JP2930621B2 - Method for producing labeled compound - Google Patents

Description

The present invention relates to a novel method for producing L-tryptophan labeled with a radioisotope such as a positron nuclide, a β ^- decay nuclide, or a stable isotope, and a derivative thereof.

BACKGROUND OF THE INVENTION Indoleamines such as serotonin and melatonin, which are important neurotransmitters, are biosynthesized via 5-hydroxytryptophan (5-HTP), which is produced by the action of tryptophan under the action of tryptophan hydroxylase.

The biosynthetic ability of the amines is reduced due to various causes,
It has been known that it causes many neuropsychiatric disorders. In these disease groups, quantitative analysis of the transport of aromatic amine acids to the brain, the transport of amine precursors such as 5-HTP, and their use in the brain can be used to objectively determine the diagnostic and therapeutic effects. Important to do. However, a method of administering such 5-HTP to a living body and quantitatively measuring its transport and utilization in the brain has not yet been established.

The present inventors have developed a positron that performs non-invasive diagnostic imaging.
L-tryptophan and L-5-HTP labeled with a positron nuclide were administered to a living body using the CT method, and it was judged that the above-mentioned object could be achieved if the kinetics were traced. By using tryptophanase in the presence of ammonium ion, it was found that tryptophan labeled with a positron nuclide or a derivative thereof could be easily produced, and the present invention was completed.

This method using tryptophanase, ¹⁴ C, ³ H
Can be used to label these compounds with radioisotopes such as, and stable isotopes such as ¹³ C, ¹⁵ N, and ² H, which will be used in future NMR diagnostics and biochemical metabolism studies. Can be used.

Accordingly, the present invention provides a compound of formula [I]: Wherein R ⁵ is H, OH, OCH ₃ , CH ₃ , F or Cl; R ⁶ and R ⁷ are each independently H, OH, OCH ₃ , CH ₃ or F
Wherein one or more carbon atoms and / or nitrogen atoms on the 3-position side chain of the compound represented by the formula are replaced with ¹¹ C and / or ¹³ N positron nuclides. And the formula [II]: In the presence of a positron nuclide labeled or unlabeled ammonium ion in the presence of tryptophanase in the presence of tryptophanase (II)
I]: Wherein R ^{5 to} R ⁷ have the same meanings as defined above (wherein at least one of pyruvate and ammonium ion is a labeled compound). Is).

The present invention further relates to a compound represented by the above formula [I], wherein any one or more carbon, nitrogen and / or hydrogen atoms on the 3-position side chain are each ¹³ C, ¹⁵ N, and / or ^A method for producing a labeled compound in which a stable isotope such as ² H or a carbon atom and / or a hydrogen atom is replaced with a radioisotope such as ¹⁴ C and / or ³ H, which is represented by the above formula [II] Reacting the consensus element-labeled or unlabeled pyruvic acid with the compound represented by the formula (III) in the presence of an isotope-labeled or unlabeled ammonium ion in the presence of tryptophanase. (Wherein at least one of pyruvate and ammonium ion is a labeling compound).

The indole derivative represented by the formula [III] used in the above-mentioned method of the present invention is commercially available or can be produced by a known method. The unlabeled compound of the formula [II] is commercially available, while the labeled compound of the formula [II] can be produced by a conventional method.
For example, pyruvate [II] in which the carbon of the methyl group is an ¹¹ C positron nuclide can be produced from labeled alanine by a known method (Appl. Radiat. Isot., 39 , 627-630, 1988). On the other hand, alanine labeled with a carboxyl group can be derived from CH ₃ CO ¹¹ CN, which is shown in Reference Example 2 below. In addition, labeled ammonium sources can be obtained from commercial sources. Tryptophanase may be purified from the cells by a known method, or a commercially available product may be further purified and used.

In order to carry out the method of the present invention, pyruvate [II] and a compound [III] as a substrate are allowed to stand at 45 ° C. for a short time (typically 1 minute to 5 minutes) in the presence of tryptophanase and ammonium ion. Let react. In this case, if it is desired to obtain a compound in which the carbon at the 3 ′ position of the side chain of the compound of the formula [I] is ¹¹ C, a labeled pyruvic acid, for example, ¹¹ CH ₃ COCOOH may be used. On the other hand, when the nitrogen of the amino group at the 2'-position of the side chain is ¹³
If a compound of N is desired, unlabeled pyruvic acid [II] and a labeled ammonium ion ¹³ NH ₄ may be used. In addition, the desired position of the side chain is ¹¹ C,
^It will be readily understood by those skilled in the art that when it is desired to obtain a compound labeled with ¹³ N, ¹⁵ N, ² H, ¹⁴ C or ³ H, the above method may be appropriately modified and carried out. Further, as a modification, if a similar isotope-labeled compound is used for the indole ring, a labeled compound at a further different position is synthesized. Further, the present invention provides a method for using two or more double-labeled and triple-labeled compounds. It is also easily understood that it is useful for manufacturing.

