JP2554878B2 - Novel betamethasone derivative, production method thereof, and enzyme immunoassay method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel betamethasone derivative and a method for producing the same,
And a hapten for producing an anti-betamethasone antibody containing this compound, and an enzyme immunoassay for betamethasone using this compound as a labeled antigen.

[Background of the Invention]

Betamethasone, which is known as one of the powerful synthetic corticosteroids, was synthesized by Taub et al. And Oliveto et al. In 1958 and has been used in various diseases as an anti-inflammatory drug, anti-allergic drug, immunosuppressant drug, etc. Widely applied. However, on the other hand, the ACTH (ad
It has a strong inhibitory effect on renocorticotropic hormone (secretory hormone) secretion, so it can be used to prevent withdrawal syndrome that appears after discontinuation of administration of this drug, to prevent accumulation of this drug in patients with liver diseases, etc. Since the difference in absorption is a particular problem, blood concentration measurement is required as a proof of the administration design.

As a method for measuring the blood concentration of betamethasone, a method by high performance liquid chromatography (HPLC) (J. Chromatography, 183 (1980), 131-139), β-galactosidase as a labeling enzyme, and a substrate have been used so far. 4-
Methylumbelliferyl-β-D-galactoside is used for enzyme immunoassay (Steroids, 37 (1
981), 303-314), a method for measurement by radioimmunoassay (J. Steroid Biochem., 7 (1976), 795-799), Journal of the Japanese Society of Endocrinology, 54 (1978), 654-665), etc. Has been done.

However, since the HPLC method requires special equipment and complicated operations, it cannot be said to be a practically preferable method. In addition, all of the immunological assays such as enzyme immunoassay and radioimmunoassay that have been reported so far are haptens for antibody production and 3- (O-carboxymethyl) oxime of betamethasone as a labeling antigen. Since the antiserum thus obtained has a low specificity and is highly cross-reactive with steroids having a similar structure, there is a problem in accuracy, and the sensitivity is not always sufficient. Absent.

[Object of the Invention]

The present invention has been made in view of the above situation, and has high specificity when used as a hapten for producing anti-betamethasone antibody, and a novel antiserum having low cross-reactivity with steroid having a similar structure is obtained. Another object of the present invention is to provide a novel betamethasone derivative, an anti-betamethasone antibody-containing hapten containing the same, and a highly accurate and highly sensitive enzyme immunoassay method for betamethasone using the hapten as a labeled antigen.

[Outline of Invention]

The present invention has the formula Represented by {[(11β, 16β) -9-fluoro-11,17,2
1-trihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-4-yl] thio} acetic acid, a method for producing the same, an anti-betamethasone antibody-containing hapten, and the same It is an invention of an enzyme immunoassay method for betamethasone used.

That is, the present inventors have conducted extensive studies in search of a highly specific antiserum in order to assemble a highly accurate and highly sensitive enzyme immunoassay for betamethasone, and as a result, have a carboxymethylthio group at the 4-position of betamethasone. Introduced {[(11β, 1
6β) -9-Fluoro-11,17,21-trihydroxy-16
-Methyl-3,20-dioxopregna-1,4-diene-4
-Yl] thio} acetic acid (hereinafter, abbreviated as 4-CMT-betamethasone) to which BSA (bovine serum albumin) was bound by, for example, the mixed acid anhydride method was used as an immunogen to immunize animals such as rabbits. In some cases, the side chain at position 17 and 16 of betamethasone
That a highly specific antiserum that recognizes the methyl group at the position A, and further recognizes the double bond between the 1,2 positions of the A ring and the carbonyl group at the position 3 can be obtained; The present inventors have completed the present invention by finding that when using 4-CMT-betamethasone as the labeled antigen, a highly accurate and highly sensitive enzyme immunoassay method for betamethasone can be easily assembled.

4-CMT-betamethasone of the present invention can be easily synthesized, for example, according to the following synthetic route.

