TWI748141B - Method of high-pressure purification of [f-18]feonm - Google Patents

Abstract

A method is provided to purify [F-18]FEONM under a high pressure. The synthesis processes of [F-18]FEONM are integrated. A separation process of non-toxic radio- high performance liquid chromatography (radio-HPLC) is used to purify the crude product. The method integrates a convention [F-18]FDG synthesizer and a novel radio-HPLC system together in a heat chamber. After radiofluorinating the precursor, the reaction product is purified with alumina solid-phase column in advance to obtain the crude product with fluorine-18 removed. Then, diphenyl semi-preparative HPLC column is used for a final purification. A non-toxic solvent is used for mobile-phase eluting to remove the unreacted precursor and the phase-transfer solvent. The radiofluorination has a reaction rate about 50 percent (%). The method has an uncorrected radiochemical yield of 10~20%. Both of the radio-HPLC and the radio-thin layer chromatography (radio-TLC) have radiochemical purity higher than 95%.

Description

[F-18] High pressure purification method of FEONM

The present invention relates to a high-pressure purification method of [F-18]FEONM, especially to a non-toxic process, especially to a non-toxic solvent purification and the precursor can be removed in this state, and the finished product can be produced It is directly used for intravenous injection into animals/humans for positron emission tomography (PET) imaging.

Nuclear medicine’s brain blood flow/metabolism examination provides data on changes in brain tissue function. For anatomical changes provided by traditional computed tomography (CT), it can provide complementary effects. Especially in the diagnosis of cerebrovascular accident (Cerebral Vascular Accident, CVA), Transient Ischemic Attack (TIA), epilepsy and dementia. As for other applications such as head trauma and mental illness, there are also positive reports.

At present, in nuclear medicine brain scans, the most commonly used imaging agents cover blood flow and glucose metabolism. Recently, foreign countries have begun to promote the use of positron scanning to replace single-photon tomography. Take the United States as an example, hospitals seldom use Tc-99m (Tc-99m) tomography, and most hospitals use higher-order positron glucose brain scans (F -18 Fluorodeoxyglucose, FDG) replaces the traditional Tc-99m-labeled imaging agent. This positron scanning method can complete the imaging in a shorter time and provide higher-resolution images and brain metabolism information. However, at present, because FDG drugs need to be manufactured by a cyclotron, the cost of the imaging agent is relatively high because inspections are not common, and the manufacturing process is not a non-toxic process, so the products cannot be directly used for intravenous injection.

Existing related technologies include Taiwan patent I541222 and US patent US 9,789,207. This patent is to add fluorine-18 ion to aminopolyether after secondary azeotropy, add precursor to fluorination reaction, and then pass through solid phase extraction column for purification. The product is made. Although this patent is the same [F-18]FEONM process, the precursors and process methods used in this patent are different from the improved pressure purification method in this case.

In view of the great market potential for the development of nuclear medicine in Taiwan, but the lack of patents related to the development of nuclear medicine in Taiwan, and there is no patent application for the same purification method as the patent in this case for such related technology, so the general users are Cannot meet the needs of users in actual use.

The main purpose of the present invention is to overcome the above-mentioned problems encountered by the conventional technology and provide a non-toxic solvent purification and the characteristics of the precursor can be removed in this state, which is similar to the traditional analogue [F-18]FDDNP It is necessary to complete a purification in a more toxic solvent. Compared with further solid-phase extraction to reduce the content of related flow wash solvents, it can effectively shorten the process time, increase the recovery rate, and reduce the process toxic solvent content [F- 18] The high-pressure purification method of FEONM.

The secondary purpose of the present invention is to provide a non-toxic manufacturing process, the finished product produced is also non-toxic, using a non-toxic solvent-ethanol to wash the product, directly diluted can be used to make injections, also because it is not a toxic substance, It can be directly used in the high-pressure purification method of [F-18]FEONM that is injected intravenously into animals/humans.

Another object of the present invention is to provide a feature that has the characteristics of being extended to the manufacturing process of positron imaging drugs and has application potential; the product is a dual-effect contrast that can simultaneously contrast two proteins related to Alzheimer’s disease [ F-18] The high-pressure purification method of FEONM.

To achieve the above objectives, the present invention is a high-pressure purification method for [F-18]FEONM, which at least includes the following steps: radiofluorination reaction: radiofluorination reaction with a precursor (TEON); high-efficiency liquid phase separation Purification: The crude product after the reaction is halfway through an injector for separation and purification by preparative high performance liquid chromatography (HPLC). The HPLC separation and purification system uses a semi-preparative diphenyl column. The mobile phase is ethanol solution, and the precursor is eluted at a flow rate of 1.6 ml/min; and filtration sterilization: the [F-18]FEONM product obtained by eluting the precursor is filtered and sterilized to form a purification The [F-18]FEONM product, wherein the purified [F-18]FEONM product has a -C ₂ H ₄ O-functional group added to its fluorine-18 end, which can provide the affinity of the [F-18]FEONM Oily.

