CA1119163A

CA1119163A - Digoxigenin derivatives

Info

Publication number: CA1119163A
Application number: CA000308753A
Authority: CA
Inventors: Ravi K. Varma; Jack Bernstein; Berthold R. Vogt; Frank L. Weisenborn
Original assignee: ER Squibb and Sons LLC
Current assignee: ER Squibb and Sons LLC
Priority date: 1977-09-01
Filing date: 1978-08-04
Publication date: 1982-03-02
Also published as: NL7808649A; JPS5448753A; GB2003480A; IT7850893A0; DE2837087A1; GB2003480B; FR2401936A1

Abstract

Case No.: RB49 Abstract DIGOXIGENIN DERIVATIVES Radiolabeled digoxigenin derivatives having the formula , wherein X is methylene or oxygen and the asterisk (*) indicates tagging with a radioisotope, are useful as tracers in digoxin radioimmunoassays.

Description

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DIGOXIGENIN DERIVATIVES
The measurement of various substances hy the use of radioimmunoassay techniques has achieved widespread acceptance in recent years.
Yalow and Berson, In Vitro Procedures With Radioisotopes In Medicine, International Atomic Energy Agency, Vienna (1970) pgs. 455 et seq., express the principle of radioimmunoassay in the following terms:
"Unlabelled antigen in unknown samples competes against labelled antigen ("tracer") for binding to antibody and therebv diminishes the binding of labelled antigen. The degree of competitive inhibition observed in unknown samples is compared with that obtained in known standard solutions for determination of concentration of antigen in unknowns~"
Radioimmunoassay tests require à specific antihody, a radioisotope-laheled (hereinafter referred to as "radiolabeled") antigen, a pure sample of the antigen to he measured to serve as a reference standard, and means for the separation of free antigen from antibody-bound antigen.
Radioimmunoassays follow the basic principal of saturation analysis, l.e., competition between labeled and unlaheled antigen for a fixed numher of antibody binding sites.

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-2-When radiolabeled antigen, unlabeled antigen, and antibody are brought together, the amount of radiolabeled antigen bound to antibody and the amount of radiolabeled antigen remaining unbound (free) has a direct relationship to the amount of unlabeled antigen present when a given amount ;`
of antibody is present. Thus, by using a constant amount of antibody and radiolabeled antigen, and using known concentrations of unlabeled antigen, a standard (calibration) curve can be plotted showing antigen concentration versus the amount of radiolabeled antigen bound or versus radiolabeled antigen unbound, or versus a ratio of the two measurements. The con-lS centration of antigen in an unknown sample canbe read from the standard curve by determining the amount of bound or free radiolabeled antigen (or ratio of the two measurements) resulting when the unknown sample is mixed with the amount of radiolabeled antigen and antibody used to prepare the curve. In all radioimmunoassay procedures it is necessary to provide means for separating the bound from the free labeled tracer material. Many widely varied procedures have -been developed and used; exemplary procedures are electrophoresis; chromatography; ion exchange;
adsorption to dextran-coated charcoal, talc, or cellulose; and a number of Solid-phase antibody techniques.

~ 163 Rs49 ~ he term "antigen", as used in the field of radioimmunoassays, may cover substances of limited immunogenicity (ability to generate antibodies). In those cases where the substance to be measured is of limited immunogenicity, the substance can be coupled with an immunogenic carrier, usually a protein, to increase its immunogenicity. ~ substance that is non-immunogenic, but acquires immunogenicity when 1~ linked with a carrier is referred to as a "hapten".
Digoxin is a hapten that is used in the treatment of congestive heart failure, atrial fibrillation, atrial flutter, and other diseases of the heart. Because many of the arrhythmias for which digoxin is indicated closely resemble the arrhythmias resulting from digoxin intoxication, it is very important that the plasma levels of digoxin in the patient's plasma be carefully monitored. Radioimmunoassay techniques have proved very adaptable to the monitoring of plasma digoxin levels.
In the development of a radioimmunoassay for digoxin, the preparation of a radiolabeled antigen is of primary concern. Possible radioisotope labels are tritium, carbon-14, iodine-125, iodine-131, and others. However, because tritium and carbon-14 must be counted by liquid scintillation (a time-consuming and expensive process), iodine-125 and iodine-131 are more desirable. For reasons well-recognized ~ ' ~

