JPH11243984A - Method for synthesizing 6- [18F] fluoro-L-dopa - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound useful as a labeling agent, etc., for diagnosing the cerebral dopamine synthetic ability by converting 4-[18 F]fluorocatechol into a 6-[18F]fluoro-L-DOPA according to a reaction with a β-tyrosinase. SOLUTION: The 4-[18F]fluofocatechol represented by formula I is converted into 6-[18F]fluoro-L-DOPA represented by formula II according to an enzymic reaction with an enzyme such as a β-tyrosinase obtained by a recombinant Escherichia coli in which a β-tyrosinase gene of Citrobacter intermedius is transferred to thereby suppress the production of an isomer and afford the objective 6-[18F]fluoro-L-DOPA in a high yield. The resultant compound is useful as a labeling agent, etc., for diagnosing the cerebral dopamine synthetic ability by positron emission tomography (PET), etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present application relates to a method for diagnosing brain dopamine synthesis ability by positron emission tomography (PET) or the like.
A method for synthesizing [18F] fluoro-L-dopa and 4- [18F] which is a novel synthetic intermediate in this synthesis method
The present invention relates to a method for synthesizing fluorocatechol.

[0002]

2. Description of the Related Art Dopamine is one of the major neurotransmitters, and its synthesis deficiency in the brain causes serious neurological diseases such as Parkinson's syndrome. This brain dopamine synthesis ability can be diagnosed by PET, and as a labeling agent therefor, conventionally, 6- [18
F] Fluoro-L-dopa has been used.

A method for synthesizing 6- [18F] fluoro-L-dopa is, for example, as follows:

[0004]

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[0005] The method of Ishiwata et al. (Appl. Radiat. Isot. 44 (4): 755-759, 1993) which has shown a reaction formula is known. Also, the method of Lemaire et al. Using [18F] F anion as a labeling material (J. Nucl. Med. 35 (12): 19)
96-2002, 1994).

[0006]

SUMMARY OF THE INVENTION However, Ishiwa
In the method of ta et al., when [18F] F ₂ or [18F] acetylhypofluorite is used as a labeling material, the positional isomer other than the intended 6- [18F] fluoro-L-dopa (2- [ 18F] Fluoro-L-dopa and 5- [18F] fluoro-L-dopa) are by-produced, and a separation operation is required to remove them. Further, there was a problem that the specific radioactivity of the target compound was low.

The method of Lemaire et al., Which uses [18F] F anion as a labeling material, has a problem that the synthesis process is complicated and the optical isomers are complex, so that the separation operation is indispensable. did. This application has been made in view of the circumstances described above, and solves the problems of the conventional synthesis methods, without generating regioisomers or optical isomers, and having a high specific activity of 6-. It is an object of the present invention to provide a new method capable of easily obtaining [18F] fluoro-L-dopa in a high yield.

Another object of the present application is to provide a novel synthetic intermediate in the above method and a method for synthesizing the same.

[0009]

According to the present invention, a first invention for solving the above-mentioned problems is represented by the following formula (1).

[0010]

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4- [18F] fluorocatechol represented by the following formula (2) is obtained by an enzymatic reaction using β-tyrosinase.

[0012]

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6- [18F] fluoro-L- represented by the formula
A method for synthesizing 6- [18F] fluoro-L-dopa, which is converted into dopa, is provided (claim 1).
In the method of the first invention, the following equation (3)

[0014]

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A preferred embodiment is to synthesize 4- [18F] fluorocatechol by elimination of methyl group of 4- [18F] fluoroveratrol represented by the formula (claim 2). According to the second aspect, the following equation (4) is satisfied.

[0016]

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By the aldehyde group elimination reaction of 6- [18F] fluoroveratraldehyde represented by the following formula, 4- [1]
In a preferred embodiment, 8F] fluoroveratrol is synthesized (claim 3). Further, in the third aspect, the following equation (5) is satisfied.

[0018]

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In a preferred embodiment, 6- [18F] fluoroveratraldehyde is synthesized by a nitro group substitution reaction of 6-nitroveratraldehyde represented by the following formula (4). This application also provides, as a second invention, the following formula (3)

[0020]

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By the elimination reaction of 4- [18F] fluoroveratrol represented by the following formula (1),

[0022]

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A method for synthesizing 4- [18F] fluorocatechol represented by the formula (5) is provided. This second
In the method of the invention, the following equation (4)

[0024]

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By the elimination reaction of 6- [18F] fluoroveratraldehyde represented by the following formula, 4- [1
A preferred embodiment is to synthesize [8F] fluoroveratrol (Claim 6). Further, in the aspect of claim 6, the following equation (5) is satisfied.