Hereinafter, an example of a synthesis flowchart of the labeling compound produced by the method of the present invention is shown.

The yield of radioactivity obtained by the production method of the present invention exemplified in Flow Chart 1 is about 25 to 50% from ¹¹ C-alanine, 40 to 70% from ¹¹ C-pyruvic acid, and ¹³ N.
30 to 50% from H _4, radiochemical purity of the labeled The reaction was purified by HPLC is respectively 97%.

Conventionally, the carboxyl group or amino group of tryptophan or its derivative in DL form is replaced with ¹⁴ C, ³ H or ² H
The method of labeling with such as was known. However, these groups are susceptible to metabolism in vivo and, therefore, could not track the mother nucleus of the tryptophan molecule. The method of the present invention makes it possible to label the 3 ′ or 2 ′ position of the side chain which is hardly metabolized, so that the tracking of the mother nucleus can be performed very easily. In addition, it was possible to measure various enzyme reaction rates by comparing and examining the metabolic rate of each group.

On the other hand, the mother nucleus beta ^- radioisotope ³ H to collapse,
Alternatively, it is possible to label over a long period of time by chemical synthesis with C ¹⁴ C or the like, but if these are used, since the radioactivity does not penetrate the body, the position of the labeled compound in the living body can be It cannot be detected. Positron nuclides such as ¹¹ C and ¹³ N used in the method of the present invention undergo β ⁺ decay and collide with body negative electrons to emit γ rays, so that they can be detected from outside the living body and thus administered to the living body. The distribution and localization of the labeled compound can be tracked externally.

For example, the present inventors administered the obtained C ¹¹ C-labeled compound to humans and monkeys, and observed that ¹¹ C-tryptophan was relatively diffusely incorporated into the brain. On the other hand, ¹¹ C−
5-HTP also accumulated in the cerebral cortex, which is considered to be a serotonin terminal. Many metastases (often multiple)
It is readily conceivable that it is particularly useful for diagnosis of carcinoid tumors (serotonin-producing tumors). In this way, the behavior of these labeled compounds in the tumor in the brain can be tracked before and after the treatment or in comparison with the clinical effect, which is extremely significant for the diagnosis and treatment of the disease.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples. However, the present invention should not be construed as being limited thereto.

In the following Reference Examples and Examples, C ¹¹ C is Uppsal
aUniversity, Svedberg Institute, series Van der Graaf
It was prepared by ¹⁴ N (p, α) ¹¹ C using a 10 MeV proton generated by an f-type accelerator and a nitrogen gas target. The obtained [ ¹¹ C] carbon dioxide was trapped in a lead-protected 4Å molecular sieve and transported to the chemical laboratory. [ ¹¹ C] methyl iodide used in the alkylation reaction was synthesized by a general method described in the literature (B. Lngstrm et al., J. Nucl. Med., 2
8 , 1037, 1987). N- (diphenylmethylene) glycine quaternary butyl ester was synthesized by the method described in the literature (WAWood et al., J. Biol. Chem., 170 , 313, 1947).

All enzymes except tryptophanase were purchased from Sigma. A suspension of D-amino acid oxidase from pig kidney (D-AAO, ECl. 4.3.3) in 3.6 M ammonium sulfate (pH 6.5) was used without treatment. The freeze-dried powder of glutamate-birpinate transaminase (GPT, EC 2.6.1.2) derived from pig heart is prepared from a 50 mM potassium phosphate buffer (pH 7.0) containing 0.2 mM pyridoxal phosphate.
Dissolved in 5). Catalase from bovine liver (EC 1.11.1.
6) A suspension of the crystals containing 0.1% thymol was dialyzed against 50 mM potassium phosphate buffer (pH 7.5) before use.

