DE2834516A1 - STEROID DERIVATIVES AND THEIR USE IN RADIOIMMUNASSAY - Google Patents

Description

"Steroid derivatives and their use in radioimmunoassay"

R.S. Yalow and S.A. Berson wrote in the book "In Vitro Procedures With Radioisotopes in Medicine ", International Atomic Energy Agency, Vienna, 1970, p. 455, the principle of Radioimmunoassays defined as follows:

An unlabelled antigen in an unknown sample competes with a radioactively labeled antigen (tracer) on binding to an antibody and thereby reduces the binding of the labeled antigen. The extent of the competitive Inhibition observed in the unknown sample is compared to the result of a known one Standard solution, and then / the antigen concentration of the unknown substance determined.

The radioimmunoassay requires a specific antibody, a radioactively labeled antigen, a pure sample of the antigen to be determined as the standard and

Antigen. The gene for the separation of antibody-bound antigen from free / Radioimmunoassay follows the basic principle of saturation analysis, i.e. the competition between labeled and unlabeled Antigen around a limited number of binding sites of the antibody.

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When a labeled antigen is brought into contact with an ST unlabeled antigen and an antibody represent the amount of labeled antigen bound to the antibody and the amount of remaining (free) labeled antigen directly related to the amount of unlabeled antigen present Antigen, when a certain amount of antibody is present. Thus, when using a constant amount Antibody and labeled antigen and, if known concentrations of unlabeled antigen are used, a calibration curve be drawn, in which the antigen concentration against the amount of bound labeled antigen or against the amount of free labeled antigen or a ratio of the two measurements is plotted. the Antigen concentration in an unknown sample can be determined from the calibration curve by determining whether it is bound or free labeled antigen or a ratio of the two measurements can be read when using the unknown sample the amount of labeled antigen and antibody used to prepare the calibration curve is mixed. With all of them Radioimmunoassay methods require the antigen-antibody complex separated from the free radioactively labeled antibody or tracer. For this purpose the various separation methods developed, for example electrophoresis, chromatography, ion exchange, Adsorption on charcoal, talc or cellulose coated with dextran. There are also a number of methods known, in which the antibody is bound to a solid phase.

The term "antigen" in the radioimmunoassay also includes substances with limited immunogenicity. In determining of such substances, the substance can be coupled to an immunogenic carrier, usually a protein, in order to increase its immunogenicity. A non-immunogenic-- A substance that assumes immunogenicity when bound to a carrier is called a hapten.

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The radioimmunity assay was used to determine the concentration various endogenous and exogenous steroids in body fluids used. In the development of the radioimmunoassay for the various steroids is the manufacture of a radioactively labeled antigen is of particular importance. Possible radioisotopes are, for example, tritium,

C, ¹²⁵ J and ¹³¹ J. Tritium and C require the measurement of the ß-radiation in the liquid scintillation counter. this

requires laborious and costly sample preparation

125 131 therefore
riding. J and J are / are preferred. For known reasons, such as the half-life, the risk of radiation and

1 25

the counting yield, the radioisotope J is particularly preferred.

In general, steroids cannot be iodized directly with the radioisotope iodine. It is therefore necessary to use a parent steroid that is easily iodinated. The choice or development of such derivatives depends mainly on the affinity of the derivative for the anti-

20 bodies of the steroid to be measured. The affinity of the

The derivative for the antibodies should of course be as close as possible to the affinity of the steroid for the antibodies.

The invention is based on the object of creating steroid derivatives that can be easily radioactively labeled, who have a strong affinity for the steroid's antibodies for the radioimmunoassay and which are in the radioimmunoassay let use. This object is achieved according to the invention. The invention thus relates to what is set out in the claims featured item.

Specific examples of hydroxysteroids for radioimmunoassay, which can be modified structurally according to the general formula I are cholesterol, cortisol, cortisone, Corticosterone, prednisolone, methyl prednisolone, triamcinolone, Betamethasone, dexamethasone, triamcinolin acetonide, betamethasone valerate, halcinonide, aldosterone, estrone, estradiol, Estriol, testosterone, 19-nortestosterone, methyl

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9098 08/08 8 1

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testosterone and pregnenolone. Specific examples of hydroxyl group-containing derivatives of steroids for radioimmunoassay which have a strong affinity for the antibodies of the steroid for radioimmunoassay are digoxigenin, digitoxigenin and 11C _v -hydroxyprogesterone.

The steroids of general formula I can be made from a hydroxysteroid of the general formula St-OH and a ß-phenylalkylglutaric anhydride of the general formula II

(CH ₀ )

VxT

-CH

¹ OL

- C

(ID

- C.

getting produced. R. denotes a C ₀ , alkanoyl radical.

ι z — o

The preferred group is the acetyl group.

The compounds of general formula II are new. You can by reacting a 4-methoxyphenylalkanal of the general Formula III

(CH ₂ ) - CH

(III)

with at least 2 molar equivalents of cyanoacetic acid in the presence a base, such as sodium hydroxide, followed by acid hydrolysis, to the corresponding glutaric acid compound of general formula IV

^CH 2

£ _ (CH ₀ ) - CH ζ η,

4L

OH

CH ₂ -C-OH

(IV)

getting produced.

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An alternative process for the preparation of the compounds of the general formula IV, in which η has the value 0 and R is a hydrogen atom means, i.e. 3- (4-methoxyphenyl) -glutaric acid, is described in J. Am. Chem. Soc., Vol. 72 (1950), p. 1877.

In this process, anisaldehyde is mixed with ethyl acetoacetate in the presence of piperidine to anisal-bis-acetic acid ethyl ester condensed. The cleavage of this compound with a boiling solution of sodium hydroxide in ethanol provides the corresponding 3- (4-methoxyphenyl) glutaric acid. The demethylation of the glutaric acid derivatives of the general Formula IV provides the glutaric acid derivatives of the general formula V.