That is, for example, first, betamethasone ([I]) is reacted with concentrated hydrochloric acid and formaldehyde in a chloroform solvent according to a conventional method to protect the 17-position dihydroxyacetone side chain as bismethylenedioxide to give compound [II], This is brominated in an appropriate solvent (eg dioxane) to give compound [III], which is then hydroxylated with potassium hydroxide, sodium hydroxide etc. in a lower alcohol such as methanol, ethanol or a mixed solvent of methanol-dioxane. The compound [IV] is reacted with thioglycolic acid in the presence of an alkali or a metal alkoxide such as sodium methoxide or sodium ethoxide, and then this is treated with an aqueous solution of an organic acid such as acetic acid or formic acid. The desired 4-CMT-betamethasone ([V]) can be obtained by removing the side chain protecting group at the 17-position. The amount of the reaction reagent, reaction solvent, etc. used in each step may be appropriately used according to common sense of the chemical reaction. The reaction conditions such as the reaction temperature and the reaction time may be appropriately selected according to those of the same kind of reaction. The post-treatment after the reaction may be appropriately carried out according to the properties of the product in accordance with the reaction of the same kind, and the purification may be repeated if necessary.

The anti-betamethasone antibody produced using the hapten for producing anti-betamethasone antibody of the present invention is a radioimmunoassay method,
It can be used in the field of immunological assay such as enzyme immunoassay.

The anti-betamethasone antibody may be prepared by a conventional method except that the hapten for preparing anti-betamethasone antibody of the present invention is used.

The enzyme immunoassay method for betamethasone according to the present invention is an anti-betamethasone antibody produced using the hapten for producing an anti-betamethasone antibody of the present invention, if necessary, it is known per se except that 4-CMT-betamethasone is further used as a labeled antigen. This may be carried out according to the operating method of the enzyme immunoassay, and the reagent used may be selected appropriately from the reagents used in the enzyme immunoassay known per se.

That is, first, 4-CMT-betamethasone was prepared by an appropriate method (eg, mixed acid anhydride method, carbodiimide method, etc.).
BSA is bound, and an appropriate animal (eg, rabbit, sheep, mouse, etc.) is immunized with this as an antigen to obtain antiserum. On the other hand, 4-CMT-betamethasone and an enzyme that is a labeling substance (for example, alkaline phosphatase, peroxidase,
β-galactosidase etc.) is bound by an appropriate method (eg carbodiimide method, mixed acid anhydride method etc.) to prepare a labeled antigen.

As the measurement procedure, first, a serum sample is treated with a sodium salicylate solution or the like to release betamethasone bound to the protein, and then a labeled antigen and an antiserum are added and reacted for a certain period of time. A second antibody is added and further reacted for a certain period of time or longer. After the reaction, the obtained precipitate was washed with water, its enzyme activity was measured by a method known per se such as a colorimetric method, a contract fluorescence method, and the same treatment was performed in advance using a standard serum having a known amount of betamethasone. The amount of betamethasone in the sample is calculated from the calibration curve prepared based on the results.

The anti-betamethasone antiserum prepared by using 4-CMT-betamethasone of the present invention as a hapten has extremely high specificity, and the cross-reactivity rate against various steroids is
0.039%, corticosterone 0.029%, progesterone 0.025%, 17-α-hydroxyprogesterone 0.047
%, 11-deoxycortisol is 0.034%, which is extremely low. In particular, when measuring serum samples, it is noteworthy that the crossover rate with cortisol, which has a high blood concentration and is considered to affect the measurement most, is 0.039%, which is completely negligible in actual measurement. It should be done.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

〔Example〕

Example 1. {[(11β, 16β) -9-fluoro-11,17,2
1-trihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-4-yl] thio} acetic acid (4-CMT-
(1) (11β, 16β) -9-fluoro-11-hydroxy-16-methyl-17,20,20,21-bis [methylenebis (oxy)]-pregna-1,4-diene-3 Synthesis of 1-one (compound [II]) 1 g betamethasone to 40 ml chloroform, 10 ml concentrated hydrochloric acid, 37
% Formalin (10 ml) was added, and the mixture was vigorously stirred at room temperature for 90 minutes. After completion of the reaction, water was added to the reaction solution while cooling with ice and extracted with chloroform. The extract was washed with water, washed with a saturated aqueous solution of sodium hydrogen carbonate until it became neutral, further washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off. The residue was crystallized with methanol to obtain 760 mg of crude crystals (yield 69%). This is recrystallized from methanol and mp 273-276 ℃ (de
to obtain colorless crystals of comp.).

