JPS6152814B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for radioimmuno assay of metanephrines, and more particularly, a novel radioactive iodophenol-metanephrine derivative and a reagent for radioimmuno assay of metanephrines comprising the metanephrine derivative. Regarding.

Catecholamines are sympathetic nervous system stimulating transmitters, and are directly involved in the nervous regulatory mechanism of the living body, and are also known to play a major role in the metabolism of various substances, including sugars and fats. Therefore, the dynamics of catecholamines most accurately reflects the function of the sympathetic nervous system, and understanding the secretion dynamics of catecholamines in the living body will help elucidate various biological phenomena and prevent various diseases associated with abnormal catecholamine secretion, such as It is extremely important for research and diagnosis of pathophysiology of Parkinson's disease, pheochromocytoma, etc.

However, since catecholamines are metabolized in various ways in vivo, there is a risk of making mistakes if the secretion dynamics of catecholamines is estimated immediately by simply quantifying catecholamines in a sample.
Therefore, by appropriately quantifying metanephrine, which is one of the metabolites, and making a comprehensive judgment, it is possible to accurately determine the function of the sympathetic nervous system.

Recently, a method for quantifying metanephrines using radioimmunoassay has been proposed.

The method is as follows [Yoshiaki Omori et al.
[Presented at the 51st General Meeting of the Japanese Endocrine Society (1978)], but because this method used tritium, which has a low energy level, as a label, the detection sensitivity was poor and it was still not desirable.

As a result of various studies on improving the sensitivity of radioimmunoassay for metanephrines, the present inventors found that the following formula In the formula, R represents a lower alkyl group; n represents an integer of 1 to 4; Z ₁ and Z ₂ are the same or different and represent a hydrogen atom or a lower alkanoyl group;
X ₁ and X ₂ are the same or different and represent a hydrogen atom or a radioactive isotope of iodine, provided that X ₁ and X ₂
By using a radioactive iodophenol-metanephrine derivative represented by , which does not simultaneously represent a hydrogen atom, as a labeling compound, the detection sensitivity of metanephrine can be increased to about 10 times or more than when using tritium-labeled metanephrine of the above formula (). was also found to improve

Thus, according to the present invention, a radioimmunoassay reagent and a method for quantifying metanephrine are provided, which use the radioactive iodophenol-metanephrine derivative of the above formula () as a labeling compound.

The labeling compound of the above formula () used in the present invention is a novel compound that has not been described in any conventional literature, and constitutes a part of the present invention.

In the above formula (), the lower alkyl group represented by the symbol R can be a linear or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, n- Or iso
-propyl group, among which methyl group is suitable. Among the integers from 1 to 4 represented by the symbol n, preferred is the case where n represents 3.
Further, when the symbols Z ₁ and Z ₂ represent a lower alkanoyl group, the lower alkanoyl group can be a linear or branched alkanoyl group having 1 to 4 carbon atoms, such as formyl , acetyl, and propionyl groups, among which acetyl group is suitable.

On the other hand, when the groups represented by the symbols X ₁ and X ₂ represent radioactive isotopes of iodine, the radioactive isotopes include all radioactive isotopes of iodine, Examples include ¹²⁵ I and ¹³¹ I. However, in the present invention
One of X ₁ and X ₂ is always limited to a radioactive isotope of iodine, except when both X ₁ and X ₂ are hydrogen atoms at the same time.

Accordingly, a preferred group of radioactive iodophenol-metanephrine derivatives of the formula () provided by the present invention include the following formula (-a): In the formula, Z ₁₁ and Z ₂₁ are the same or different and represent a hydrogen atom or an acetyl group; X ₁ and X ₂ have the above meanings;

Thus, representative examples of the radioactive iodophenol-metanephrine derivatives represented by the formula () or (-a) provided by the present invention are as follows.

1-[N-[2-hydroxy-3-methoxy-5
-(1-acetoxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-
3-(3-iodo-4-hydroxyphenyl)propane- ¹²⁵ I (or ¹³¹ I), 1-[N-[2-hydroxy-3-methoxy-5
-(1-acetoxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-
3-(3,5-diiodo-4-hydroxyphenyl)propane- ¹²⁵ I (or ¹³¹ I), 1-[N-[2-hydroxy-3-methoxy-5
-(1-hydroxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-
3-(3-iodo-4-hydroxyphenyl)propane- ¹²⁵ I (or ¹³¹ I), 1-[N-[2-hydroxy-3-methoxy-5
-(1-hydroxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-
3-(3,5-diiodo-4-hydroxyphenyl)propane- ¹²⁵ I (or ¹³¹ I), 1-[N-[2-hydroxy-3-methoxy-5
-(1-hydroxy-2-methylaminoethyl)
phenylmethyl]-N-methylamino]-3-(3
-Iodo-4-hydroxyphenyl)propane-
¹²⁵ I (or ¹³¹ I), 1-[N-[2-hydroxy-3-methoxy-5
-(1-hydroxy-2-methylaminoethyl)
phenylmethyl]-N-methylamino]-3-
(3,5-diiodo-4-hydroxyphenyl)
Propane- ¹²⁵ I (or ¹³¹ I), 1-[N-[2-hydroxy-3-methoxy-5
-(1-hydroxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-
2-(3-iodo-4-hydroxyphenyl)ethane- ¹²⁵ I (or ¹³¹ I), etc.