¹⁵ ^ ^ V CH-C-OH

HO // \ _(CH ν I ^Z

\ / ^{2 n} I (V)

W / CH ₂ -C-OH

This cleavage can be carried out by various known processes for demethylating aryl methyl ethers. In one of these processes, which is carried out in Aust. J. Chem., Vol. 25 (1972), p. 1719, is the aryl methyl ether with Thioethylate ions (produced in situ from ethanethiol and sodium hydride) in a polar aprotic solvent, preferably dimethylformamide, implemented.

The phenolic hydroxyl group of the compounds of the general formula V can be replaced by an alkanoyl radical in a customary manner to be protected. Such a process consists in the reaction of the glutaric acid derivative with the corresponding acid anhydride, preferably acetic anhydride. The preferred method for making the glutaric anhydride derivatives of general formula II as the glutaric acid derivatives of the general Formula V consists in the conversion of the acid to the acid anhydride and the protection of the phenolic hydroxyl group in

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a single step. If the protective group R _{1 represents} an acetyl group, the glutaric acid derivative of the general formula V is heated in acetic anhydride.

Implementation of the starting steroids of the general formula St-OH with the glutaric anhydride derivatives of the general formula II provides the steroids of the general formula VI

Il

St-OC-CH ₀ -CH-CH ₀ -C-OH 2 j 2

^(C ? ^H 2> n

(VI)

This reaction can be carried out in the presence of an organic base. Examples of usable organic Bases are nitrogen-containing heterocyclic compounds such as pyridine and tertiary amines such as triethylamine. Preferably the reaction is carried out at elevated temperatures.

If the initial steroid is more than. Contains a hydroxyl group, it can with a glutaric anhydride derivative of the general Formula II can be monoacylated because the hydroxyl groups of the steroid have different reactivity demonstrate. If you want to acylate a hydroxyl group that is less reactive than the particularly reactive one Hydroxyl group, the more reactive hydroxyl group can be blocked in the usual way. The split the protective group for the hydroxyl group in the compounds of the general formula VI provides the corresponding compounds of the general formula I.

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The compounds of general formula I can with a

125 131 radioisotope, preferably J and J, in particular 1 25

Jj can be marked using standard procedures. It will

radioactively labeled haptens of the general formula Ia ⁵

Π ii

St-OC-CH ₂ - CH-CH ₂ -C-OH (CH ₂ )

(I-a) 10

obtain. The asterisk (*) in the formula Ia shows the marking with a radioisotope. An example of the known methods is the method of Hunter and Greenwood, Nature, Vol. 194 (1962), p. 495. The labeled compounds of the general formula Ia are the subject of the invention.
20th

The labeled compounds of the general formula I-a can be used as tracers in the customary radioimmunoassay. Such methods are described, for example, by B.M. Jaffe and H.R. Behrmann, Methods of Hormones Radioimmunoassay, Academic Press, New York, 1974, and S.A. Berson et al., Methods in Investigative and Diagnostic Endocrinology, Vol. 3, Steroid Hormones, North-Holland, Publ. Comp., Amsterdam, 1975.

The radiolabeled compounds of the invention are particularly useful as reagents in the automated Radioimmunoassay described in U.S. Patent 4,022,577.

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₁ example 1

(3β, 5β, 12β) ^ - /. I-Carboxy ^ - (4-hydroxyphenyl) -i-oxobutoxy / -12,14-dihydroxycard-20 (22) -enolide

5 A) 3- (4-methoxyphenyl) glutaric acid

A mixture of 27.2 g of p-anisaldehyde, 52.1 g of ethyl acetoacetate and 4 ml of piperidine in 10 ml of 95 percent ethanol is stirred for 5 hours at room temperature. The resulting solid is filtered off, washed with 25 percent ethanol and recrystallized from 95 percent ethanol. There are 31.4 g of 2,2 '- (4-Methoxybenzal) -bis-ethyl acetoacetate obtained, mp 138 to 141 ⁰ C. After diluting the filtrate with the same amount of water, a further amount of the compound is obtained which

_{15 is} recrystallized from 95proζentigem ethanol. Yield 8.5 g, mp 137-142 ° C.

A mixture of 30 g of 2 , 2 '- (4-methoxybenzal) -bis-acetacetic acid ethyl ester, 450 ml of ethanol and 450 g of 50 percent sodium hydroxide is heated vigorously under reflux for 1 hour. 150 ml of water are then added and most of the ethanol is distilled off under reduced pressure. The concentrate is acidified with concentrated hydrochloric acid and then extracted with ethyl acetate. The ethyl acetate extract is washed with brine, dried and evaporated. The residue is recrystallized from a mixture of benzene and methanol. It will be 3.3 g of 3- (4-methoxyphenyl) glutaric acid obtained, mp 147-150 ⁰ C.

B) 3- (4-hydroxyphenyl) glutaric acid

A suspension of 6.45 g of a 57 percent dispersion of sodium hydride in paraffin oil in 70 ml of anhydrous dimethylformamide will prefer stirring slowly with a solution of 11.89 ml of ethanethiol in 20 inL of anhydrous dimethylformamide offset. The resulting slurry is 15 minutes

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stirred and then treated with a solution of 3.0 g of 3- (4-methoxyphenyl) glutaric acid in 20 ml of anhydrous dimethylformamide. The suspension is heated ^{to 165 °} C. in an oil bath for 5 hours. Most of the solvent is then distilled off under reduced pressure. The residue is diluted with water, acidified with concentrated hydrochloric acid and extracted twice with diethyl ether. The ether extracts are discarded. The solution is then saturated with sodium chloride and extracted with ethyl acetate.

The ethyl acetate extract is washed once with brine, then dried and evaporated. The residue is recrystallized from a mixture of chloroform and hexane. It will be 2.3 g of 3- (4-hydroxyphenyl) glutaric acid obtained, mp 168-170 ⁰ C.

