A substituted aldosterone of the formula: ##STR1## wherein either one of R.sup.1 and R.sup.2 is hydrogen and the other is --S(CH.sub.2).sub.m COR.sup.3 or --OCO(CH.sub.2).sub.n COR.sup.3, provided that when R.sup.1 is hydrogen, R.sup.2 is --S(CH.sub.2).sub.m COR.sup.3 or --OCO(CH.sub.2).sub.n COR.sup.3 and when R.sup.2 is hydrogen, R.sup.1 is --S(CH.sub.2).sub.m COR.sup.3 ; m being an integer from 1 to 3, n being an integer from 1 to 5 and R.sup.3 being hydroxyl, lower alkoxy or a residue of tyramine, tyrosine lower alkyl ester, histamine, histidine, 7-aminoheptanoyltyrosine lower alkyl ester or .beta.-D-galactosidase as optionally iodinated, or its (18-20)-acetal 20,21-ketonide, which is useful as the reagent in determination of aldosterones by radioimmunoassay or enzyme immunoassay.

The present invention relates to substituted aldosterones, which are useful 
in determination of aldesterone (hereinafter referred to as "ALD") by 
radioimmunoassay (hereinafter referred to as "RIA") or enzyme immunoassay 
(hereinafter referred to as "EIA"). 
Among various steroid hormones, determination of ALD has been considered 
most difficult, and conventional immunoassay is not satisfactory for 
determination of ALD. In general, the adequacy of immunoassay depends much 
on aptitude of antihapten antiserum employed therein, characteristics of 
which vary with the structure of immunogen. For immunogen, compounds 
having functional groups in a free state as many as possible are 
considered to be desirable. 
As the hapten of ALD, there are known its 21-hemisuccinate, 
3-(O-carboxymethyl)oxime, 18,21-bishemisuccinate, etc.; however, all of 
them are at least partly blocked in their functional groups and hence are 
not satisfactory. 
Considering the above situation, the present inventors have attempted to 
provide haptens of ALD which have all the functional groups of ALD in a 
free state and now succeeded in providing such haptens. 
Accordingly, a main object of the present invention is to provide 
substituted aldosterones available as haptens of aldosterones. Another 
object of this invention is to provide a process for preparation of said 
substituted aldosterones. A still other object of the invention is to 
provide anti-hapten antisera by the use of said haptens. A further object 
of the invention is to provide labelled antigens by the use of said 
haptens. A still further object of the invention is to provide a method of 
RIA or EIA of ALD using said anti-hapten antisera or labelled antigens. A 
still further object of the invention is to provide a kit for RIA or EIA 
examination using said anti-hapten antisera or labelled antigens. These 
and other objects will be apparent to those skilled in the art from the 
foregoing and subsequent descriptions. 
The substituted aldosterones of the invention are represented by the 
formula: 
##STR2## 
wherein either one of R.sup.1 and R.sup.2 is hydrogen and the other is 
--S(CH.sub.2).sub.m COR.sup.3 or --OCO(CH.sub.2).sub.n COR.sup.3, provided 
that when R.sup.1 is hydrogen, R.sup.2 is --S(CH.sub.2).sub.m COR.sup.3 or 
--OCO(CH.sub.2).sub.n COR.sup.3 and when R.sup.2 is hydrogen, R.sup.1 is 
--S(CH.sub.2).sub.m COR.sup.3 ; m being an integer from 1 to 3, n being an 
integer from 1 to 5 and R.sup.3 being hydroxyl, lower alkoxy or a residue 
of tyramine, tyrosine lower alkyl ester, histamine, histidine, 
7-aminoheptanoyltyrosine lower alkyl ester or .beta.- D-galactosidase as 
optionally iodinated (particularly radioiodinated). Specific examples of 
--S(CH.sub.2).sub.m COR.sup.3 are carboxymethylthio, carboxylethylthio, 
carboxypropylthio, etc. and specific examples of --OCO(CH.sub.2).sub.n 
COR.sup.3 are hemimalonyloxy, hemisuccinyloxy, hemiglutaryloxy, 
hemiadipoyloxy, hemipimeloyloxy, etc. 
Of these substituted aldosterones (I), the compounds wherein R.sup.3 is a 
hydroxyl group may be combined with proteins such as bovine serum albumin. 
Immunization of rabbits with the resulting products as antigens gives anti 
ALD antiserum. They may be also combined with enzymes for labelling such 
as horseradish peroxidase, alkalinephosphatase, .beta.-D-galactosidase and 
glucosidase to give labelled products for EIA. Examples of suitable 
substituted aldosterones (I) are 
(11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-dioxo-4-pregnen-4-ylthio)ac 
etic acid, 
(11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-dioxo-4-pregnen-6.beta.-ylt 
hio)acetic acid, 
11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-dioxo-4-pregnen-6.alpha. or 
6.beta.-yl hemisuccinate, etc. 