According to the present invention, the radioactive iodophenol-metanephrine derivative represented by the formula () is:
The following formula () In the formula, R, n, Z ₁ and Z ₂ have the above-mentioned meanings, and if a lower alkanoyl group is present in the resulting reaction product, it may be hydrolyzed if necessary. It can be manufactured by

Radioiodination of the compound of the above formula () can be carried out by means known per se.

As the radioiodinating agent, in addition to radioactive iodine itself, for example, a combination of a water-soluble radioactive iodine compound and an oxidizing agent can be used. Preferred examples of water-soluble radioactive iodine compounds include radioactive sodium iodide, radioactive potassium iodide, and radioactive lithium iodide. Examples of oxidizing agents include chloramine T, chloramine B, N-chlorosuccinimide, and N-bromsuccinic acid. Imide, N-
Examples include chloracetamide and N-bromoacetamide, among which a combination of radioactive sodium iodide and chloramine T is preferred.

To radioiodinate a compound of formula () using the above radioiodinating agent, for example, first dissolve the compound of formula () in an inert solvent, such as water; an alcohol such as methanol or ethanol. , after adding acid to make the whole acidic, the above-mentioned radioactive iodinating agent is added, usually in slight excess,
The reaction is carried out at a temperature of 0 to 50°C, preferably at room temperature.
After completion of the reaction, the generated radioactive iodophenol-metanephrine derivative of formula () can be isolated by conventional methods such as distillation, recrystallization, chromatography, etc.; It can also be used as a mixture with the compound of formula () in this reaction for the above-mentioned purposes.

If a lower alkanoyl group is present in the radioactive iodophenol-metanephrine derivative of the formula () thus obtained, it may be hydrolyzed as necessary.

The hydrolysis is carried out using an alkali or acid in the presence or absence of water or a water-miscible organic solvent, such as alcohols such as methanol and ethanol, ketones such as acetone and methyl ethyl ketone, and ethers such as tetrahydrofuran and dioxane. can be done.

Examples of the alkali that can be used in the hydrolysis include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and barium hydroxide, alkaline earth metal hydroxides, and alkali metal carbonates such as sodium carbonate and potassium carbonate. Acids that can be used include hydrochloric acid, sulfuric acid,
Examples include p-toluenesulfonic acid, phosphoric acid, trifluoromethanesulfonic acid, and the like.

These acids or alkalis are compound 1 of formula ()
It can be used generally in a proportion of at least 2 equivalents, preferably 5 to 50 equivalents, based on mole.

Hydrolysis can be carried out at about 0°C to the reflux temperature of the reaction mixture, but generally at 20-100°C.
A temperature of .

The compound of formula () used as a starting material in the method of the present invention described above is a novel compound not described in conventional literature, and can be produced, for example, as shown in reaction formula A below. .

In each of the above formulas, R and n have the above meanings, and Z
represents a lower alkanoyl group, and Z' and Z'' are the same or different and are a hydrogen atom or a lower alkanoyl group, provided that Z' and Z'' do not simultaneously represent a lower alkanoyl group.

In the above reaction formula A, metanephrine of formula () is first reacted with a lower alkanoic acid or a reactive derivative thereof. This reaction is usually carried out in an inert organic solvent, such as organic bases such as triethylamine, dimethylaniline, and pyridine; halogenated hydrocarbons such as chloroform and carbon tetrachloride; and aromatic hydrocarbons such as benzene and toluene. The reaction can be carried out in a system containing these organic solvents at room temperature to elevated temperature, preferably at a temperature of about 20 to 100°C. Examples of lower alkanoic acids or reactive derivatives thereof that can be used include acetic anhydride, acetyl chloride, formic acid, acetic acid, and propionic anhydride. The ratio of the metanephrine of the formula () and the lower alkanoic acid or its reactive derivative is not particularly limited, but generally at least one mole of the lower alkanoic acid or its reactive derivative is used per mole of the metanephrine of the formula (). 3
It is desirable to use an amount of equivalents, preferably 5 to 50 equivalents.

