FI66593C - N '- (HYDROXIALKYL) - OCH N' - (DIHYDROXIALKYL) AMIDER AV HYDROXIFENYL- OCH DIHYDROXIPHENYLALAN SOM KAN ANVAENDAS FOER FRAMSTAELLNING AV REAGENSER MAERKTA MED JOD OCH FOERFARANDE F - Google Patents

Description

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Federal Republic of Germany Förbundsrepubliken Tyskland (DE) P 2616724.8 (71) Boehringer Mannheim GmbH, Sandhofer Strasse 112-132, D-6800 Mannheim-Waldhof, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (72) Hans-Georg Batz, Tutzing, Klaus Stellner, Bernried, Hans-Ralf Linke, Wieienbach, Gunter Weimann, Tutzing, Federal Republic of Germany Förbundsrepubl iken Tyskland (DE) (74) Berggren Oy Ab (54) Hydroxy- and dihydroxyphenylalanine in N '- (hydroxyalkyl) - and N' - (C 1-4 hydroxyalkyl) amides which can be used for the preparation of iodine-labeled reagents and process for the preparation of these amides - N '- (hydroxyalkyl) - and N' - (dihydroxyalkyl) amide av hydroxyphenyl- and dihydroxy-phenylalanine, This is done for the purpose of reacting with a reagent not used for the purpose of the reagent.

The invention relates to hydroxyalkylamides of amino acids which can be labeled with iodine and to a process for their preparation.

When preparing reagents that are added to aqueous or physiological solutions, such as plasma, serum, or urine, in immunological assays, they are often labeled with substances labeled with radioactive substances, primarily iodine isotopes J and J. Because iodine isotopes can seldom be introduced directly into a molecule under study or determination without irreversible alteration or inactivation of the molecule, readily iodinated substances such as amino acids tyrosine, dihydroxyphenylalanine, 5-hydroxytryptophan, or their histidine are generally incorporated into the test substance. esters, biogenic amines or hydroxy derivatives.

Thus, for example, J. Clin. Investig. 47, pages 1035-1042, an immunoassay for the determination of digitoxin and digoxin using a compound containing tyrosine methyl ester as a radiolabeled substance.

Conversion of digitoxygenin derivatives and tyrosine, i.e. conjugates with a free carboxyl group, is known from DE-A-2 66593 DE 2 142 421.

Admittedly, these known conjugates have different drawbacks. Thus, these coupling products formed with amino acid esters or biogenic amines are highly hydrophobic and either poorly or not at all soluble in water. Conjugates with a free amino acid, on the other hand, have the disadvantage that they are difficult to synthesize and the yield is poor because the solubility of the amino acids in organic solvents is too low and, moreover, it is difficult to perform selective hydrolysis of amino acid lower alkyl esters with another molecule. ester group. In addition, the free carboxyl function can cause undesired side reactions with the test serum under study and, in some cases, sensitively interfere with the test.

The object of the invention is to avoid these disadvantages and to prepare hydrophilic, non-polar amino acid derivatives which are highly soluble in water and physiological fluids, to which iodine can be added and which can be prepared more economically because of the better yield and are suitable for the preparation of immunoassay reagents.

This object can be solved according to the invention by the N1- (hydroxyalkyl) and N '- (dihydroxyalkyl) amides of hydroxy and dihydroxyphenylalanine of the general formula I and their water-soluble salts:

H..N-CH-C

2 \ 2 NH-R

1 2 wherein R is a hydrogen atom or a hydroxy group and R is a mono- or dihydroxyalkyl group having 1 to 4 carbon atoms.

The hydroxyphenyl group is preferably a p-hydroxyphenyl group and the dihydroxyphenyl group is preferably a 3,4-dihydroxyphenyl group.

3,66593

Preferred mono- and dihydroxyalkyl groups are, for example, hydroxymethyl, 2-hydroxyethyl, 1,3-dihydroxypropyl, 1,4-dihydroxybutyl and dihydroxy-tert-butyl groups. Suitable water-soluble salts include salts with mineral acids, but readily crystallizing hydrohalides are preferred.

A particularly preferred compound of the invention is N '- (2-hydroxyethyl) -tyrosinamide hydrochloride.

The compounds of the invention are hydrophilic, readily soluble in water and physiological fluids and, due to their reactive amino group, can be easily conjugated to radioactive iodine-containing tracers, and are particularly suitable for attachment to acid derivatives to form an amide bond. for the preparation of reagents for testing in aqueous or physiological solutions. The compounds according to the invention are particularly suitable for use in the preparation of reagents for the study of cardiac glycosides (digitalis glycosides). For this purpose, the alkylamides according to the invention are reacted with digoxin, digoxigenin, digitoxin or digitoxigenin derivatives, for example digoxin or digitoxin dicarboxylic acid esters, in accordance with German Application Publication No. P 25 37 129.3. When the compounds of the invention are used in the preparation of such reagents, no undesired side reactions occur, for example in a radioirunung test with test serum, e.g. because there is no free carboxyl group in the molecule of the alkylcholamides of the invention.

Compounds of formula I may be prepared by first protecting the amino group of the hydroxyphenylalanine starting material in a manner known per se, then esterifying the carboxyl group with a reactive aminolytically cleavable solvent and reacting the ester thus obtained with the group-containing alkanolamine to give the corresponding amide. a protecting group in an acidic medium or by reduction, and the resulting amide is optionally converted into a water-soluble salt.

This method comprises, in more detail, the use of a trifluoroacetyl, carbobenzoxy or trityl group as a protecting group 4 66593 and, as a reactive, aminolytically cleavable compound, hydroxyacetonitrile, hydroxysuccinimide or N-hydroxybenzate esters and for example chloroacetonitrile, hydroxysuccinimide sulfonate, N-hydroxybenztriazole tosylate, N-hydroxybenztriazole trifluoromethyl sulfonate or 2,4,5-trichlorophenyl sulfonate.