(2) (6β, 11β, 16β) -6-Bromo-9-fluoro-11-hydroxy-16-methyl-17,20,20,21-bis [methylenebis (oxy)] pregna-1,4-diene-
Synthesis of 3-one (compound [III]) 63 mg of compound [II] was dissolved in 2 ml of dioxane, and
3.6 ml of 0.65% bromine-dioxane solution was slowly added dropwise, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure at room temperature, saturated aqueous sodium hydrogen carbonate solution was added to the residue while cooling with ice, and the mixture was extracted with diethyl ether. 0.5M extract
It was washed with an aqueous solution of sodium pyrosulfate and then with a saturated aqueous solution of sodium chloride, and dried over anhydrous sodium sulfate. The solvent was evaporated and the obtained residue was crystallized from diethyl ether-acetone to obtain 37 mg of crude crystals (yield 69%). This was recrystallized from diethyl ether to give colorless crystals with mp 195 to 197 ° C (decomp.).

MS m / z (EI): 512 (M ⁺ ), 514 (M ⁺ +2), (CI): 513 (M ⁺ +1), 515 (M ⁺ +3).

¹ H-NMR δppm (CDCl ₃ -CD ₃ OD): 7.39 (1H, d, J = 10Hz, 1
-H), 6.37 (1H, br.d, J = 10Hz, 2-H), 6.36 (1H, b
rs, 4H), 5.18~4.93 (4H , m, (OCH 2 O) × 2), 5.1
3 (1H, br.t, J = 3.5Hz, 6α-H), 4.37 (1H, m, 11-
H), 4.28,4.06 (each 1H, ABq, J = 9Hz, 21−H ₂ ), 2.80
(1H, m, 8-H), 1.92 (3H, s, 10-Me), 1.22 (3H, s, 13
-Me), 1.14 (3H, d, J = 7Hz, 16-Me).

Elemental analysis value (C ₂₄ H ₃₀ BrFO ₆ ): Calculated value (%) C, 56.14; H, 5.89 Measured value (%) C, 56.24; H, 5.99.

▲ [α] ²² _D ▼ -40.0 ° (c = 0.50, CH ₃ OH).

(3) {[(11β, 16β) -9-Fluoro-11-hydroxy-16-methyl-17,20,20,21-bis [methylenebis (oxy)]-3-oxopregna-1,4-diene-4
Synthesis of -yl] thio} acetic acid (Compound [IV]) 236 mg of Compound [III] was dissolved in 94 ml of methanol, 0.7 ml of thioglycolic acid was added, and then 6% potassium hydroxide-methanol solution was added to this solution while refluxing. 19 ml was dropped slowly. After refluxing for 72 hours, the solvent was evaporated, water was added to the residue while cooling with ice, and the alkaline substance and raw materials were extracted and removed with chloroform.

The aqueous layer was adjusted to pH 1 with 10% hydrochloric acid, extracted with chloroform, washed with water, dried over anhydrous sodium sulfate, the solvent was distilled off, and the residue was crystallized from diethyl ether and petroleum ether to give crude crystals (133 mg, yield 55%). ) Got. This was recrystallized from diethyl ether to give colorless crystals with mp 230 to 231.5 ° C (decomp.).

MS m / z (EI): 524 (M ⁺ ), (CI): 525 (M ⁺ +1).

