention provides a method for purifying a precursor compound targeting FAP, wherein the structure of the precursor compound NOTA-FAPI is shown in formula (I):

,

The precursor compound NOTA-FAPI is obtained by the following preparation process:

The purification method comprises three high-pressure preparative liquid phase purification methods. Specifically, the purification method comprises the following steps:

(1) The first high pressure preparative liquid phase purification process focuses on effectively controlling the phosphorus impurity content in the product. The process may specifically include the following steps:

S1. Prepare the mobile phase: mobile phase A ₁ is 0.02M ammonium acetate solution, mobile phase B ₁ is acetonitrile;

S2. Prepare the sample solution a to be purified: take the sample to be purified, calculate the final solution volume a of the sample to be purified according to the formula W _{mass of the sample to be purified} : V _{final solution volume a of} the sample to be purified = 1:4 (w/v, g/ml), use a mixed solution of mobile phase _A1 and mobile phase _B1 (V _{mobile phase A1} : V _{mobile phase B1} = 9:1, v/v) to dissolve the sample to be purified and quantify it to the final solution volume a of the sample to be purified, pass the dissolved feed solution through a 0.2 μm filter membrane, collect the filtrate, and use it as the sample solution a to be purified. The final concentration of the sample solution a to be purified is 250±10 mg/ml;

S3, sample loading:

Instrument: Shimadzu LC-20AP,

Chromatographic column: Yuexu XB-Phenyl (50*250mm, 10μm),

Flow rate: 55ml/min,

Sample volume range: 5±1ml,

Detection wavelength: 254nm;

S4, elution, collection of target peak components, sampling for HPLC detection, and merging of preparation solutions with purity > 95%;

S5, vacuum rotary evaporation to obtain the first prepared concentrate a;

(2) Second high pressure preparative liquid phase purification, this process focuses on further removing impurities and improving product purity. The process may specifically include the following steps:

S6. Prepare the mobile phase: mobile phase A ₂ is 0.1% trifluoroacetic acid in water, mobile phase B ₂ is 0.1% trifluoroacetic acid in acetonitrile;

S7, prepare the sample solution b to be purified: take the first prepared concentrate a, calculate _{the final solution volume b of the sample to be} purified according to the formula W _{first prepared concentrate a} : V final solution volume b of the sample to be purified = 1:4 (g/ml), use a mixed solution of mobile phase _A2 and mobile phase _B2 (V _{mobile phase A2} : V _{mobile phase B2} = 9:1, v/v) to dissolve the first prepared concentrate a and quantify it to the final solution volume b of the sample to be purified, after dissolution, pass the feed solution through a 0.2 μm filter membrane, collect the filtrate, and use it as the sample solution b to be purified, the final concentration of the sample solution b to be purified is 250 ± 10 mg/ml;

S8, sample loading:

Instrument: Shimadzu LC-20AP,

Chromatographic column: Yuexu XB-C18 (50*250mm, 10μm),

Flow rate: 55ml/min,

Sample volume range: 15±3ml,

Detection wavelength: 254nm;

S9, eluting, collecting target peak components, sampling for HPLC detection, and combining preparation solutions with purity > 99%;

S10, vacuum rotary evaporation to obtain the second prepared concentrate b;

(3) The third high-pressure preparative liquid phase purification process focuses on reducing the TFA content in the product, thereby improving the purity and stability of the product. The process may specifically include the following steps:

S11, prepare mobile phase: mobile phase A ₃ is purified water, mobile phase B ₃ is acetonitrile;

S12, preparing the sample solution c to be purified: taking the second preparation concentrate b, calculating _{the final solution volume c of the sample to be purified} according to the formula W _{second preparation concentrate b} : V final solution volume c of the sample to be purified = 1:4 (g/ml), dissolving the second preparation concentrate b with a mixed solution of mobile phase A ₃ and mobile phase B ₃ (V _{mobile phase A3} : V _{mobile phase B3} = 9:1, v/v) and quantifying to the final solution volume c of the sample to be purified, passing the dissolved feed solution through a 0.2 μm filter membrane, collecting the filtrate as the sample solution c to be purified, and the final concentration of the sample solution c to be purified is 250±10 mg/ml;

