RU2583371C1 - Method of producing o-(2'-[18f]fluoroethyl)-l-tyrosine - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to method of producing O-(2′-[¹⁸F]fluoroethyl)-L-tyrosine, which can be used in synthesis of radiopharmaceuticals for positron emission tomography. Proposed method is implemented in synthesis module with vessels for reagents, reaction vessel, capillaries and column with sorbent C18. Method involves nucleophilic fluorination of precursor Ni-(S)-BPB-(S)-TyrO-CH₂CH₂OX (X=Ms, Ts, Tf; BPB=[N-2-(N′-benzyl-prolyl)amino]benzophenone) in the presence of interphase catalyst in reaction vessel, hydrolysis of fluorinated precursor and cleaning of the end product by means of column with sorbent C18 to produce preparation and subsequent purification of module. Method is characterised by fact that final semi-finished product is further purified from residue formed during neutralisation of reaction mass after hydrolysis of fluorinated precursor by means of coarse and fine purification filters arranged between reaction vessel and column with sorbent C18. Cleaning of synthesis module is performed in two steps with removal of formed during synthesis nickel compounds and organic impurities using deionised water, acetonitrile and one molar hydrochloric acid at the first stage and trace amounts of water and hydrochloric acid by means of acetonitrile, deionised water and ethanol - at the second. At that given solutions are arranged in the module in eight vessels for reagents.

EFFECT: method enables to obtain end product with stable high radiochemical yield and high radiochemical and enantiomeric purity, as well as provides complete automation of synthesis.

3 cl, 3 dwg, 1 ex

Description

The method relates to the production of medications, namely to the synthesis of radiopharmaceuticals (RFPs) and can find application in radiopharmaceutical production in centers of positron emission tomography (PET).

PET - one of the most rapidly developing areas of modern nuclear medicine, allows you to detect malignant neoplasms, foci of inflammation and other pathological processes, as well as visualize myocardial metabolism.

The use of PET requires the use of radiopharmaceuticals (RFPs) labeled with ultrashort-lived positron-emitting radionuclides, which are obtained using cyclotrons. The half-lives of such radionuclides are minutes or tens of minutes. The most widely used radionuclide for PET imaging is fluorine-18 (more than 90% of patient examinations are performed with radiopharmaceuticals labeled with fluorine-18).

The fluorine-18 radionuclide is produced by the nuclear reaction ¹⁸ O (p, n) ¹⁸ F when irradiated with 18-18 MeV protons of oxygen enriched with the ¹⁸ O isotope in the chemical form H ₂ ¹⁸ O. The radionuclide has a half-life of 109 minutes.

The capabilities of the PET method are largely determined by the arsenal of available radiopharmaceuticals. It is the choice of a suitable radiopharmaceutical that allows one to study various processes such as metabolism, transport of substances, ligand-receptor interactions, gene expression, etc. using PET. A wide range of different classes of radiopharmaceuticals makes PET a fairly versatile tool in modern molecular biology and clinical medicine. Therefore, the radiochemical synthesis of new radiopharmaceuticals and the development of effective synthesis techniques of already proven radiopharmaceuticals are currently becoming one of the most important problems to be solved for the development of the PET method.

The most common radiopharmaceutical for PET is 2- [ ¹⁸ F] fluoro-2-deoxy-D-glucose (FDG), with this drug, up to 95% of all examinations are performed. FDG allows you to evaluate the regional rate of glucose utilization. Replacing the hydroxyl group in the glucose molecule with a fluorine atom leads to the fact that FDG is transported in the tissue similarly to glucose, however, in cells, FDG metabolism stops at the phosphorylation stage, which leads to the accumulation of FDG in areas of increased energy metabolism of glucose. Therefore, the distribution of FDH in the body tissues is described in the framework of a simple pharmacokinetic model, and the data of PET studies can be easily interpreted. However, this radiopharmaceutical has a number of limitations in the diagnosis of brain tumors associated with the fact that glucose is the main energy substrate for the brain and, moreover, is utilized in macrophages produced in abscesses or foci of inflammation in the postoperative period, resulting in differentiation of the focus of inflammation and tumor using FDG presents certain difficulties. In this regard, in recent years, an intensive search for radiopharmaceuticals has been conducted, the accumulation mechanism of which is specific to tumors. In this aspect, amino acids that could be used in PET are widely studied.