[0026]

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In a preferred embodiment, 6- [18F] fluoroveratraldehyde is synthesized by a nitro group substitution reaction of 6-nitroveratraldehyde represented by the following formula (7).

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION The synthesis method of the first invention of this application is represented by the following formula:

[0029]

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The reaction formula can be exemplified as follows. Also,
Preferred embodiments of the first invention are as follows:

[0031]

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[0032]

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[0033]

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An example of the reaction formula is shown below. These preferred embodiments correspond to the method of the second invention described later and a preferred embodiment thereof for the method for synthesizing 4- [18F] fluorocatechol. 6 of the first invention
The method of synthesizing [18F] fluoro-L-dopa is as follows. Specifically, a solution of 4- [18F] fluorocatechol (for example, an ethanol solution) is prepared by adding ammonium sulfate, pyridoxal phosphate, Tris-HCl buffer, sodium pyruvate, ascorbin Β-tyrosinase (1
(Approximately 10 Unit / ml), the mixture is heated at 25 to 45 ° C. for 1 to 10 minutes, and the reaction is stopped by adding an aqueous solution of trichloroacetic acid or the like. Next, the total amount of the reaction solution was filtered with a filter, and the obtained filtrate was passed through a column for separation or the like, so that 6- [18F] fluoro-L-dopa was converted to about 2
~ 5% can be obtained.

As β-tyrosinase, a commercially available product can be used, or, as shown in Examples described later, a gene whose gene is expressed in a host-vector system can also be used. 4- [18F] fluorocatechol can be obtained by a known method (Appl. Radiat. Isot. 42 (7): 673-681, 1991).
) Thus, it is also possible to use those synthesized or, according to the method of the second invention of this application, 4- [18F]
A compound synthesized by a methyl group elimination reaction of fluoroveratrol can also be used.

The method of the second invention can be performed, for example, as follows. That is, after distilling hexane from 4- [18F] fluoroveratrol by drying under reduced pressure or the like, hydroiodic acid is added to the residue, and the mixture is refluxed to give 4- [18F] fluorocatechol by acid hydrolysis. Generate. After the reaction, the whole amount of the reaction solution is diluted with distilled water, the whole amount is passed through a reversed-phase column to adsorb 4- [18F] fluorocatechol, and 4- [18F] fluorocatechol is extracted from the column with ethanol or the like. 4- [18F] fluorocatechol obtained by this method has a radiochemical yield of about 10% and a radiochemical purity of 99% or more.

4- [18F] fluoroveratrol, which is a precursor of 4- [18F] fluorocatechol, can be synthesized, for example, by a method such as an aldehyde group elimination reaction of 6- [18F] fluoroveratraldehyde. Further, 6- [18F] fluoroveratraldehyde can be synthesized by, for example, a nitro group substitution reaction of 6-nitroveratraldehyde. This synthesis of 6-nitroveratraldehyde → 6- [18F] fluoroveratraldehyde → 4- [18F] fluoroveratrol was carried out by the method of Plenevaux et al. Described in detail in Example 1 (Ap.
pl. Radiat. Isot. 43 (8): 1035-1040, 1992). Of course, each synthetic intermediate can be obtained and obtained by another method. For example, 4- [18
F] Fluorocatechol is obtained by a method using 6-nitropepyronal as a starting material (Journal of Fluorine Chemistry 4
8: 189-205, 1990).

[0038]

EXAMPLES Hereinafter, the present invention of this application will be described in more detail and concretely with reference to examples, but the present invention is not limited to the following examples. The materials used in the following examples are as follows. (1) [18F]: Prepared by a conventional method using a cyclotron. (2) β-tyrosinase (EC 4.1.99.2): Citrobacter inte
It was obtained from a recombinant Escherichia coli obtained by introducing the β-tyrosinase gene of rmedius. The specific activity of β-tyrosinase was measured by a lactate dehydrogenase conjugate method using L-tyrosine as a substrate, and the specific activity was 1.5 (μmol / min / min).
mg protein). Protein concentration 50-50
It was 0 mg / ml. In order to maintain the activity of the enzyme, the prepared enzyme solution of β-tyrosinase was stored in the freezer little by little. After thawing, it can be stored in a refrigerator for about one month while maintaining the enzyme activity. (3) Liquid chromatography: Performed using liquid chromatography equipped with a UV detector in series with a NaI radioactivity detector, using the following columns.