C) 3- (4-Acetyloxyphenyl) -glutarsäureanhydrid A solution of 800 mg of 3- (4-hydroxyphenyl) glutaric acid in 15 ml of acetic anhydride is heated to 100 ⁰ C and then evaporated under reduced pressure to dryness Λ12 2 hours. The remaining solid is recrystallized from a mixture of chloroform and hexane. There are 600 mg of 3- (4-Acetyloxyphenyl) -glutarsäureanhydrid obtained, mp 140-143 ⁰ C.

D) 12ß- (acetyloxy) - (3ß, 5ß) -3-Z'4-carboxy-3-Z4- (acetyloxy) -

phenyl / -1-oxobutoxy / -14-hydroxycard-20 (22) -enolide A solution of 130 mg digoxigenin-12-acetate and 286 mg 3- (4-acetyloxyphenyl) -glutaric anhydride in 4.0 ml anhydrous pyridine is made under nitrogen heated ^{to 120 °} C. as a protective gas for 12 hours in an oil bath. 0.5 ml of water are then added and after 5 minutes the mixture is evaporated under reduced pressure. The residue is dissolved in chloroform, washed with 10 percent hydrochloric acid and sodium chloride solution, dried and evaporated. The residue obtained is subjected to preparative thin layer chromatography on a silica gel plate (2.0 × 200 × 200 mm)

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■ j subjected to a 9: 1 mixture of chloroform and methanol as the eluent. 110 mg of the title compound are obtained as an amorphous solid with a melting point of 118 to 135 ^° C.

E) (33/56, 12 [beta]) -3- / ~ 4-carboxy - 3- (4-hydroxyphenyl) -1-oxobut-

oxy7-12,14-dihydroxycard-20 (22) -enolide

A solution of 68 mg of 12ß- (acetyloxy) - (3ß, 5ß) -3- / 4-Ca ^ OXy-3- / 4- (acetyloxy) -phenyl7 ~ 1-oxobutoxy7 ~ 14-hydroxycard-20 (22) - A solution of 83 mg 1Q potassium carbonate in 1.0 ml water is added to enolide in 3.0 ml of methanol, and the mixture is stirred at room temperature for 2 1/2 hours. The resulting solution is then acidified with 5 percent hydrochloric acid and the methanol is distilled off under reduced pressure. The residue is diluted with 10 ml of water and extracted with ethyl IS acetate. The ethyl acetate extract is washed in brine, dried over magnesium sulfate and evaporated, and the residue is subjected to preparative thin-layer chromatography on silica gel plates measuring 1 × 200 × 200 mm with a 3: 1 mixture of chloroform and methanol as the developing solvent. 22 mg of the title compound are obtained as an amorphous solid with a melting point of 110 to 118 ° C. The connection is completely melted ^{at 144 ° C.}

25 Example 2

(3 [beta], 5 [beta], 12 [beta]) -3- / 4-carboxy-3 - / "(4-hydroxyphenyl) -methyiy-ioxobutoxy7-12,14-dihydroxycard-20 (22) enolide

A) 3 - / * (4 -Methoxypheny 1) -methy 1 / - glut aric acid

A solution of 3.57 g of cyanoacetic acid in 20 ml of water is mixed with a solution of 2.03 g of sodium hydroxide in 20 ml of water offset. The solution obtained is with 70 ml of ethylene glycol dimethyl ether diluted, then mixed with 3.0 g of 4-methoxyphenylacetaldehyde and left to stand for 6 hours at room temperature. ether distilled off under reduced pressure, the residue mixed with 200 ml of 10 percent hydrochloric acid and 6 hours

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Ί heated to reflux. After cooling, the mixture is extracted with ethyl acetate. The ethyl acetate extract is washed once with brine, then dried and evaporated, and the residue is chromatographed on silica gel with mixtures of chloroform and ethyl acetate. The eluates are evaporated. 2.2 g of 3- / T (4-methoxyphenyl) methyl / glutaric acid are obtained. After recrystallization from a mixture of benzene, ethyl acetate and hexane, the compound melts at 109 to 110.degree
10

B) 3 - £ ( 4-hydroxyphenyl) methyl7 ~ glutaric acid

A suspension of 1.4 g of a 57 percent dispersion of sodium hydride in paraffin oil in 30 ml of anhydrous dimethylformamide a solution of 4.0 ml of ethanethiol in 10 ml of anhydrous dimethylformamide is added dropwise. After 15 minutes, a solution of 800 mg of 3- / ~ (4-methoxyphenyl) methyl / glutaric acid is obtained added in 20 ml of anhydrous dimethylformamide. The resulting slurry is 20 hours heated in an oil bath to 165 ° C and then under vermin-20

evaporated under the current pressure. The residue becomes with 20 percent Hydrochloric acid acidified and extracted with diethyl ether. The ether extracts are discarded. Then the aqueous solution saturated with sodium chloride and extracted with ethyl acetate. The ethyl acetate extracts are combined, once with saline washed, dried and evaporated. The residue is made from a mixture of ethyl acetate, chloroform and hexane recrystallized. There are 600 mg of 3-Z '(4-hydroxyphenyl) methyl / glutaric acid obtained from 117 to 119 ° C.

30 C) 3 - // _ (4-acetyloxy) -phenyl7-methyl7-glutaric anhydride

500 mg of 3-yf (4-hydroxyphenyl) methyl7-glutaric acid in 15 ml of acetic anhydride are heated to 120 ° C. in an oil bath for 2 hours. The solution is then evaporated to dryness under reduced pressure urtö the residue is digested with a mixture of chloroform and hexane. Crystallization takes place and 490 mg of the title compound with a melting point of 88 to 89 ^° C. are obtained.