In the foregoing embodiment of the present invention, the radiofluorination reaction rate is 50±5%.

In the foregoing embodiment of the present invention, the ethanol solution is diluted to 20% by adding 95% ethanol to physiological saline.

In the above embodiment of the present invention, the filtration sterilization step is to filter the purified crude product with a filter cartridge to remove impurities and bacteria. The [F-18]FEONM product after filtration and sterilization is stored in sterile glass Keep in the bottle for later use.

In the foregoing embodiment of the present invention, the filter cartridge is 0.15-0.25 micrometers (μm).

In the above embodiments of the present invention, the radiochemical yield of the [F-18]FEONM product is 10-20%, and the radiochemical purity is greater than 95%.

In the foregoing embodiment of the present invention, after the precursor is radiofluorinated, the fluorine-18 fluoride is purified by an alumina solid phase extraction column in advance to obtain the crude product.

S11~S13: Sub-step

Figure 1 is a schematic diagram of the high-pressure purification process of [F-18]FEONM of the present invention.

Figure 2 is a schematic diagram of the brain biodistribution uptake rate of [F-18]FEONM in the 12-13-month-old P301S/PS19 gene transgenic mice of the present invention.

Figure 3 is the HPLC chart of the [F-18]FEONM precursor reference standard and its mixture of the present invention.

Figure 4 is a schematic diagram of the ultraviolet absorption peaks of the precursors of the present invention after radiofluorination.

Figure 5 is a schematic diagram of the radiochemical purity analysis results of [F-18]FEONM of the present invention.

[F-18]FEONM is a naphthol derivative and an analog of [F-18]FDDNP, which is specially designed for brain positron emission tomography (PET) imaging. It is compared with [F-18]FDDNP and a new kind of The effective Tau Tangle imaging agent has higher lipophilicity. In the method proposed in the present invention, the synthesis process of [F-18]FEONM is integrated, and a non-toxic radio-High performance liquid chromatography (radio-High performance liquid chromatography, radio-HPLC) separation process is used to purify its crude product. The whole process combines a traditional [F-18]FDG synthesizer with a new radioactive HPLC system in a hot cell. After the radiofluorination of the precursor, the reaction product was previously purified by an alumina solid phase extraction column to obtain a crude product to remove fluorine-18 fluoride, and then a semi-preparative diphenyl HPLC column was used for final purification by using a non-toxic solvent Used as a mobile phase to remove unreacted precursors and phase transfer solvents. The radiofluorination reaction rate is about 50%. The uncorrected radiochemical yield of the whole process is 10-20%. The radiochemical purity of radio-HPLC and radio-Thin layer chromatography (radio-TLC) is higher than 95%. Hereinafter, the present invention will be described in detail through examples, but the present invention is not limited to these examples.

Please refer to "Figures 1 to 5", which are schematic diagrams of the high-pressure purification process of [F-18]FEONM of the present invention, and the use of P301S/PS19 gene transgenic mice in the present invention with 12 to 13 months old [ F-18] Schematic diagram of the uptake rate of the biodistribution of FEONM in the brain, the HPLC diagram of the [F-18] FEORM precursor of the present invention, the reference standard and its mixture, the schematic diagram of the ultraviolet absorption peak of the precursor after the radiofluorination of the present invention, and the present invention Schematic diagram of the radiochemical purity analysis results of the invention [F-18]FEONM. As shown in the figure: the present invention is a high-pressure purification method of [F-18]FEONM, which at least includes the following steps: radiofluorination reaction step s11: a precursor (2-(1-{6-[(2- 2'-p-toluenesulfonic acid-ethoxyethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile, TEON) undergo radiofluorination reaction, wherein the structural formula of the precursor is:

High-efficiency liquid phase separation and purification step s12: the crude product after the aforementioned reaction is half-shot with an injector A for separation and purification by preparative high-efficiency liquid chromatography (HPLC), and the separation is performed by HPLC The column used for purification uses Waters semi-preparative diphenyl column (5μm), 250x10 millimeters (mm), the mobile phase is 95% ethanol as the eluent, and the flow rate is 1.6ml/min. The precursor is eluted.

Filtration sterilization step s13: filter the [F-18]FEONM product obtained from the elution of the precursor with a 0.22 micron (μm) filter cartridge to remove impurities and bacteria to form a purified [F-18]FEONM(2) -(1-{6-[(2-2'-[ ¹⁸ F]Fluoroethoxyethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile) product, after filtering and sterilizing [F-18]FEONM product Stored in a sterile glass bottle for later use, where the purified [F-18]FEONM product has a -C ₂ H ₄ O-functional group added to its fluorine-18 end, which can provide the affinity of the [F-18]FEONM Oily, the structural formula of the [F-18]FEONM product is:

If so, a new high-pressure purification method of [F-18]FEONM is formed by the above-disclosed process.