S13, sample loading:

Instrument: Shimadzu LC-20AP,

Chromatographic column: Yuexu XB-Phenyl (50*250mm, 10μm),

Flow rate: 55ml/min,

Sample volume range: 15±3ml,

Detection wavelength: 254nm;

S14, eluting and collecting target peak components;

S15, rotary evaporation under reduced pressure to obtain a purified sample.

Further, the mobile phase gradient of the elution in step S4 is:

.

Further, the mobile phase gradient of the elution in step S9 is:

.

Further, the mobile phase gradient of the elution in step S14 is:

.

Further, the sample loading volume described in step S3 is 4 ml, 5 ml, 6 ml or any value in the range of 4 ml-6 ml; preferably, the sample loading volume described in step S3 is 4 ml, 5 ml, 6 ml; more preferably, the sample loading volume described in step S3 is 6 ml.

Further, the loading volume described in step S8 is 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml or any value in the range of 12 ml-18 ml; preferably, the loading volume described in step S8 is 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml; more preferably, the loading volume described in step S8 is 15 ml.

Further, the loading volume described in step S13 is 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml or any value in the range of 12 ml-18 ml (such as 14.375 ml); preferably, the loading volume described in step S13 is 12 ml, 13 ml, 14 ml, 14.375 ml, 15 ml, 16 ml, 17 ml, 18 ml; more preferably, the loading volume described in step S13 is 14.375 ml.

Furthermore, the vacuum rotary evaporation temperature in step S5 is 35-45°C.

Furthermore, the vacuum rotary evaporation temperature in step S10 is 35-45°C.

Furthermore, the vacuum rotary evaporation temperature in step S15 is 35-45°C.

Furthermore, steps S1-S4 in the first high pressure preparative liquid phase purification process may be repeated 2-5 times.

Furthermore, steps S6-S9 in the second high pressure preparative liquid phase purification process may be repeated 2-5 times.

Furthermore, steps S11-S14 in the third high pressure preparative liquid phase purification process may be repeated 2-5 times.

Further, the HPLC detection conditions described in step S4 and step S9 are as follows:

Chromatographic column: YMC-Triart C18 (4.6×150mm, 3µm);

Mobile phase A ₄ : 0.1% trifluoroacetic acid solution;

Mobile phase B ₄ : 0.1% trifluoroacetic acid in acetonitrile

Flow rate: 0.8ml/min

Column temperature: 30°C

Injection volume: 10µl

Detection wavelength: 254nm

Elution procedure:

.

Further, the HPLC process described in step S4 and step S9 is as follows: according to the above detection conditions, take a sample for injection, use the last needle spectrum as a blank chromatogram, inject one needle of the test solution, and record the chromatogram. If there are impurity peaks in the chromatogram of the test solution, deduct the chromatographic peaks in the blank chromatogram, and ignore the peaks less than 0.01% of the main peak area in the test solution. Calculate the purity of the target peak in the test sample by peak area normalization method.

The present invention provides a method for purifying NOTA-FAPI, a precursor compound of a ¹⁸ F-NOTA-FAPI tracer. The method can effectively control the content of phosphorus-containing impurities in the product, significantly improve the purity and quality of the product, and can effectively control the content of TFA introduced in the synthesis process and the purification process, thereby improving the product quality and the product stability, and ultimately improving the quality and stability of the drug, reducing the clinical safety risk caused by excessive impurities, ensuring the safety of patients' medication, and being suitable for large-scale production and promotion of drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an HPLC spectrum of the crude precursor compound NOTA-FAPI after purification.