¹ H-NMR δ ppm (CDCl ₃ ): 7.39 (1 H, d, J = 10 Hz, 1-H),
6.52 (1H, d, J = 10Hz, 2-H), 5.17 to 4.90 (4H, m, (OCH
₂ O) x 2), 4.30 (1H, m, 11-H), 4.24,4.03 (each 1
H, ABq, J = 10Hz, 21−H ₂ ), 3.74 (1H, br.d, J = 14Hz, 6α
-H), 3.46, 3.37 (each 1H, ABq, J = 15Hz, SCH ₂ CO),
1.60 (3H, s, 10-Me), 1.14 (3H, s, 13-Me), 1.12 (3
H, d, J = 7Hz, 16-Me).

Elemental analysis _{(C 26 H 33 FO 8 S} ): Calculated (%) C, 59.52; H , 6.34 Found (%) C, 59.34; H , 6.44.

▲ [α] ^21.5 _D ▼ + 20.0 ° (c = 0.50, MeOH).

The above compound [IV] shows a molecular ion peak at m / z 524 in MS, and shows absorption of a carboxylic acid at 3700 to 2500 cm ⁻¹ in an IR spectrum. Further, in the NMR spectrum, the signal of the olefin hydrogen at the 4-position of δ6.36 found in the raw material (III) disappeared, and δ6.37 was obtained in (III).
2nd olefin hydrogen which appeared as br.d (J = 10Hz) in δ6.52 appears as d (J = 10Hz) in long range coupling between hydrogens in 2nd and 4th positions based on W law. Since it disappeared, it was found that a substituent was introduced at the 4-position. In addition, new methylene signals appeared at δ3.46 and 3.37.

From the above, it was confirmed that a carboxymethylthio group was introduced at the 4-position.

(4) Synthesis of 4-CMT-betamethasone Compound (IV) (80 mg) was dissolved in 50% acetic acid aqueous solution (18 ml), and the mixture was refluxed at 100 ° C for 5 hr while blowing nitrogen gas.
The solvent was distilled off under reduced pressure and the resulting residue was preparative TLC.
Purified (developed with chloroform: methanol 5: 3) and crystallized with ethyl acetate-isooctane to give white crystals.
mg (54% yield) was obtained.

MS m / z (FD): 482 (M ⁺ ).

¹ H-NMR δ ppm (CDCl ₃ -CD ₃ OD): 7.38 (1H, d, J = 10Hz, 1
-H), 6.42 (1H, d, J = 10Hz, 2-H), 4.50,4.34 (eac
h 1H, ABq, J = 20Hz, 21−H ₂ ), 3.75 (1H, br.d, J = 14Hz, 6
α-H), 3.30 (2H, s, SCH ₂ CO), 2.56 (1H, td, J = 14,6)
Hz, 6β-H), 1.61 (3H, s, 10-Me), 1.31 (3H, d, J = 7
Hz, 16-Me), 1.08 (3H, s, 13-Me).

Calculated (%) C, 56.56; H, 6.73, Found (%) C, 56.79; H, 6.72.

Example 2. Measurement of betamethasone by enzyme immunoassay (1) Preparation of 4-CMT-betamethasone-BSA conjugate 4-CM
10 mg of T-betamethasone was added to N, N-dimethylformamide (DM
F) Dissolve in 0.2 ml and add tri-n-butylamine 5μ
Was added, and the mixture was cooled to 10 ° C., 3 μm of chromium isobutyl carbonate was added, and the mixture was reacted at 4 ° C. for 20 minutes. This reaction solution was added to BSA40m
g slowly into a solution of DMF (1 ml) and water (1.15 ml) and adjust to pH 9.5 with 1N sodium hydroxide solution.
The reaction was carried out at 0 ° C overnight. This was applied to Sephadex G-25 column chromatography (internal diameter 1.5 cm, length 20 cm) and eluted with physiological saline, and Tonein reagent (Coomassie Brilliant Blue G-
250, consisting of hydrochloric acid, sodium acetate, methyl cellulose. ) Was used to obtain a protein-positive fraction by the dye-binding method. The yield is 6 ml.