Figure 2 shows the brain biodistribution uptake rate of [F-18]FEONM in P301S/PS19 transgenic mice between 12 and 13 months old. [F-18] FEONM is purified by alumina solid phase extraction column. In the figure, BS is the brain stem; ST is the striatum; MB is the midbrain; HP is the hippocampus; CTX is the cortex; CB is the cerebellum. From the results shown in the figure, it can be found that the staging of Alzheimer's disease is effective. The separation of precursors and reference standards is basic work. As shown in Figure 3, the present invention tests the residence time of the reference standard and the precursor separately, as shown in Figure 3 (b), (a), and uses a radioactive HPLC system and carbon 18 (C-18) , Germini C-18, silica gel and Hydrophilic Interaction Chromatography (Hydrophilic Interaction Chromatography, HILIC) column uses acetonitrile (acetonitrile) and ethanol as the mobile phase to separate them, but before using the diphenyl column, from the standard In the flowchart of whether to add or not, it is shown that all have the same residence time. This may be due to their similar structure, the main difference is that fluorine comes from oxygen. The residence time of [F-18]FEONM is about 12 minutes. The precursor is eluted at about 13 minutes using a semi-preparative diphenyl column with 95% ethanol at 1.6ml/min. Although the difference between the two residence times is only 1 minute, the UV absorption peaks of the mixture of [F-18]FEONM precursor TEON and the reference standard FEON do not overlap, as shown in Figure 3(c). Therefore, the present invention applies this condition to the separation process of the product collection bottle of the automatic synthesizer, and can successfully separate the precursor from the reference standard, ensuring that the precursor can be removed by collecting the fraction of the final product.

Compared with nitro-aromatic precursors such as nitrofluorobenzonitrile, [F-18]FEONM and its precursor TEON are relatively unstable. Therefore, the precursors may be degraded during the high-temperature radiofluorination process. As shown in Figure 4, it shows that the same volume of precursor and crude product samples were injected. Figure (a) shows that the amount of [F-18]FEONM precursor (5mg) added exceeds that of the UV detector. Sensing limit; Figure (b) shows that the ultraviolet absorption peak of the precursor is greatly reduced after radiofluorination, indicating that the precursor has been decomposed in a large amount during the radiofluorination process. Therefore, when the same amount of precursor and crude product are injected, its The ultraviolet absorption peak (residence time: 13 minutes) is very small. This indicates that most of the precursors will degrade during the reaction. This is because the molecular structure of the precursor contains a toluene sulfonate leaving group, so it will be greatly degraded during the high-temperature radiofluorination process. This is completely different from the nitro aromatic precursor because it is a resonance structure combined with the nitro group and the diphenyl ring. This resonant electron orbit is strong enough to resist the breaking of a covalent bond during a fluorination reaction on another molecule. The toluene-based precursor degradation compound can be read from the UV absorption chart, and its peak value starts at 3 minutes, and the residence time is 11 minutes. The most recent by-product residence time was 11 minutes. This makes it the most difficult impurity to remove in this product. The quantity can be controlled by chemical impurities as one of the specifications of the final product. [F-18] The final product of FEONM was collected by collecting fractions from the eluent of radioactive HPLC. The radiochemical yield of the final product is 10-20%. Radiochemical purity was checked by radioactive HPLC (C-18 column, 95% acetonitrile elution) and radioactive TLC (silica gel plate, mobile phase 95% acetonitrile). Both test results show radiochemical purity higher than 95%.

Figure 5 shows the radiochemical purity of the final product [F-18]FEONM. Figure (a) shows the results of radioactive HPLC analysis. The HPLC column is Cogent C18 100A 5μm, 150×4.6mm, and the eluent is 95% acetonitrile, 0.3 ml/min. Figure (b) shows the results of radioactive TLC analysis. The TLC plate is Merck TLC Silica gel 60 F254, and the eluent is 95% acetonitrile. The result is shown in the figure, there will be radioactive products and non-radioactive products, and there is no precursor, so this method The effect is good.

It can be seen from the above that, based on the high-pressure separation and purification test method, the results of the present invention show that using ethanol as the eluent in the new high-pressure separation and purification system can successfully remove the precursors and achieve the effect of improving chemical purity. In addition, organic solvents are also Can be removed at the same time. Moreover, the present invention designs a new naphthol analogue [F-18]FEONM to increase lipophilicity. After being tested by the same shake flask gold standard test method, its lipophilicity value is higher than [F-18]FDDNP, As shown in Table 1. This is in line with the structural design concept of the present invention. The lipophilicity of [F-18]FEONM is increased by _{adding -C 2} H _{4 O- functional group to its fluorine-18 end, and it becomes a potential new brain imaging agent.} The high-pressure separation and purification method developed by the present invention can achieve the goal of non-toxic substance purification [F-18]FEONM.

The entire [F-18]FEONM process of the present invention is combined with the traditional [F-18]FDG synthesizer and an additional radioactive HPLC system. The purification conditions of the semi-preparative HPLC column of diphenyl were successfully developed, and the final product without precursor content can be collected. This is more advantageous than using semi-preparative HILIC and HPLC columns in previous studies. Compared with other HPLC columns, the high-pressure purification method of [F-18]FEONM proposed in the present invention is also a non-toxic process, and the finished product produced is also non-toxic. It uses a non-toxic solvent-ethanol to elute the product, and it can be used directly after dilution. It is made into an injection, and because it is not a poison, it can be used directly for intravenous injection. Therefore, after 95% ethanol eluate is added to physiological saline and diluted to 20% ethanol solution, the final product can be injected directly into animals/humans by intravenous injection for positron emission tomography (PET) imaging. Therefore, the present invention has the characteristics of being extended to the manufacturing process of positron imaging drugs, and has application potential; The dual-effect contrast power of the protein related to Hymer's disease.

In summary, the present invention is a high-pressure purification method of [F-18]FEONM, which can effectively improve the various shortcomings of conventional use. It has the characteristics of non-toxic solvent purification and the ability to remove precursors in this state, which is more traditional The analogue [F-18]FDDNP needs to be purified once in a more toxic solvent. Compared with further solid phase extraction to reduce the content of related flow wash solvents, the present invention can effectively shorten the process time, increase the recovery rate, and Reduce the high solvent content of the process toxicity, so that the production of the present invention can be more advanced, more practical, and more in line with the needs of users. It has indeed met the requirements of an invention patent application, and a patent application is filed in accordance with the law.

However, the above are only preferred embodiments of the present invention, and should not be used to limit the scope of implementation of the present invention; therefore, all simple equivalent changes and modifications made in accordance with the scope of the patent application of the present invention and the content of the description of the invention , Should still fall within the scope of the invention patent.

S11~S13: steps

Claims (7)

A high-pressure purification method for [F-18]FEONM, which at least includes the following steps: radiofluorination reaction: with a precursor (2-(1-{6-[(2-2'-p-toluenesulfonic acid-ethoxyethyl) (methyl)amino]-2-naphthyl}ethylidene)malononitrile, TEON) undergo a radiofluorination reaction, wherein the structural formula of the precursor is:

High-efficiency liquid phase separation and purification: the crude product after the reaction is pumped halfway through an injector with a preparative high-efficiency liquid chromatography (HPLC) for separation and purification. The HPLC separation and purification system uses semi-preparative Diphenyl column, the mobile phase of which is ethanol solution, the precursor is eluted at a flow rate of 1.6ml/min; and filter sterilization: the [F-18]FEONM product obtained by eluting the precursor is filtered out Bacteria, forming a purified [F-18]FEONM(2-(1-{6-[(2-2'-[ ¹⁸ F]Fluoroethoxyethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile) product, The purified [F-18]FEONM product has a -C ₂ H ₄ O-functional group added to its fluorine-18 end, which can provide the lipophilicity of the [F-18]FEONM, the [F-18]FEONM The structural formula of the product is:

According to the high-pressure purification method of [F-18]FEONM described in item 1 of the scope of patent application, the reaction rate of the radiofluorination reaction is 50±5%. According to the high-pressure purification method of [F-18]FEONM described in item 1 of the scope of patent application, the ethanol solution is diluted to 20% by adding 95% ethanol to physiological saline. According to the high-pressure purification method of [F-18]FEONM described in item 1 of the scope of patent application, the filtration and sterilization step is to filter the purified crude product with a filter cartridge to remove impurities and bacteria. The [F-18]FEONM product after bacteria is stored in a sterile glass bottle for later use. According to the high-pressure purification method of [F-18]FEONM described in item 4 of the scope of patent application, the filter cartridge is 0.15~0.25 microns ( μm ). According to the high-pressure purification method of [F-18]FEONM described in item 1 of the scope of patent application, the radiochemical yield of the [F-18]FEONM product is 10-20%, and the radiochemical purity is greater than 95% . According to the high-pressure purification method of [F-18]FEONM described in item 1 of the scope of patent application, after the precursor is radiofluorinated, the fluorine-18 fluoride is purified by an alumina solid phase extraction column in advance. The crude product is obtained.

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