The invention is illustrated by the following examples.

Example 1 - N 1 - (2-hydroxyethyl) -tyrosinamide hydrochloride a) F. Weygand and R. According to Geiger’s original literature, Chem. Ber. 8j), page 647 (1956) prepared N-trifluoroacetyltyrosine by reacting tyrosine with trifluoroacetic anhydride in a mixture of trifluoroacetic acid and diethyl ether.

b) To a solution of 10 g (36 mmol) of trifluoroacetyltyrosine and 4.6 g (40 mmol) of N-hydroxysuccinimide in absolute tetrahydrofuran was added dropwise with stirring and ice-cooled a solution containing 8.5 g (41.2 mmol) of dicyclohexylcarbodi -imide in absolute tetrahydrofuran. After stirring for 12 h at room temperature, the precipitated dicyclohexyl urea was filtered off, the filtrate was evaporated and dissolved in ethyl acetate. The ethyl acetate solution was shaken with water, bicarbonate solution and again with water, dried over sodium sulfate and evaporated to dryness. The remaining N-trifluoroacetyltyrosyl-hydroxysuccinimide ester was recrystallized from petroleum ether. Yield: 11.3 g (84% of theory), m.p. 145-147 ° C.

Analysis: C H N

C 15 H 13 O 6 N 2 F 3 <374-27) calculated: 48.14 3.50 7.48 obtained: 48.68 3.23 7.28 c) 7.6 g (20.3 mmol) of N-trifluoroacetyl-tyrosyl-hydroxy- the succinimide ester was dissolved in 60 ml of dioxane and a solution of 2.1 g (34.4 mmol) of ethanolamine in water was added. The pH of the solution was then adjusted to 5,66593 7.5 with 5% CO 2 solution and stirred for 4 days at room temperature. The solution was then evaporated to dryness, the residue was dissolved in methanol and fractionated on a silica gel column (eluent: methanol / chloroform 1: 1). The homogeneous fractions were evaporated to dryness and recrystallized from ether. Yield: 3.9 g (45% of theory) of N-trifluoroacetyl-tyrosyl-ethanolamide, m.p. 153-155 ° C.

Analysis: C H N

C 13 H 15 O 4 N 2 F 3 (320.27) calculated: 48.75 4.72 8.74 obtained: 47.91 4.07 8.40 d) 4.3 (13.4 mmol) of N-trifluoroacetyl-tyrosylethanolamide were dissolved in 30 ml of: aan abs. ethanol and 2 g (53 mmol) of sodium borohydride were added and stirred for 1 h. Then 30 ml of acetone were added and stirring was continued for 30 min. The reaction solution was evaporated to dryness, dissolved in water and shaken with a small amount of ethyl acetate. The aqueous solution was evaporated to dryness and the residue was dissolved in a mixture of ethanol and chloroform, filtered and evaporated to dryness. The residue was dissolved in ethanol. Hydrogen chloride was introduced into this solution to precipitate tyrosylethanolamide hydrochloride. Yield: 2.5 g (70% of theory). Sp. 220-225 ° C (dec.).

Use of a compound of Example 1 for the preparation of a reagent for use in the study of cardiac glycosides, which may be labeled with radioactive iodine:

Preparation of digoxin-4,111-glutaryl-N1- (2-hydroxyethyl) tyrosinamide 200 mg of digoxin-41 '' - glutaryl-hydroxysuccinimide ester prepared according to Example 4 of German Patent Application No. P 25 37 129.3 were dissolved in 20 ml. to absolute ethanol and 60 mg of tyrosylethanolamide hydrochloride prepared according to Example 1 in 5 ml of water was added. The pH of the solution was then adjusted to 8.0 with 5% CO 2 solution and the solution was stirred for 3 days at room temperature. It was then evaporated to dryness, the residue was dissolved in absolute ethanol, filtered and re-evaporated to dryness. When water was added to the residual oil, the product precipitated as a precipitate which was filtered and dried. For purification, the product was dissolved in a mixture of ethanol and ethyl acetate and chromatographed on a silica gel column. The impurities were eluted with ethyl acetate, pure product with ethanol.

Claims (3)

6,66593 Yield: 135 mg (63% of theory) Analysis: CHN C 57 H 85 N 2 O 19 (1 * 101) calculated: 62.1 7.7 2.5 obtained: 60.84 7.78 2.21 The digoxin-4 thus prepared Glutaryl-tyrosyl-ethanolamide, after being labeled with 125 radioactive J by conventional methods, is an excellent non-polar reagent for the determination of digoxin in aqueous or physiological saline. N1- (hydroxyalkyl) and N '- (dihydroxyalkyl) amides of hydroxy and dihydroxyphenylalanine which can be used for the preparation of iodine-labeled reagents of the formula? H2 O H_N-CH-C 2 \ 2 NH-R 1. wherein R is a hydrogen atom or a hydroxy group, and R is a mono- or dihydroxyalkyl group having 1 to 4 carbon atoms, and water-soluble salts thereof. Compound according to Claim 1, characterized in that it is N '- (2-hydroxyethyl) -tyrosinamide hydrochloride. Process for the preparation of compounds according to Claim 1 or 2, characterized in that the amino group of the hydroxyphenylalanine starting material is first protected in a manner known per se, then the carboxyl group is esterified with a reactive aminolytically cleavable compound and the ester 2 thus obtained is reacted with R-containing alkanolamine, the corresponding amide is obtained, the protecting group is cleaved in an acidic medium or by reduction, and the resulting amide is optionally converted into a water-soluble salt.