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D) 12ß- (Acetyloxy) - (3ß, 5ß) -3-Z'4-carboxy-3-Z7 ~ 4- (acetyloxy) -phenyl7-methyl7-1 -oxobutoxy_7-1 4-hydroxycard-20 (22) - enolid

A solution of 216 mg of digoxigenin-12-acetate and 500 mg of 3 - / ^ (4 ~ acetyloxy) -phenyl ^ -methylj-glutaric anhydride in 6.0 ml of anhydrous pyridine is refluxed for 20 hours. Most of the pyridine is then distilled off under reduced pressure, the residue is diluted with 20 ml of water and acidified with concentrated hydrochloric acid. The mixture is then extracted with ethyl acetate. The ethyl acetate extract is washed twice with small amounts of sodium chloride solution, then dried and evaporated, and the residue is subjected to the preparative thin-layer chromatography according to Example 1 D). 323 mg of the title compound with a melting point of 70 to 79 ^° C. are obtained.

E) (3β, 5β, 12β) - Z4-carboxy-3- / f (4-hydroxyphenyl) methyl / -

1-oxobutoxy7-12,14-dihydroxycard-20 (22) -enolide A mixture of 160 mg 12ß- (acetyloxy) - (3ß, 5ß) -3- / ~ 4-carboxy-3 - // 4- (acetyloxy) -phenyl7-methyl-1 / -1--oxobutoxy_7-14 hydroxycard-20 (22> -enolide, 20 ml of methanol, 7.0 ml of water and 2Ο7 mg of potassium carbonate is stirred for 2 1/2 hours at 15 ⁰ C. Thereafter the The mixture is acidified with concentrated hydrochloric acid and most of the methanol is distilled off under reduced pressure at room temperature. The concentrate is extracted with ethyl acetate. The ethyl acetate extract is washed with small amounts of sodium chloride solution, dried over magnesium sulfate and evaporated. The residue obtained is according to Example 1 E) subjected to preparative thin layer chromatography on silica gel plates. 55 mg of the title compound with a ^{temperature of -155 to 165 °} C. are obtained.

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1 example 3

(3 [beta], 5 [beta], 12 [beta]) -3- / 7 * 3- (carboxymethyl) -5- (4-hydroxyphenyl) -1 -oxopentyl7-oxy7-12,14-dihydroxycard-20 (22) enolide

5 A) 3- / 2- (4-methoxyphenyl) ethyl7 ~ glutaric acid

A solution of 850 mg of cyanoacetic acid in 5.0 ml of water is made mixed with a solution of 440 mg sodium hydroxide in 10 ml water. The solution obtained is mixed with 15 ml of ethylene glycol dimethyl ether diluted and with 820 mg 3- (4-methoxyphenyl) propionaldehyde offset. The solution is left to stand for 10 hours at room temperature. Then the ethylene glycol dimethyl ether distilled off. The residue obtained is mixed with 50 ml of 10 percent hydrochloric acid and 6 hours heated under reflux and stirred. Thereafter, the reaction mixture cooled and extracted with ethyl acetate. The ethyl acetate extract is washed with brine, over Magnesium sulfate dried and evaporated. The residue obtained is then poured onto 15 g of silica gel and with an 8O: 20 Chromatographed mixture of chloroform and ethyl acetate as eluent. 600 mg of the title compound are obtained. After recrystallization from a mixture of ethyl acetate and benzene melts the compound at 98 to 100 ° C.

B) 3- / 2- (4-hydroxyphenyl) ethylZ-glutaric acid

A suspension of 2.2 g of a 57 percent dispersion of sodium hydride in paraffin oil in 70 ml of anhydrous dimethylformamide is slowly mixed with 4.0 ml of ethanethiol under nitrogen as an inert gas. The mixture is then stirred at room temperature until a clear solution has formed. The solution obtained is mixed with a solution of 1.2 g of 3- / 2- (4-methoxyphenyl) ethy / glutaric acid in 10 ml of anhydrous dimethylformamide. The mixture is then ^{heated to 165 °} C. in an oil bath and stirred for 6 hours. The mixture is then evaporated to dryness under reduced pressure. The residue obtained is dissolved in 50 ml of water and extracted twice with 50 ml of diethyl ether each time.

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The ether extracts are discarded. The aqueous solution is then acidified with concentrated hydrochloric acid and with Ethyl acetate extracted. The ethyl acetate extract is washed with brine and dried over magnesium sulfate and evaporated. After recrystallization of the residue from a mixture of chloroform and hexane, 1.0 g of the title compound are obtained obtained from the mp 142 to 144 ° C.

C) 3- / 2- (4-acetyloxy) -phenylZ-äthyl7-glutaric anhydride A solution of 600 mg 3- / 2- (4-hydroxyphenyl) -äthyl7 ~ glutaric acid in 15 ml of acetic anhydride for 2 hours in an oil bath at 120 ⁰ C heated. The reaction mixture is then evaporated under reduced pressure and the residue is recrystallized from a mixture of dichloromethane and hexane. There are 495 mg of the title compound from

F. 97 to 99 ⁰ C obtained.

D) 12ß- (acetyloxy) - (3ß, 5ß) -3- / 5- / 4- (acetyloxy) -phenylj-

-3- (carboxymethyl) -1-oxopentyl-7-oxycard-20 (22) enolide

A solution of 220 mg digoxigenin-12-acetate and 470 mg 3- / 2- / 4- (acetyloxy) phenyl7-ethyl7-glutaric anhydride in 8.0 ml of anhydrous pyridine is refluxed for 20 hours heated. The pyridine is then distilled off under reduced pressure, the residue is diluted with 25 ml of water,

acidified with concentrated hydrochloric acid and extracted with chloroform. The chloroform extract is with a low Amount of brine washed, dried and evaporated. The residue obtained is subjected to preparative thin-layer chromatography according to Example 1D). It will

253 mg of the title compound were obtained.

