JPH0257560B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel substituted aldosterones,
These substituted aldosterones are produced by radioimmunoassay (hereinafter sometimes abbreviated as RIA) or enzyme assay (hereinafter sometimes abbreviated as EIA).
It is useful for measuring ALD (sometimes abbreviated as ALD). Conventionally, the measurement of ALD has been one of the most difficult steroid hormone measurements, and known immunoassays have not always had sufficient characteristics. In other words, the quality of the immunoassay method is largely determined by the suitability of the anti-hapten antiserum used.
The properties of this antiserum depend on the structure of the immunogen. In the case of anti-hapten antiserum, it is said that it is desirable that the compound used as the immunogen should have as many functional groups as possible in a free state. Haptens for ALD that have been reported so far include 21-hemisuccinate, 3-(O-carboxymethyl)oxime, and 18,21-bishemisuccinate;
Some of the functional groups of ALD itself are blocked, so it cannot necessarily be said to be a satisfactory hapten. In view of the above situation, the present inventors proposed that ALD
We attempted to synthesize a hapten in which all functional groups were free, and succeeded. Therefore, an object of the present invention is to provide the above-mentioned hapten, a method for producing the same, and an aldosterone testing reagent containing the above-mentioned hapten. The test reagents include anti-hapten antiserum and labeled substances using the above-mentioned hapten, and if these are used in combination, it can be used for ALD RIA method or EIA method.
It is possible to measure by method. In addition to these, if you combine standard ALD, RIA or
We can provide kits for EIA. According to the present invention, to achieve the above object, the formula (However, in the formula, either R ¹ or R ² is hydrogen, and when R ¹ is hydrogen, R ² is -S-(CH ₂ ) _1-3 COR ³
or a group represented by -O-CO-(CH ₂ ) _1-5 COR ³ , when R ² is hydrogen, R ¹ is -S-
A group represented by (CH ₂ ) _1-3 COR ³ , where R ³ is a hydroxy group, or a tyramine residue, which may each be iodinated, a tyrosine lower alkyl ester residue, a histamine residue, or a histidine residue. Alternatively, substituted aldosterones represented by 7-aminoheptanoyl tyrosine lower alkyl ester residue are provided as haptens. Of these substituted aldosterones (),
When R ³ is a hydroxy group, an anti-ALD antiserum can be obtained by immunizing a rabbit, for example, with an antigen bound to a protein such as bovine serum albumin (BSA). Furthermore, it can be combined with other proteins, such as labeling enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, or glycosidase, to form a label for EIA. Preferred substituted aldosterones () are (11β,
18-epoxy-18α,21-dihydroxy-3,20
-dioxo-4-pregnene-4-thio)acetic acid,
(11β,18-epoxy-18α,21-dihydroxy-
Examples include 3,20-dioxo-4-pregnen-6β-ylthio)acetic acid, 11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnen-6α or 6β-yl hemisuccinate. In addition, among the above substituted aldosterones (),
When R ³ is other than a hydroxy group, it can be labeled with radioactive iodine, such as ¹²⁵ I or ¹³¹ I, by the commonly used chloramine T method, enzyme method, etc., and then used as a labeled product for RIA. Suitable examples can be exemplified as follows. That is, N-(p-hydroxyphenethyl)-2-
(11β,18-epoxy-18α,21-dihydroxy-
3,20-dioxo-4-pregnen-4-ylthio)acetamide, 11β,18-epoxy-18α,
Examples include 21-dihydroxy-3,20-dioxo-4-pregnene-6α (or 6β)-yl 4-(p-hydroxyphenethylamino)-4-oxobutyrate. Also according to the present invention, the formula (wherein R ¹ and R ² have the same meanings as above, and X represents a glycol protecting group) is subjected to a deprotecting group reaction, Provided is a method for producing substituted aldosterones () represented by the formula (wherein R ¹ and R ² have the same meanings as above). In the above formula, -S-(CH ₂ ) _1-3 COR ³ is carboxymethylthio, carboxyethylthio, carboxypropylthio group, -O-CO-(CH ₂ ) _1-5 COR ³ is hemimalonyloxy, hemisuccinyloxy , hemiglutaryloxy, hemiadipoyloxy, hemipimeloyloxy group. The general formula for the above production method, including the synthetic route of the starting material, is as follows: (Here, R ¹ , R ² and X have the same meanings as above, R ⁴
(represents a lower alkyl group, Y represents a hydroxy group or bromine) That is, first, hemiacetal type ALD,
11β,18-epoxy-18α,21-dihydroxy-
The 3-position carbonyl of 4-pregnene-3,20-dione () is enol etherified, and the 20 and 21 positions are protected with acetonide to obtain the compound (). When this compound () is brominated, a compound () in which Y is bromine is obtained. On the other hand, when treated with a peracid (for example, m-chloroperbenzoic acid or monoperphthalic acid), a compound () in which Y is a hydroxy group is obtained. When the compound () where Y is bromine (Y=Br) is reacted with sodium methylthioglycol,
A corresponding acetylthio derivative () (one of R ¹ and R ² is H and the other is SCH ₂ COOCH ₃ ) is obtained. When one of the acetylthio derivatives () obtained in this way is hydrolyzed under basic conditions (in aqueous methanol, potassium carbonate, 1 to 6 hours) at room temperature in a nitrogen stream, the corresponding carboxylic acid (') (R ¹ , R ² , one of which is H and the other is SCH ₂ COOH). On the other hand, the compound () (Y
=OH) under basic conditions (in pyridine, in the presence of a 4-dimethylaminopyridine catalyst, 24
~63 h) and under heating (30-70 °C), the corresponding hemisuccinyl derivative () (R ¹
=H, R ² = OCOCH ₂ CH ₂ COOH) is obtained. These acetylthio derivatives () (one of R ¹ and R ² is H and the other is SCH ₂ COOH) or hemisuccinyl derivatives () (R ¹ = H, R ² =
When tyramine, 1 hydroxybenzotriazole and dicyclohexylcarbodiimide are sequentially added to OCO-CH ₂ CH ₂ COOH) in tetrahydrofuran under ice-cooling and reacted for 7 to 21 hours, the corresponding tyramine adducts () [(R ¹ or
Either one of R ² is H, the other is

[Formula] or (R ¹ = H,

[Formula] is obtained. If these compounds () are subjected to a deprotection reaction under acidic conditions, preferably at room temperature in an inert gas flow, the corresponding target compounds (), for example, (11β,18-epoxy-18α,21- Dihydroxy-3,20-dioxo-4-pregnene-4
(or 6β)-ylthio)acetic acid, or 11β,18
-Epoxy-18α,21-dihydroxy-3,20-
Dioxo-4-pregnene-6β (or 6α)-yl hemisuccinate and the like are obtained. This deprotecting group reaction is carried out in a well-known manner regarding steroids in general, such as Greene, TW, “Protective Groups in Organic Synthesis”.
(1981).For example,
Deacetonidation conditions using mineral acids such as hydrochloric acid, sulfuric acid, and perchloric acid; organic acids such as formic acid, acetic acid, propionic acid, and p-toluenesulfonic acid; other Lewis acids such as BCI ₃ ; and acid type ion exchange resins. is applicable. More specifically, 70%
Conditions for reacting with acetic acid for 2 to 12 hours, conditions for reacting with 1N-hydrochloric acid (addition concentration: 2 to 20%) in dioxane or tetrahydrofuran, or conditions for reacting with p-toluenesulfonic acid (concentration; 0.1 to 1%) in methanol or acetone. %), and under any of these conditions, the compound () can be obtained advantageously. These are used to make a conjugate with bovine serum albumin by the method described later and use this as an immunogen to produce antiserum, and are also labeled with an enzyme and used as a tracer for EIA. In addition, the compound () (one of R ¹ or R ² is H, the other is