Column (A): 4.6 × 150 mm C-18 (Crest-
Pak C18S) column. Column (B): 20 × 250 mm C-18 (YMC-Pak ODS S-5) column. Column (C): 4 × 150 mm (Crownpak CR (+)) column. Mobile phase (D): 0.1% containing methanol / 1 mM sodium octyl sulfate and 1 mM ethylenediaminetetraacetic acid (EDTA)
Acetic acid (60:40 (V / V)).

Mobile phase (E): 0.1% acetic acid containing methanol / 1 mM sodium octyl sulfate and 1 mM EDTA (20:80 (V /
V)). Mobile phase (F): 0.1% acetic acid containing 0.1% ascorbic acid and 1 mM EDTA. Mobile phase (G): 0.01M perchloric acid aqueous solution (pH 2). All other chemical reagents were used without purification. Example 1: Synthesis of 4- [18F] fluorocatechol The process up to the synthesis of 4- [18F] fluorocatechol was carried out by the method of Plenevaux et al. (Appl. Radiat. Isot. 43 (8):
1035-1040, 1992).

[18F] F obtained from cyclotron
An anion aqueous solution (0.5 mL) was placed in a test tube with a screw cap, and potassium carbonate (2.44 mg) and Kryptofix 222 (22 mg) were added thereto. Acetonitrile (0.5 mL) was further added thereto, and the mixture was dried under a nitrogen stream while heating to 120 ° C. in an oil bath. To the residue after drying was added dimethyl sulfoxide (1.0 mL) containing 6-nitroveratraldehyde (15 mg), and the mixture was heated to 145 ° C. for 20 minutes in an oil bath with the screw cap stoppered. Then, 6- [18F] fluoroveratraldehyde was produced by an exchange reaction between the nitro group and the [18F] F anion. After the reaction, add the entire amount of the reaction solution to distilled water (20 mL).
And dilute the entire amount of the diluent with a Waters C18 SepPak cartridge.
After the column was adsorbed with 6- [18F] fluoroveratraldehyde, the column was washed with distilled water (5 mL) and dried while heating with a dryer under a nitrogen stream for 5 minutes. After drying, the column was treated with hexane (10 mL)
[18F] Fluoroverertraldehyde was extracted.

After the hexane was distilled off under reduced pressure to dryness, 1,4-dioxane (2 mL) was added to the residue, which was then redissolved. After that, the whole amount was subjected to a pressure test containing chlorotrisrhodium (I) (150 mg). Transferred to tube. Keep in a sealed oil bath for 20 minutes.
And then heated to 150 ° C for 4 min.
[18F] Fluoroveratrol was produced. After the reaction, the entire amount of the reaction solution was diluted with distilled water (40 mL), and the entire amount of the diluted solution was passed through a new Waters C18 SepPak cartridge to adsorb 4- [18F] fluoroveratrol. 4- [18F] with hexane (5-10 mL) from this column
Fluorveratrol was extracted.

After hexane was distilled off under reduced pressure to dryness, 57% hydroiodic acid (0.5 mL) was added to the residue, and the mixture was refluxed for 20 minutes to produce 4- [18F] fluorocatechol by acid hydrolysis. I let it. After the reaction, the entire amount of the reaction solution was diluted with distilled water (5 mL), and the whole amount was replaced with a new Waters C18 Sep.
4- [18F] fluorocatechol was adsorbed through a Pak cartridge. Ethanol (0.5 to 2.
5- [5]) to extract 4- [18F] fluorocatechol.

The radiochemical purity of 4- [18F] fluorocatechol obtained by the above method was measured by analytical liquid chromatography using column (A).
Chromatography conditions were as follows. Flow rate: 1.5 ml / min Solvent: mobile phase (E) Column temperature: room temperature Wavelength: 280 nm Retention time of 4- [18F] fluorocatechol: 5.5 to
6.5 minutes 4- [18F] with non-radioactive 4-fluorocatechol standard
FIG. 1 shows the results of measurement by diluting fluorocatechol. The position of the UV detection signal (A) coincided with the position of the radiation detection signal (B), thereby confirming that the compound was 4- [18F] fluorocatechol. Example 2: Synthesis of 6- [18F] fluoro-L-dopa An ethanol solution (1.0 mL) of 4- [18F] fluorocatechol obtained in Example 1 above was mixed with 300 mM ammonium sulfate, 0.08 mM pyridoxal phosphate, 100 mM Tris-HCl buffer (pH 9), 100 mM sodium pyruvate,
It was added to a mixture (19 mL) containing 4 Unit / mL β-tyrosinase and 9.3 mM sodium ascorbate. After heating the whole mixture at 45 ° C for 5 minutes, trichloroacetic acid (1
g) was added to stop the reaction. The total volume of the reaction solution is
The solution was filtered through a 0.22 μm membrane filter, and the obtained filtrate (20 mL) was passed through a separation column to give 6- [18F].
Fluoro-L-dopa was fractionated and purified. The radiochemical purity of 6- [18F] fluoro-L-dopa obtained as the final product was measured by analytical liquid chromatography using column (A). Chromatography conditions were as follows.