The resulting compound of formula () is then partially hydrolyzed. Partial hydrolysis of the compound of formula () can be carried out in the same manner as described above for hydrolysis. Examples of the acid or alkali that can be used include those mentioned above, but in this hydrolysis reaction, in order to selectively hydrolyze only the lower alkanoyloxy group substituted on the benzene nucleus, the acid or alkali used is The alkali is preferably used in the form of a dilute acid or dilute alkali.

The compound of formula () obtained by the above partial hydrolysis is converted into the compound of formula () by subjecting it to Mannitz reaction. The Mannitz reaction between the compound of formula () and the compound of formula () is usually carried out by bringing the compound of formula () into contact with the amine of formula () in the presence of formaldehyde or paraformaldehyde in an inert solvent. Examples of inert solvents that can be used include:
Water; alcohols such as methanol, ethanol and iso-propanol; organic acids such as acetic acid, propionic acid and butyric acid. The reaction is carried out at room temperature to elevated temperature, preferably from about 20°C to the reflux temperature of the reaction mixture, using formaldehyde or paraformaldehyde in an amount of at least 1 equivalent, preferably 1.1 to 2 equivalents, per mol of the compound of formula (). It is desirable to use and do this. formula()
The ratio of the compound of formula () to the amine of formula () is not particularly limited, but generally 1 to 1 mole of the compound of formula () is used.
Preferably, it is used in an amount of 1.5 equivalents. Thus, under the conditions described above, the Mannitz reaction can be completed in about 5 to 50 hours.

In this way, a compound of the formula () is obtained, and this compound is then hydrogenolyzed to obtain a compound of the formula (
-1). This hydrogenolysis is
Usually in an inert solvent, for example, alcohols such as methanol and ethanol, ethers such as diethyl ether and tetrahydrofuran, etc., in the presence of a metal catalyst, at room temperature to elevated temperature, preferably for about 100 min.
It can be carried out at a temperature of -50 DEG C. and a hydrogen pressure of 1 to 100 atmospheres, preferably 1 to 10 atmospheres. Usable metal catalysts include ordinary hydrogenation catalysts,
Examples include palladium, rhodium, platinum, Raney nickel, U-nickel, etc. Among them, when palladium on carbon is used as a catalyst, it is advantageous because hydrogenolysis can be carried out under relatively mild conditions.

The compound of formula (-1) obtained in this way can itself be subjected to the above-mentioned radioiodination reaction as a starting material of the present invention, but by further hydrolysis, the compound of formula (-1) included in the above formula () can be obtained. It can be changed to the compound (-2).
This hydrolysis can be carried out in the same manner as described above. That is, this hydrolysis reaction can be completed by using a strong acid or strong alkali, such as concentrated hydrochloric acid, at a temperature of about 10 to 50° C. for about 5 to 50 hours.

Formula () used in the above Mannitz reaction
The amine of the formula () is also a new compound that has not been described in conventional literature. For example, in the above formula (), when R represents a methyl group and n represents 3, the amine of the formula () is represented by the following reaction formula. It can be manufactured as shown in B.

In the above reaction formula B, the carboxylic acid of formula () is amidated. This amidation is advantageously carried out by first converting the carboxylic acid of formula () into its reactive derivative and then contacting it with an amine. Examples of the reactive derivative of the carboxylic acid of formula () include acid halides, active esters, mixed acid anhydrides, and the like. formula()
The conversion of carboxylic acid into a reactive derivative can be easily carried out according to methods known per se, for example, in the case of acid chlorides, by contacting the compound of formula () with thionyl chloride. Although the reactive derivative of the carboxylic acid of the formula () obtained by the above reaction can be isolated, it can be easily converted into the amide of the formula () by subsequent contact with an amine. The reaction with the amine can be carried out, for example, by bringing the chloride of the carboxylic acid of formula () into contact with an aqueous solution of methylamine for about 1 to 5 hours under ice cooling.

The resulting amide of formula () is subsequently converted into a compound of formula (XI) by hydrogenation.
This hydrogenation can be carried out in the same manner as described for the hydrogenolysis of formula () above. This hydrogenation yields a compound of formula (XI) in which the double bond of the compound of formula () is saturated and the benzyl group is removed at the same time, and this compound is subsequently treated with benzyl chloride to obtain a compound of formula ( XII). This benzylation is also a method known per se, for example, the compound of formula (XI) and benzyl chloride are mixed in a solvent such as methanol or ethanol in the presence of an acid binder such as potassium carbonate at room temperature to the reflux temperature of the reaction mixture. This can be easily carried out by reacting for about 5 to 20 hours.

The thus obtained compound of formula (XII) is reduced with lithium aluminum hydride to form a compound in which R represents a methyl group in the general formula (),
Compounds where n represents 3, that is, the formula (-
It can be changed to the compound of 1). This reduction is carried out, for example, by treating the compound of formula (XII) with an excess amount of lithium aluminum hydride in a solvent such as diethyl ether or tetrahydrofuran. The reaction can be completed in about 5 to 20 hours at room temperature to elevated temperature, preferably under reflux.

The case where R represents a methyl group and n represents 3 in the above formula () has been described above, but other compounds included in the formula () can also be produced according to the above synthesis method. for example,
In the reaction formula B, if another carboxylic acid, such as 4-benzyloxyphenylacetic acid, is used in place of the carboxylic acid of formula (), a compound of formula () in which n represents 2 can be obtained, and for example, If another amine, such as ethylamine, is used instead of methylamine as the amine to be brought into contact with the carboxylic acid of formula (), a compound of formula () in which R represents an ethyl group can be obtained.

The compound of formula () produced as described above is extremely suitable as a labeling compound in the determination of metanephrines by radioimmunoassay as described above.

Therefore, when using the compound of the present invention, a known amount of anti-metanephrine antibody and a known amount of the radioactive iodophenol-metanephrine derivative of the formula () are added to a test solution, and incubation is performed to detect free or Metanephrine in the test liquid can be quantified by measuring the amount of radioactivity of the bound radioactive iodophenol-metanephrine and comparing the amount of radioactivity with a standard curve.

The test liquid that can be used in this method is not limited to those collected from living organisms, but also samples collected from other than living organisms, but this method is particularly effective for quantifying trace amounts of metanephrines, and It can be advantageously applied to the collected test liquid. The above-mentioned test fluid can be collected, for example, from body fluids such as blood, lymph, urine, saliva, etc. of mammals and poultry, as well as biological tissues, organs, and the like.

These collected biological samples may be subjected to assay as they are, but prior to assay, the biological samples may be subjected to protein removal treatment, and if necessary, further separation and purification may be performed to some extent. This purification and protein removal treatment can be carried out by methods known per se, for example Seki, Wada: S.
Chromatogr, 114 , 227 (1975); Chemistry Special Issue No. 114 (Application of Fluorescence Measurement to Biochemical Research), No. 1
- 11 pages (1976) (Nankodo) and the like can be used.

Since such pretreatment is an operation to remove impurities that are undesirable for quantifying metanephrines, it is of course possible to omit the pretreatment when such impurities are not contained or even if they are contained, the amount thereof is small. can.

A sample that has been collected and pretreated if necessary can be subjected to the quantification described below as is or after being diluted or concentrated, and is usually used in the form of a diluted solution in a buffer solution.

According to this quantitative method, a known amount of an anti-metanephrine antibody and a compound of the formula () are added to a test liquid to be determined, and incubation is performed.

The anti-metanephrine antibody (antiserum) used in this quantitative method can be produced by a known method. Metanephrine is combined with an antigenic substance, and an animal is immunized with this as an antigen by a conventional method to produce an antibody (antiserum). In this case, antibodies (antiserum) can be effectively produced by immunizing an animal with metanephrine bound to an antigenic substance in combination with an adjuvant such as complete Freund's adjuvant. Examples of antigenic substances include bovine serum albumin (BSA), rabbit serum albumin (RSA), human serum albumin (HSA), bovine γ-globulin, rabbit γ-globulin, human γ-globulin, tetanus toxoid, Pneumococcus polysaccharide and the like can be used. Animals that can be used for immune formation are not particularly limited as long as they are non-human animals, such as cows, horses, sheep, goats, etc.
It can be appropriately selected from rabbits, mice, rats, guinea pigs, chickens, etc.

To immunize such an animal, an injection solution containing the metanephrine-antigenic substance conjugate is administered by subcutaneous, intradermal, intraperitoneal, intramuscular, or a combination thereof, most preferably by intradermal administration. do.
After confirming that the antibody concentration (antibody titer) of anti-metanephrine antibodies in the animal's blood has increased sufficiently, the blood is collected.

Blood cells and other solid components are removed from the collected blood by a method known per se to collect antiserum, and anti-metanephrine antibodies are extracted from the antiserum by fractionation using a conventional method, for example, by ammonium sulfate precipitation. It can be separated and obtained.

The anti-metanephrine antibody and the labeled compound of formula () can generally be added to the test solution in the form of a solution in a buffer. The order of addition is not critical; the anti-metanephrine antibody and the labeled compound of formula () may be added at the same time, or the anti-metanephrine antibody may be added first and then the labeled compound of formula () may be added.

A reaction mixture consisting of the test solution thus prepared, the anti-metanephrine antibody, and the labeled compound of formula () is incubated.

Incubation is usually in the range of about 0 to 80℃,
It can be carried out preferably at a temperature in the range of about 4 to 40°C for at least 5 minutes, preferably from 1 hour to 10 hours. It is also advantageous to maintain the pH of the reaction mixture during incubation generally in the range of 6 to 12, preferably in the range of 7 to 8.

After incubation for a certain period of time, the reaction mixture is subjected to a separation operation using a method known per se, such as the Millipore filter method, centrifugation method, gel filtration method, dextran charcoal method, etc., to remove unreacted formulas. Although the labeled compound in () can be separated,
Among these, the dextran charcoal powder method is suitable.

The thus obtained sample is used to measure the radioactivity of the bound radioactive iodophenol-metanephrine derivative, or to measure the radioactivity of the free radioactive iodophenol-metanephrine derivative remaining in the solution after precipitation separation, and to calculate the measured value. By comparing this with a standard curve and calculating the amount of metanephrine corresponding to the measured value, the amount of metanephrine in the test liquid can be determined.

As is conventionally known, the standard curve is prepared by using a standard sample containing a known amount of metanephrine to be changed in place of the test solution in the above assay, and using the same amount of anti-metanephrine antibody and labeled compound of formula () as described above. It can be created by operating under the same conditions as above, measuring the amount of radioactivity, and plotting it on a graph.

According to this quantitative method described above, it is possible to quantify metanephrines in a sample solution with a sensitivity approximately 10 to 20 times higher than that of the conventionally proposed radioimmunoassay using tritium-labeled metanephrine. can.

Therefore, according to this quantitative method, it is possible to quantify a small amount of metanephrine, and there are advantages such as, for example, the amount of biological samples collected from a patient can be much reduced compared to the conventional method.

Next, the present invention will be further explained by examples.

The NMR measurements were conducted in a deuterated chloroform solution using tetramethylsilane as an internal standard. In addition, confirmation of the iodination reaction (NMR) was carried out using a non-radioactive iodine because it was impossible with a radioactive iodine.

Example 1 Production of labeled compound (Part 1) (a) Metanephrine hydrochloride (1 g), 4-dimethylaminopyridine (1.4 g) in chloroform (10
ml) Add acetic anhydride (1.4 ml) to the solution and stir at room temperature.
After stirring for 30 minutes, add water and stir for another 30 minutes. After the reaction, the chloroform layer was separated, washed with 5% sodium carbonate, water, 10% sulfuric acid, and water in this order, and the solvent was distilled off to obtain triacetylmetanephrine (1.8 g).

NMRδ; 2.06 (3H, singlet), 2.10 (3H, singlet), 2.30 (3H, singlet), 2.93 and 2.98
(3H, two singlets), 3.4-3.9 (2H, multiplet), 3.92 (3H, singlet), 5.79-6.14 (1H,
multiplet), 6.8-7.1 (3H, multiplet).

(b) Add 10% hydrochloric acid (1.5 ml) to a solution of triacetylmetanephrine (1 g) in methanol (50 ml) and reflux for 30 minutes. Sodium bicarbonate (480
mg), most of the solvent is distilled off under reduced pressure. Add chloroform and anhydrous magnesium sulfate to the residue and stir. After removing the insoluble matter, the solvent was distilled off to obtain 2-methoxy-4-(1-acetoxy-2-acetylmethylaminoethyl)phenol (688 mg).

NMRδ: 1.96 and 2.07 (3H, two singlets), 2.97 (3H, singlet), 3.1-3.6 (2H, multiplet), 3.22 (3H, singlet), 3.88 (3H, singlet), 4.0 ~4.5 (1H, multiplet), 5.38 (1H,
wide singlet), 6.6 to 7.0 (3H, multiplet).

(c) 3-(4-benzyloxyphenyl)propanamine (623 mg), paraformaldehyde (75
A solution obtained by heating a mixture of 2-methoxy-4-(1
-acetoxy-2-acetylmethylaminoethyl)phenol (685 mg) in ethanol, and after refluxing for 24 hours, the solvent was distilled off. The residue was dissolved in benzene and washed successively with 5% sodium carbonate and water. The crude product (1.17 g) obtained by distilling off the solvent was subjected to thin layer chromatography on silica gel (Merck GF ₂₅₄ ) (benzene: methanol =
9:1) to give 1-[N-[2-hydroxy-3-methoxy-5-(1-acetoxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-3-(4 -benzyloxyphenyl)propane (500 mg) is obtained.

NMRδ; 1.6-2.2 (2H, multiplet), 1.93 and
2.06 (3H, two singlets), 2.26 (3H, singlet), 2.3-2.8 (4H, multiplet), 2.93 (3H, singlet), 3.1-3.7 (2H, multiplet), 3.20 (3H ,
singlet), 3.67 (2H, singlet), 3.87 (3H,
singlet), 4.0 to 4.5 (1H, multiplet), 5.00
(2H, singlet), 6.45 and 6.54 (1H, two singlets, J = 2Hz), 6.65 to 6.85 (1H, multiplet), 6.83 (2H, doublet, J = 8.5Hz),
7.10 (2H, double line, J=8.5Hz), 7.3~7.5
(5H, wide single line).

The starting compound 3-(4-benzyloxyphenyl)propanamine is synthesized as follows.

N-[1-oxo-3-(4-benzyloxyphenyl)propyl]-N-methylamine (1
The tetrahydrofuran solution of g) was added dropwise to a suspension of lithium aluminum hydride (0.8 g) in tetrahydrofuran (8 ml) over 5 minutes while stirring in an ice-water bath. After stirring at room temperature for 15 minutes, reflux for 14 hours. Cold water (5 ml) was gradually added to the reaction mixture, and then chloroform (10 ml) was added to the reaction mixture.
After stirring for an hour, insoluble matter is removed by decanting. The chloroform layer is dried over potassium carbonate and concentrated under reduced pressure to obtain 3-(4-benzyloxyphenyl)propanamine.

NMRδ; 1.22 (1H, wide singlet), 1.5~
2.0 (2H, multiplet), 2.35 to 2.80 (4H, multiplet), 2.41 (3H, singlet), 5.00 (2H, singlet), 6.84 (2H, doublet, J=8.5Hz), 7.09
(2H, double line, J=8.5Hz), 7.36 (5H, wide single line).

(d) 1-[N-[2-hydroxy-3-methoxy-
A solution of 5-(1-acetoxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-3-(4-benzyloxyphenyl)propane (160 mg) in ethanol (5 ml) was added to 5% palladium-carbon. (30 mg),
Contact with hydrogen gas at normal pressure for 15 hours. After leaving the catalyst, the solvent is distilled off. The residue is dissolved in chloroform and extracted with 4% aqueous sodium hydroxide solution. The aqueous layer is acidified with 10% sulfuric acid and then made alkaline again with sodium bicarbonate.

After extraction with chloroform and drying with potassium carbonate, the resulting crude product was purified using silica gel (Merck
GF ₂₅₄ ) by thin layer chromatography (chloroform:methanol = 9:1),
1-[N-[2-hydroxy-3-methoxy-5
-(1-acetoxy-2-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-3-(4-hydroxyphenyl)propane (hereinafter referred to as "phenol compound") (104 mg) is obtained.

NMRδ; 1.6-2.2 (2H, multiplet), 1.95 and
2.09 (3H, two singlets), 2.26 (3H, singlet), 2.3-2.8 (4H, multiplet), 2.97 (3H, singlet), 3.1-3.7 (2H, multiplet), 3.20 (3H ,
single line), 3.66 (2H, wide single line),
3.87 (3H, singlet), 4.0-4.5 (1H, multiplet), 6.46 and 6.52 (1H, doublet, J
= 2Hz), 6.65 to 6.85 (1H, multiplet), 6.73
(2H, doublet, J = 8.5Hz), 6.97 (2H, doublet, J = 8.5Hz), 8.68 (2H, wide singlet) (e-1) Obtained in step (d) above. Phenol (100
A solution of chloramine T (90 mg) in water (3 ml) is added to a solution of chloramine T (90 mg) and sodium iodide (50 mg) in ethanol (3 ml). Then 2% sulfuric acid (1
ml) and stir for 50 minutes at room temperature. After the excess iodine is destroyed with sodium bisulfite, sodium bicarbonate is added to make it alkaline. Extract with chloroform and extract the chloroform layer with 4% sodium hydroxide. The following steps were carried out in the same manner as in step (d) above, and the resulting crude product was separated by thin layer chromatography (benzene:methanol = 5:1) on silica gel (Merck GF ₂₅₄ ).
Raw material (18mg) was recovered from the Rf0.28 part, Rf0.36
1-[N-[2-cedroxy-3-methoxy-5-(1-acetoxy-2-acetylmethylaminoethyl)phenylmethyl]-N-
Methylamino]-3-(3-iodo-4-hydroxyphenyl)propane (31 mg) is obtained.