[Formula] or ) (R ³ = tyramine residue) For example, N-(p-hydroxyphenethyl)-2-(11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo- 4-pregnen-4-ylthio)acetamide, or 11β,18
-Epoxy-18α (or 18β), 21-dihydroxy-3,20-dioxo-4-pregnen-6β (or 6α)-yl 4-(p-hydroxyphenethylamino)-4-oxobutyrate, As described later, it is labeled with radioactive iodine and used as a tracer for RIA. At least one of the above-mentioned substituted ALDs is
It can be used as a hapten for producing anti-hapten antiserum or as a labeling antigen (tracer) for immunoassay of serum ALD. These can also be combined with standard ALD
It can be a kit for RIA or EIA. The anti-hapten antiserum used in the immunoassay method is prepared by first preparing the compound () in which R ³ is a hydroxyl group (R ³ =OH) by a conventional method known per se, such as the mixed acid anhydride method. The BSA conjugate is then made into a conjugate with bovine serum albumin (BSA conjugate), which is injected into rabbits several times as an immunogen, and then blood is collected from the rabbit. In the mixed acid anhydride method, the compound () (R ³ = H) is
For example, by dissolving it in dioxane, adding a small amount of tri-n-butylamine and isobutyl chlorocarbonate, and stirring at 8 to 10°C for 30 minutes, the active ester of the compound ()-COOCOOC ₄ H ₉ can be obtained.
Add to this aqueous dioxane solution of BSA (pH with NaOH).
8.5) and continue stirring to obtain a BSA conjugate of compound (). If the immunogen is insufficient, purification procedures such as dialysis in cold water, adjustment of pH, collection of precipitate by centrifugation, dissolution of the collected precipitate in a sodium bicarbonate solution, and re-dialysis in cold water are performed to prepare the immunogen. In addition, a carbodiimide method (using ethylcarbodiimide or morphocarbodiimide) or an isoxazolium method may also be used. Furthermore, these methods can be used in exactly the same way when labeling with the following enzymes. BSA simply replaces the enzyme. (For example, Kazuo Shizume et al. eds. [New Edition Radio Immunoassay] published by Asakura Shoten in 1997, and Eiji Ishikawa et al. "Enzyme Immunoassay" Igaku Shoin 1978)
In the case of EIA, the antigen for labeling is prepared by labeling the compound () in which R ³ is a hydroxyl group with an enzyme using a known conventional method, such as the active ester method. . In the case of RIA, R ³ of the compound () other than hydroxyl is labeled with radioactive iodine ( ¹²⁵ I or ¹³¹ I) by conventional means known per se, such as the chloramine T method or the enzyme method. Make it. That is, by oxidizing iodine ions with chloramine T or by using atomic iodine obtained by oxidizing with a combination of hydrogen peroxide and lactoperoxidase,
For example, a method is adopted in which iodine is introduced into the meta position of hydroxyphenyl (see the above-mentioned "New Edition Radioimmunoassay"). The immunoassay method is carried out according to a method known per se, and in the case of EIA, the amount is determined by comparing the measured values of the standard solution and the serum to be measured in, for example, fluorescence intensity measurement. In the case of RIA, it is determined, for example, by comparing the radioactivity of a standard solution measured with a well-type scintillation counter and the test serum. The immunoassay kit using the hapten provided by the present invention includes a standard material.
Includes antisera obtained from ALD and activation of compound () or enzyme-labeled products or compound (). Either of the latter two may be derived from known ALD derivatives. In addition, buffers, F/B (free/bound) separation reagents [(e.g.
PEG) or a second antibody (when the above is used as the first antibody in a two-antibody method)]. As described above, it has been found that immunoassays with excellent sensitivity and cross-reactivity can be performed using the hapten provided by the present invention, and as will be explained in more detail in the following Examples. This confirmed the superiority of the present invention over known techniques. In the following examples and reference examples, the compounds are given relevant numbers in principle based on the above reference numbers. In addition, while describing the physical property values of the compound,
NMR shows spectra at 60 MHz unless otherwise specified, thin layer chromatography (TCL)
The RF value was obtained using "Pre-coated Silica Gel-60-Plate 0.25 mm" (manufactured by Merck & Co.). Example 1 (11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnene-4-
ylthio)acetic acid () (R ¹ = SCH ₂ COOH, R ²
- Production of H) Methyl (11β, 18; 18α, 20β-bisepoxy-
20α,21-isopropylidenedioxy-3-oxo-4-pregnen-4-ylthio)acetate () (R ¹ = SCH ₂ COOCH ₃ , R ² = H, 20, 21 (S)
To a mixture of 82 mg of acetonide, 8 ml of methanol, and 4 ml of water, 90 mg of potassium carbonate was added at room temperature in a nitrogen gas stream, and the mixture was reacted for 2 hours and 30 minutes. The reaction mixture was neutralized with acetic acid and extracted with ethyl acetate. The extract was washed repeatedly with saturated saline, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure to obtain a candy-like (11β,18;18α,20β-bisepoxy-20α,21-isopropylidenedioxy- 3-
Oxo-4-pregnen-4-ylthio)acetic acid (') (R ¹ = SCH ₂ COOH, R ² = H, 20, 21 (S)
acetonide) was obtained. (') (R ¹ = SCH ₂ COOH, R ² = H, 20, 21 (S)
acetonide) NMR (CDCl ₃ , δ) 1.34, 1.50 (9H, 19H and

[Formula]) 3.38 (2H, s, -SCH ₂ CO) 3.92, 4.03 (2H, ABq, J _AB = 9Hz, ν〓 _AB = 6.3
Hz, 21-CH ₂ -) 4.87 (1H, d, J = 6Hz, 11-H) 5.33 (1H, S, 18-H) 7.22 (1H broad-COOH) Add this directly to 3 ml of 70% acetic acid solution, After stirring at room temperature in a nitrogen gas stream for 6 hours, ice water was added and the mixture was extracted with ethyl acetate. The extract was washed with saturated brine, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The residue was recrystallized from methanol to give the title compound () as colorless needles (R ¹ = SCH ₂
-COOH, R ² = H) 34 mg was obtained. (Yield 46.7% from ()) mp.121-123℃ Elemental analysis: _{C23H30O7S (} 450.536) Calculated value: C, _61.31 ; H, 6.71; S, 7.12 Experimental value: C, 61.11 ; H, 6.95; S, 6.88 [α] ²³ _D +136.8±1.7 (c=1.068, chloroform; methanol=
1:1) UVλ ^EtOH _nax (nm; ε) 245.5 (10700) 305 (2250) IRν ^Nujol _nax (cm ^-1 ) 3470 (sh), 3415, 3238, 2660
，
1710, 1672, 1550. NMR (CD ₃ OD, δ) 1.31, 1.35 (3H, 19-H) 3.44 (2H, s, -SCH ₂ CO-) 3.70 (1H, m, 6α-H) 4.58 (1H, ^{d .} M ⁺ .−108 4%) 〃 44 (100%) 〃 18 (97%) In addition, this product contains methyl (11β, 18; 18α, 20α
-Bisepoxy-20β,21-isopropylidenedioxy-3-oxo-4-pregnen-4-ylthio)acetate () (R ¹ = SCH ₂ COOCH ₃ ,
R ² = H, 20, 21 (R) acetonide) was treated in the same way, and (11β, 18; 18α, 20α-bisepoxy-20β,
21-isopropylideneoxy-3-oxo-4-
Pregnen-4-ylthio)acetic acid (') (R ¹ =
SCH ₂ COOH, R ² = H, 20, 21 (R) acetonide)
obtained by obtaining. (Reference physical property values) (') (R ¹ = SCH ₂ COOH, R ² = H, 20, 21 (R)
Acetonide mp.168-170℃ Elemental analysis: as C ₂₆ H ₃₄ O ₇ S (490.598) Calculated value: C, 63.65; H, 6.99; S, 6.54 Experimental value: C, 63.72; H, 6.85; S, 6.64 [ α] ²³ _D +104.1±1.4 (c=1.00, chloroform) NMR (CDCl ₃ , δ) 1.34, 1.40, 1.50 (9H, 19-H and