E) ( 3 [beta], 5 [beta], 12 [beta]) -3- / 7 "3- (carboxymethyl) -5- (4-hydroxyphenyl) -1-oxoypentyl7-oxy7-12,14-dihydroxycard-20 (22) enolide

A solution of 230 mg of 12ß- (acetyloxy) - (3ß _; 5ß) -3 - / "/ 5- / 4-

(acetyloxy) -phenyl ^ ⁷ -3 ^ (carboxymethyl) -i-oxopentylj-oxyj-35

card-20 (22) enolide in a mixture of 10 ml of methanol and 2.5 ml of water containing 210 mg of potassium carbonate will be

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Stirred at 10 to 15 ^° C. for 2 1/2 hours. The solution is then acidified with concentrated hydrochloric acid. After working up according to Example 1E), 44 mg of the title compound with a melting point of 126 ° to 134 ° C. are obtained.

Example 4

(3ß, 5ß, MQ) -3-113- (Carboxymethyl) -6- (4-hydroxyphenyl) -1-oxohexyl7-oxy7-12,14-dihydroxycard-20 (22) enolide

A) 3- / 3- (4-methoxyphenyl) propy! / - glutaric acid

5.34 g of 4- (4-methoxyphenyl) -butyraldehyde be interspersed with 5.15 g of cyanoacetic acid and 2.74 g of sodium hydroxide in a mixture of 50 ml ethylene glycol dimethyl ether and 60 ml of water vice-.J. ₅ The reaction mixture is treated with hydrochloric acid and worked up according to Example 2 A). 2.1 g of the title compound are obtained which, after recrystallization from a mixture of ethyl acetate, chloroform and hexane, melts at 71 to 73.degree.

B) 3- / 3- (4-hydroxyphenyl) propy! / - glutaric acid

2.0 g of 3- / 3- (4-methoxyphenyl) -propy 1-7-glutaric acid are according to Example 1 B) demethylated. After recrystallization. of the product from a mixture of ethyl acetate, chloroform and Hexane, 1.2 g of the title compound with a melting point of 107 to 108 ° C. are obtained.

C) 3- / 3- / 4- (acetyloxy) -phenyiy-propylZ-glutaric anhydride 533 mg 3- / 3- (4-hydroxyphenyl) -propy17-glutaric acid are

3Q measured example 1 C) reacted with acetic anhydride. The title compound with a melting point of 55 to 57 ^° C. is obtained.

D) 1 2β- (Acetyloxy) - (3β, 5β) -3- / Z6- / 4- (acetyloxy) -phenyU-3- (carboxymethyl) -1, -oxohexyl7-oxy7 ~ card-2Q (22) -enolide

35 A mixture of 216 mg digoxigenin-12-acetate and 610 mg

3 - // 3- (4-acetyloxy) -phenyl7-propyl_7-glutaric anhydride in

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9.0 ml of anhydrous pyridine is reacted for 18 hours. That Product is isolated and purified according to Example 1 D). 245 mg of the title compound with a melting point of 98 ° to 113 ° C. are obtained.

E) (3β, 5β, 12β) -3- / 73- (carboxymethyl) -6- (4-hydroxyphenyl) -

1-oxohexyl7-oxy7-12,14-dihydroxycard-20 (22) -enolide A mixture of 150 mg of 12ß- (acetyloxy) - (3ß, 5ß) -3- / Z6 - / "4- (acetyloxy) -phenyl7- 3- (carboxymethyl) -1 -oxohexyl_7-oxy_ / - _1n card-20 (22) -enolide and 172 mg potassium carbonate are reacted in a mixture of 5.0 ml methanol and 2.0 ml water E) worked up and purified, 58 mg of the title compound with a melting point of 115 to 128 ^° C. are obtained.

Example 5

(3β, 5β, 12β) -3-ZZ3- (carboxymethyl) -7- (4-hydroxyphenyl) -1-oxoheptyiy-oxy-12,14-dihydroxycard-20 (22) -enolide Example 4 is with 5- ( 4-methoxyphenyl) valerianaldehyde repeated _2Q. The title compound is obtained.

Description of the labeling of digoxigenin derivatives with

radioactive iodine.

' 125

10 ml (about 6 mCi) of an aqueous solution of sodium J are placed in a reaction vessel containing 10 ml (1 mg / ml of a solution of (3β, 5β, 12β) -3- / 4-carboxy-3 (4-hydroxyphenyl) -1 -oxobutoxy7 ~ 1 2, 1 4-dihydroxycard-20 (22) -enolide in methanol contains. The vessel is closed with a stopper, and 25 ml (2 mg / ml in

₃ q 0/5 m phosphate buffer, pH 7.5) of a chloramine T solution is injected. The contents of the vessel are mixed well and left to stand for 5 minutes. 20 ml (3 mg / ml in 0.5 M phosphate buffer, pH 7.5) of a solution of sodium metabisulphite are then inoculated through the stopper in order to stop the reaction.

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The reaction mixture is applied to a column filled with 40 ml of Sephadex G-10 and eluted with 0.05 M Trxsacetatbuffer, pH 6.5. A fraction collector is used to collect 44 drops per tube. Free iodine escapes from the column at tube No. 38. The iodized product comes out in two fractions, namely a first fraction in tubes 110 to 130 and a second fraction in tubes 131 to 150. Fraction 2 is superior and is used in radioimmunoassay.
10

Example 6

21- / 4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxyj-i1ß, 18-epoxy-18-hydroxypregn-4-ene-3,20-dione

15 A) 21- / 3- (4-acetyloxyphenyl) -4-carboxy-1-oxobutoxy / -

11β, 18-epoxy-18-hydroxypregn-4-en-3,20-dione A mixture of 100 mg of aldosterone in 6.0 ml of anhydrous pyridine is refluxed with 300 mg of 3- (4-acetyloxyphenyl) -glutaric anhydride for 2 hours . The solution is then cooled to room temperature, mixed with water, left to stand for a few minutes and then evaporated under reduced pressure. The residue is taken up in 20 ml of ethyl acetate, washed with 6.0 ml of 15 percent hydrochloric acid and sodium chloride solution, dried and evaporated. There remains 408 mg of a goo. This product is examined by thin layer chromatography. There are three steroid products. The product is then subjected to preparative thin layer chromatography on two 2.0 mm silica gel plates. The plates are developed twice with a 9: 1 mixture of chloroform and methanol. 135 mg of the title compound with a melting point of 145 to 160 ° C. are isolated. The spectral data agree with the given formula.