The substituted aldosterones (I) wherein R.sup.3 is other than hydroxyl may 
be labelled with radioiodine such as .sup.125 I or .sup.131 I according to 
a conventional Chloramine-T or enzymatic method to give a labelled product 
for RIA. Examples of suitable substituted aldosterones (I) are 
N-(p-hydroxyphenethyl)-2-(11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-di 
oxo-4-pregnen-4-ylthio)acetamide, 
11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-dioxo-4-pregnen-6.alpha. or 
6.beta.-yl 4-(p-hydroxyphenethylamino)-4-oxobutyrate, etc. 
The substituted aldosterones (I) can be produced, for instance, by 
subjecting a compound of the formula: 
##STR3## 
wherein R.sup.1 and R.sup.2 are each as defined above and X is a glycol 
protective group to elimination of the protective group. 
The conversion of the compound (II) into the compound (I) as well as the 
production of the compound (II) as the starting material are summarized 
shown in the following scheme: 
##STR4## 
wherein R.sup.1, R.sup.2 and X are each as defined above, R.sup.4 is a 
lower alkyl group and Y is a hydroxyl group or a bromine atom. 
In the above scheme, the starting hemiacetal-type ALD, i.e. 
11.beta.,18-epoxy-18.alpha.,21-dihydroxy-4-pregnene-3,20-dione (III), is 
enol-etherified on the carbonyl group at the 
3-position and simultaneously protected on the hydroxyl groups at the 20- 
and 21-positions as an acetonide to give the compound (IV). The compound 
(IV) is converted into the compound (V) wherein Y is a bromine atom by 
bromination, or into the compound (V) wherein Y is a hydroxyl group by 
treating with a peracid such as m-chloroperbenzoic acid or monoperphthalic 
acid. 
The compound (V) wherein Y is a bromine atom is reacted with sodium methyl 
thioglycolate (NaSCH.sub.2 COOCH.sub.3) to give the corresponding acetate 
derivative (II') wherein either R.sup.1 or R.sup.2 is hydrogen and the 
other is SCH.sub.2 COOCH.sub.3. The acetate derivative (II') thus obtained 
is hydrolyzed under basic conditions, for instance, by treatment with 
potassium carbonate in aqueous methanol under nitrogen stream at room 
temperature 1-6 hours to give the corresponding acetic acid derivative 
(II) wherein either R.sup.1 or R.sup.2 is hydrogen and the other is 
SCH.sub.2 COOH. 
Also, the compound (V) wherein Y is a hydroxyl group is reacted, for 
instance, with succinic anhydride under basic conditions, e.g. by 
treatment with succinic anhydride in the presence of 
4-dimethylaminopyridine as a catalyst in pyridine while warming 
(30.degree.-70.degree. C.) for 24 to 63 hours to give the corresponding 
hemisuccinyl derivative (II) wherein R.sup.1 is hydrogen and R.sup.2 is 
OCOCH.sub.2 CH.sub.2 COOH. 
The acetic acid or hemisuccinyl derivative (II) as prepared above is 
reacted with tyramine in the presence of 1-hydroxybenzotriazole and 
dicyclohexylcarbodiimide in tetrahydrofuran while ice-cooling for 7 to 21 
hours to give the tyramine-conjugated compound (II) wherein either R.sup.1 
or R.sup.2 is hydrogen and the other is 
##STR5## 
or R.sup.1 is hydrogen and R.sup.2 is 
##STR6## 
In this reaction, tryamine may be replaced by any other compound having an 
amino group so that the corresponding carbonamide compound is obtainable 
as the compound (II). 
The thus obtained compound (II) is subjected to elimination of the 
protective group, preferably at room temperature in an inert gas under 
acidic conditions, to give the corresponding unprotected compound (I) such 
as (11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-dioxo-4-pregnen-4 or 
6.beta.-ylthio)acetic acid or its amide with tyramine or 
11.beta.,18-epoxy-18.alpha.,21-dihydroxy-3,20-dioxo-4-pregnen-6.beta. or 
6.alpha.-yl hemisuccinate or its amide with tyramine. 