[Formula]) 3.37 (2H, s, -SCH ₂ CO-) 3.89, 4.18 (2H, ABq, J _AB = 9Hz, ν〓 _AB = 17
Hz, 21-CH ₂ -) 4.83 (1H, d, J = 6Hz, 11-H) 5.30 (1H, s, 18-H) 5.90 (1H, broad, -COOH) Mass spectrometry: m/z489 (M ⁺・−1,16%) 〃 471 (M ⁺・−19,7%) 〃 400 (M ⁺・−90, 20%) 〃 43 (100%) Example 2 (11β, 18-epoxy-18α, 21 -dihydroxy-3,20-dioxo-4-pregnene-6β
-ylthio)acetic acid, (6β-carboxymethylthioALD), ()(R ¹ = H, R ² = β・
Manufacturing of SCH ₂ COOH) The same operation as in Example 1 was carried out with methyl (11β, 18;
18α,20β-bisepoxy-20α,21-isopropylidenedioxy-3-oxo-4-pregnene-
6β-ylthio)acetate () (R ¹ = H, R ² =
β・SCH ₂ COOCH ₃ , 20, 21 (S) acetonide, (11β, 18; 18α, 20β-bisepoxy-
20α,21-isopropylidenedioxy-3-oxo-4-pregnen-6β-ylthio)acetic acid (′) (R ¹ = H, R ² = β・SCH ₂ COOH, 20, 21
(S) acetonide), the title compound ()
(R ¹ =H, R ² =β·SCH ₂ COOH) was obtained. (Physical property value) (′) (R ¹ = H, R ² = β・SCH ₂ COOH, 20, 21
(S) acetonide) mp.210-213℃ NMR (CD ₃ OD, δ) 1.50, 1.54 (9H, 19-H and

[Formula]) 5.41 (1H, s, 18-H) 5.76 (1H, s, 4-H) () (R ¹ = H, R ² = β・SCH ₂ COOH),
mp.164-166℃ Elemental analysis: C ₂₃ H ₃₀ O ₇ S (450.536) as H ₂ O Calculated value: C, 58.95; H, 6.85; S, 6.84 Experimental value: C, 58.38; H, 6.65; S, 6.31 Mass spectrometry: m/z414 (M ⁺・-36<1%) 〃 44 (45%) 〃 18 (100%) This product also contains methyl (11β, 18, 18α, 20α
-Bisepoxy-20β-isopropylidene-3-
Oxo-4-pregnene-6β-ylthio)acetate () (R ¹ = H, R ² = β・SCH ₂ ―
COOCH ₃ , 20, 21 (R) acetonide) was treated in the same manner, (11β, 18; 18α, 20α-bisepoxy-20β
-isopropylidene-3-oxo-4-pregnen-6β-ylthio)acetic acid (') (R ¹ = H, R ² =
β・SCH ₂ COOH, 20, 21 (R) acetonide). (Reference physical property values) (′) (R ¹ = H, R ² = β.SCH ₂ COOH, 20, 21
(R) acetonide), mp.164-166℃ Elemental analysis: _C26H34O7S4 ( _490.598 ) Calculated value: C, _63.65 ; H, _6.99 ; S, 6.54 Experimental value: C, 63.90; H, 6.76; S, 6.46 [α] ²⁷ _D +144.6±1.0 (c=1.010, chloroform) UVλ ^EtOH _nax (nm; ε) 243 (13200) IR: ν ^Nujol _nax (cm ^-1 ) 3300 to 3000, 2628, 1730,
1722, 1649, 1599, 1063, 886. NMR (CDCl ₃ , δ) 1.40, 1.50, 1.54 (9H, 19-H and

[Formula]) 3.16 (2H, s, -SCH ₂ CO-) 3.88, 4.16 (2H, ABq, J _AB = 9Hz, ν〓 _AB =
1.68Hz, 21-CH ₂ -) 4.81 (1H, d, J = 6Hz, 11-H) 5.31 (1H, S, 18-H) 5.81 (1H, s, 4-H) Mass spectrometry: m/z472 ( M ⁺・−18 1%) 〃 414 (M ⁺ .−76 7%) 〃 44 (100%) Reference example 1 (Production of raw materials for Examples 1 and 2) Used as starting materials in Examples 1 and 2 above Compound () [(R ¹ = SCH ₂ COOCH ₃ , R ² =
H, 20, 21 (S) and (R) acetonide) and (R ¹ = H, R ² = β・SCH ₂ COOCH ₃ , 20, 21
(S) acetonide)] were obtained as follows. That is, first, in dimethylformamide, hemiacetal type ALD, that is, 11β,18-epoxy-18α,21-dihydroxy-4-pregnene-3,20-dione () in the presence of p-toluenesulfonic acid By reacting with 2,2-dimethoxypropane, 11β,18;18α,20β-bisepoxy-20α,21-isopropylidenedioxy-3-methoxy-3,5-pregnadiene ()
(20, 21 (S) acetonide and 11β, 18; 18α,
20α-bisepoxy-20α,21-isopropylideneoxy-3-methoxy-3,5-pregnathien() (20,21(R) acetonide). Next, these compounds ( ) [(20,21(S)acetonide) and (20,21(R)acetonide)] were individually reacted with N-bromoacetamide to form 6β-bromo-11β,18;18α, respectively. ,20β-bisepoxy-20α,21-isopropylidenedioxy-4
-Pregnen-3-one () (R ¹ = H, R ² =
β・Br,20,21(S)acetonide) or 6α-bromo-11β,18;18α,20β-bisepoxy-20α,
21-isopropylidenedioxy-4-pregnen-3-one () (R ¹ = H, R ² = α・Br, 20, 21
(S) acetonide) or 6β-bromo-11β,
18; 18α, 20α-bisepoxy-20β, 21-isopropylidenedioxy-4-pregnen-3-one () (R ¹ = H, R ² = β・Br, 20, 21 (R) acetonide) or 6β -Bromo-11β,18;18α,-bisepoxy-20β,21-isopropylidenedioxy-4-pregnen-3-one () (R ¹ = H,
R ² =α·Br, 20, 21 (R) acetonide).
As a result of reacting these with sodium methylthioglycolate at room temperature without isolation, the main products were () [(R ¹ = SCH ₂ COOCH ₃ ,
R ² = H, 20, 21 (S) and (R) acetonide)]
is obtained, and at the same time a small amount of () [(R ¹ = H, R ² =
β・SCH ₂ COO―CH ₃ , 20, 21 (S) and (R)
acetonide)] was obtained. Note that both of the latter are 6β
The 6α form was not obtained in the body. The physical property values of these intermediates will be described below. () (20, 21 (S) acetonide) mp.164-168℃
(Recrystallized from acetone/hexane) Elemental analysis: As C ₂₅ H ₃₄ O ₅ (441.522) Calculated value: C, 72.43; H, 8.27 Experimental value: C, 72.39; H, 8.31 [α] ²⁶ _D −32.4±1.0 (c=0.701, chloroform) UV: λ ^EtOH _nax (nm; ε) 240.5 (19500) IRν ^Nujol _nax (cm ^-1 ) 1650, 1625, 1246, 1236,
1168, 1048, 1020, 996 NMR (CDCl ₃ , δ) 1.08, 1.34, 1.51 (9H, 19-H and

[Formula]) 3.58 (3H, s, - OCH ₃ ) 3.88, 4.04 (2H, ABq, J _AB = 9Hz, ν〓 _AB = 6.3
Hz, 21-CH ₂ ) 4.90 (1H, d, J = 6Hz, 11-H) 5.14 (2H, 4H and 6-H) 5.33 (1H, s, 18-H) () (20, 21 (R) Acetanide) mp.126-129
°C (from dichloromethane/methanol) Elemental analysis: C ₂₅ H ₃₄ O ₅ (414.522) Calculated value: C, 72.43; H, 8.27 Experimental value: C, 72.38; H, 8.27 [α] ²⁶ _D −86.1±2.0 ( c=0.634, chloroform) UV: λ ^EtOH _nax (nm; ε) 240.5 (19150) IR: ν ^Nujol _nax (cm ^-1 ) 1648, 1622, 1233, 1180,
1168, 1071, 1053, 989, 725 NMR (CDCl ₃ , δ) 1.09, 1.40, 1.50 (9H, 19-H and

[Formula]) 3.59 (3H, s, - OCH ₃ ) 3.90, 4.20 (2H, ABq, J _AB = 9Hz, ν〓 _AB =
17.9Hz, 21-CH ₂ -) 4.90 (1H, d, J = 6Hz, 11-H) 5.15 (2H, 4H and 6-H) 5.30 (1H, s, 18-H) () (R ¹ = SCH ₂ COOCH ₃ , R ² = H, 20, 21
(S) acetonide) mp.118-120℃ (acetone/
(from hexane) Elemental analysis: as C ₂₇ H ₃₆ O ₇ S (504.624) Calculated value: C, 64.26; H, 7.19; S, 6.35 Experimental value: C, 64.09; H, 7.07; S, 6.07 [α] ²⁴ _D +169.3±2.1 (c=0.984, chloroform) UV: λ ^EtOH _nax (nm; ε) 242 (11570) 303 (2550) IR: ν ^Nujol _nax (cm ^-1 ) 1732, 1667, 1551, 1226,
1192, 1050, 1012, 990, 885, 815 NMR (CDCl ₃ , δ) 100MHz 1.34, 1.49 (9H, 19-H and

[Formula]) 3.36, 3.42 (2H, ABq, J _AB = 14Hz, ν〓 _AB = 5.9
Hz, ―SCH ₂ CO―) 3.64 (3H, s, ―OCH ₃ ) 3.90, 4.02 (2H, AB _q , J _AB = 9Hz, ν〓 _AB = 12
Hz, 21-CH ₂ ) 4.86 (1H, d, J = 6Hz, 11-H) 5.30 (1H, s, 18-H) Mass spectrometry: m/z504 (M ⁺ .29%) m/z446 (M ⁺ .−58, 37%) m/z43 (100%) () (R ¹ = H, R ² = β・SCH ₂ COOCH ₃ , 20,
21 (S) acetonide) mp. 206-208℃ (recrystallized from acetone/hexane) Elemental analysis: as C ₂₇ H ₃₆ O ₇ S (504.624) Calculated value: C, 64.26; H, 7.19; S, 6.35 Experimental value :C, 64.90; H, 7.37; S, 7.15 [α] ²⁴ _D +210.3±3.2 (c=0.787, chloroform) UV: λ ^EtOH _nax (nm; ε) 241 (15400) IR: ν ^Nujol _nax (cm ^-1 ) 1732, 1682, 1605, 1051,
1028, 999 NMR (CDCl ₃ , δ) 100MHz (diethyl ether mixed in sample) 1.42, 1.49, 1.52 (9H, 19-H and

[Formula]) 3.14 (2H, ―SCH ₂ CO) 3.72 (3H, s, ―OCH ₃ ) 3.92, 4.03 (2H, AB _q , J _AB = 9Hz, ν〓 _AB = 11
Hz, 21-CH ₂ -) 4.83 (1H, d, J = 6Hz, 11-H) 5.34 (1H, s, 18-H) 5.70 (1H, s, 4-H) Mass spectrometry: m/z504 (M ⁺ ., 80%) m/z446 (M ⁺ .−58, 72%) m/z43 (100%) () (R ¹ = SCH ₂ COOCH ₃ , R ¹ = H, 20, 21
(R) acetonide) mp.140-142℃ (acetone/
(from hexane) Elemental analysis: as C ₂₇ H ₃₆ O ₇ S (504.624) Calculated value: C, 64.26; H, 7.19; S, 6.35 Experimental value: C, 64.34; H, 7.24; S, 6.32 [α] ²⁶ _D +95.5±1.1 (c=1.034, chloroform) UV: λ ^EtOH _nax (nm; ε) 241.5 (11760) 303 (2540) IR: ν ^Nujol _nax (cm ^-1 ) 1749, 1730, 1721, 1556,
1202, 1146, 1068, 1050, 1020, 997, 882 NMR (CDCl ₃ , δ) 100MHz 1.35, 1.39, 1.49 (9H, 19-H and

[Formula]) 3.36, 3.42 (2H, ABq, J _AB = 14Hz, ν〓 _AB = 6.1
Hz, ―SCH ₂ CO―) 3.65 (3H, s, ―OCH ₃ ) 3.88, 4.16 (2H, ABq, J _AB = 9Hz, ν〓 _AB = 28
Hz, 21―CH ₂ -) 4.83 (1H, d, J = 6Hz, 11―H) 5.28 (1H, s, 18―H) Mass spectrometry: m/z504 (M ⁺・, 62%) m/z72 ( 100%) () (R ¹ = H, R ² = β・SCH ₂ COOCH ₃ , 20,
21 (R) acetonide) mp.192-194℃ (from acetone/hexane) Elemental analysis: As C ₂₇ H ₃₆ O ₇ S (504.624) Calculated value: C, 64.26; H, 7.19; S, 6.35 Experimental value: C , 64.53; H, 7.19; S, 6.61 [α] ²⁴ _D +150.1±1.9 (c=0.997, chloroform) UV: λ ^EtOH _nax (nm; ε) 241.5 (15300) IR: ν ^Nujol _nax (cm ^-1 ) 1720, 1682, 1607, 1160,
1077, 1063, 1023 NMR (CDCl ₃ , δ) 100MHz 1.39, 1.49, 1.53 (9H, 19-H and

[Formula]) 3.10, 3.19 (2H, ABq, J _AB = 15Hz, ν〓 _AB = 19
Hz, ―SCH ₂ CO―) 3.73 (3H, s, ―OCH ₃ ) 3.86, 4.16 (2H, ABq, J _AB = 9Hz, ν〓 _AB = 28
Hz, 21-CH ₂ -) 4.81 (1H, d, J=6Hz, 11-H) 5.30 (1H, s, 18-H) 5.71 (1H, s, 4-H)

[Table] Example 3 N-(p-hydroxyphenethyl)-2-
(11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnen-4-ylthio)acetamide () Production of R ² =H N-(p-hydroxyphenethyl)-2-
(11β, 18; 18α, 20β-bisepoxy-20α, 21-
Isopropylidenedioxy-3-oxo-4-pregnen-4-ylthio)acetamide () Add 3 ml of 70% acetic acid to 27 mg of R ² = H, 20, 21 (S) acetonide, and add 5 ml of 70% acetic acid at room temperature in a nitrogen gas stream.
Allowed time to react. Ice water is added to the reaction mixture, extracted with ethyl acetate, washed repeatedly with brine, dried over sodium sulfate, and then the solvent is distilled off under reduced pressure. The residue was dissolved in methanol and treated with activated carbon powder, and ether was added to this to give the title compound () in powder form. R ² =H) 21 mg were precipitated. Softens at 114-117℃. NMR (d ₆ -acetone, δ) 1.22, 1.30, 1.35 (3H, 19-H) 4.54, 4.78 (1H each, d, J = 6Hz, 11-H) 5.06, 5.40 (1H each, s, 18-H ) 6.77, 7.05 (4H, A ₂ B ₂ q, J _AB = 8Hz, ν〓 _AB =
17Hz,

[Formula]) NMR (CD ₃ OD, δ) 1.23, 1.34 (3H, 19-H) 5.03, 5.45 (1H each, s, 18-H) 6.74, 7.05 (4H, A ₂ B ₂ q, J _AB = 8Hz, ν〓 _AB =
18.4Hz,

[Formula]) UV: λ ^EtOH _nax (nm; ε) 225.5 (13800) 242 (8900) 279 (3000) 285.5 (300) 299 (2300) TLC Rf=0.32 (dichloromethane:acetone 1:1) Also, the compound () is N-(p-hydroxyphenethyl)-2-(11β,18-18;18α,20β-bisepoxy-20α,21-isopropylidenedioxy-3-oxo-4-pregnen-4-ylthio) Acetamide () ( It was also obtained by similarly treating R ² =H, 20, 21 (R) acetonide). Example 3-1 N-(p-hydroxy-α-methoxycarbonylphenethyl)-2-(11-β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnene-4- ylthio)acetamide()( Production of R ² =H) N-(p-hydroxy-α-methoxycarbonylphenethyl)-2-(11β,18;18α,20β-bisepoxy-20-α,21-isopropylideneoxy-3-oxo-4-pregnene-4- 2 ml of 70% acetic acid was added to 27 mg of ylthio)acetamide (20,21 (R) acetonide), and the mixture was reacted for 7 hours at room temperature in a nitrogen gas stream. Ice water was added to the reaction mixture, extracted with ethyl acetate, washed repeatedly in saturated brine, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The residue was analyzed using a reverse phase chromatograph (Merck Rover column, RP).
-8, size B; eluent, 65% methanol)
Ether was added to the obtained main fraction to obtain 18 mg of the title compound () in powder form. Yield, 70.9%. NMR (d ₆ -acetone, δ) 1.23, 1.33 (3H, 19-H) 3.31 (2H, s, -SCH ₂ CO) 3.65 (3H, s, -COOCH ₃ ) 4.51 (1H, m, NHC H COO- ) 4.56, 4.77 (1H each, d, 11-H) 5.06, 5.40 (1H each, s, 18-H) 6.77, 7.06 (4HA ₂ B _2q , J _AB = 8Hz, ν〓 _AB =
17.2,

[Formula]) 7.63 (1H, d, J = 7.5Hz, -CONH) Mass spectrometry: m/z627 (M ⁺ 62%) 〃 178 (100%) TCL: Rf, 0.19 (Chloroform: Acetone = 1:1 ) R, 0.33 (chloroform:methanol=9:
1) Example 3-2 N-[2-(1H-imidazol-4-yl)
ethyl]-2-(11β, 18-epoxy-18α,
21-dihydroxy-3,20-dioxo-4-pregnen-4-ylthio)acetamide ()
(

Production of [Formula] R ² = H) N-[2-(1H-imidazol-4-yl)
ethyl]-2-(11β,18;18α,20β-bisepoxy-20-α,21-isopropylideneoxy-3
-Oxo-4-pregnen-4-ylthio)acetamide (20,21(R)acetonide (), 12 mg
1 ml of 70% acetic acid was added, and the mixture was reacted for 6.5 hours at room temperature in a nitrogen gas stream. The reaction mixture was treated in the same manner as in Example 3-1 to obtain 6 mg of the title compound () in powder form (softened at 87°C). Yield, 53.7
%. NMR (d ₆ -acetone, δ) 1.22, 1.33 (3H, 19-H) 4.55, 4.77 (1H each, d, 11-H) 5.06, 5.41 (1H each, s, 18-H) 6.85, 7.62 (2H Each, broad s,

[Formula]) 7.55 (1H, -NH-) Mass spectrometry: m/z543 (M ⁺・1%) 〃 94 (100%) TCL: Rf, 0.19 (Chloroform: methanol = 5:1) Reference example 2 (Implementation Preparation of Starting Materials for Example 3) Starting Materials for Example 3 Above () [( R ² = H) 20, 21 (S) or (R) acetonide] is the compound () described in Reference Example 1 above [(R ¹ = SCH ₂ COOCH ₃ , R ² = H) 20, 21 (S)
or (R)acetonide] to the corresponding carboxylic acid obtained by demethylating tyramine, 1-hydroxybenzotriazole, and dicyclohexylcarbodiimide in tetrahydrofuran under ice cooling (W. Ko¨nig, et al.: Chem. .Ber.103,
788 (1970)) and then purified.

[Table] Mass spectrometry: m/z 609 (M ⁺・3%) 〃 594 (M ⁺・−153%) 〃 120 (100%) TCL: Rf, 0.30 (ethyl acetate: benzene = 4:1) 0.41 (chloroform : Acetone=3:1) Reference Example 2-1 (Production of starting material for Example 3-1) Also, starting material for Example 3-1 () [ R ² = H, 20, 21 (S) or (R) acetonide]
is the compound () [(R ¹ =
SCH ₂ COOCH ₃ , R ² =H), 20, 21 (S) or (R) acetonide] in the same manner as above. In the case of (R) acetonide, 28 mg of the title compound in powder form (softening point 108°) was obtained from 35 mg of ester. Yield 60.5% (20, 21 (R) acetonide) NMR (CDCl ₃ δ) 1.26, 1.31, 1.40, 1.49 (19-H and

[Formula]) 3.26 (2H, s, -SCH ₂ CO-) 3.85, 4.13 (2H, ABq, J _AB = 9Hz, ν〓 _AB =
16.6, 21―CH ₂ ―) 4.63 (1H, m, ―NH CH COOCH ₃ ) 4.78 (1H, d, J=6Hz, 11―H) 5.24 (1H, s, 18H) 6.66, 6.96 (4H, A ₂ B ₂ q, J _AB = 8.5Hz, ν〓 _AB =
17.7,

[Formula]) 7.64 (1H, d, J = 7.5Hz, -CONH-) IR: ν ^CHCl3 _nax (cm ^-1 ) 3584, 3280 (broad) 1745
，
1672, 1613, 1594. Mass spectrometry: m/z667 (M ⁺ 2%) 〃 107 (100%) TCL: Rf, 0.41 (chloroform:acetone = 3:1) Reference example 2-2 (Example 3-2 (Manufacture of the starting raw material of)) The starting raw material of Example 3-2 above () [

[Formula] R ² = H, 20, 21 (S) or (R) acetonide] is
The compound described in Reference Example 1 [(R ¹ =
SCH ₂ COOCH ₃ , R ² =H) 20, 21 (S) or (R) acetonide] in the same manner as above. However, the carboxylic acid obtained in the middle (the amount obtained from 22 mg of the corresponding methyl ester) was dissolved in 2 ml of dimethylformamide instead of tetrahydrofuran, and 9 mg (1.1 mol) of histamine hydrochloride, 15.4 μg of N-methylmorpholine ( 3.5 mol) and 6.5 mg (1.1 mol) of 1-hydroxybenztriazole, followed by 11.7 mg of dicyclohexylcarbodiimide.
(1.3 mol) were sequentially added and reacted for 17 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was treated with chromatography (Lover column, size A, manufactured by Merck & Co.). Ether was added to the obtained main fraction to obtain a powder (softening point, 122 13 mg of the title compound () was obtained (~124°C). Yield 51.1%. (20, 21 (R) acetonide) NMR (CDCl ₃ , δ) 1.30, 1.39, 1.49 (19-H, and

[Formula]) 3.87, 4.15 (2H, ABq, J _AB = 9Hz, ν〓 _AB =
16.8Hz, 21-CH ₂ -) 4.80 (1H, d, J = 6Hz, 11-H) 5.27 (1H, S, 18-H) 6.81, 7.55 (2H each, broad s,

[Formula]) 6.41, 7.92 (2H, -NH-) Mass spectrometry: m/z 583 (M ⁺ 1%) 〃 94 (100%) TCL: Rf 0.39 (chloroform:methanol = 6:1) Example 4 Compound () (Preparation of immunogen and antiserum with R ¹ = SCH ₂ COOH, R ² = H) Immunogen Compound described in Example 1 () (R ¹ =
SCH ₂ COOH, R ² = H) 18 mg in dioxane 500μ
Dissolve tributylamine 9.3 μ and isobutyl chlorocarbonate while keeping at 10-12 °C.
Add 4.9μ and stir for 30 minutes to obtain an active ester solution. On the other hand, dissolve 69 mg of bovine serum albumin (BSA) in 2 ml of water, add 2 ml of dioxane, mix, and then adjust the pH to 8.5 with 1N sodium hydroxide. After adding the above ester solution dropwise under ice-cooling, stirring was continued for 4 hours while maintaining the pH at 8.5, the mixture was coupled, dialyzed against water, and then freeze-dried to obtain the title compound ().
50 mg of BSA conjugate was obtained. The binding molar ratio of hapten to carrier protein was 15. ) Dissolve 250 μg of the above immunogen in 250 μg of physiological saline and add 250 μg of Freund's Combination Adjuvant to make an emulsion. This is injected intradermally into the back of the rabbit, and this is repeated 5 times at 3-week intervals. On the 10th day after the final immunization, whole blood was collected to obtain antiserum. When the titer of this antiserum was measured using the RIA system described below, the value expressed as the dilution factor of the antiserum required to bind 50% of 2.2 x 10 ⁴ dpm of ¹²⁵ I-labeled ALD was 4.6 x 10. It was ^four times as hot. Example 5 Compound ()( Labeling of R ² =H) Compound ( ) ( ) prepared according to the method described in Example 3 R ² = H) 500ng, dimethylformamide 5μ,
0.5M - Phosphate buffer (PH7.5) 25μ and ^125I
-Pour 1mCi of sodium iodide into a glass tube, add 5μ of phosphate buffer (PH7.5) containing 20μg of chloramine T, stir at room temperature for 45 seconds, then add 5μ of an aqueous solution containing 100μg of sodium metabisulfite. The reaction was stopped. Add 500 μg of potassium iodide to this, extract with 1 ml of dichloromethane, dry with sodium sulfate, and perform chromatography (Sephadex LH-20, 0.7
cmφ×20cm, dichloromethane:methanol=9:
1) was carried out to obtain 1000-1500 μCi/μg of the title compound. The chromatogram (elution curve) was as shown in FIG. Example 5-1 Compound () [ R ² =H] and the compound ()
[

Label of [Formula] R ² = H] According to the description in the first half of Example 5, Example 3-
The title compounds () obtained in 1 and 3-2 were labeled. The labeled substances were each purified by chromatography (Sephadex LH-20, 9 mmφ x 90 mm, ethanol-M/10 citrate buffer pH 2.2), and tyrosine methyl ester with a radioactivity of 500 μCi/μg and tyrosine methyl ester with a radioactivity of 400 μCi /μg of histamine bodies were obtained. Chromatogram (elution curve)
Shown in FIGS. 5 and 6, respectively. Example 6 11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnene-6β-
Il-hemisuccinate () (R ¹ = H, R ² =
Production of β・OCOCH ₂ CH ₂ COOH) 11β, 18; 18α, 20β-Bisepoxy-20α, 21
-isopropylidenedioxy-3-oxo-4-
Pregnene-6β-yl hemisuccinate ()
(R ¹ = H, R ² = β・OCOCH ₂ CH ₂ COOH) 35 mg
The mixture was dissolved in 4 ml of 70% acetic acid and stirred at room temperature for 16 hours in a nitrogen gas stream. Water was added to the reaction mixture, extracted with ethyl acetate, washed repeatedly with saturated brine, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The resulting residue was dissolved in methanol and treated with activated carbon powder. Add ether to this to obtain the powdered title compound () (R ¹ = H, R ² = β・OCOCH ₂ CH ₂ COOH)
16 mg was precipitated. Yield 49.6%. Also, the compound () (R ¹ = H, R ² = β・
OCOCH ₂ CH ₂ COOH, 20, 21 (S) acetonide)
() (R ¹ = H, R ² = β・
OCOCH ₂ CH ₂ COOH, 20, 21 (R) acetonide)
79 mg was treated in the same manner to obtain 34 mg of the title compound (). () (R ¹ = H, R ² = β・OCOCH ₂ CH ₂ COOH) Elemental analysis: C ₂₅ H ₃₂ O ₉ 1/2 H ₂ O (485,514) Calculated value: C, 61.84; H, 6.85 Experimental value: C, 62.09; H, 7.30 Mass spectrometry: m/z458 (M ⁺ -18, 2%), m/z429 (M ⁺ -
47, 11%) m/z 358 (M ⁺・−118, 57%), m/z 149 (100
%) NMR (CDCl ₃ δ) 1.28, 1.39, (3H, 19-H) 2.66 (4H, s,

[Formula]) 5.33 (1H, s, 18-H) 5.45 (1H, w1/2=8Hz, 6α-H) 5.94 (1H, s, 4-H) TLC Rf=0.17 (Ethyl acetate: cyclohexane: acetic acid: Ethanol = 20:10:1:2) Example 7 11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnene-6α-
Il-hemisuccinate () (R ¹ = H, R ² =
Production of α・OCOCH ₂ CH ₂ COOH) Similar operations to those described in Example 6 were carried out.
11β, 18; 18α, 20β-Bisepoxy-20α, 21-
Isopropylidenedioxy-3-oxo-4-pregnen-6α-yl hemisuccinate ()
(R ¹ = H, R ² = α・OCOCH ₂ CH ₂ COOH, 200,
21 (S) acetonide) to give the title compound () (R ¹ = H, R ² = α・OCOCH ₂ CH ₂ COOH)
Obtained 10 mg. Yield 47.1%. This product also contains 11β, 18; 18α, 20α-bisepoxy-20β, 21-isopropylidenedioxy-3-
Oxo-4-pregnene-6α-yl hemisuccinate () (R ¹ = H, R ² = α・
OCOCH ₂ CH ₂ COOH, 20, 21 (R) acetonide)
Even when 49 mg was treated in the same manner, 26 mg was obtained. () (R ¹ = H, R ² = α・OCOCH ₂ CH ₂ COOH) Elemental analysis: C ₂₅ H ₃₂ O ₉・2H ₂ O (512, 538) Calculated value: C, 58.58; H, 7.08 Experimental value :C, 57.99; H, 6.78 Mass spectrometry: m/z458 (M ⁺ -18, <1%) m/z429 (M ⁺ -47, 20%) m/z358 (M ⁺ -118, 57%) m/z28 (100%) NMR (CDCl ₃ δ) 1.27 (19-H) 2.70, 2.67 (sh) (4H,

[Formula]) 5.32 (1H, s, 18-H) 5.94 (1H, d, J = 2Hz, 4-H) TLC Rf = 0.17 (ethyl acetate: cyclohexane: ethanol:
Acetic acid = 20:10:2:1) Reference Example 3 (Production of raw materials for Examples 6 and 7) Compound () [(R ¹ = H, R ² = β・OCOCH ₂ CH ₂ COOH),
20, 21 (S) or (R) acetonide, and (R ¹ = H, R ² = α・OCOCH ₂ CH ₂ COOH), 20,
21 (S) or (R) acetonide] can be obtained from the compound ( ) [20, 21 (S) or (R) acetonide] described in Reference Example 1 as follows. First, the compound () [20, 21 (S) or (R)
11β, 18; 18α, and 20β-bis, respectively, were dissolved in 10% aqueous tetrahydrofuran, reacted with m-chloroperbenzoic acid under stirring under ice-cooling, and subjected to chromatographic fractionation. Epoxy-6β-hydroxy-2α,21-isopropylidenedioxy-4-pregnen-3-one () (R ¹ = H, R ² = β・OH, 20, 21 (S) acetonide) and 11β, 18; 18α, 20β-Bisepoxy-
6α-hydroxy-20α,21-isopropylidenedioxy-4-pregnen-3-one () (R ¹ =
H, R ² = α・OH, 20,21(S)acetonide), and 11β,18;18α,20α-bisepoxy-6β-hydroxy-20β,21-isopropylidenedioxy-4-pregnen-3-one ()(R ¹ = H, R ²
= β・OH, 20, 21 (R) acetonide) and 11β,
18; 18α, 20α-bisepoxy-6α-hydroxy-20β, 21-isopropylidenedioxy-4-pregnen-3-one () (R ¹ = H, R ² = α・
OH, 20, 21 (R) acetonide) is obtained.

[Table] Next, these compounds () were dissolved separately in pyridine, succinic anhydride and 4-dimethylaminopyridine were added, and heated (67℃ for β-OH form, α-
The OH form was reacted at 38°C), and the corresponding compound () [(R ¹ = H, R ² = β・
OCOCH ₂ CH ₂ COOH), 20, 21 (S) or (R)
acetonide, and (R ¹ = H, R ² = α・
OCOCH ₂ CH ₂ COOH), 20, 21 (S) or (R)
acetonide] was obtained.

[Table] Example 8 Compound () [(R ¹ = H, R ² = β and α・
Preparation of immunogen and antiserum by OCOCH ₂ - CH ₂ COOH)] Immunogen; The title compound () produced by the method described in Examples 6 and 7 was individually treated in the same manner as in Example 4. () (R ¹ = H, R ² = β・OCOCH ₂ CH ₂ COOH)
-BSA and () (R ¹ = H, R ² = α・
OCOCH ₂ CH ₂ COOH)-BSA conjugate was obtained. The amounts of conjugates obtained from 14.5 mg of the starting material were 65 mg and 68 mg, respectively.
The binding molar ratio of hapten to carrier protein was 11 and 20, respectively. ) Antiserum Immunization was performed using the above immunogen in the same manner as described in Example 4, to obtain antiserum. The titers of these antisera, shown in the same expression as above, were 9×10 ⁴ times and 6×10 ³ times, respectively. Example 9 11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnene-6β-
yl=4-(p-hydroxyphenethylamino)-4-oxobutyrate () R ¹ =H,

[Formula]) and 11β,18-epoxy-18α,21-dihydroxy-3,20-dioxo-4-pregnene-6α-
yl=4-p-hydroxyphenethylamino)
-4-oxobutyrate () (R ¹ = H,

Production of [formula]) 11β, 18; 18α, 20β-Bisepoxy-20α, 21
-isopropylidenedioxy 3-oxo-4-pregnen-6β-yl=4-(p-hydroxyphenethylamino)-4-oxobutyrate () (R ¹ =H,

[Formula] 20, 21 (S) acetonide) 13 mg and 11β, 18; 18α,
20α-bisepoxy-20α, 21-isopropylidenedioxy-3-oxo-4-pregnene-6β
-yl=4-(p-hydroxyphenethylamino)-4-oxobutyrate () (R ¹ =H,

[Formula] 20, 21 (R) acetonide) 21mg combined, 70% acetic acid 2ml
After stirring at room temperature for 16 hours in an argon gas stream, ice water was added and the mixture was extracted with ethyl acetate. After washing this repeatedly with saturated saline,
It was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in methanol and treated with activated carbon to give the title compound () (R ¹ = H,

[Formula]) 24 mg was obtained. Similarly, 11β,18;18α,20β-bisepoxy-20α,21-isopropylidenedioxy-3-oxo-4-pregnen-6α-yl=4-(p-
Hydroxyphenethylamino)-4-oxobutyrate () (R ¹ = H,

[Formula] 20, 21 (S) acetonide) 6 mg and 11β, 18; 18α,
20α-bisepoxy-20β, 21-isopropylidenedioxy-3-oxo-4-pregnene-6α
-yl=4-(p-hydroxyphenethylamino)-4-oxobutyrate () (R ¹ =H,

[Formula] From 8 mg of 20, 21 (R) acetonide), the title compound () (R ¹ = H,

[Formula]) Obtain 10mg Ta.

【table】

[Table] Reference Example 4 (Production of raw material for Example 9) Compound () which is the starting material for Example 9 above
[(R ¹ = H) (R ² = β or

[Formula]) 20, 21 (S) or (R) acetonide] is Reference Example 3
Compound () [(R ¹ = H) (R ²
= β or α・OCOCH ₂ CH ₂ COOH), 20, 21
(S) or (R) acetonide] were combined with tyramine by the dicyclohexylcarbodiimide/1-hydroxybenzotriazole method (the same method as described in Reference Example 2).

【table】

[Table] Example 10 Compound () according to ¹²⁵ I (R ¹ = H, R ² = β or

Labeling of [Formula]) Compound () prepared according to the method described in Example 9 (R ¹ = H, R ² = β or

[Formula]) was subjected to the same operation as in Example 5 to obtain ¹²⁵ -
()(R ¹ =H,

[Formula]) 330 μCi and ¹²⁵ () (R ¹ = H,

[Formula]) 600μCi was obtained. The chromatograms (elution curves) were as shown in FIGS. 2 and 3, respectively. Reference Example 5 In addition to the ALD labeling compounds described in Examples 5 and 10, known 11β,18-epoxy, 18α,21-
Dihydroxy-4-pregnene-3,20-dione = 3-{O-[N-(p-hydroxylphenethyl)carbamoylmethyl]oxime} () is iodinated by the chloramine T method to give ¹²⁵ -()
was prepared and its specific radioactivity was confirmed to be 680Ci/mmol. Reference Example 6 RIA of serum ALD 1) Procedure 100μ of serum was collected in a test tube, and ¹²⁵ I-labeled ALD (2.2×10 ⁴ dpm) obtained by either of Examples 5 and 10 or Reference Example 5 above was collected. buffer (0.1M containing 0.1% bovine serum γ-globulin)
Citrate buffer, PH5.0) 100μ, 8-anilinonaphthalene-1-sulfonic acid solution (625mg/
ml) 400μ and various antiserum solutions (prepared according to Examples 4 and 8) at the dilution ratios listed below: 100μ
After incubation at 4°C for 16 hours, add 1 ml of 25% polyethylene glycol 6000 solution, shake for 10 seconds, centrifuge (2250 x g, 20 minutes), and aspirate the supernatant. The radioactivity of the precipitate was measured using a well-type scintillation counter.

[Table] 2) Standard curve: Using the procedure described in 1) above, the ALD standard solution (0
The standard curve prepared for 5000 pg/ml) was as shown in FIG. Here, B ₀ is the radioactivity of the antigen-antibody conjugate obtained in the absence of non-radioactive ALD, and B is the radioactivity of the conjugate obtained when non-radioactive ALD is added. 3) Cross-reactivity: The results of the cross-reactivity test (method described in 1 above) of the various substituted aldosterones obtained according to the present invention with various steroids are summarized in the following table.

[Table] Reference Example 6-1 RIA of serum ALD According to the procedure described in Example 1 above, the labeled substance obtained in Example 5-1 and the anti-( ) (R ¹ = H, R ² = α・
The B/B ₀ standard curve prepared using OCOCH ₂ CH ₂ COOH)-BSA was as shown in FIG. Reference Example 7 EIA of serum ALD 1) Preparation of enzyme-labeled antigen: The above-mentioned carboxylic acid derivative of ALD [shown in Example 1 () (R ¹ = SCH ₂ COOH, R ² =
H), () shown in Example 2 (R ¹ = H, R ² =
β・SCH ₂ COOH), as shown in Example 6 ()
(R ¹ = H, R ² = β・OCOCH ₂ CH ₂ COOH), () (R ¹ = H, R ² = α・
OCOCH ₂ CH ₂ ―COOH) and the known 11β, 18
-Epoxy-18α,21-dihydroxy-4-pregnene-3,20-dione=3-(O-carboxymethyl)oxime ()] 6 μmol each was individually dissolved in 400 μm of dioxane, containing 3.3 μmol of tri-n-butylamine. Add 50μ of dioxane and add 50μ of dioxane containing 6μmol of isobutyl chlorocarbonate while cooling to 10-12℃.
The mixture was stirred for 30 minutes to obtain an active ester solution. On the other hand, β-D-galactosidase (30U/
mg, manufactured by Boehringer Mannheim) 2 mg for 10
Dissolve in 3.5ml of dioxane and adjust the pH to 9.2 with 0.1N sodium hydroxide. The above active ester solution was added dropwise to the solution under ice cooling and stirring, and after 5 minutes, the pH was adjusted to 8.5. After further stirring at 4℃ for 4 hours, 0.05% sodium azide was added.
The β-D-galactosidase obtained by dialysis against 0.01M phosphate buffer (PH7.4) for 2 days
ALD derivatives/conjugates (according to the order of the labeled compounds, a, b, c, d, respectively)
or e) was used as an enzyme-labeled antigen. 2) EIA: Take 100μ of ALD standard solution into a test tube and add 0.1M
Phosphate buffer (PH7.4) 300μ and enzyme-labeled antigen dilution solution (enzyme-labeled antigen diluted 100,000 times)
After adding 100μ of the solution and mixing, 50μ of the antiserum solution was added and allowed to stand at 4°C for 5 hours. Next, Immunobead (Bio-
After adding 100μ of Rad's product name, 1 mg/ml) and leaving it at 4℃ for 1 hour, it was centrifuged (2000 Washed twice with liquid (PH7.4). Add this to 0.1M-sodium chloride, 0.001M.
Magnesium chloride, 0.05% sodium azide,
0.01M phosphate buffer (PH7.3) containing 0.5% BSA
and further suspended in 4-methylumbelliferyl-β-D-galactoside solution (80 μg/ml).
After adding 400μ and incubating at 37℃ for 30 minutes, add 0.1M glycine buffer (PH10.5).
The reaction was stopped by adding 3 ml of the solution, and the fluorescence intensity (excitation: 360 nm, fluorescence: 448 nm) was measured. When measuring ALD in serum, use 100μ of test serum and 8-anilinonaphthalene-1-sulfonic acid solution (800μg/ml) instead of 100μ of standard solution and 300μ of buffer solution, respectively.
Using 300μ, proceed in the same manner as above.
Further, possible combinations (marked with ○) of the enzyme-labeled antigen and antiserum to be used are shown in Table 1.

【table】 [Brief explanation of drawings]

Figures 1, 2 and 3 are respectively ¹²⁵ I-()
( R ² = H), ¹²⁵ I-() (R ¹ = H,

[Formula]) and ¹²⁵ I-() (R ¹ = H,

This is a column chromatographic elution curve of [Formula]). In addition, Figure 4 shows either the labeled substance or the anti-ALD antiserum.
Alternatively, both are B/B ₀ standard curves prepared using the substituted ALD according to the present invention. Figures 5 and 6 are ¹²⁵ I-(), respectively. R ² = H and ¹²⁵ I—()
(

[Formula] R ² = H) is a column chromatographic elution curve. FIG. 7 is a B/B ₀ standard curve where both the label and the anti-ALD antiserum were substituted ALD according to the invention.

Claims (1)

[Claims] 1 formula (However, in the formula, either R ¹ or R ² is hydrogen, and when R ¹ is hydrogen, R ² is -S-(CH ₂ ) _1-3 COR ³
or a group represented by -O-CO-(CH ₂ ) _1-5 COR ³ , when R ² is hydrogen, R ¹ is -S-(CH ₂ ) _1-3 COR ³
A group represented by R ³ is a hydroxy group, or a tyramine residue, a tyrosine lower alkyl ester residue, a histamine residue, a histidine residue, or a 7-aminoheptanoyl tyrosine lower alkyl ester, each of which may be iodinated. Substituted aldosterones represented by (representing a residue). 2 formulas (However, in the formula, either R ¹ or R ² is hydrogen,
When R ¹ is hydrogen, R ² is a group represented by -S-(CH ₂ ) _1-3 COR ³ or -O-CO-(CH ₂ ) _1-5 COR ³ ,
When R ² is hydrogen, R ¹ is a group represented by -S-(CH ₂ ) _1-3 COR ³ , where R ³ is a hydroxy group, or a tyramine residue which may be iodinated, or a lower alkyl tyrosine group. Represents an ester residue, histamine residue, histidine residue, or 7-aminoheptanoyltyrosine lower alkyl ester residue. Also, X represents a glycol protecting group)
characterized by subjecting a compound represented by the formula to a deprotecting group reaction A method for producing substituted aldosterones represented by the formula (wherein R ¹ and R ² have the same meanings as above). 3 formulas (However, in the formula, either R ¹ or R ² is hydrogen, and when R ¹ is hydrogen, R ² is -S-(CH ₂ ) _1-3 COR ³
or a group represented by -O-CO-(CH ₂ ) _1-5 COR ³ , when R ² is hydrogen, R ¹ is -S-(CH ₂ ) _1-3 COR ³
A group represented by R ³ is a hydroxy group, or a tyramine residue, a tyrosine lower alkyl ester residue, a histamine residue, a histidine residue, or a 7-aminoheptanoyl tyrosine lower alkyl ester, each of which may be iodinated. An aldosterone testing reagent containing substituted aldosterones represented by (representing a residue). 4. The aldosterone testing reagent containing substituted aldosterones according to claim 3, wherein R ³ is a residue labeled with radioactive iodine. 5. The aldosterone testing reagent containing substituted aldosterones according to claim 3, wherein R ^{3 is} a reactive residue that binds to a biologically active protein. 6. The aldosterone testing reagent containing substituted aldosterones according to claim 5, wherein the biologically active protein is bovine serum albumin (BSA). 7. The aldosterone testing reagent containing substituted aldosterones according to claim 6, wherein the biologically active protein is a labeling enzyme. 8. The reagent for aldosterone testing containing substituted aldosterones according to claim 7, wherein the labeling enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-D-galactosidase and glycosidase.