In addition, it was confirmed by UV absorption method that 9 mol of betamethasone was bonded to 1 mol of BSA.

(2) Preparation of anti-betamethasone antiserum The 4-CMT-betamethasone-BSA conjugate physiological saline solution obtained in (1) was supplemented with Freund's complete adjuvant to make a uniform emulsion, and the number of rabbits on the back It was injected subcutaneously at a place. Three rabbits were used, and as an immunization method, booster immunization was performed 4 times every 2 weeks after the first immunization, and test blood was collected from the ear vein every 1 week after immunization. The whole blood was collected from the carotid artery 9 weeks after the initial immunization in which the antibody titer was observed, and the antiserum obtained was found to have the least cross-reactivity with other steroids. The serum was used for measurement.

(3) Binding of 4-CMT-betamethasone and alkaline phosphatase (Alp) (preparation of Alp-labeled antigen) Centrifugation of 0.25 mg / 50μ Alp suspension (50μ) gave a precipitate, which was pH 8.0 phosphate buffer solution. After dissolving in 0.3 ml, 5 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloric acid (EDAC) was added. 4-CMT-betamethasone (5 mg) dissolved in DMF (0.1 ml) was added thereto, and the mixture was reacted at room temperature for 4 hours and then applied to Sephadex G-25 column chromatography (internal diameter 1.5 cm, length 20 cm) to give 0.05. Elution with M phosphate buffer (pH 7.4) gave the fraction with the highest measured Alp activity by the modified Kind-King method. The yield is 2 ml.

It was confirmed by UV absorption that 4 mol of betamethasone was bonded to 1 mol of Alp.

(4) Measurement of betamethasone by enzyme immunoassay (preparation of calibration curve) [Reagent] 0.25% BSA solution BSA 0.25g was dissolved in 0.05M phosphate buffer (pH7.4) to obtain 10
It was set to 0 ml.

Anti-betamethasone antiserum The anti-betamethasone antiserum obtained in (2) was diluted with a 0.25% BSA solution 5000 times before use.

Alp-labeled antigen (3) Alp-labeled antigen obtained in (3) is used in 0.25% BSA solution 800
It was diluted 1-fold and used by adding 0.1% normal rabbit serum (NRS).

Second antibody Anti-rabbit IgG goat antiserum (manufactured by Eiken Immunochemical Laboratories) was diluted to 2% with a 0.25% BSA solution.

About 140g of activated carbon washed with water was added to steroid-free serum pool serum 1,
Stir overnight at 4 ° C. Activated carbon was separated by centrifugation (10000 rpm, 20 minutes), and the serum that had been passed through a Millipore filter twice was centrifuged again, and the obtained serum was used as a steroid-free serum.

1% aqueous solution of sodium salicylate 1 mg of sodium salicylate was dissolved in distilled water to give a 1% aqueous solution.

Betamethasone standard serum The betamethasone standard solution was evaporated to dryness, and then steroid-free serum was added thereto to prepare a standard serum.

Alp activity measuring reagent (manufactured by Wako Pure Chemical Industries, Ltd.) 1v / v when using 30mM magnesium chloride aqueous solution as a substrate buffer
% Added and used.

〔sample〕

1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 of standard serum with betamethasone concentration of 1000 ng / dl and serum containing no steroid
Samples were serially diluted.

〔Operating procedure〕

0.1 ml of 1% sodium salicylate aqueous solution for 10μ of sample
Was added and allowed to stand at room temperature for 1 hour, 0.1 ml of Alp-labeled antigen was added and mixed, and 0.5 ml of anti-betamethasone antiserum was further added and mixed and reacted at room temperature for 1 hour. To this, the second antibody
After adding 0.1 ml and reacting at 4 ° C overnight, wash with 2 ml of water,
After centrifugation, the supernatant was removed by decantation. After repeating this washing operation twice, the Alp activity of the obtained precipitate was measured by the Kind-King modified method using an Alp activity measuring reagent.