B) 21- / 4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7 ~ 11ß, 18-epoxy-18-hydroxypregn-4-en-3,20-dione A solution of 130 mg 21- / 3- (4-Acetyloxyphenyl) -4-carboxy-1-oxobutoxy7 ~ 11β, 18-epoxy-18-hydroxypregn-4-en-3,20-dione in

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15 ml of anhydrous methanol containing 0.2 ml of triethylamine is left to stand for 24 hours at room temperature. The methanol is then distilled off under reduced pressure, the residue is diluted with 5.0 ml of water, acidified with 10 percent hydrochloric acid and extracted three times with 5.0 ml of ethyl acetate each time. The ethyl acetate extracts are combined, washed with brine, dried over magnesium sulfate and evaporated. The residue is subjected to preparative thin-layer chromatography on silica gel plates with a 9: 1 mixture of chloroform and methanol as the developing solvent. 83 mg of the title compound from F-145 to 152 ^° C. are obtained. The spectral data agree with the formula.

Description of the labeling of the aldosterone derivative with radioactive iodine

A solution of 5.0 µg of 21- / 4-carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy / -11β, 18-epoxy-18-hydroxypregn-4-ene-3,20-dione in 50 μΐ of a 1: 9 mixture of dioxane and 0.5 M borate buffer from pH 8.5 is successively with 20 μΐ an aqueous

125

Solution of sodium J (4 mCi) and 80 μg of freshly prepared chloramine-T were added. After 90 seconds, the reaction is terminated by adding 80 μg of sodium bisulfite in 20 μΐ of water. The solution is placed on a silica gel plate measuring 5 × 20 cm. The plate is then developed with a 9: 1 mixture of chloroform and methanol. The band of the labeled product is located with a scanner and extracted with ethanol. The concentrated ethanol solution is kept at 15 ⁰ C.

909808/0881 _i

1 example 7

21 - / 4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-11β, 17-dihydroxypregn-4-en-3 _f 20-dione

5 A) 21- / 3- (4-acetyloxyphenyl) -4-carboxy-1-oxobutoxy ^ -Π ß, 17-dihydroxypregn-4-en-3,20-dione

A solution of 346 mg of cortisol and 496 mg of 3- (4-acetyloxy-

ml of phenyl) glutaric anhydride in 5.0 / anhydrous pyridine Heated under reflux for 40 minutes. The pyridine is then distilled off under reduced pressure, the residue with 30 ml of ethyl acetate diluted with 10 ml of 15 percent hydrochloric acid and sodium chloride solution, dried over magnesium sulfate and evaporated. It becomes 840 mg of a powder obtain. On the basis of the NMR spectrum and the behavior in the thin-layer chromatogram it is found that the product is a 1: 1 mixture of the title compound and 3- (4-acetyloxyphenyl) glutaric acid is.

B) 21- / 4-carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-11ß, 17-

20 dihydroxypregn-4-en-3,20-dione

700 mg of the mixture obtained are dissolved in 30 ml of anhydrous methanol containing 1.0 ml of triethylamine. The solution is refluxed for 5 hours, then cooled and acidified with 10 percent hydrochloric acid. Most of the methanol is then distilled off under reduced pressure. The remaining slurry is extracted with ethyl acetate. The ethyl acetate extract is washed with brine, dried and evaporated. The residue is subjected to preparative thin-layer chromatography on two 2.0 mm thick silica gel plates with a 9: 1 mixture of chloroform and methanol as the developing solvent. The main band is isolated with an 8: 2 mixture of chloroform and methanol. 300 mg of the title compound with a melting point of 98 to 110 ^° C. are obtained.

909808/0881

Description of the labeling of the cortisol derivative with radioactive iodine
In the manner described above for the aldosterone derivative, the 21 - ^ - carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-

1 25 11β, 17-dihydroxypregn-4-en-3,20-dione with . s. J marked.

Example 8

(38,56,12β) -3- / 4-Ca ^ QXy-B- (4-hydroxy-3-methy! Phenyl) -1 -oxobutoxy_7-12,14-dihydroxycard-20 (22) enolide 10

A) 3- (4-methoxy-3-methylphenyl) glutaric acid

A mixture of 100 mmol of 4-methoxy-3-methylbenzaldehyde, 210 mmoles of cyanoacetic acid and 250 mmoles of potassium hydroxide in 500 ml of water are stirred for 20 hours at room temperature. Then the solution obtained is acidified with 100 ml of concentrated hydrochloric acid and refluxed for 60 hours. After this After cooling, the solution is saturated with sodium chloride and extracted with ethyl acetate. The ethyl acetate extract is evaporated. The title compound remains.

20 ■

B) 3- (4-Hydroxy-3-methy! Phenyl) glutaric acid

The title compound is obtained according to Example 1 B), but using 3- (4-methoxy-3-methylphenyl) glutaric acid.
25th

C) 3- / 4- (Acetyloxy) -3-methylphenyl) _7-glutaric anhydride According to Example 1C), but using 3- (4-hydroxy-3-methylphenyl) -glutaric acid, the title compound is obtained.

D) 1 2ß- (Acetyloxy) - (3ß, 5ß) ^ - / ^ - carboxy ^ - /! - (acetyloxy) -3-methylpheny ^ 7-1-oxobuboxy _ / - 1 4-hydroxycard-20 (22) - enolid

According to example 1 D), but using 3- / 4- (acetyloxy) -3-methy! Phenyl> i7-glutaric anhydride, the Title compound obtained.