Tryptophanase (EC 4.1.9.1) is a modification of the method of Kagamiyama et al. (T. Honda et al., J. Biochem., 100 , 679, 1986).
And obtained from E. coli according to Purification of the resulting enzyme was performed by Kagamiya
(H. Kagamiyama et al., J. Biol. C
hem., 247, 1571,1972). The specific activity of tryptophanase is determined by So-nitrophenyl-L-cysteine (SOPC).
Was measured by spectrophotometry using CH as a substrate (CH
Suelter et al., FEBS Lett., 66 , 230, 1976), whose specific activity is 4
8 μmol / min / mg protein. Protein concentration of 4
It was 2.4 mg / ml. GPT, to maintain the activity of the enzyme
The prepared enzyme solutions of catalase and tryptopase were stored in small portions in a freezer. After thawing, it can be stored in a refrigerator for about one month while maintaining the enzyme activity.

Analytical LC (liquid chromatography) was performed using a Hewlett-Packard 1090 liquid chromatograph equipped with a UV-diode array detector in series with a β ⁺ -flow detector (B. Lngstrm et al., Radiochem. Radioanal. Letter.
s, 41 , 375, 1979), and the following columns were used.

Column (A): 250 × 4.6 mm LC-NH ₂ (Nucleosil) 10 μm
column.

Column (B): 250 × 4.6mm C-18 (Spherisorb ODS2) 5
μm column.

Column (C): 150 × 4.6 mm C-18 (Supelco) 3 μm column.

Mobile phase (D): 0.01 M potassium dihydrogen phosphate (pH 4.6).

Mobile phase (E): acetonitrile / water (500: 70, v / v).

Mobile phase (F): 17 mM acetic acid Mobile phase (G): 0.05 M ammonium formate (pH 3.5).

Mobile phase (H): methanol.

Preparative (LC) preparative LC was performed using a Waters M-45 pump and a 250 × 10 mm C-18 (Nucleoosil) 10 μm semi-preparative column in series with a Pharmacia dual path monitor UV-2 and β ⁺ -70-detector. (B. Langstr et al., Supra).

Before use, the solid phase extraction C-18 (Supelco) column should be dichloromethane (3 ml), ethanol (10 ml), distilled water (20 m
Pretreated in l). ^{Sep-Pak R C-18 (} Waters) columns were pre-treated with ethanol (10ml) and distilled water (20ml). Cation exchange resin (Bio-Rad Lboratories) AG50
W-X4 (200-400 mesh) was pretreated with distilled water.

Tetrahydrofuran (THF) was dehydrated by distillation from sodium / benzophenone. All other chemical reagents were used without purification.

From Reference Example 1 ^[11 C] molecular sieve produced lead protection methyl iodide, by heating ^[11
C] A specially designed reaction flask (B. Lngstrm et al., J. Nucl.
ed., 28 , 1037, 1987) in THF (0.8 M, 0.5 ml) in lithium aluminum hydride. After evaporation of the THF, hydrochloric acid (57%, 2 ml) was added and refluxed to form the product [
^[ 11C] Methyl iodide was distilled off and transferred to a reaction vessel.

Reference Example 2 Production of DL- [3- ¹¹ C] alanine N- (diphenylmethylene) glycine quaternary butyl ester (3 mg, 10 μm) was placed in a 1.5 ml vial provided with a partition wall.
ol) was dissolved in DMF (450 μl) and DMSO (50 μl). An aqueous potassium hydroxide solution (5 M, 2 μl) was added to this solution, and then [ ¹¹ C] methyl iodide was added to the reaction vessel in a nitrogen stream. After heating the reaction mixture at 80 ° C. for 2 minutes, distilled water (20 ml) connected to a C-18 solid phase extraction column
This mixture was transferred to a syringe containing. After passing the aqueous solution mixture through a column and washing with water (3 ml), the radioactive substance adsorbed on the column was eluted with dichloromethane (2 ml) and received in a 7 ml glass container containing hydrochloric acid (6 M, 0.8 ml). . The mixture was heated and shaken at 130 ° C. for 5 minutes, and the racemic [3- ¹¹ C] alanine solution was diluted with water (1 ml).
Purified through a -Pak ^R C-18 column. The radiochemical purity of this eluate was controlled by analytical liquid chromatography using column A. The chromatography conditions were as follows.

Flow rate: 2 ml / min, solvent: D / E, gradient: 0.8 min 95-60%
E, Column temperature: 40 ° C., wavelength: 230 nm, retention time of [3- ¹¹ C] alanine: 6.1 minutes.