~19~.63 RB4~

in the art (e.g., half-life, radiation hazard, counting efficiency and others) iodine-125 has become the radioisotope of choice for use in digoxin radioimmunoassays.
S The chemical structure of digoxin is such that it is not possihle to radioiodinate it directly. It is, therefore, necessary to utilize a derivative of digoxin which can be readily iodinated. In choosing or developing such a derivative, the primary concern is the affinity of the derivative for the digoxin antibodies; it should, of course, be as close to the affinity of digoxin for the digoxin antibody as possihle.
United States patent 3,855,208 discloses certain digoxigenin derivatives which are usieful (after labeling with a radioisotope) as tracers in digoxin radioimmunoassavs. Among the digoxigenin derivatives disclosed are those having the formula j :, ~
. ~ '. .

~9~63 _r)~

Ra(~o S ~

O ~
H 1l B-NH-CH-C -O--Rb OH

wherein Ra is hydroxy or acetyloxy and Rh is hydrogen or alkyl of 1 to 6 carbon atoms. The B group is defined most broadly as "a diacyl radical of a dicarboxylic acid in which the carboxy groups are substituted on ad~acent carbon atoms, such as succinyl, maleyl, fumaryl or o-phthaloyl, preferably succinyl".
Belgian patent 839,hO6 discloses, inter alia, that the following haptens may he radio-laheled and used as tracers in a radio-immunoassay for digoxin:

3-succinyldigoxigenin-L-tyrosine 3-succinyldigitoxigenin-L-tyrosi.ne 3-adipyldigoxigenin-L-tyrosine :-.

.' , ", ~: , '.

, 3-adipyldigitoxigenin-L-tyrosine 3-carhodigoxigenin-glycyl-L-tyrosine 3-carhodigitoxigenin-glycyl-L-tyrosine.
Compounds having the formula o~o OEI ~
~
~ ~OH
,~
1 H o 1l O=C-CH2-X-CH2-C-NH-CH-C~OH

H

are readily radioiodinated and can be used (when radiolabeled) as a tracer in radioimmunoassay procedures for the determination of digoxin levels in a body fluid. In formula I, and throughout the specification, X can he methylene or oxygen.
The compounds of formula I can be prepared.
from a digoxigenin derivative having the formula II
O~r Rl-O ~J
s ,~
I ¦ OH
HO~`-- É ~'~

and an anhydride having the formula III

/ ~ C
X O
C/

In formula II, and throughout the specification, Rl is an alkanoyl group having 2 to 6 carbon atoms, acetyl heing the preferred group.
~igoxigenin-12-acetate (the preferred starting material of formula II), glutaric anhydride (formula III) and diglycolic acid anhydride (formula III) are all well known ~n the art. The other digoxigenin derivatives of formula II having the 12-hydroxy group pro-tected can be prepared using the known proceduresfor preparing digoxigenin-12-acetate.
A digoxigenin derivative of formula II
and a glutaric anhydride derivative of formula III can be reacted in the presence of an organic hase to yield a compound having the formula .. .

~19~3 RB49 . IV

Rl-O ~

lo ~ R
O=C-CH2-X~CH2-C-OH

Exemplary organic ~ases are nitrogen containing heterocyclics, e.g., pyridine, and tertiary amines, _~., triethylamine. The reaction will preferably be run at an elevated temperature.
Reaction of a digoxigenin derivative of formula IV with an ester of L-tyrosine (L-tyrosine methyl ester is preferred), or an acid-addition salt of the tyrosine ester, yields a compound having the formula 2s ~' .' ` .