Currently, fluorinated tyrosine analogues containing fluorine-18 in the aromatic ring, O- (2 ′ - [ ¹⁸ F] fluoroethyl) -L-tyrosine, are being intensively studied in a number of PET centers in Europe, Japan, and the USA. It is assumed that fluorinated analogs of amino acids (FAA), in particular tyrosine fluoride derivatives, accumulate to a much lesser extent in the foci of inflammation compared to FDG, and they are considered the most promising agents for the differentiation of tumor and inflammatory processes. The optimal characteristics of such an agent are a low accumulation index in an abscess with a relatively high accumulation in malignant cells.

The precursor of O- (2-tosyloxyethyl) -N-trityl-L-tyrosine tert-butyl ester [K. Hamacher, N.N. Coenen / Applied Radiation and Isotopes 57 (2002) 853-856]. The tosyl group acts as a “leaving” group, the substitution of which on [ ¹⁸ F] fluoride proceeds in high yield. With this precursor, a synthesis method was developed that was adapted for the GE Healthcare TracerLab FX FN module [Bourdier T., Greguric I., Roselt P., Jackson T, Faragalla J., Katsifis A. Fully automated one-pot radiosynthesis of O- ( 2- [ ¹⁸ F] fluoroethyl) -L-tyrosine on the TracerLab FXFN module. // Nucl. Med. Biol. - 2011. - V. 38. - No. 5. - P. 645-651]. According to this method, the radiochemical yield of the final product is 35% without correction for decomposition at a synthesis time of about 60 minutes, the enantiomeric purity of [ ¹⁸ F] PET is more than 99%.

However, the method has significant disadvantages, namely: the use of the HPLC method (high performance liquid chromatography) of the purification of the drug makes the system cumbersome, and the synthesis method itself is long and laborious.

Purification of the final product is possible by a simpler and more convenient method of solid-phase extraction (TFE). This greatly simplifies the automation of the process, allows for synthesis in a shorter time and the use of disposable, inexpensive components (cartridges) [Mueller D., Klette I., Kalb F., Baum RR Synthesis of O- (2- [ ¹⁸ F] fluoroethyl) - L-tyrosine based on a cartridge purification method. // Nucl. Med. Biol. - 2011. - V. 38. - No. 5. - R. 653-658].

The reaction mixture containing [ ¹⁸ F] PET was passed through two disposable Chromafix C18 cartridges (Macherey Nagel, phase adsorbent inverted), the cartridges were washed successively with a solution of ammonium acetate and 5% ethanol in 0.9% NaCl, after which [ ¹⁸ F] PET was eluted a solution of 0.9% sodium chloride, but containing 10% ethanol.

The synthesis time is significantly lower with respect to the HPLC method of PET purification and is 35 minutes; the radiochemical yield is about 45%. However, despite the good results obtained by individual groups of authors, the synthesis of PET, even in combination with purification by the TFE method, has not been widely used in routine PET practice, since fluorine-18-ethyl- is used as an alkylating agent in this methodology for the synthesis of PET. bromide. The purification of fluoro-18-ethyl bromide is achieved by distillation, which, on the one hand, is an advantage, since there is no need for additional purification of the alkylating agent, but, on the other hand, makes great demands on the tightness of the automated synthesis system. This method often leads to a loss of radioactivity precisely at the stage of the distillation of fluoro-18-ethyl bromide and can lead to irreproducible radiochemical yields. From this it follows that such a method for the synthesis of PET is unsuitable for its mass production.

The present invention relates to the synthesis of O- (2 ′ - [ ¹⁸ F] fluoroethyl) -L-tyrosine in order to use it for mass production of this drug and its widespread use in clinical practice.

Closest to the proposed is a method for producing O- (2 ′ - [ ¹⁸ F] fluoroethyl) -L-tyrosine (FET), described in [Bioorg. Med. Chem. 16 (2008) 4994-5003, however, the synthesis itself is presented in more detail in a Diploma made in the same organization, the Institute of the Human Brain of the Russian Academy of Sciences (copy attached)].

The method involves nucleophilic fluorination of Ni- (S) -BPB- (S) -TyrO-CH ₂ CH ₂ OX (X = Ms, Ts, Tf; BPB = [N-2- (N′-benzyl-prolyl) amino] benzophenone) (precursor) (Fig. 1), hydrolysis of the fluorinated precursor and purification of the final intermediate in the synthesis module, followed by purification of the module itself.