Reference Example 3 [3- ¹¹ C] obtained in Reference Example 2 of pyruvate DL- [3- ¹¹ C] alanine solution was evaporated to dryness, the residue triethylene hydroxyamine / HCl (Tris / HCl) buffer The solution was dissolved in a liquid (pH 8.5, 0.1 M, 0.8 ml). This solution was treated with α-ketoglutarate (α-KG) (0.
2M, 50μl), pyridoxal phosphate (PLP) (10mM, 1
0 μl), and flavin adenine dinucleotide (FA
D) Transferred to a 5 ml glass tube containing (1.7 mM, 10 μl). After adjusting the pH to 8.5, D-AAO (1.9 units), GPT
(12 units), and catalase (3600 units) were added and the reaction mixture was kept at 45 ° C. for 2.5 minutes. Hydrochloric acid (6 M, 0.2 ml) was added to stop the reaction, and the mixture was filtered through a 0.22 μm pore filter, and the radiochemical yield was measured by analytical liquid chromatography under the same conditions as those described in Reference Example 2. . [3- ¹¹ C] the retention time of pyruvic acid was 3.7 minutes.

Evaporated to dryness eluate containing [3- ¹¹ C] alanine purified racemate obtained in Example 1 ^L-[beta-11 C] Reference Example 2 of tryptophan, the residue Tris / HCl
Redissolved in buffer (pH 9.0, 0.1 M, 0.6 ml). This solution was treated with ammonium sulfate (AmS), Tris / HCl (pH 9.0), α
-KG, FAD and PLP, adjust the pH to 9.0 with 1 M potassium hydroxide and add enzyme to make the composition of this final solution as follows: D-AAO (1.9 units), GPT ( 12 units), catalase (3600 units), tryptophanase (100 units; 1 unit = 1 μmol SOPC / min), Tris / HCl (pH 9.
0, 0.1 M), AmS (0.15 M), α-KG (10 mM), PLP (0.1 m
M) and FAD (17 μM). The reaction mixture was thermostated at 45 ° C. for 15 seconds and an indole ethanol solution (0.5 M, 10 μl) was added.
l) was added. After keeping the reaction mixture at 45 ° C. for 3 minutes,
Hydrochloric acid (6M, 0.2ml) was added to stop the reaction. The denatured enzyme was removed by filtration through a 0.22 μm pore filter, and the resulting clear solution was purified by preparative liquid chromatography. As the mobile phase, 17 mM acetic acid / ethanol (80: 2
0, v / v) and the flow rate was 8.0 ml / min. The radioactive fraction containing the desired compound was collected and evaporated. The residue was treated with saline (5 ml) and phosphate buffer (pH 7.4, 1 ml)
And then adjusted the pH to about 6. 0.22
Sterilized through a μm pore filter and received in sterile vials. This vial is used for human or animal administration.

Radiochemical purity was analyzed by HPLC under the following conditions.

Condition 1 Column: Column A, flow rate: 2.0 ml / min, solvent: D / E, gradient: 0-8 min 95-60% E, column: 40 ° C., wavelength: 230 n
m. Retention time was 4.1 minutes.

Condition 2 Column: column B, flow rate: 2.0 ml / min, solvent: F / H, isocratic: 40% H, column temperature: 60 ° C., wavelength: 278 n
m. Retention time was 4.5 minutes. Non-radioactive L- tryptophan preparation L- ^[beta-11 C] Results of measurement by diluting the tryptophan shown in Figure 1. The position of the UV-detection signal and the position of the radiation detection signal were in agreement, confirming that the compound produced by this method was L-tryptophan.

Enantiomeric purity The enantiomeric purity was measured as follows.

After completion of the enzyme reaction, the reaction mixture was loaded on a cation resin exchange column containing about 2 ml of AG50W-X4 resin. The column was washed with distilled water (4 ml), and the ¹¹ C-labeled amino acid was eluted with ammonium hydroxide (2M). The eluate was evaporated to dryness, and the residue was dissolved in an aqueous solution of DL-Trp (0.05 M, 0.1 ml). This solution was transferred to a 1.5 ml glass vial and N- (2,4-dinitro-5-fluorophenyl) -L-
Alanine amide (DFAA) solution (1%, 0.2 ml) and sodium bicarbonate solution (1 M, 40 μl) were added. The vial was sealed with a septum and reacted at 60 ° C. for 15 minutes, after which hydrochloric acid (2 M, 20 μl) was added. The obtained solution was analyzed by HPLC using column C under the following conditions. Flow rate: 1.0ml
/ Min, solvent: G / H, gradient: 0-15 min 25-50% H, column temperature: 50 ° C., wavelength: 340 nm.

The retention times of the DFAA derivatives of L-Trp and D-Trp were 12.0 minutes and 16.4 minutes, respectively (the retention time of unreacted DFAA was 8.8 minutes).