~1:19~ RB4 9 _g_ ~

C~' ' H O O
100=C-CH2-X-CH2-C-NH- ICH-C-OCH3 OH

The reaction can be carried out in an inert organic solvent under anhydrous conditions, in the presence of an organic base and in the preser-ce of an acylating agent, e.g., pivaloyl chloride, isobutyl chloroformate, or _-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.
The reaction should be run in the temperature range of about -10C to about -15C.
Saponification of a compound of formula V yields the corresponding compounds of formula I.
The compounds of formula I can be labeled ("tagged") with a radioisotope, preferably iodine-125 or iodine-131, and 9~
Rs49 most preferably iodine-125, using procedures well known in the art, to yield a radiolabeled hapten having the formula OH ~
~J`~l ~
~\1~\~
l ¦ OH
O ~/ \/
1 ~ O o O=C-CH2-X-CH2-C-NH-CH-C-OH

~*

OH

The asterisk (*) in formula VI indicates tagging with a radioisotope. Exemplary of the methods known in the art is the method of Hunter and Greenwood; see Nature, 194:495 (1962). The radiolabeled compounds of formula VI form an integral part of this invention.
The radiolabeled compounds of formula VI can be used as tracers in radioimmunoassay 1~9~3 RB49 procedures following the general principles set forth hereinabove. Exemplary detailed procedures are described in Jaffe et al., "Methods of Hormone Radioimmunoassay", Academic Press, New York tl974) and Berson et al., "Methods in Investigative and Diagnostic Endocrinology", Vol, 3 on "Steroid Hormones", North Holland, Amsterdam (1975). the radiolabeled compounds of this invention are particularly useful as reagents in the automated radioimmunoassay system of Brooker et al. disclosed in United States patent

4,022,577 issued May 10, 1977.
The following examples are specific embodiments of this invention.

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, ~6 3 RB49 I~'xaml) 1 e ( ,r)~l2~)-3-[5-[~ Carboxy-2-(4-hydroxyphenyl)-ethyl]amino]-1,5-dioxopentyloxy]-12,14-dihydroxycard-20(22)-enolide

- 5 A) (3~,5~,12~)-12-(Acetvloxy)-3-(4-carboxy oxo-butoxy)-14-hydroxycard-20(22)-enolide ~ solution of digoxigenin-12-acetate (303 mg) and distilled glutaric anhydride (285 mg) in dry pyridine (5.0 ml) is heatea in a bath at 125-13~C for 7.0 hours. The solution is then c.~oled, poured into cold 10% hydrochloric acid and extracted with chloroform. The chloroform extracts arr combined, washed with hrine, dried, cvaporated and the residue (350 mg) is su~ected to a preparative thin layer chromatography (TLC) on silica gel plates using chloroform-methanol (95:5) for development to afford 282 mg of the title compound, melting point ~3-96C.
~) (3~,5~,12~)-12-(Acetyloxy)-14-hydroxy-3-[[5-[[1-[(4-hydroxyphenyl)methyl]-2-methoxy 2-oxoethyl]amino]-1,5-dioxopentyl]oxy]card-20(22)-enolide A solution of (3~,5~,12~)-12-(acetyloxy)-3-(4-carboxy-1-oxo-butoxy)-14-hydroxycard-20(22)-enol.ide (220 mg) in dry dichloromethane containing tri.ethylamine (n.04 ml) and pivaloyl chloride (n . ns2 ml) is prepared and cooled to -10 to -15C.
To the ahove solution is added a solution of L-tyro.sine methyl ester hydrochloride (88 mg) in a mixtur~ of tri-_-butylamine (O.n72 ml) and ~ 163 RB49 pyridine (2.0 ml) that is pre-cooled to -15C.
The mixture is then stirred at -10 to -15C for 10 minutes and at room temp. for 2.5 hours. It is then added to cold water, acidified with 6N
hydrochloric acid and extracted with dichloro-methane. The dichloromethane extract is washed with brine, dried, evaporated and the residue (342 mg) is subjected to a preparative TLC on silica gel plates using chloroform-methanol (90:10) to isolate the title compound, melting point 116-120C.
C) (3~,5~,12~)-3-[5-l[L-l-Carboxy-2-(4-hydro~y-phenyl)ethyl ~amino]-1,5-dioxopentyloxy]-12,14-dihydroxycard-20(22)-enolide lS A solution of (3~,5~,12~)-12-(acetyloxy)-14-hydroxy-3-[[5-[[1-[(4-hydroxyphenyl)methyl]-2-methoxy-2-oxoethyl] amino]-1,5-dioxopentyl]-oxy]card-20(22)-enolide (100 mg) in methanol (10 ml) and water (6.0 ml) is stirred with a 3% potassium carbonate solution (10 ml) for 3.5 hours at room temperature. It is then aoidified with 10% hydrochloric acid and the methanol is evaporated in vacuo and extracted with ethyl acetate. The ethyl acetate solution is washed once with a small amount of brine, dried and evaporated to afford a gum (89 mg). This is su~jected to a preparative TLC on a 1.0 mm silica gel plate using chloroform-methanol (3:1) for development to afford 48 mg of the title compound, melting point 208-215C and 21 mg of its 12-acetate derivative.