To carry out the synthesis in a reaction vessel containing a complex of [TBA] ⁺¹⁸ F ^-, or [K / 2.2.2.] ⁺¹⁸ F ^-, the precursor solution is added (4-5 mg of Ni- (S) -BPB- (S) - TyrO-CH ₂ CH ₂ OX (X = Ms, Ts, Tf) in 0.5 ml of acetonitrile). The mixture was kept at 80 ° С for 5 min without stirring, after which 0.5 ml of an aqueous solution of 0.5 M HCl was added to the reaction vessel, and hydrolysis and deprotection were carried out at a temperature of 120 ° С for 5 min.

According to the prototype, the best fluorination yields of the intermediate complex were achieved using the tosyl (Ts) leaving group and cryptofix 2.2.2 ([K / 2.2.2.] ⁺ ) As an interphase catalyst, 57.3 ± 8.5% (n = 3).

Purification of unreacted fluorine and by-product of inorganic and organic impurities and isolation of the intermediate were carried out using columns with sorbent С18 SepPak.

To automate the synthesis, according to the prototype, a synthesis module was constructed at the IPM RAS using standard commercially available components: valves from Burkert Compromatic (Germany), thermostat TPM1A, gas flow regulator, pneumatic piston SMC Pneumatics for raising and lowering the needles into the reaction vessel, 5 standard conical vessels with a volume of 5 ml.

The liquids were transported by a nitrogen current, and the pneumatic piston for the needle-raising device was controlled by compressed air. To flush the module lines (1/16 ″ Teflon capillaries with an inner diameter of 0.75 mm), the reagent vessels were filled with solvents that were transported by a stream of nitrogen in various directions. The module included 15 three-way and two-way valves, five vessels with a volume of 1-5 ml for adding reagents, the main vessel for carrying out the reactions, hermetically sealed with a Teflon lid equipped with four tube inlets and one needle. The main reaction vessel was in a heating device, which provided a heating range of up to 200 ° C. Blowing the reaction vessel with compressed air made it possible to quickly cool it. In addition, the module was equipped with a filtering device, which made it possible to purify the preparation by solid-phase extraction and sterile filtration. The HPLC purification system of the product is not integrated into the synthesis system, but is located separately. The process was controlled remotely using switches installed outside the hot zone.

As the authors noted, the synthesis time was 55 minutes, the radiochemical yield was 40-45% (with correction for decay), the enantiomeric purity was more than 98%, and the radiochemical purity was more than 95%.

For a better understanding, we present the synthesis flow chart according to the prototype (Fig. 2), where 1 is a column with an anion-exchange sorbent, 2 is a vessel for regenerate, 3 is vessels for reagents, 4 are valves (two- and three-way), 5 is a reaction vessel, 6 - heating block, 7 - flow regulator, 8 - vessel for liquid waste, 9 - capillaries.

This method, according to the authors, allows to obtain a PET with a high radiochemical yield and high radiochemical and enantiomeric purity.

However, our repeated repetition of this synthesis by the prototype method showed that the radiochemical yield of the drug is unstable, namely 5–35% without correction for decay. This, in our opinion, is due to the fact that, as the authors note, they did not achieve complete automation of the synthesis (it is still under development) and therefore the purification of the synthesis module, in which various impurities accumulate during multiple syntheses, was partially carried out manually.

In addition, the precipitation during the neutralization of the reaction mass after hydrolysis of the fluorinated precursor is likely to cause clogging of communications (standard capillaries with an inner diameter of 0.75 mm) and lead to the loss of the entire synthesis, which significantly reduces the value of the prototype method.

The technical result of the present invention is to improve the method for producing O- (2 ′ - [ ¹⁸ F] fluoroethyl) -L-tyrosine due to additional purification of the synthesis module and the final intermediate, which ensures a stable high yield of the drug and complete automation of the process.

This result is achieved by the fact that in the known method for producing O- (2 ′ - [ ¹⁸ F] fluoroethyl) -L-tyrosine in the synthesis module with reagent vessels, a reaction vessel, capillaries and a column with sorbent C18 nucleophilic fluorination of Ni- (S ) -BPB- (S) -TyrO-CH ₂ CH ₂ OX (X = Ms, Ts, Tf) (precursor), in the presence of an interfacial catalyst in the reaction vessel, hydrolysis of the fluorinated precursor and purification of the final intermediate by means of a C18 sorbent column to obtain preparation and subsequent cleaning of the module itself, while according to The final intermediate is additionally purified from the precipitate formed during the neutralization of the reaction mass after hydrolysis of the fluorinated precursor by means of coarse and fine filters installed between the reaction vessel and the C18 sorbent column, and the synthesis module is cleaned in two stages with the removal of nickel compounds formed during synthesis and organic impurities using deionized water, acetonitrile and unimolar hydrochloric acid - in the first stage and trace amounts of water and hydrochloric acid acid through acetonitrile, deionized water and ethyl alcohol - on the second, and these solutions are placed in the module in eight vessels for reagents.

