CS254288B1 - A method for preparing cardiotonic glycoside conjugates with L-tyrosine - Google Patents

Abstract

The solution relates to a method for preparing conjugates of cardiotonic glycosides with L-tyrosine of the general formula I, .* where R is H or^H. It is prepared by reacting hemisuccinate of cardiotonic glycosides such as digitoxin or digoxin in an organic solvent with L-tyrosine methyl ester and with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, the reaction mixture is diluted with trichloromethane, washed with an aqueous solution of an organic acid, dried, evaporated and the resulting product is purified. Substances of this type are used after labeling with the radionuclide 125i to prepare components of analytical and diagnostic RIA kits.

Description

(54) A method for preparing cardiotonic glycoside-L-tyrosine conjugates

The invention relates to a process for the preparation of conjugates of cardiotonic glycosides with L-tyrosine of the general formula I,

wherein R is H or 1 H. It is prepared by reacting a cardiotonic glycoside hemisuccinate such as digitoxin or digoxin in an organic solvent with L-tyrosine methyl ester and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, the reaction mixture is diluted with trichloromethane, washed with an aqueous solution of organic acid. It is dried, evaporated and the product is purified. Substances of this type serve for the preparation of components of analytical and diagnostic RIA kits after radionuclide 125i labeling.

The invention relates to a process for the preparation of conjugates of cardiotonic glycosides with L-tyrosine.

125

These conjugates are used to prepare components (labeled with iodine radionuclide 1) (analytical and diagnostic RIA kits) (see, e.g., Polyevaya 0. Yu.). et al.,

Chim. Farm. OF. 17, (5) 533 (1983) or Calbiochem-Hoechst 1983 Biochemical and Immunochemioal Catalog. 5 and 114] These kits are used in clinical practice to determine the content of cardiotonic glycosides, mainly digitoxin and digoxin, in blood serum and urine.

There are not many digoxin derivatives suitable for iodine radionuclide labeling. The first work in this area is the publication of G. C. Oliver et al. / J. Clin. Invest. 47, (1968), comprising reacting succinic anhydride with digoxigenin in pyridine and utilizing the thus obtained 3-hemisuccinate to prepare the amide with tyrpsin methyl ester. However, this procedure is elaborated only on steroid aglycone.

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A patent is described by R. N. Piasio and J. E. Woiszwilly (DE P 2505267) consisting in oxidizing the sugar moiety of digoxin with periodate and in the subsequent condensation of the resulting dialdehyde with tyrosine methyl ester. A variation of this procedure is the work of N. D. Blazey / Clin. Chim. Asta 80, 403 (1977)] using hintamine instead of tyrosine methyl ester and sodium borohydride to reduce aldimine. The resulting intermediates in this publication are not chemically characterized.

The dialdehyde formed by the oxidation of the sugar moiety of the digoxin molecule is the starting compound of a further patented process by K. C. Tovey and J. W. Findlay (DE P 252 6984.5). However, the dialdehyde is condensed with aminoxyacetic acid and the resulting derivative reacted with tyramine or histamine to provide the desired substrate for iodination.

U.S. Pat. No. 3,963,697 discloses the reaction of digoxin or digitoxin 4-hemisuccinate with tyrosine methyl ester hydrochloride in the presence of Ν, Ν'-dicyclohexylcarbodiimide in pyridine. However, this method of preparation does not guarantee the purity of the product, which is characterized only by the UV spectrum. However, purity has a considerable effect on the specificity of the assay when used as a radiligand in analytical RIA kits.

The latest journal publication by E. W. Weiler and Μ. H. Zenenka / Clinical Chem. 25, No 1, (1979) discloses another procedure: digoxin is first activated with cyanogen bromide to form a cyclic imidocarbonate at C 1 and C 1, which reacts with tyramine or p-hydroxyphenylpropionylputresoin to provide a substrate suitable for iodination. However, working with cyanogen bromide is unpleasant and dangerous.

The disadvantages of these processes are eliminated by the process for preparing the cardiotonic glycoside-L-tyrosine conjugates of the invention.

The present invention provides a process for the preparation of conjugates of cadriotonic glycosides with L-tyrosine of the general formula I,

wherein R is H or OH. The compounds are prepared by reacting a cardiotonic glycoside hemisuccinate such as digitoxin or formula II,

wherein R is H or OH in an organic solvent, preferably tetrahydrofuran or dimethoxymethane at 0 to +40 ° C with L-tyrosine methyl ester and with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for 10 to 70 hours, reaction mixture Dilute with dichloromethane, wash with an aqueous solution of organic acid, preferably citric, tartaric or malic acid, dry over anhydrous sodium or magnesium sulphate and evaporate, and purify the product.

Purification is best accomplished by chromatography on a silica gel column using dichloromethane-methanol. The most potent seems to be the interaction of cardiogenic glycoside 4-hemisuccinate with L-tyrosine in a 3 to 6 molar excess and with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in a 3 to 6 molar excess.

The following are examples of the preparation process of the invention.