NMRδ; 1.6-2.2 (2H, multiplet), 1.95 and
2.09 (3H, two singlets), 2.28 (3H, singlet), 2.3-2.8 (4H, multiplet), 2.98 (3H, singlet), 3.1-3.7 (2H, multiplet), 3.22 (3H ,
single line), 3.66 (2H, wide single line), 3.87
(3H, singlet), 4.0~4.5 (1H, multiplet), 6.4
~6.6 (1H, multiplet), 6.65~6.85 (1H, multiplet), 6.83 (2H, doublet, J=8.5Hz), 7.00
(2H, two sets of double lines, J=2Hz, J=8.5
Hz), 7.44 (1H, double line, J=2Hz), 8.01
(2H, wide single line).

From the Rf0.41 part, 1-[N-[2-hydroxy-3-methoxy-5-(1-acetoxy-2
-acetylmethylaminoethyl)phenylmethyl]-N-methylamino]-3-(3,5-diiodo-4-hydroxyphenyl)propane (24
mg).

NMRδ; 1.6-2.2 (2H, multiplet), 1.94 and
2.07 (3H, singlet), 2.28 (3H, singlet),
2.3 to 2.8 (4H, multiplet), 2.96 (3H, singlet), 3.1 to 3.7 (2H, multiplet), 3.22 (3H, singlet), 3.68 (2H, wide singlet), 3.87
(3H, singlet), 4.0~4.5 (1H, multiplet), 6.4
~6.6 (3H, multiplet), 6.72 and 6.78 (1H,
Two doublets, J = 2Hz), 7.45 (2H, singlet).

(e-2) Phenol compound (0.30
mg) in ethanol (0.05 ml) is added an aqueous solution of sodium iodide- ¹²⁵ I (30μ, 3 mCi), and to this mixture is added a solution of chloramine T (0.05 mg) in water (0.05 ml). Then 2% sulfuric acid (0.1
ml), seal, and let stand at room temperature for 10 minutes. Hereinafter, the same process as in step (e-1) above was carried out, and methanol (0.2 ml) was added to the obtained crude product.
Add concentrated hydrochloric acid (0.1 ml), seal tightly, and let stand overnight at room temperature. The radioactive iodophenol-metanephrine derivative obtained by distilling off the solvent under reduced pressure is developed using a thin plate scanner.
It exhibits 95% or more radioactivity in the Rf0.03-0.09 region and is used as a labeling compound for metanephrine radioimmunoassay.

Example 2 Production of labeled compound (Part 2) (a) The phenol compound (38 mg) obtained in step (d) of Example 1 is dissolved in methanol (0.8 ml), and concentrated hydrochloric acid (0.4 ml) is added. Seal tightly and leave at room temperature overnight, then make alkaline with sodium bicarbonate. After chloroform extraction, the chloroform layer is extracted with a 4% aqueous sodium hydroxide solution. The following treatment was carried out in the same manner as in step (d) of Example 1, and the obtained crude product (16 mg) was difficult to purify, but
From the NMR spectrum, the main product is 1-[N-
[2-Hydroxy-3-methoxy-5-(1-hydroxy-2-acetylmethylaminoethyl)
phenylmethyl]-N-methylamino]-3-
It was found to be (4-hydroxyphenyl)propane.

NMRδ; 1.6-2.2 (2H, multiplet), 2.2-2.9
(4H, multiplet), 2.27 (3H, singlet), 2.47
(3H, singlet), 3.20 (3H, singlet), 3.64
(2H, wide singlet), 3.85 (3H, singlet), 4.0-4.4 (1H, multiplet), 6.09 (3H, wide singlet), 6.44 (1H, doublet, J=2
Hz), 6.64 (1H, double line, J=2Hz), 6.68
(2H, double line, J=8.5Hz), 6.99 (2H, double line, J=8.5Hz).

In addition, from qualitative thin layer chromatography,
1-[N-[2-hydroxy-3-methoxy-5
-(1-hydroxy-2-methylaminoethyl)phenylmethyl]-N-methylamino]-3
-(4-Hydroxyphenyl)propane is observed. (Chloroform: methanol =
In the 3:1 system, the former Rf is 0.09, and the latter Rf
was 0.03. ) (b) Using the crude product obtained in step (a) above, the radioactive iodophenol-metanephrine derivative obtained by the same procedure as in step (e-2) of Example 1 was prepared using a thin plate scanner. Expanding by
It exhibits 95% or more radioactivity in the Rf0.03-0.09 region and is used as a labeling compound for metanephrine radioimmunoassay.