To remove nickel compounds and organic impurities from the synthesis module, it is advisable to place deionized water in the first and fourth reagent vessels, acetonitrile in the second, third and fifth, unipolar hydrochloric acid in the sixth.

It is also advisable after the first stage of module cleaning to remove traces of water and acid in it, acetonitrile is placed in the first, second, third, fourth and fifth vessels for reagents, deionized water in the sixth, ethanol in the seventh and eighth.

The use of two-stage purification for purification of communications and a reaction vessel makes it possible to completely remove nickel compounds and organic impurities formed during synthesis, which ensures the achievement of a consistently high yield of the drug in serial production.

The use of coarse and fine filters in the capillary between the reaction vessel and the column with С18 sorbent during the synthesis of the preparation ensures the purification of the intermediate from the precipitate falling out in the reaction vessel when neutralizing the reaction mixture with alkali after hydrolysis. This prevents clogging of the pores of the column with the sorbent C18 formed by the precipitate and ensures the safe and reliable passage of reagents through the capillaries of the synthesis module, allowing the synthesis to be carried out in an automated mode.

For a better understanding, we present a diagram of the synthesis module according to the invention (Fig. 3), where 1 is a column with anion exchange resin, 2 is a vessel for regenerate, 3 are vessels for reagents, 4 are valves (two- and three-way), 5 is a reaction vessel, 6 - heating block, 8 - vessel for liquid waste, 9 - capillaries, 10 - coarse and fine filters, 11 - column with sorbent C18, 12 - column with cation-exchange sorbent, 13 - vessel for the product, 14 - pressure sensor.

An example of the synthesis.

1. Obtaining radionuclide (PH) ¹⁸ F.

To obtain the pH, the protons are irradiated with water enriched in the ¹⁸ O isotope in the target of the accelerator (cyclotron). The target pH ¹⁸ F is obtained by the nuclear reaction ¹⁸ O (p, n) ¹⁸ F.

2. Isolation of pH.

The irradiated material is transported through a capillary into the synthesis module on a column with an anion exchange resin (1), while the enriched water from which the target pH is isolated is collected in a regenerate vessel (2).

The pH adsorbed on the column is eluted into the reaction vessel (5). As an eluent, an aqueous solution of potassium carbonate coming from a vessel for reagents (vessel 1) is used.

The transportation of liquids through capillaries (9) is carried out by an inert gas current, and the distribution of flows is carried out using valves (4). The inert gas pressure is monitored by a pressure sensor (14).

3. The formation of the pH complex with IFC and its azeotropic dehydration.

From the reagent vessel (vessel 2), IFC (interfacial catalyst) in acetonitrile is added to the reaction vessel (5). For azeotropic drying, the obtained pH complex with MPC is heated to a temperature of 85 ° C in an inert gas stream for 10 minutes using a heating block (6).

4. Fluorination of the precursor.

A solution of the precursor (commercially available) in acetonitrile is added to the reaction vessel (5) from the reagent vessel (vessel 3). The resulting mixture is heated to a temperature of 85 ° C for 5 minutes using a heating block (6).

5. Hydrolysis and neutralization of the reaction mass.

0.6 ml of a 0.5 M aqueous hydrochloric acid solution is added to the reaction mass from the reagent vessel (vessel 4), the resulting mixture is heated to 125 ° C for 5 minutes. Then the reaction mass is cooled to a temperature of 40 ° C using a heating block (6).

To the reaction mixture from the reagent vessel (vessel 5) add 10 ml of deionized water, then from the reagent vessel (vessel 6) add 4 ml of a 0.1 M aqueous solution of sodium hydroxide. The resulting mixture was stirred by bubbling with an inert gas for 5 seconds.

6. Purification of the reaction mixture from sediment.

The reaction mass is passed through coarse and fine filters (10) to remove the precipitate formed at the stage of neutralization of the reaction mass with sodium hydroxide solution.