〔result〕

The betamethasone concentration, which was created based on the obtained results, and
A calibration curve showing the relationship with the absorbance at 500 nm is shown in FIG.

As is clear from FIG. 1, the measurable range of the present application is 100.
~ 10000 ng / dl with minimum detection sensitivity of approximately 10 pg / t
It was ube.

(5) Measurement of betamethasone by enzyme-linked immunosorbent assay (preparation of calibration curve-use of methanol standard solution) As a sample, a solution of betamethasone standard solution in methanol (methanol standard solution) was used instead of betamethasone standard serum. Measurement was performed in exactly the same manner as in (4) using the same reagents, and a calibration curve exactly similar to (4) was obtained.

(6) Measurement of betamethasone by enzyme immunoassay (confirmation of quantification) [Reagent] Same as (4).

〔sample〕

Two different specimens and serial dilutions of these with steroid-free serum to 1/2, 1/4, 1/8, 1/16 were used as samples.

〔Operating procedure〕

Alp for each sample exactly as in (4)
After measuring the activity, the betamethasone concentration was determined from the calibration curve obtained in (4).

〔result〕

 Results are shown in FIG.

As is clear from FIG. 2, the origins of all the samples were connected by connecting the points plotted along the vertical axis with the betamethasone concentration (μg / dl) values obtained for each dilution ratio on the horizontal axis. It is a straight line passing through, and it can be seen that the measuring method of the present invention is excellent in quantitativeness.

〔The invention's effect〕

As described above, the present invention provides a novel betamethasone derivative, a hapten for producing an anti-betamethasone antibody containing the same, and a novel enzyme immunoassay method for betamethasone using this compound as a labeled antigen. The antibody produced by using the hapten for producing anti-betamethasone antibody has high specificity, and the cross-reactivity rate against various steroids expected to coexist in a sample is extremely low, and the assay method of the present invention is It has a remarkable effect in that the measurable range is wide and the detection altitude is extremely high.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the calibration curve obtained in (4) of Example 2, and 500 obtained for each betamethasone concentration (ng / dl) on the horizontal axis.
It is the connection of the points where the absorbance values in nm are plotted along the vertical axis. FIG. 2 shows the results obtained in (6) of Example 2, in which the horizontal axis represents the sample dilution ratio and the vertical axis represents the betamethasone concentration (μ
g / dl) respectively.

Claims (4)

(57) [Claims] 1. A formula Represented by {[(11β, 16β) -9-fluoro-11,17,2
1-Trihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-4-yl] thio} acetic acid. 2. A formula The betamethasone represented by is protected with the side chain of dihydroxyacetone at the 17-position as bismethylenedioxide, and then brominated to give the formula: (6β, 11β, 16β) -6-Bromo-9-fluoro-11-hydroxy-16-methyl-17,20,20,21-bis [methylenebis (oxy)] pregna-1,4-diene- 3-one, which is then reacted with thioglycolic acid to give the formula Represented by {[(11β, 16β) -9-fluoro-11-hydroxy-16-methyl-17,20,20,21-bis [methylenebis (oxy)]-3-oxopregna-1,4-diene-4 -Yl] thio} acetic acid, followed by removal of the side chain protecting group at the 17-position Represented by {[(11β, 16β) -9-fluoro-11,17,2
Process for producing 1-trihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-4-yl] thio} acetic acid. 3. A formula Represented by {[(11β, 16β) -9-fluoro-11,17,2
A hapten for producing an anti-betamethasone antibody, which comprises 1-trihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-4-yl] thio} acetic acid. 4. A formula Represented by {[(11β, 16β) -9-fluoro-11,17,2
An enzyme immunoassay method for betamethasone, which comprises using 1-trihydroxy-16-methyl-3,20-dioxopregna-1,4-dien-4-yl] thio} acetic acid as a labeling antigen.