Reference example 1 Quantification of metanephrines (1) Correlation between the concentration of catecholamines and catecholamine metabolites and the binding amount of radioactive iodophenol metanephrine derivatives and creation of a metanephrine standard curve Catecholamines and catecholamine metabolites, namely adrenaline, normetanephrine, noradrenaline, and dopamine , percent decrease in counts based on standard samples at various concentrations in 10 mM phosphate buffer (containing 10 mM ascorbic acid and 0.5% BSA) for l-dopa, phenylalanine and metanephrine;
That is, the binding rate was measured as follows. Here, the binding rate (%) can be expressed as B/Bo x 100, where B represents the number of counts when incubated using a binding mixture system containing a standard sample, and Bo represents a binding rate without a standard sample. It represents the number of counts when incubating using a mixture system.

Add ¹²⁵ I-phenol-metanephrine derivative (5000 cpm) to 200 μl of antiserum diluted 1000 times.
μ and 100 μ of the standard sample were added, and the binding mixture system consisting of 200 μ of the above phosphate buffer was incubated at 4°C for 4 hours. After that, 0.5 ml of dextran charcoal powder (0.05% dextran, 0.2% charcoal powder) was added and 4°C I left it for 20 minutes. Next, centrifugation was performed at 4° C. and 3000 rpm for 15 minutes, and free unreacted substances adsorbed on the dextran charcoal powder were separated by decantation, and the radioactivity of the supernatant was measured using a scintillation counter.

The results are shown in FIG. The value of Bo is a measured value obtained by operating in the same manner as above except for adding 100μ of the phosphate buffer solution instead of the standard solution.

As is clear from this table, no significant cross-reactivity was observed between the anti-metanephrine antibody and catecholamines or catecholamine metabolites.

(2) Quantification of urinary metanephrines (2-1) 100 samples of urine from 5 healthy men aged 37 to 45 each.
Add 100μ of ¹²⁵ I-phenol-metanephrine derivative (5000cpm), 200μ of anti-metanephrine antibody and 10mM phosphate buffer (10mM ascorbic acid and 0.5%
After adding 100μ of BSA (containing BSA) and incubating at 4°C for 4 hours, 0.5ml of dextran charcoal powder was added and the mixture was further left at 4°C for 20 minutes. Next, centrifugation was performed at 4°C and 3000 rpm for 15 minutes, the supernatant was removed by decantation, and the radioactivity was measured using a scintillation counter. As a result of comparison with a standard curve, the amount of metanephrine contained in daily urine was 7 to 12 μg.

(2-2) When the procedure was performed in the same manner as in (2-1) above, except that the urine of three patients with pheochromocytoma was used instead of the urine of healthy men, the amount of metanephrine in the urine of these patients per day was 450 to 5,600 μg. It was hot.

[Brief explanation of the drawing]

FIG. 1 is a standard curve of catecholamines and catecholamine metabolites shown in the above examples,
(1) represents the standard curves of metanephrine, (2) adrenaline, (3) normetanephrine, (4) noradrenaline, (5) dopamine, (6) l-dopa, and (7) phenylalanine. .

Claims (1)

[Claims] 1 formula In the formula, R represents a lower alkyl group; n represents an integer of 1 to 4; Z ₁ and Z ₂ are the same or different and represent a hydrogen atom or a lower alkanoyl group;
X ₁ and X ₂ are the same or different and represent a hydrogen atom or a radioactive isotope of iodine, provided that X ₁ and X ₂
A radioactive iodophenol-metanephrine derivative represented by: does not represent a hydrogen atom at the same time. 2 formulas In the formula, Z ₁₁ and Z ₂₁ are the same or different and represent a hydrogen atom or an acetyl group; X ₁ and X ₂ are the same or different and represent _a hydrogen atom or a radioactive isotope of iodine, _provided that The radioactive iodophenol-metanephrine derivative according to claim 1, which does not simultaneously represent a hydrogen atom. 3 formulas In the formula, R represents a lower alkyl group; n represents an integer of 1 to 4; Z ₁ and Z ₂ are the same or different and represent a hydrogen atom or a lower alkanoyl group;
X ₁ and X ₂ are the same or different and represent a hydrogen atom or a radioactive isotope of iodine, provided that X ₁ and X ₂
A reagent for radioimmunoassay of metanephrine, comprising a radioactive iodophenol-metanephrine derivative represented by: , which does not simultaneously represent a hydrogen atom.