909808/0881

- 26 -

E) (3 [beta] _f 5 [beta], 12 [beta]) -3-Z'4-carboxy-3- (4-hydroxy-3-methylphenyl) -1-

oxobutoxy_7-12,14-dihydroxycard-20 (22) -enolide

According to Example 1 E), but using 12ß- (acetyl oxy) - (3ß, 5ß) -3-Z £ -carboxy-3- / 4- (acetyloxy) -3-methylphenyl / -1-oxobutoxy7-14- hydroxycard ~ 20 (22) -enolide, the title compound is obtained.

Examples 9 to 26

According to Example 6, but using the following in Column I listed steroids and the anhydrides listed in column II are those listed in column III Products received.

J 35

L 909808/0881 _j

■ is

ro

cn

cn

cn

Column I.

Column II Column III

9) 10) 11)

ο 12)

13)

- ^ 14)

16)

cholesterol

Cortisone

Corticosterone

Prednisolone

Methylprednisolone

Triamcinolone

15) betamethasone

Dexamethasone

3-f4-acetyl oxyphenyl) glutaric anhydride

3- (4-acetyl oxyphenyl) glutaric anhydride

3- / T (4-acetyloxy) -3-

methylphenyl7-glutaric acid

anhydride

3- (4-acetyloxyphenyl) glutaric anhydride

3- (4-acetyloxyphenyl) glutaric anhydride

3- (4-acetyloxyphenyl) ■ glutaric anhydride

3- (4-acetyloxyphenyl) ■ glutaric anhydride

3- (4-Acetyloxyphenyl) ■ glutaric anhydride y-S- (4-hydroxyphenyl) -1-oxobutoxy7-cholest-5-en

21- / 4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxyj-17-hydroxypregn-4-ene-3,11,20-trione

21 - / "4-carboxy-3- (4-hydroxy-3-methylphenyl) -

1-oxobutoxy7-11ß-hydroxypregn-4-en-3,20-dione

21- / 4-carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-11.33 / 1 7-dihydroxypregna-1,4-diene-3,20-dione

21 - / "4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxyi / -11 β, 1 T-dihydroxy-ooC-methylpregna-i, 4-diene-3,20-dione

21- / 4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy_7-9-f luor-11 [beta], 16 [beta], 17-trihydroxypregna-1,4-diene-3,20-dione :

21 - / pi -carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy-7-9-fluoro-11β, 17-dihydroxy-16β-methyl-pregna-1,4-diene-3 , liO-dione

/ y-S- (4-hydroxyphenyl) -1-oxobutoxy7-9-f luor-11β, 17-dihydroxy-16o6-methyl pregna-1,4-diene-3,20-dione

ro

cn

17) Column I. Column II 18) Triamcinolone
acetonide 3- / 4- (acetyloxyphenyl)
methyl7-glutaric acid
anhydride 19) Betamethasone
valerate 3- (4-acetyloxyphenyl) -
glutaric anhydride 9098 Halcinonide 3- (4-acetyloxyphenyl) -
glutaric anhydride CD
CO 20) 880, oestrone 3- (4-acetyloxyphenyl) -
glutaric anhydride

21) estradiol

22) estriol

23) testosterone

24) 19-nortestosterone

3- / 7 "(4-acetyloxy) pheny 17-met hy l7-glut aric acid anhydride

3- (4-acetyloxyphenyl) glutaric acid anhydride

3- / Γ4- (acetyloxy) -3-methylphenyl / glutaric anhydride

3 = (4-acetyloxyphenyl) glutaric anhydride column III

21- / 4-Carboxy-3- / ~ (4-hydroxyphenyl) -methyl_7-1-oxobutoxy _ / - 9-f luor-11β, 16β, 17-trihydroxypregna-1,4-diene-3,20-dione-16,17-acetonide

21 - / "4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-9-fluoro-11ß-hydroxy-16ß-methyl-

17- (1-oxopentyloxy) -pregna-1,4-diene-3,20-dione

11beta - / "4-carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-21-chloro-9-fluoro-16ss, 17-dihydroxypregna-1, 4-diene-3,20-dione-16,17- acetonide

3 - / "4-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7 ~ oestr-1,3,5 (10) -trien-17-one

17β- / 1-carboxy-3- _j T ( ^4- hydroxyphenyl) -methyl7-1-oxobutoxy-7-ö "stra-1, 3,5 (10) -trien-3-ol

3- / l-Carboxy-3- (4-hydroxyphenyl) -1-oxobutoxy7-estra-1 , 3,5 (10) -triene-16Oi, 17β-diol

17ß- / 4-carboxy-3- (4-hydroxy-3-methy! Phenyl) 1 -oxobutoxy7 "-androsta ~ 4-en-3-one

17β ~ ^ 4-Carboxy-3- (4-hydroxyphenyl) -1 -oxobutoxy / "19-norandrosta-4-en-3-one

co cn UI

Oil

Column I.

25) methyltestosterone

26) pregnenolone

27) 1 ioC-hydroxy

progesterone

Column II

3- (4-Acetyloxyphenyl) -glutaric anhydride column III

Zy-S- (4-hydroxyphenyl) -1-oxobutoxyi7 ~ 17o {-niethylandrosta-4-en-3-one

3 - / "(4-acetyloxyphenyl) - 3ß- / £ 3- (carboxymethyl) -5- (4-hydroxyphenyl) ethyl7-glutaric anhydride oxopentyl7-oxy7-pregn-5-en-20-one

3 - / "4- (acetyloxy) -phenyl / -glutaric anhydride 110 | - / ~ 4-Carboxy-3 -. (4-hydroxyphenyl) -1-oxobutoxy_7-pregn-4-ene-3,20-dione

O CO OO

Claims (1)

10 UZ: M 82Ο (Vo / kä)
Case: T-824 016-S ER SQUIBB & SONS ₇ INC.
Princeton, NJ, V.St.A. 7 AUG. 1378 "Steroid derivatives and their use in radioimmunoassay" Priority: August 12, 1977, 'V-St.A. , No. 824 016 May 1 , 1978, V.St. A., No. 9-1952 15 20 Claims 'f 1
1. j steroid derivatives of the general formula I. 0 Ό St-OC-CH ₉ -CH-CH ₇ -C-OH 25th (D 30th in which R represents a hydrogen atom or a C 1 -C alkyl radical, η has the value 0, 1, 2, 3 or 4 and St represents the remainder of a deshydroxysteroids or a hydroxysteroids. 35 2. Steroid derivatives according to claim 1 of the general formula I, in which η has the value 0. 3098C8 / 0881 3. Steroid derivatives according to claim 1 of the general formula I, in which η is 1. 4. Steroid derivatives according to claim 1 of the general form I, in which η has the value 2. 5. Steroid derivatives according to claim 1 of the general formula I, in which η has the value 3. 6. Steroid derivatives according to claim 1 of the general formula I, in which η has the value 4. 7. Steroid derivatives according to claim 1 of the general formula I, in which R represents a hydrogen atom. 8. Steroid derivatives according to claim 1 of the general formula I, in which R represents a C .__- alkyl radical. 9. Steroid derivatives according to claim 1 of the general formula I, different from cholesterol, cortisol, cortisone, corticosterone, prednisolone, methylprednisolone, triamcinolone, Betamethasone, dexamethasone, triamcinolone acetonide, betamethasone valerate, halcinonide, aldosterone, estrone, estradiol, estriol, testosterone, 19-nortestosterone, methyltestosterone, pregnenolone, digoxigenin, or digitoxigenin 11Ot-hydroxyprogesterone derived. 10. (3 [beta], 5 [beta], 12 [beta]) ^ - / I-Carboxy-S- (4-hydroxyphenyl) -1-oxobuto xy7-12,14-dihydroxycard-20 (22) -enolide. 30th 11. (3 [beta], 5 [beta], 12 [beta]) -3- / 4-carboxy-3-Z. "(4-hydroxyphenyl) -methyl_7-1-oxobutoxy7-12,14-dihydroxycard-20 (22) -enolide. 12. (3 [beta], 5 [beta], 12 [beta]) -3- / 73- (carboxymethyl) -5- (4-hydroxyphenyl) -1-oxopentyl7-oxy7-12,14-dihydroxycard-20 (22) enolide. ^LC J 0 9 8 ' ⁿ / 0 BH 1 13. (3β _/ 5β _f 12β) -3 / '/ "3- (carboxymethyl) -6- (4-hydroxyphenyl) -1-oxohexyl_7 ^ oxy_ / -12,14-dihydroxycard-20 (22) -enolide. 14. (3 [beta], 5 [beta], 12 [beta]) -3- / 73- (carboxymethyl) -7- (4-hydroxyphenyl) -1-oxohepty Ij- 12,1 4-dihydrox'ycard-20 (22) -encolide. 15. 21- / l-Carboxy-S- (4-hydroxyphenyl) -1-oxobutoxy_7 ~ 11 β, 18-epoxy-18-hydroxypregn-4-ene-3,20-dione. 16. 2i - ^ - Carboxy-S- (4-hydroxyphenyl) -1-oxobutoxyy-Hß, 17-dihydroxypregn-4-ene-3,20-dione. 17. ß-Phenylalkylglutaric anhydrides of the general
Formula II
15th (CH ₀ ) - CH (I DO i j 2 in which R is a hydrogen atom or a C .._ -alkyl radical
and R _{1 is} a C ₂ _g-alkanoyl radical and η is the value O,
1,2,3 or 4 has. 18. Compounds according to claim 17, wherein R _{1 is} an acetyl group and R is a hydrogen atom. 19. Compounds according to claim 17, wherein η has the value 0. 20. Compounds according to claim 17, wherein η is 1. 21. Compounds according to claim 17, wherein η is 2. 22. Compounds according to claim 17, wherein η is 3. 35 23. Compounds according to claim 17, wherein η is 4. L- 909808/0881 24. ß-Phenylalkylglutaric acids of the general formula IV (IV) in which R is a hydrogen atom or a. C ₁ denotes _-, - alkyl radical and η has the value 0, 1, 2, 3 or 4. 25. ß-Phenylalkylglutaric acids of the general formula V, II CH ₂ -C-OH (CH ₂ ) -CH CH ₂ -C-OH in which R denotes a hydrogen atom or a C ₁ alkyl radical and η has the value 0, 1, 2, 3 or 4. 26.Radioactively labeled steroid derivatives of the general formula I-a _ fr StO-C-CH ₂ -CH-CH ₂ -C-OH (CH ₂ ) n OH (I-a) in which R, St and η have the meaning given in claim 1 and the asterisk (*) indicates the marking with a radioisotope. _> 909808/0881 -s- 2834518 27. Steroid derivatives according to claim 26 of the general formula 125 mel I-a, where the radioisotope. J or J is. 28. Process for the preparation of steroid derivatives according to 5 claim 1, characterized in that a hydroxy steroid or a hydroxyl group-containing derivative of a Steroid, which has a strong affinity for antibodies of the steroid for radioimmunoassay, with a ß-Phenylalkylglutaric anhydride of the general formula II 10 / ^CH 2 - C ^ O (II) -CH wherein R is a hydrogen atom or a C. -. means "alkyl radical and R _{1 is} a C" _ _fi alkanoyl and η is 0, T, 2, 3 or 4, and reacting the steroid obtained is saponified. 20 29. Use of the compounds according to claim 26 as tracers in radioimmunoassay. 909808/0881