The elimination of the protective group may be accomplished by per se 
conventional procedures, for instance, as described in T. W. Greene, 
"Protective Groups in Organic Synthesis" (1981). Applicable conditions are 
treatment with mineral acids (e.g. hydrochloric acid, sulfuric acid, 
perchloric acid), organic acids (e.g. formic acid, acetic acid, propionic 
acid, p-toluenesulfonic acid), Lewis acids (e.g. BCl.sub.3), acid-type 
ion-exchange resins, etc. More specifically, there may be adopted 
treatment with 70% acetic acid for 2-12 hours, treatment with 1N HCl 
(concentration: 2-20%) in dioxane or tetrahydrofuran, treatment with 
p-toluenesulfonic acid (concentration: 0.1-1%) in methanol or acetone, or 
the like. 
These compounds (I) may be combined with bovine serum albumin (BSA) to make 
BSA-conjugates. Using these BSA-conjugates as immunogens, antisera are 
obtainable. Alternatively, they may be labeled with enzymes to make 
tracers for EIA or with radioiodine to make tracers for RIA. 
For instance, the compound (I) wherein R.sup.3 is a hydroxyl group is 
combined with bovine serum albumin by a per se conventional binding 
procedure such as the mixed anhydride method to make its BSA conjugate. 
The BSA conjugate is injected as an immunogen into rabbits several times, 
and then the blood is collected from the rabbits to obtain an anti-hapten 
antiserum. The mixed anhydride method as herein stated may be carried out, 
for instance, by dissolving the compound (I) in dioxane, adding 
tri-n-butylamine and isobutyl chlorocarbonate thereto and stirring the 
resultant mixture at a temperature of 8.degree. to 10.degree. C. for 30 
minutes. To the reaction mixture containing the active ester, an aqueous 
dioxane solution containing BSA (adjusted to pH 8.5 with sodium hydroxide) 
is added, and stirring is continued to give the BSA conjugate of the 
compound (I). Application of conventional purification procedures to the 
resulting product affords the material usable as an immunogen. Examples of 
the purification procedures are dialysis against cold water, adjustment of 
pH, centrifugation, dissolution into sodium bicarbonate solution, 
re-dialysis against cold water, etc. 
In the above operation, the mixed anhydride method may be replaced by any 
other binding procedure such as the carbodiimide method (using 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or 
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide or the isoxazolium method. 
Still, these binding procedures may be likewise applied when labelling is 
to be achieved with enzymes. Namely, the only difference is the use of an 
enzyme in place of BSA [cf. K. Shizume et al.: "Radioimmunoassay, New 
Edition" published by Asakura Shoten (1977) and E. Ishikawa et al.: 
"Enzyme immunoassay" published by Igakushoin (1978)]. 
The labelled antigen for EIA may be prepared, for instance, by labelling 
the compound (I) wherein R.sup.3 is a hydroxyl group with an enzyme by a 
per se conventional procedure such as the activated ester method. The 
labelled antigen for RIA may be prepared, for instance, by labelling the 
compound (I) wherein R.sup.3 is other than hydroxyl with radioiodine 
(.sup.125 I or .sup.131 I) by a per se conventional procedure such as the 
Chloramine-T method or the enzyme method. In the latter case, atomic 
iodine obtained by oxidizing iodine ion with Chloramine-T or with the 
combination of hydrogen peroxide and lactoperoxidase may be introduced 
into the meta-position of a hydroxyphenyl group [cf. "Radioimmunoassay, 
New Edition" recited supra]. 
The immunoassay may be conducted by a method known per se. In case of EIA, 
determination is achieved, for instance, by comparing the obtained values 
of the standard solution and a serum to be assayed in measurement of 
intensity of fluorescence. In RIA, determination is accomplished by 
comparing the radioactivities of the standard solution and a serum to be 
assayed, as obtained by the use of a well-type scintillation counter. 
Based on the above development, this invention can provide a kit for 
immunoassay, which comprises (1) ALD as the standard substance, (2) the 
labelled product of the compound (I) with a radioisotope or an enzyme and 
(3) an antiserum obtained from the compound (I). Either one of the latter 
two may be replaced by the one obtained from the known ALD derivative. 
Optionally, it may contain a buffer solution and an F/B 
(free/bound)-separating reagent (e.g. PEA) or, in case of the antiserum 
(3) being used as the first antibody in the two antibody method, a second 
antibody. 
The immunoassay according to the present invention is excellent in 
sensitivity and cross-reactivity. 
The present invention will be illustrated more in detail by the following 
examples wherein the NMR values indicate those in the spectrum determined 
with 60 MHz and the Rf values in thin layer chromatography (TLC) show the 
ones obtained by the use of a precoated silica gel-60 plate (0.25 mm) 
(manufactured by Merck) unless otherwise indicated.