Example 1

To a solution of digitoxin 4-hemisuccinate (1.58 g, 1.82 mmol) in tetrahydrofuran (35 mL) was added L-tyrosine methyl ester (1.54 g, 7.88 mmol) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.95 g, 7.87) mmol /. After stirring at room temperature for 70 hours, the reaction mixture was diluted with dichloromethane (400 mL) and washed with 5% aqueous citric acid, water, and dried over anhydrous sodium sulfate. The residue was chromatographed on a silica gel column (200 g) in dichloromethane-methanol (98: 2). 310 mg (16%) of an amorphous digitoxin-L-tyrosine conjugate were obtained. IR (chloroform, cm cm): 3600 (OH), 3,425, 1,668, 1,521 (CONH), 1,745 (Lactone + ester). For C 7^H H^N NOO ^ g (1042.2) calculated: 63.38% C, 7.64% H,

N, 1.34; Found:% C, 63.25;% H, 7.55;% N, 1.45.

Example 2

To a solution of digoxin 4-hemisuccinate (200 mg, 0.23 mmol) in tetrahydrofuran (5 mL) was added L-tyrosine methyl ester (195 mg, 1 mmol) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (247 mg, 1 mg). mmol /. After stirring at room temperature for 35 hours, the reaction mixture was diluted with dichloromethane (60 mL), washed with 5% aqueous citric acid, water, dried over anhydrous sodium sulfate, and evaporated. The residue was chromatographed on a silica gel column (30 g) in dichloromethane-methanol (95: 5). 58 mg (24%) of an amorphous digoxin-L-tyrosine conjugate was obtained. IR (chloroform, cm ^ /): 3 605 (OH), 3440, 1671, 1510 (COHN), 1745 (ester + lactone).

To 55 ^ ^{Η Ν θΐ9} 79/1 059.2 / calculated: 62.43% C, 7.52% H, 1.32% N; Found: C, 32.57; H, 7.77; N, 1.12.

Example 3

The preparation was carried out as in Example 1 except that the reaction was carried out at +40 ° C for 10 hours. 305 mg (16%) of the amorphous digosine-L-tyrosine conjugate were obtained. Analysis found 63.19% C, 7.47% H, 1.27% K.

Example 4

To a solution of digitoxin-4-hemisuccinate (1.58 g, 1.82 mmol) in 1,2-dimethoxyethane (35 mL) was added L-tyrosine methyl ester (1.54 g, 7.88 mmol) and N-ethoxycarbonyl-2. -ethoxy-1,2-dihydroquinoline (1.95 g, 7.87 mmol). After stirring at room temperature for 70 hours, the reaction mixture was diluted with dichloromethane (400 mL) and washed with 5% aqueous citric acid, water, and dried over anhydrous magnesium sulfate. The residue was chromatographed on a silica gel column (200 g) in dichloromethane-methanol (98: 2). 300 mg (15.8%) of an amorphous digitoxin-L-tyrosine conjugate were obtained. IR (chloroform, cm < -1 >): 3600 (OH), 3425, 1668,

521 (CONH), 1745 lactone + ester). For Η 0 _{55? 9 Ν0} 1θ (1 042.2) calculated: 63. 38% C, 7.64% H, 1.34% N; Found: C, 63.20; H, 7.45; N, 1.50.

Example5

The preparation was carried out as in Example 3 except that a 5% aqueous tartaric acid solution was used for washing. 55 mg (23%) of an amorphous digoxin-L-tyrosine conjugate was obtained. Analysis found: C 62.64, H 7.75, N 1.54.

Example 6

The preparation was carried out as in Example 1 except that digitoxin 4-hemisuccinate was reacted with a 6 molar excess of L-tyrosine methyl ester and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline at 0 ° C for 70 hours. 305 mg (15.9%) of an amorphous digoxin-L-tyrosine conjugate were obtained. Analysis found: C, 63.18; H, 7.46; N, 1.25.

Claims (3)

OBJECT OF THE INVENTION Process for the preparation of conjugates of cardiotinic glycosides with L-tyrosine of the general formula I, wherein R is H or OH, characterized by reacting digoxin or digitoxin 4'-hemisuccinate of the general formula II, wherein R is H or OH in an organic solvent with With L-tyrosine methyl ester and with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, the reaction mixture is diluted with dichloromethane, washed with an aqueous solution of organic acid, dried and evaporated and the product purified. 2. The method of furan or dimethoxymethane. 1 marked team 3. The method according to item new, wine or apple. 1 marked team 4. The method of magnesium. 1 marked team 5. The method of item for 10 to 70 hours. 1 marked team that tetrahydro is used as the organic solvent, citric acid is used as the organic acid, anhydrous sodium sulfate is used as the desiccant or the reaction is carried out at a temperature of 0 to +40 ° C for 6. The process of claim 1 wherein the cardiovascular glycoside 4 '-hemisuccinate is reacted with the L-tyrosine methyl ester in a 3-6 molar excess and the N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in a 3-6 molar excess. 7. The process of claim 1 wherein the product is purified by chromatography on a silica gel column using dichloromethane / methanol.