7. Cleaning the final intermediate.

The intermediate product purified from the sediment enters the column with С18 sorbent (11), while the target product is retained on the sorbent, and the waste enters the vessel for liquid waste (8). The column with sorbent C18 (11) is washed with 3 ml of deionized water from the reagent vessel (vessel 7).

The final intermediate is eluted from the column with the sorbent C18 (11) 5 mm solution of sodium acetate with 5% ethanol (pH = 4), from the vessel for reagents (vessel 8).

The eluate is passed through a cation exchange sorbent column (12) and collected in a product vessel (13) containing 2 ml of a 0.85 M sodium acetate solution to give isotonicity. With stirring by bubbling a purified intermediate and a solution of sodium acetate, the product (preparation) is obtained.

The synthesis time is 35 minutes, the radiochemical yield is 45-50% (with correction for decay), the enantiomeric purity is more than 98%, the radiochemical purity is more than 95%.

After synthesis, the synthesis module is cleaned, which consists of two stages. In the first stage, the vessels with reagents (3) are charged as follows: vessel 1 - deionized water 3 ml, vessel 2 - acetonitrile 3 ml, vessel 3 - acetonitrile 2 ml, vessel 4 - deionized water 1 ml, vessel 5 - acetonitrile 15 ml, vessel 6 - 1M hydrochloric acid 14 ml. Reagents are subsequently transported from the reagent vessels (3) to the reaction vessel (5) and then transferred to the liquid waste vessel (8).

In the second stage, the vessels with reagents (3) are charged as follows: vessel 1 - acetonitrile 3 ml, vessel 2 - acetonitrile 3 ml, vessel 3 - acetonitrile 1 ml, vessel 4 - acetonitrile 1 ml, vessel 5 - acetonitrile 15 ml, vessel 6 - deionized water 14 ml, vessel 7 - ethanol 5 ml, vessel 8 - ethanol 5 ml. Reagents are subsequently transported from the reagent vessels (3) to the reaction vessel (5) and then transferred to the liquid waste vessel (8).

After such cleaning, the module is ready for the next synthesis.

All parameters of the synthesis technology were obtained experimentally and allowed to achieve good results.

Currently, the drug obtained by the described method is undergoing preclinical trials in the Russian Center for Science and Technology.

The proposed method in comparison with the known has several significant advantages:

1) full automation of the synthesis that the prototype cannot achieve;

2) the achievement of a stably high technological yield of the final product, which does not allow the prototype;

3) in comparison with other known methods differs in the simplicity of synthesis with a higher technological yield of the drug, not inferior to them in enantiomeric and radiochemical purity.

The method was developed at the Russian Center for Science and Technology and was tested in 50 syntheses with a positive result.

Claims (3)

1. A method of producing O- (2 ′ - [ ¹⁸ F] fluoroethyl) -L-tyrosine in a synthesis module with reagent vessels, a reaction vessel, capillaries, and a C18 sorbent column, including nucleophilic fluorination of the Ni- (S) -BPB- precursor (S) -TyrO-CH ₂ CH ₂ OX (X = Ms, Ts, Tf; BPB = [N-2- (N′-benzyl-prolyl) amino] benzophenone) in the presence of an interphase catalyst in the reaction vessel, hydrolysis of the fluorinated precursor and cleaning the final intermediate by means of a column with a sorbent C18 to obtain the drug and subsequent cleaning of the module itself, characterized in that the final half the product is additionally purified from the precipitate formed during the neutralization of the reaction mass after hydrolysis of the fluorinated precursor by means of coarse and fine filters installed between the reaction vessel and the C18 sorbent column, and the synthesis module is cleaned in two stages with the removal of nickel compounds and organic impurities using deionized water, acetonitrile and unimolar hydrochloric acid in the first stage and trace amounts of water and hydrochloric acid by acetonitrile, deionized water and ethyl alcohol - on the second, and these solutions are placed in the module in eight vessels for reagents. 2. The method according to claim 1, characterized in that to remove nickel compounds and organic impurities from the synthesis module, deionized water is placed in the first and fourth reagent vessels, acetonitrile in the second, third and fifth, unipolar hydrochloric acid in the sixth. 3. The method according to claim 1 or 2, characterized in that after the first stage of cleaning the module to remove traces of water and acid in it, acetonitrile is placed in the first, second, third, fourth and fifth vessels for reagents, deionized water in the sixth, ethanol - in the seventh and eighth.

Images