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Example 2 (3~,5~,12R)-3-1~[[2-[1-Carboxy-2-(4-hydroxyphenyl)-ethyl]amino]-2-oxoethoxy]acetylloxy]-12,14-dihydroxy-card-20(22)-enolide A) (3~,5~,12~)-12-(Acetyloxy)-3-[[(carboxymethoxy)-acetyl]oxy]-14-hydroxycard-20(22)-enolide Digoxigenin-12-acetate (800 mg) and diglycolic acid anhydride (624 mg) are refluxed in dry pyridine (25 ml) under nitrogen for 7 hours in pre-dried equipment. The mixture is cooled, concentrated to one-half of its original volume and treated with a solution of potassium carbonate (5.3 g) in water (122 ml). The reaction mixture is stirred for 30 minutes and acidified to pH 3-4 with 6N hydro-chloric acid. The precipitate that forms is filtered off, washed with cold water (10 ml) and dried at 50C for 2 hours to yield 1.2g of material.
The crude product is chromatographed on five preparative silica gel plates, developing the plates with CHC13:MeOH(3:1). The desired band is then scraped off and extracted with four 300 ml portions of CHC13:MeOH(l:l). The extracts are combined and stripped to dryness yielding 1.016 g of the title compound. Re-crystallization of a small amount from CH3OH:H2Ogives fluffy white material, melting point 200-201C.
B) (3~,5~,12~)-12-(Acetyloxy)-14-hydroxy-3-[[[2-[[1-[(4-hydroxyphenyl)methyl]-2-methoxy-2-oxoethyl]amino]-2-oxoethoxy]-acetyl]oxy]card-20(22)-enolide (3~,5~,12~)-12-(Acetyloxy)-3[[(carbo-methoxy)acetyl]oxy]-14-hydroxycard-20(22)-enolide :: ~

.

~ 3 RB49 (700 mg) is taken up in methylene chloride (12.6 ml) and treated with pivaloyl chloride (0.163 ml) and triethylamine (0.126 ml). The resulting solution is cooled down to -5 to 0C, treated with a cold solution (-5 to 0C) of L-tyrosine methyl ester hydrochloride (276 mg) and tri-n-butylamine (0.224 ml) in pyridine (6.27 ml) and stirred for 10 minutes. The mixture is then allowed to warm to room temperature and stirring is continued for another 2.5 hours.
The reaction mixture is then acidified with 6N hydrochloric acid to pH 4-5 and extracted with three 50 ml portions of methylene chloride. The organic extracts are washed successively with 1%
sodium bicarbonate (50 ml) and water (50 ml), dried over anhydrous sodium sulfate, filtered and stripped to dryness yielding 1.01 g of crude product. The crude product is then chromatographed twice on preparative silica gel plates, eluting the plates with CHC13:MeOH(9:1). The desired band is extracted with two 300 ml portions of CHC13:MeOH(9:1) and three 300 ml portions of CHC13:MeOH(5:1). The extracts are combined and stripped to dryness to give 714 mg of the title compound as an amorphous solid, melting point 121-125C.
C) (3~,5~,12~)-3-~[[[2-[1-Carboxy-2-(4-hydroxy-phenyl)ethyl]amino]-2-oxoethoxy]acetyl]oxy]-12,14-dihydrooxycard-20(22)-enolide (3~,5B,12~)-12-(acetyloxy)-14-hydroxy-3-[[[2-[[1-[(4-hydroxyphenyl)methyl]-2-methoxy-2-oxoethyl]amino]-2-oxoethoxy]acetyl]oxy]card-20(22)-enolide (100 mg) is taken up in 10 ml of methanol, cooled to 10C and treated with potassium .