DETAILED DESCRIPTION

The embodiments of the present invention are described in detail below in conjunction with examples. Those skilled in the art should understand that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

【Abbreviations and compounds】

Embodiment 1

Preparation of the precursor compound NOTA-FAPI

Weigh NOTA-Bis(tBuEster) (1.0 eq) and HATU (1.2 eq), add them into a reaction bottle, add an appropriate amount of DMF, stir for 30 min, and prepare an active ester solution.

Take an appropriate amount of DMF, add it to the reaction bottle, mix and stir with compound A (1.0 eq), add DIPEA (5.2 eq), stir for 10 min, add the above prepared active ester solution, control the temperature at 20-30 °C, and stir and react for 2 h.

Purified water (based on compound A, 20 ml/g) was added to the system, stirred, and then DCM (based on compound A, 20 ml/g) was added each time for extraction, and extracted 3 times in total. After extraction, the organic phases were combined and washed once with purified water (based on compound A, 20 ml/g) and saturated sodium chloride solution (based on compound A, 20 ml/g) in sequence. When washing with purified water, an appropriate amount of 0.5 mol/L NaHCO ₃ solution was used to neutralize the excess acid in the system until the pH of the system was 7-8. The washed organic phase was dried with anhydrous sodium sulfate, and then vacuum evaporated at 35-45°C to obtain a crude compound B. The crude compound B was purified by silica gel column chromatography (DCM: MeOH = 50: 1-10: 1) to obtain purified compound B.

TFA (based on compound B, 20 ml/g) was added to the reaction bottle, mixed with compound B (1.0 eq), the temperature was controlled at 33-37°C, and the reaction was stirred for 20 min.

The reaction solution was added dropwise to MTBE (based on compound B, 140 ml/g), stirred while adding dropwise, solid was precipitated, and slurry was made. After the addition, suction filtration was performed, and the filter cake was rinsed with MTBE (based on compound B, 20 ml/g). During the rinsing, the MTBE on the filter cake should not be completely removed; after rinsing, the filter cake was transferred to a single-mouth bottle with MTBE (based on compound B, 20 ml/g), and vacuum rotary evaporation was performed at 35-45°C to obtain a crude product of the compound of formula (I) (i.e., the precursor compound NOTA-FAPI).

Embodiment 2

The first study of parameters for high pressure preparative liquid phase purification

1) Final concentration of the sample solution to be purified

The process parameters are as follows:

The preparation method of the sample solution to be purified is as follows: take the sample to be purified (i.e., the crude precursor compound NOTA-FAPI), dissolve it with a small amount of a mixed solution of mobile phase _A1 and mobile phase _B1 (Vmobile _{phase A1} : Vmobile _{phase B1} = 9:1, v/v), pass the dissolved solution through a 0.2 μm filter membrane, collect the filtrate, and then dilute it with the above-mentioned mobile phase mixed solution to the target concentration as the loading solution.

Two groups were set up in the experiment. The initial purity of the sample to be purified in Group 1 was 93.24% (10.92 grams), and the final concentration a of the sample solution to be purified was 191 mg/ml; the initial purity of the sample to be purified in Group 2 was 93.14% (5.26 grams), and the final concentration a of the sample solution to be purified was 250 mg/ml. The test results are shown in Table 1. The results show that after the first high-pressure preparative liquid phase purification, the purity of NOTA-FAPI in Group 1 was 99.66%, and the purity of NOTA-FAPI in Group 2 was 99.84%. The purity of NOTA-FAPI after the first high-pressure preparative liquid phase purification in both groups was >95%, but Group 2 had certain purification advantages over Group 1. Therefore, it was determined that the final concentration of the sample solution to be purified during the first high-pressure preparative liquid phase purification was 250±10 mg/ml.