Flow rate: 2 mL / min Solvent: mobile phase (E) Column temperature: room temperature Wavelength: 283 nm Retention time of 6- [18F] fluoro-L-dopa: 4.5
FIG. 2 shows the results obtained by diluting 6- [18F] fluoro-L-dopa with a non-radioactive 6-fluoro-L-dopa standard and a 4-fluorocatechol standard. The position of the UV detection signal (A) coincided with the position of the radiation detection signal (B), thereby confirming that the compound was 6- [18F] fluoro-L-dopa.

Further, the purity of the optical isomer was determined using the column (C).
Was determined by analytical liquid chromatography using Chromatography conditions were as follows. Flow rate: 0.8 mL / min Solvent: mobile phase (G) Column temperature: room temperature Wavelength: 283 nm Retention time of 6- [18F] fluoro-L-dopa: 7.5
~ 8.5 minutes.

The radiochemical yield of each reaction in the series of synthesis reactions and the radiochemical yield of each reaction product with respect to [18F] anion are as shown in Table 1.
The final product, purified 6- [18F] fluoro-L
-The specific activity value of dopa was 10.3 GBg / μmol (EOB).

[0048]

[Table 1]

[0049]

As described above in detail, according to the present invention, 6- [18F] fluoro-L- having a high specific activity can be obtained without producing regioisomers and optical isomers.
Dopa can be easily obtained in high yield from easily available raw materials. This makes it possible to efficiently perform PET diagnosis and the like of brain dopamine synthesis ability related to various neurological diseases.

[Brief description of the drawings]

FIG. 1. Non-radioactive 4-fluorocatechol standard
[18F] Fluorocatechol diluted and measured, (A) UV detection signal of non-radioactive 4-fluorocatechol sample, (B) radiation detection signal of 4- [18F] fluorocatechol.

FIG. 2. Non-radioactive 6-fluoro-L-dopa preparation and 4-
6- [18F] fluoro-L with fluorocatechol standard
And (A) UV detection signals of each of a non-radioactive 6-fluoro-L-dopa sample and a 4-fluorocatechol sample, and (B) 6- [18F ] Is the radiation detection signal of fluoro-L-dopa.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 49/00 A61K 49/00 A C07C 45/63 C07C 45/63 47/575 47/575 C07M 5:00 (72) Inventor Yuji Furuya 20-9 Jusankenya, Kanbe-cho, Shenzhen-gun, Hiroshima Prefecture

Claims (7)

[Claims] 1. The following formula (1) 4- [18F] fluorocatechol represented by the formula
By the enzymatic reaction with tyrosinase, the following formula (2) is obtained. A method for synthesizing 6- [18F] fluoro-L-dopa, which is converted to 6- [18F] fluoro-L-dopa represented by the following formula: 2. The following formula (3): 6- [18F] fluoro-L- according to claim 1, wherein 4- [18F] fluorocatechol is synthesized by a methyl group elimination reaction of 4- [18F] fluoroveratrol represented by the formula:
Dopa synthesis method. 3. The following formula (4): The method for synthesizing 6- [18F] fluoro-L-dopa according to claim 2, wherein 4- [18F] fluoroveratrol is synthesized by an aldehyde group elimination reaction of 6- [18F] fluoroveratraldehyde represented by the following formula: 4. The following formula (5): 6- [18F] fluoroveratraldehyde is synthesized by a nitro group substitution reaction of 6-nitroveratraldehyde represented by the formula:
-Dopa synthesis method. 5. The following formula (3): By the methyl group elimination reaction of 4- [18F] fluoroveratrol represented by the following formula (1): A method for synthesizing 4- [18F] fluorocatechol represented by the formula: 6. The following formula (4): The method for synthesizing 4- [18F] fluorocatechol according to claim 5, wherein 4- [18F] fluoroveratrol is synthesized by an aldehyde group elimination reaction of 6- [18F] fluoroveratraldehyde represented by the following formula: 7. The following formula (5): 9. The method for synthesizing 4- [18F] fluorocatechol according to claim 8, wherein 6- [18F] fluoroveratraldehyde is synthesized by a nitro group substitution reaction of 6-nitroveratraldehyde represented by the formula: