JPH0255434B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring pterin derivatives and a novel pterin derivative useful therein.

Pteridine derivatives, represented by folic acid, have recently been shown to play a role as coenzymes in amino acid metabolism, and knowing their quantitative changes in the body is important from both basic and clinical medical perspectives to understand pathological conditions such as various enzyme deficiency diseases. It has come to be considered indispensable for analysis and diagnosis.

In particular, a relationship between phenylketonuria and biopterin has recently been reported (Ann. Neurol. 3) .
224-230 (1978),
TheNewEnglandJournalofMedicine 299 673~
679 (1978) and Clinina Chimica Acta 93 251~
262 (1979)), quantitative changes in the body of pterin, or 2-amino-4-hydroxypteridine, have attracted attention.

The inventor is Radio Immunoassay (hereinafter referred to as RIA)
biopterin (2-amino-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)pteridine) and neopterin (2
-amino-4-hydroxy-6-(D or L-
We planned to develop a method for measuring pterins mainly containing erythro-1,2,3-trihydroxypropyl) pteridine, and produced pterin derivatives represented by the following general formula.

(In the formula, R ₆ and R ₇ each mean H, alkyl or hydroxyalkyl, and R means H or radioactive iodine.) This compound is basically produced by the method shown in the following reaction formula. be able to.

That is, 4-amino-6-hydroxy-2-
The nitroso group of methylthio-5-nitrosopyrimidine () is reduced using an appropriate catalyst, and the 4,5-diamino product () is purified, or the crude product is purified with arabinose/phenylhydrazone etc. in a solvent. Condensation and ring closure are performed by heating to produce 4-hydroxy-2-methylthiopteridine ().

When this product is heated in a solvent with tyramine in the presence of ammonium acetate, a pterin derivative () in which a (p-hydroxyphenyl)ethylamino group is substituted for the methylthio group at the 2-position is obtained; When labeled with radioactive iodine, it becomes a compound of formula (), which can be used as a tracer for RIA. As a labeling method, it is appropriate to use the most common chloramine T method, in which radioactive sodium iodide and chloramine T are used to react, and the reaction is stopped with sodium metabisulfite.

The antigen for producing antibodies used in RIA may be a substance in which the pterin to be measured is bound to a protein such as bovine serum albumin (BSA). For example, the 2-amino group of pterin is ε-aminocaproic acid. A condensate of a substituted compound and BSA is suitable.

To obtain antibodies from condensates of pterins and proteins, etc., the antigen is suspended in Freund's adjuvant and injected into mammals (rabbits, guinea pigs, sheep, goats, etc.) using a general antibody production method. After the appropriate booster immunization, blood is collected and blood cells are removed to obtain antiserum.

Piopterin antibodies include tetrahydrobiopterin, dihydrobiopterin, neopterin, 6,
Cross-reactivity with 7-dimethylpterin, pterin, and folic acid was extremely small, and the neopterin antibody and dimethylpterin antibody also had almost no cross-reactivity with other pterins, indicating high specificity.

For RIA, samples such as urine are pretreated as necessary. For example, in RIA of biopterin, the reduced dihydrobiopterin that coexists,
It is preferable to oxidize tetrahydrobiopterin to form biopterin before subjecting it to the assay.

In RIA, antiserum diluted to an appropriate concentration, a buffer containing a specimen or standard, and a labeling substance as a tracer are mixed and incubated at an appropriate temperature, for example, room temperature, and the resulting antigen-antibody reaction is combined with two antibodies. The radioactivity of one of the two is measured, a standard curve is created from the values for the standard, and the amount of pterins in the sample is determined from this.

The conventional method for measuring biopterin is the bioassay method (Methods in Enzymol.18B 618 (1971)).
and the RIA method previously developed by the present inventor (Proc.
Japan Acad. 55 319 (1979)), but the present invention is far superior to them and is useful as a clinical testing method. That is, the bioassay method takes a long time to obtain results, and in the RIA described above, the tracer and the antigen for antibody production are bound to the labeling moiety or BSA through the same type of bridge (e.g., ε-aminocaproic acid). When using this method, a step is required to remove antibodies against this bridge portion, making processing operations complicated. The RIA of the present invention does not require this step, the overall processing time is short, and above all, its sensitivity is far superior to these, which is a major advantage.

Next, an example will be given and explained.

Example 1 18 g of 4-amino-6-hydroxy-2-methylthio-5-nitrosopyrimidine () was dissolved in 500 ml of 0.5M potassium hydroxide aqueous solution, 10 g of 5% palladium-carbon was added, and the theoretical amount of hydrogen was dissolved at room temperature and normal pressure. was returned until it was consumed. After removing the catalyst by filtration, the pH of the filtrate was adjusted to 3 to 4 with formic acid, and the 5-amino compound ()
Colorless needle crystals precipitate. The crystals were added to a mixture of 32 g of 5-deoxy-L-arabinose phenylhydrazone and 400 ml of methanol without being isolated, and stirred for 90 minutes at 25 DEG C. under nitrogen gas and then for 30 minutes under reflux. Next, it was cooled in an ice bath, and 100 g of potassium hexacyanoferrate () and an aqueous potassium iodide solution (5 g/500 ml) were added to the mixture and the pH was adjusted to 25
The mixture was stirred for 20 hours while bubbling oxygen. The reaction solution is 500
It was concentrated under reduced pressure to ml, the pH was adjusted to 9-10 with ammonia, and the non-fluorescent solid was filtered off. The filtrate was passed through a Florisil column (5 x 60 cm) and eluted with water to obtain fractions with two types of blue fluorescence. The main fraction was concentrated to 150 ml and subjected to the same treatment again on the Florisil column. The eluate was dried under reduced pressure, the residue was extracted with 500 ml of hot methanol, the extract was concentrated to 50 ml, and upon cooling, 4-hydroxy-
5.5 g of colorless needles of 6-(L-erythro-1,2-dihydroxypropyl)-2-methylthiopteridine (a) were obtained. Decomposes (crystallizes from water) above 210℃.

Elemental analysis C ₁₀ H ₁₂ N ₄ O ₃ S・2H ₂ O Calculated value C 39.46, H 5.31, N 18.41 Experimental value C 40.27, H 4.45, N 18.65 Instead of 5-deoxy-L-arabinose phenylhydrazone D- or L-arabinose
Using phenylhydrazone, 4-hydroxy-6-(D-erythro-1,2,3-trihydroxypropyl)-2-methylthiopteridine (b) and its L-isomer (c) were obtained, respectively. When crystallized from water, both isomers showed a decomposition point of 156-158℃, and the elemental analysis was C ₁₀ H ₁₂ N ₄ O ₄ S・H ₂ O
For the calculated values C39.72, H4.68, N18.54,
The experimental values are C39.63, H4.68, N18.03, L-
The body was C39.75, H4.63, N18.17.

Example 2 Compound (a) 0.2g, ammonium acetate 0.6g
and 5 ml of concentrated aqueous ammonia were mixed and heated under reflux for 4 hours. After adjusting the pH to 2 with hydrochloric acid, it was applied to a Florisil column (3.5 x 20 cm) and eluted with water. The eluate was dried under reduced pressure, and the residue was extracted with 50 ml of 1% aqueous ammonia. The extract was concentrated to 10 ml, adjusted to pH 3, and then cooled to obtain 90 mg of biopterin (a) as ivory needle-shaped crystals. The physical properties of this product matched those of the standard product.

Similarly, D-erythro-neopterin (b) and L-erythro-neopterin (c) were obtained from compounds (b) and (c), respectively.

Example 3 Compound (a) 1.0g, tyramine 3.0g, acetic acid 0.8
g and 50% aqueous solution of 2-methoxyethanol were mixed and heated to 100-105°C for 6 hours. of reaction solution
The pH was adjusted to 1-2 with hydrochloric acid, applied to a Florisil column (3.5 x 40 cm), unreacted starting material was eluted with 500 ml of 2M aqueous formic acid solution, then 500 ml of water, and then eluted with 1000 ml of 0-2% aqueous ammonia. The eluate was concentrated to 150 ml and treated with the Florisil column again in the same manner. The eluate was concentrated to dryness, extracted with 200 ml of 2% aqueous ammonia, and concentrated to 100 ml. Adjust the pH of this substance to 3-4 with formic acid and cool it to 4-
Yellow needles of hydroxy-2-[p-hydroxyphenyl)ethyl]amino-6(L-erythro-1,2-dihydroxypropyl)pteridine (a) were obtained. Decomposition point 165℃ (crystallized from water).

Elemental analysis C ₁₇ H ₁₉ N ₅ O ₄・H ₂ O Calculated value C 54.39, H 5.64, N 18.66 Experimental value C 54.56, H 5.62, N 18.38 Similarly, compound (b) or compound (
A compound (b or c) in which each 2-amino group was substituted with a 2-(p-hydroxyphenyl)ethyl group was obtained from c) and tyramine. Decomposition point (b): 180-182℃ (crystallized from water), (c):
182-184℃ (crystallized from water), elemental analysis value is
Calculated value as C ₁₇ H ₁₉ N ₅ O ₅・2H ₂ O C49.87,
H5.66, N16.72 (b): C49.78, H5.05,
N16.72, (c): C49.88, H5.23, N16.90.

Example 4: Iodine labeling Contains 10 μg of compound (a) obtained in Example 3
DMF 10μ 20μ 0.05M phosphate buffer (PH
7.4), to which was added 10 μg of Na ¹²⁵ I (1.2 mCi), and then 20 μg of chromilan T dissolved in 10 μg of the same buffer. This was reacted at 25℃ for 30 seconds,
The reaction is terminated by adding 40μg of sodium metabisulfite dissolved in 10μ of the same buffer. The product was purified by electrophoresis in cellulose acetate-0.05M phosphate buffer (PH 7.6) and then extracted with phosphate buffer containing 5% BSA to produce ¹²⁵ I-labeled 4-hydroxy-2-[2-( p-hydroxyphenyl)ethyl]amino-6-(L-erythro-1,2-dihydroxypropyl)pteridine (a) was obtained.
Rf0.48 (silica gel thin layer; ethyl acetate-methanol-5% aqueous ammonia (1:1:0.2).

Example 5: Production of BSA condensate for antibody production 10 g of 4-amino-6-hydroxy-2-methylthio-5-nitrosopyrimidine () and 20 g of ε-aminocaproic acid were added to 400 ml of water and heated under reflux for 1 hour. The reaction solution was adjusted to pH 2 to 3 with formic acid, cooled, and the resulting precipitate was collected by filtration to give 4-amino-2-
8.5 g of (5-carboxypentylamino)6-hydroxy-5-nitrosopyrimidine was obtained. Dissolving this substance in hot dilute ammonia water and acidifying it with formic acid yields orange-red needle-shaped crystals. of this
Add 4.3 g to 60 ml of 2M sodium hydroxide solution and perform catalytic reduction using 2 g of palladium on carbon. After hydrogen absorption is completed, 20 ml of concentrated hydrochloric acid is added and the catalyst is filtered off. The filtrate was concentrated and the resulting crystals were dried over phosphorus pentoxide to obtain 9.5 g of 4,5-diamino-2-(5-carboxypentylamino)-6-hydroxypyrimidine dihydrochloric acid monohydrate. of this
Add 5.0 g and 3.7 g of 5-deoxy-L-arabinose phenylhydrazone to 400 ml of 50% methanol, and reflux for 20 minutes in a nitrogen stream. The reaction solution was adjusted to pH 5 and air was passed through it for 6 hours. Evaporate the reaction solution to dryness, add 100 ml of water, adjust the pH to 2.0 with formic acid, apply to a 400 ml Florisil column, wash with 0.25M formic acid, and elute with water. Evaporate the eluate to dryness and dilute with methanol 250
ml and dry the extract. This product was crystallized from 10 ml of ethanol to obtain 465 mg of 2-(5-carboxypentylamino)-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)pteridine. Add 74mg of this product and 168μ of triethylamine to 1ml of DMF, cool to -5℃,
Add 87μ of ethyl chloroformate and stir for 15 minutes. This reaction solution was mixed with 114 mg of bovine serum albumin.
Add to a mixture of 2 ml of water and 200μ of 1M sodium hydroxide solution and heat at -5℃ for 30 minutes while adjusting the pH to 9.
Stir at 0°C for 1 hour and then at room temperature for 1 hour. next
Add 5 ml of 1M sodium hydroxide and leave at room temperature for 30 minutes. The reaction solution is poured into a cellophane tube and dialyzed in running water. Next, this cellophane tube is placed in a beaker containing 300 ml of water containing 1 g of sodium acetate, the external solution is adjusted to pH 4.5, and dialyzed for 6 hours. Centrifuge the internal solution, freeze-dry the obtained precipitate,
2-(5-carboxypentylamino)-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)pteridine/BSA condensate (-NH ₂ at the 2-position of biopterin is -NH(CH ₂ ) ₅ CO・
(which became BSA) 95mg was obtained.

Example 6: Production of antibodies The condensate of biopterin and BSA obtained in Example 5 was injected intradermally (1 mg/mouse) into New Zealand white rabbits together with complete Freund's adjuvant.
One month later, booster immunization (0.4 mg/mouse) was given in the same manner, and thereafter 1
A total of four booster immunizations were carried out in the same manner every week,
Blood was collected 10 days after the final booster to obtain antiserum.

Example 7: Pretreatment of sample urine Add 2M hydrochloric acid to 0.1-0.5ml of fresh urine to 1/10 of the sample urine volume.
and 1/10 volume of 2% _I2-4 % KI solution were added, and the mixture was left at room temperature in the dark for 1 hour. Next, 1/10 volume of a 2% ascorbic acid solution was added to the sample urine volume to stop oxidation. After evaporating hydrochloric acid by freeze-drying the reaction solution (neutralization with sodium hydroxide may be used instead of freeze-drying), 0.3% BSA was added.
This solution was redissolved in 0.02M phosphate buffer (PH7.5) containing 100% phosphate and used in the assay.

Example 8: A 0.02M phosphate buffer (PH7.4) containing 0.1% BSA was used for RIA assay of biopterin. Buffer 100μ and approx. 4000~
Mix 100μ of 5000-fold diluted antiserum with 100μ of buffer containing standard biopterin (0-300 pmol) or
Add 100 µl of 50-100-fold diluted urine for 30 min at 37 °C.
After incubating for 10 minutes, 100μ (10000-20000cpm) of the compound (a) solution obtained in Example 4 was added to each
was added and left at 4°C for 1 to 2 hours. To this was added 100μ of anti-rabbit IgG as a second antibody, and the mixture was left at room temperature for 10 minutes. Next, 100μ of 4% dextran T-70 (manufactured by Pharmacia) was added, mixed vigorously, and heated at 4°C.
The mixture was centrifuged at 2500 rpm for 15 minutes, and the radioactivity of the precipitate was measured.

The standard curve (A) created based on the measured values for standard biopterin is shown in the drawing. B-N/B ₀ -N (%) on the vertical axis of the drawing indicates that the amount of biopterin is 0.
It is the value obtained by dividing the radioactivity of the precipitate for each biopterin concentration by the radioactivity of the precipitate at . Control standard curve (B)
The 2- _NH2 of biopterin is used as a tracer. This is an old method using a compound, and it is recognized that the RIA of the present invention has high sensitivity.

[Brief explanation of the drawing]

The drawing is the RIA standard curve.

Claims (1)

[Claims] 1. General formula (In the formula, Q means [formula], and R ₁ means H or radioactive iodine.
R ₆ means dihydroxypropyl and R ₇ means H. ) pterin derivatives. 2 General formula (In the formula, Q means [formula], R ₁ and R ₂ mean H or radioactive iodine, R ₁ and R ₂ are not both H, R ₆ is dihydroxypropyl, R ₇ is H A radioimaging assay method for pterins using a pterin derivative represented by the following as a tracer.