Table 1

2) Sample volume parameters

The process parameters are as follows:

The preparation method of the sample solution to be purified is as follows: take the sample to be purified (i.e., the crude precursor compound NOTA-FAPI), dissolve it with a small amount of a mixed solution of mobile phase _A1 and mobile phase _B1 (Vmobile _{phase A1} : Vmobile _{phase B1} = 9:1, v/v), pass the dissolved solution through a 0.2 μm filter membrane, collect the filtrate, and then dilute it to 250 mg/ml with the above-mentioned mobile phase mixed solution as the loading solution.

The experiment was divided into 3 groups. In the first group, 5.26 g of the sample to be purified (phosphorus content was 2.8%) was taken, dissolved and diluted to the specified concentration, and 15 ml and 6 ml were measured for loading respectively; in the second group, 6.38 g of the sample to be purified (phosphorus content was 3.0%) was taken, dissolved and diluted to the specified concentration, and 10 ml, 8 ml and 6 ml were measured for loading respectively; in the third group, 18.01 g of the sample to be purified (phosphorus content was 2.3%) was taken, dissolved and diluted to the specified concentration, and 6 ml and 4 ml were measured for loading respectively; the phosphorus content of each group after purification is shown in Table 2.

Table 2

As can be seen from Table 2, when the sample volume was 15 ml, the phosphorus content of Group 1 after the first high-pressure preparative liquid phase purification was 0.63%, and when the sample volume was 6 ml, the phosphorus content after the first high-pressure preparative liquid phase purification was only 0.0099%. Compared with 15 ml, the sample volume of 6 ml can significantly remove phosphorus impurities in the product and ensure the final quality of the product; Group 2 focused on the effect of the sample volume gradient between 6 ml and 15 ml on the phosphorus content. As can be seen from Table 2, when the sample volume was 10 ml, the phosphorus content after the first high-pressure preparative liquid phase purification was 0.46%, and when the sample volume was 8 ml, its The phosphorus content after the first high-pressure preparative liquid phase purification was 0.34%, while when the sample volume was 6 ml, the phosphorus content after the first high-pressure preparative liquid phase purification was only 0.075%, that is, the phosphorus content after the first high-pressure preparative liquid phase purification with a sample volume of 6 ml had an order of magnitude advantage over that of 10 ml and 8 ml, and the phosphorus removal effect with a sample volume of 10 ml and 8 ml was not as obvious as that of 6 ml; the third group focused on the phosphorus removal effect of the lower limit volume of 6 ml. The results showed that the phosphorus content of the purified products with a sample volume of 6 ml and 4 ml was not much different. Therefore, the sample volume for the first high-pressure preparative liquid phase purification was determined to be 5±1 ml.

In summary, the process parameters for the first high-pressure preparative liquid phase purification are determined as follows:

Embodiment 3

Study on the parameters of the second high pressure preparative liquid phase purification

1) Different loading volumes

The process parameters are as follows:

The preparation method of the sample solution to be purified is as follows: take the sample after the first high-pressure preparative liquid phase purification, dissolve it with a small amount of a mixed solution of mobile phase _A2 and mobile phase _B2 (Vmobile _{phase A2} : Vmobile _{phase B2} = 9:1, v/v), pass the dissolved liquid through a 0.2 μm filter membrane, collect the filtrate, and then dilute it to 250 mg/ml with the above-mentioned mobile phase mixed solution as the loading solution.

The experiment was divided into 3 groups. The sample volume of the first group was 12 ml, the sample volume of the second group was 15 ml, and the sample volume of the third group was 18 ml. The test results are shown in Table 3.

Table 3

The experimental results show that when the loading volume is 12, 15, and 18 ml, the purity after purification is greater than 99.9%, which can effectively improve the purity of the product. Therefore, the loading volume in the second high-pressure preparative liquid phase purification process is determined to be 15±3 ml.

2) Final concentration of the sample solution to be purified

The process parameters are as follows:

The preparation method of the sample solution to be purified is as follows: take the sample after the first high-pressure preparative liquid phase purification, dissolve it with a small amount of a mixed solution of mobile phase _A2 and mobile phase _B2 (Vmobile _{phase A2} : Vmobile _{phase B2} = 9:1, v/v), pass the dissolved liquid through a 0.2μm filter membrane, collect the filtrate, and then dilute it with the above-mentioned mobile phase mixed solution to the target concentration as the loading solution.