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i3 -carbonate ~77.4 mg) in water (2.2 ml). The reaction mixture is stirred at 10-15C for 2 hours, acidified with lN hydrochloric acid and extracted with three 50 ml portions of ethyl acetate. The organic extract is dried over anhydrous sodium sulfate, filtered and stripped to dryness yielding 100 mg of crude product.
The crude product is chromatographed on a preparative silica gel plate eluting the plate with CHC13:MeOH(3:1). The desired band is extracted - 10 three times with 100 ml portions of CHC13:MeOH(4:1) yielding 19 mg of the title compound, melting point 138-145C.
Example 3 Radioiodination of (3~,5~,12B)-3-[5-[[L-l-carboxy-2-(4-hydro~y_henyl)ethyl~amino]-1,5-dioxopentyloxy]-12,14-dihydroxycard-2o(22)-enolide Sodium radioiodide (I125) aqueous solution (10 ml) approximately 6 mCi is added to a reaction vial containing a methanolic solution of (3B,5B, 12~)-3-15-[[L-l-carboxy-2-(4-hydroxyphenyl)ethyl]-amino]-1,5-dioxopentyloxy]-12,14-dihydroxycard-20(22)-enolide (10 mg; 1 mg/ml). The vial is stoppered and a chloramine T solution (25 ml; 2 mg/ml in 0.5M phosphate buffer, pH 7.5) is injected through the stopper. The vial is mixed well by shaking and allowed to stand for five minutes.
Sodium metabisulfite solution (20 ml; 3 mg/ml in 0.5M phosphate buffer, pH 7.5) is injected through the stopper to quench the reaction.
The reaction mixture is applied to ~ 20 ml Sephadex G-10 column and eluted with tris acetate ~ Bi Trade Mark .
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- 111916;~
. -17-~, buffer (0.05M, pH 6.5). Fractions of 30 drops per - . tube are collected. Free iodide comes off around tube ~ 20 and the radioiodinated product elutes off.in fractions # 40 thru 70.
Example 4 Radioiodination of (3~,5~,12B)-3-[[[[2-[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-2-oxoethoxy]acetyl]-oxv]-12,14-dihYdroxYcard-20(22)-enolide ~ .
Sodium radi¢iodide (I125) aqueous solution ~ (10 ml) approximately 6 mCi is added to a reaction vial containing a methanolic solution of (3~,5B,12B)-3-[[[[2-[1-carboxy-2-(4-hydroxyphenyl)ethyl~amino]-2-oxoethoxy]acetyl]oxy~-12,14-dihydroxycard-20(22)-enolide (10 mg; 1 mg/ml). The vial is stoppered and a chloramine T solution (25 ml; 2 mg/ml in 0.5M
phosphate buffer, pH 7.5) i8 injected thx~ugh the stopper. The vial i~ mixed well by shaking and allowed to stand for five minutes. Sodium meta-bisulfite solution (20 ml; 3 mg/ml in 0.5M phosphate buffer pH 7.5) is injected through the stopper to quench the reaction.
. The reaction mixture is applied to a 20 ml Sephadex G-10 column and eluted with ~ris acetate buffer (0.05M, pH 6.5). Fractions of 30 drops per tube are collected. Free iodide comes off around 25 tube # 20 and the radioiodinated product elutes off in fractions # 40 thru 70.
.

* Trade Mark . . .

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A compound having the formula wherein X is methylene or oxygen, or its labelled radioiso-tope counterpart.

2. A compound in accordance with claim 1 having the formula

3. A compound in accordance with claim 1 having the formula

4. A labelled radioisotope compound in accordance with claim 1 having the formula wherein X is methylene or oxygen and the asterisk (*) indicates labeling with a radioisotope.

5. A compound in accordance with claim 4 wherein the radioisotope label is iodine-125 or iodine-131.

6. A compound in accordance with claim 4 wherein the radioisotope label is iodine-125.

7. A compound in accordance with claim 6 wherein X is methylene.

8. A compound in accordance with claim 6 wherein X is oxygen.

9. A process for preparing compounds having the formula wherein X is methylene or oxygen, which comprises reacting a digoxigenin derivative having the formula wherein R1 is an alkanoyl group of 2 to 6 carbon atoms, and a glutaric anhydride derivative having the formula to yield a compound having the formula reacting said compound with an ester of L-tyrosine or an acid-addition salt of said tyrosine ester to yield a compound having the formula and saponifying said last-mentioned compound.