Evaporated to dryness eluate containing [3- ¹¹ C] alanine purified racemate obtained in Example 2 5-hydroxy -L- ^[beta-11 C] Reference Example 2 of tryptophan, the residue Tris / HCl
Redissolved in buffer (pH 9.0, 0.1 M, 0.6 ml). This solution was treated with ammonium sulfate (AmS), Tris / HCl (pH 9.0), α
-Into a mixture of KG, FAD and PLP, adjust the pH to 9.0 with 1 M potassium hydroxide, add enzyme and make up the composition of this final solution as follows: D-AAO (1.9 units), GPT (12 units) ), Catalase (3600 units), tryptophanase (100 units; 1 unit = 1 μmol SOPC / min), Tris / HCl (pH 9.
0, 0.1 M), AmS (0.15 M), α-KG (10 mM), PLP (0.1 m
M) and FAD (17 μM). The reaction mixture was thermostated to 45 ° C. for 15 seconds and a solution of 5-hydroxyindole in ethanol (0.5 M, 10 μl) was added. After maintaining the reaction mixture at 45 ° C. for 3 minutes, hydrochloric acid (6M, 0.2 ml) was added to stop the reaction. The denatured enzyme was removed by filtration through a 0.22 μm pore filter, and the resulting clear solution was purified by preparative liquid chromatography. The mobile phase used was 17 mM acetic acid / ethanol (92: 8, v / v) and the flow rate was 8.0 ml / min. The radioactive fraction containing the desired compound was collected and evaporated. The residue was dissolved in saline (5 ml) and phosphate buffer (pH 7.4, 1 ml), then the pH was adjusted to about 6.
The solution was sterilized through a 0.22 μm pore filter and placed in a sterile vial. This vial is used for human or animal administration.

Radiochemical purity was analyzed by HPLC under the following conditions.

Condition 1 Column: Column A, Flow rate: 2.0 ml / min, Solvent: D / E, Gradient: 0-8 min 95-60% E, Column temperature: 40 ° C., Wavelength:
230 nm. Retention time was 4.1 minutes.

Condition 2 Column: Column B, flow rate: 2.0 ml / min, solvent: F / H, isocratic: 10% H, column temperature: 60 ° C., wavelength: 278 n
m. Retention time was 5.8 minutes. In the same manner as in Example 1,
The structure of the product was confirmed by comparison with a non-radioactive standard (FIG. 2).

Enantiomeric purity The enantiomeric purity was measured as follows.

After completion of the enzyme reaction, the reaction mixture was loaded on a cation resin exchange column containing about 2 ml of AG50W-X4 resin. The column was washed with distilled water (4 ml), and the ¹¹ C-labeled amino acid was eluted with ammonium hydroxide (2M). The eluate was evaporated to dryness and the residue was dissolved in an aqueous solution of DL-5-HTP (0.05 M, 0.1 ml). This solution was transferred to a 1.5 ml glass vial and N- (2,4-dinitro-5-fluorophenyl) in acetone
-L-alanine amide (DFAA) solution (1%, 0.2 ml) and sodium bicarbonate solution (1 M, 40 μl) were added.
The vial was sealed with a septum and reacted at 60 ° C. for 15 minutes, after which hydrochloric acid (2 M, 20 μl) was added. The obtained solution was analyzed by HPLC using column C under the following conditions. Flow rate: 1.0 ml / min, solvent: G / H, gradient: 0-15 minutes 25-50
% H, column temperature: 50 ° C, wavelength: 340 nm.

The retention times of the DFAA derivatives of L-5-HTP and D-5-HTP were 7.0 minutes and 8.3 minutes, respectively (unreacted DFAA
Retention time is 8.8 minutes).

[Brief description of the drawings]

1 and 2 show that the radioactive product was diluted with the corresponding non-radioactive substance sample and subjected to HPLC analysis, and that the retention times indicated by the radiation detection signal and UV detection signal were the same. FIG. 1 is a chromatogram showing
FIG. 2 is a graph for 5-hydroxytryptophan.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Peter Burling, Uppsala, Sweden, 752-27, Norteljegatan No. 23 (58) Field surveyed (Int.Cl. ⁶ , DB name) C12P 13 / 00-17/18 CA (STN)

Claims (1)

(57) [Claims] (1) Formula [I]: Wherein, R ⁵ represents H or OH] 3-position carbon atom on the side chain of the compound represented by (3 ') is ¹¹
A process for preparing a labeled compound replaced with a C positron nuclide, comprising the step of formula [II]: Formula [II in the ¹¹ C-labeled pyruvate represented, the presence of ammonium ions in the presence of tryptophanase
I]: [Wherein R ⁵ has the same meaning as described above].