The experiment was set up in two groups. The final concentration of the sample solution to be purified in Group 1 was 100 mg/ml; the final concentration of the sample solution to be purified in Group 2 was 250 mg/ml. The test results are shown in Table 4.

Table 4

As can be seen from Table 4, the purity of the product of Group 1 before purification was 99.66%, and after the second high-pressure preparative liquid phase purification, the purity of the product of Group 1 was 99.72%; the purity of the product of Group 2 before purification was 99.81%, and after the second high-pressure preparative liquid phase purification, the purity of the product of Group 2 was 99.92% (up to 99.9%). Group 2 has a better advantage over Group 1. Therefore, the final concentration of the sample solution to be purified during the second high-pressure preparative liquid phase purification was determined to be 250±10 mg/ml.

In summary, the parameters of the second high pressure preparation liquid phase are determined as follows:

Embodiment 4

The third study on parameters of high pressure preparative liquid phase purification

1) Chromatographic column

The process parameters are as follows:

The preparation method of the sample solution to be purified is as follows: take the sample after the second high-pressure preparative liquid phase purification, dissolve it with a small amount of a mixed solution of mobile phase A ₃ and mobile phase B ₃ (V _{mobile phase A3} :V _{mobile phase B3} = 9:1, v/v), pass the dissolved liquid through a 0.2 μm filter membrane, collect the filtrate, and then dilute it to 250 mg/ml with the above mobile phase mixed solution as the loading solution.

Two groups of experiments were set up, focusing on the removal effect of TFA by chromatographic columns with the same specifications (21.2×250mm, 5μm) and different fillers (Phenyl, C18). The results are shown in Table 5.

Table 5

The results in Table 5 show that the content of trifluoroacetic acid in the product of group 1 before purification was as high as 44.1%, and the content of trifluoroacetic acid in the product after purification was 14.1%. The content of trifluoroacetic acid in the product of group 2 before purification was as high as 44.1%, and the content of trifluoroacetic acid in the product after purification was reduced to 3.2%. This indicates that Yuexu XB-Phenyl (21.2×250mm, 5μm) can more effectively reduce the TFA content in the product compared with Yuexu XB-C18 (21.2×250mm, 5μm). Therefore, Yuexu XB-Phenyl (21.2×250mm, 5μm) was determined to be used for the third high-pressure preparative liquid phase purification.

In summary, the parameters of the third high pressure preparation liquid phase are determined as follows:

Embodiment 5

Effect of secondary high-pressure preparative liquid phase purification on product stability compared with tertiary high-pressure preparative liquid phase purification

The secondary high-pressure preparative liquid phase purification was carried out according to the process parameters and methods confirmed in Examples 2 and 3; the tertiary high-pressure preparative liquid phase purification was carried out according to the process parameters and methods confirmed in Examples 2, 3 and 4. Table 6 shows the trifluoroacetic acid content of the final product after secondary high-pressure preparative liquid phase purification (Group 3 and Group 4) and after tertiary high-pressure preparative liquid phase purification (Group 1 and Group 2).

Table 6

As can be seen from Table 6, after three purifications, the content of trifluoroacetic acid in the final product of Group 1 and Group 2 was 8.7% and 8.8%, respectively, while the content of trifluoroacetic acid in the final product of Group 3 and Group 4 was as high as 34.4% after only two purifications, which indicates that three high-pressure preparative liquid phase purifications can significantly reduce the content of trifluoroacetic acid in the final product compared with two high-pressure preparative liquid phase purifications.

This example further studies the stability of the above four groups of products, as shown in Table 7.

Table 7

As can be seen from Table 7, whether it is 0 day, within 6 months at 5°C, or within 6 months at -20°C, the total impurities of Group 1 and Group 2 products are significantly lower than those of Group 3 and Group 4. In terms of product stability, the total impurity content of Group 1 and Group 2 at 5°C for 6 months is still significantly lower than that of Group 3 and Group 4, which shows that Group 1 and Group 2 have good product stability at 5°C for 6 months compared with Group 3 and Group 4. Similarly, at -20°C for 6 months, Group 1 and Group 2 have good product stability compared with Group 3 and Group 4. This shows that three high-pressure preparative liquid phase purification can significantly reduce the content of trifluoroacetic acid in the final product compared with two high-pressure preparative liquid phase purification, and ultimately significantly improve the stability of the product.

Embodiment 6

Purification of crude precursor compound NOTA-FAPI

The crude NOTA-FAPI obtained in Example 1 was purified according to the high-pressure preparative liquid phase purification method provided in Examples 2 to 4.

[The first high pressure preparative liquid phase purification]

S1. Prepare the mobile phase: mobile phase A ₁ is 0.02 M ammonium acetate solution, mobile phase B ₁ is acetonitrile.

S2. Prepare the sample solution a to be purified: take 49.76 g of the crude precursor compound NOTA-FAPI to be purified, calculate the final solution volume a=199 ml of the sample to be purified according to the formula W _{mass of the sample to be purified} : V _{final solution volume a of the sample to be purified} =1:4 (w/v, g/ml), use a mixed solution of mobile phase _A1 and mobile phase _B1 (Vmobile _{phase A1} : Vmobile _{phase B1} =9:1, v/v) to dissolve the sample to be purified and quantify it to the final solution volume a=199 ml of the sample to be purified, pass the dissolved feed solution through a 0.2 μm filter membrane, collect the filtrate as the sample solution a to be purified, and the final concentration of the sample solution a to be purified is 250 mg/ml.

S3, sample loading:

Instrument: Shimadzu LC-20AP,

Chromatographic column: Yuexu XB-Phenyl (50*250mm, 10μm),

Flow rate: 55 ml/min, use the initial mobile phase A:B = 9:1 (v/v) to balance the column for 10 min,

Sample volume: 6ml,

Detection wavelength: 254nm.

S4, elution (eluent gradient is as follows), collect the target peak components, sample the collected target peak components for HPLC detection, combine the preparations with purity > 95%, repeat S1-S4 steps until the purification is completed, and combine all the preparations with purity > 95%

.

S5. The collected preparation solution was subjected to reduced pressure rotary evaporation at 35-45°C to obtain 30.92 g of the first purified product.

【Second high pressure preparative liquid phase purification】

S6. Prepare the mobile phase: mobile phase A ₂ is 0.1% trifluoroacetic acid in water, mobile phase B ₂ is 0.1% trifluoroacetic acid in acetonitrile;

S7, prepare the sample solution b to be purified: take the above 30.92g of the first purification product, calculate the final solution volume b=123ml of the sample to be purified according to the formula W _{first preparation concentrate a} : V _{final solution volume b} of the sample to be purified =1:4 (g/ml), use the mixed solution of mobile phase _A2 and mobile phase _B2 (Vmobile _{phase A2} : Vmobile _{phase B2} =9:1, v/v) to dissolve the first preparation concentrate a and quantify it to the final solution volume b=123ml of the sample to be purified, pass the dissolved feed solution through a 0.2μm filter membrane, collect the filtrate, and use it as the sample solution b to be purified. The final concentration of the sample solution b to be purified is 251mg/ml;

S8, sample loading:

Instrument: Shimadzu LC-20AP,

Chromatographic column: Yuexu XB-C18 (50*250mm, 10μm),

Flow rate: 55 ml/min, use the initial mobile phase A:B = 9:1 (v/v) to balance the column for 10 min,

Sample volume: 15ml,

Detection wavelength: 254nm;

S9, elution (eluent gradient is as follows), collect the target peak components, sample the collected target peak components for HPLC detection, combine the preparation solutions with purity > 99%, repeat steps S6-S9 until the purification is completed, and combine all the preparation solutions with purity > 99%

.

S10, the collected preparation solution was subjected to reduced pressure rotary evaporation at 35-45°C to obtain 28.66 g of the second purified product.

【Third high pressure preparative liquid phase purification】

S11, prepare mobile phase: mobile phase A ₃ is purified water, mobile phase B ₃ is acetonitrile;

S12, prepare the sample solution c to be purified: take the above 28.66g of the second preparation concentrate b, calculate _{the final solution volume c=115ml of the sample to be} purified according to the formula W _{second preparation concentrate b} : V final solution volume c of the sample to be purified =1:4 (g/ml), use the mixed solution of mobile phase A ₃ and mobile phase B ₃ (V _{mobile phase A3} : V _{mobile phase B3} =9:1, v/v) to dissolve the second preparation concentrate b and quantify it to the final solution volume c=115ml of the sample to be purified, pass the dissolved feed solution through a 0.2μm filter membrane, collect the filtrate as the sample solution c to be purified, and the final concentration of the sample solution c to be purified is 249mg/ml;

S13, sample loading:

Instrument: Shimadzu LC-20AP,

Chromatographic column: Yuexu XB-Phenyl (50*250mm, 10μm),

Flow rate: 55 ml/min, use the initial mobile phase A:B = 9:1 (v/v) to balance the column for 10 min,

Sample volume: 14.375 ml,

Detection wavelength: 254nm;

S14, elution (elution gradient is as follows), collect the target peak components, repeat S11-S14 steps until the purification is completed, and combine all the preparation solutions

.

S15. The collected preparation liquid was subjected to reduced pressure rotary evaporation at 35-45°C to obtain 21.75 g of the final purified product (i.e., purified NOTA-FAPI). A sample was taken for HPLC detection (see Figure 1). The product purity was 99.92%, the TFA content was 8.7%, and no phosphorus was detected.

Subsequently, a product stability study was conducted on the purified NOTA-FAPI. As shown in Table 8, the results showed that the purified product (i.e., purified NOTA-FAPI) obtained by the purification method provided by the present invention still had good product stability after being placed at 5°C and -20°C for 6 months.

Table 8

Various modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art. Although the present invention has been described by way of specific preferred embodiments, it should be understood that the present invention is not limited to the above specific embodiments. For those skilled in the relevant art, various variations of the modes described herein are still within the scope of protection.

Claims (9)

1. A method for purifying a precursor compound targeting FAP, characterized in that the structure of the precursor compound is as shown in formula (I), and the precursor compound is obtained by the following preparation process: , Wherein, the purification method comprises the following steps: First high pressure preparative liquid purification S1. Prepare the mobile phase: mobile phase A ₁ is 0.02M ammonium acetate solution, mobile phase B ₁ is acetonitrile; S2. Prepare the sample solution a to be purified: take the sample to be purified, calculate the final solution volume a of the sample to be purified according to the formula W _{mass of the sample to be purified} : V _{final solution volume a of} the sample to be purified = 1:4 (g/ml), use a mixed solution of mobile phase _A1 and mobile phase _B1 (V _{mobile phase A1} : V _{mobile phase B1} = 9:1, v/v) to dissolve the sample to be purified and quantify it to the final solution volume a of the sample to be purified, pass the dissolved feed solution through a 0.2 μm filter membrane, collect the filtrate, and use it as the sample solution a to be purified. The final concentration of the sample solution a to be purified is 250±10 mg/ml; S3, sample loading: Instrument: Shimadzu LC-20AP, Chromatographic column: Yuexu XB-Phenyl (50*250mm, 10μm), Flow rate: 55ml/min, Sample volume range: 5±1ml, Detection wavelength: 254nm; S4, elution, collection of target peak components, sampling for HPLC detection, and merging of preparation solutions with purity > 95%; S5, vacuum rotary evaporation to obtain the first prepared concentrate a; Second high pressure preparative liquid purification S6. Prepare the mobile phase: mobile phase A ₂ is 0.1% trifluoroacetic acid in water, mobile phase B ₂ is 0.1% trifluoroacetic acid in acetonitrile; S7, prepare the sample solution b to be purified: take the first prepared concentrate a, calculate _{the final solution volume b of the sample to be} purified according to the formula W _{first prepared concentrate a} : V final solution volume b of the sample to be purified = 1:4 (g/ml), use a mixed solution of mobile phase _A2 and mobile phase _B2 (V _{mobile phase A2} : V _{mobile phase B2} = 9:1, v/v) to dissolve the first prepared concentrate a and quantify it to the final solution volume b of the sample to be purified, after dissolution, pass the feed solution through a 0.2 μm filter membrane, collect the filtrate, and use it as the sample solution b to be purified, the final concentration of the sample solution b to be purified is 250 ± 10 mg/ml; S8, sample loading: Instrument: Shimadzu LC-20AP, Chromatographic column: Yuexu XB-C18 (50*250mm, 10μm), Flow rate: 55ml/min, Sample volume range: 15±3ml, Detection wavelength: 254nm; S9, eluting, collecting target peak components, sampling for HPLC detection, and combining preparation solutions with purity > 99%; S10, vacuum rotary evaporation to obtain the second prepared concentrate b; The third high pressure preparative liquid purification S11, prepare mobile phase: mobile phase A ₃ is purified water, mobile phase B ₃ is acetonitrile; S12, preparing the sample solution c to be purified: taking the second preparation concentrate b, calculating _{the final solution volume c of the sample to be purified} according to the formula W _{second preparation concentrate b} : V final solution volume c of the sample to be purified = 1:4 (g/ml), dissolving the second preparation concentrate b with a mixed solution of mobile phase A ₃ and mobile phase B ₃ (V _{mobile phase A3} : V _{mobile phase B3} = 9:1, v/v) and quantifying to the final solution volume c of the sample to be purified, passing the dissolved feed solution through a 0.2 μm filter membrane, collecting the filtrate as the sample solution c to be purified, and the final concentration of the sample solution c to be purified is 250±10 mg/ml; S13, sample loading: Instrument: Shimadzu LC-20AP, Chromatographic column: Yuexu XB-Phenyl (50*250mm, 10μm), Flow rate: 55ml/min, Sample volume range: 15±3ml, Detection wavelength: 254nm; S14, eluting and collecting target peak components; S15, rotary evaporation under reduced pressure to obtain a purified sample. 2. The purification method according to claim 1, characterized in that the mobile phase gradient of the elution in step S4 is: . 3. The purification method according to claim 1, characterized in that the mobile phase gradient of the elution in step S9 is: . 4. The purification method according to claim 1, characterized in that the mobile phase gradient of the elution in step S14 is: . 5. The purification method according to claim 1, characterized in that the sample loading volume in step S3 is 6 ml. 6. The purification method according to claim 1, characterized in that the sample loading volume in step S8 is 15 ml. 7. The purification method according to claim 1, characterized in that the sample loading volume in step S13 is 14.375 ml. 8. The purification method according to claim 1, characterized in that the vacuum rotary evaporation temperature in steps S5, S10 and S15 is 35-45°C. 9. The purification method according to claim 1, characterized in that the HPLC detection conditions described in step S4 and step S9 are as follows: Chromatographic column: YMC-Triart C18 (4.6×150mm, 3µm); Mobile phase A ₄ : 0.1% trifluoroacetic acid solution; Mobile phase B ₄ : 0.1% trifluoroacetic acid in acetonitrile; Flow rate: 0.8ml/min; Column temperature: 30°C; Injection volume: 10 µl; Detection wavelength: 254nm; Elution procedure: .