JP2000063398A - Production of labeled steroid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a high-purity labeled steroid from an obtainable general-purpose compound as a raw material useful for elucidating an organism function, etc., in a high yield by catalytically deuterating or catalytically tritiating a specific compound prepared by a reaction under prescribed conditions. SOLUTION: A compound of formula I [St is a steroid skeleton; R is H or a lower alkyl; X and Y are each H or a (protected) hydroxyl group] is oxidized to give a compound of formula II. The obtained compound of formula II is dehydrated to give a compound of formula III. The prepared compound is catalytically deuterated or catalytically tritiated to give a labeled steroid of formula IV (Z is deuterium or tritium). The compound of formula III the compound of formula IV and a compound of formula V [W1 and W2 are each a (protected)hydroxyl group] are new.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a steroid compound labeled with deuterium or tritium and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Steroids are a general term for compounds having a structure closely related to cyclopentanoperhydrophenanthrene, and those having various substituents in the basic skeleton of cyclopentanoperhydrophenanthrene, The existence of various compounds such as those containing a double bond in the ring is known.
Steroids are widely present in animal and plant tissues as biofunctional substances represented by biological constituents or hormones. A large number of steroids having various physiological activities are known, and extensive research has been conducted for the purpose of elucidating biological functions and developing new drugs or pesticides. Isotope-labeled steroids are widely used in such studies, particularly in studies aimed at elucidating biosynthesis, metabolism, or receptor mechanism of steroids.

Conventionally, isotope-labeled steroids have been synthesized by various methods, but most of them are produced by multistep synthesis. For example, the isotope-labeled brassinosteroids used in studies to elucidate the function of brassinosteroids, which are plant hormones, are described by Yokota et al.
t al., J. Plant Growth Regul., 9, pp.151-159, 199
Except for 0, all are synthesized in multiple stages (Marqua
rdt, V. and Adam, G., Chemistry of Plant Protectio
n, Ebing, W. Ed.-in-Chief; Springer-Verlag, New Yo
rk, 7, pp. 103-139, 1991 and references cited therein). The method of Yokota et al. Has only a trace amount in nature.
D25 (28) -brassinosteroid, which is a substrate, is used to obtain the desired tritium-labeled castasterone or tritium-labeled brassinolide by tritiation using drichosterone or dricholide as a substrate, which is not a practical method. Therefore, there has been a demand for development of a method for more easily producing an isotope-labeled steroid.

[0004]

An object of the present invention is to provide a method for easily producing an isotope-labeled steroid. More specifically, it is an object of the present invention to provide a method for efficiently producing an isotope-labeled steroid by a simple process using a commercially available general-purpose compound as a raw material. It is also an object of the present invention to provide a production method that can be generally used for labeling various steroids and is industrially applicable. Furthermore, another object of the present invention is to provide a novel isotope-labeled steroid.

[0005]

[Means for Solving the Problems] The present inventors hydrate the 25-position carbon of the steroid side chain and then introduce a double bond by a dehydration reaction to deuterate or tritiate the double bond. Therefore, it was found that the isotope-labeled steroid can be produced extremely efficiently.

That is, the present invention provides the following formula (I):

[Chemical 6] (In the formula, St represents a steroid skeleton, R represents a hydrogen atom or a lower alkyl group, X and Y each independently represent a hydrogen atom or a hydroxyl group which may have a protecting group, Z
Represents a deuterium atom or a tritium atom) and is a method for producing a labeled steroid represented by the following steps: (A) the following formula (II):

[Chemical 7] (In the formula, St, R, X, and Y are as described above.)
The compound represented by the following formula (III):

[Chemical 8] (In the formula, St, R, X, and Y are as described above.)
(B) dehydrating the compound represented by the above formula (III) to obtain a compound represented by the following formula (I)
V):

[Chemical 9] (In the formula, St, R, X, and Y are as described above.)
And a step (C) of subjecting the compound represented by the above formula (IV) to catalytic deuteration or catalytic tritiation.

Further, according to the present invention, there is provided a method for producing a labeled steroid represented by the above formula (I), which comprises a step of catalytic deuteration or tritiation of a compound represented by the above formula (IV). A method; and a method for producing the labeled steroid represented by the above formula (I), wherein the compound represented by the above formula (III) is dehydrated to obtain the compound represented by the above formula (IV); And a step of catalytic deuteration or catalytic tritiation of the compound represented by the above formula (IV) obtained in the above step.

According to the preferred embodiments of these inventions, R
Is a hydrogen atom, a methyl group, or an ethyl group;
There is provided the above method of catalytic deuteration or tritiation of a compound of formula (IV), wherein X and Y are hydroxyl groups; the above method wherein catalytic deuteration or catalytic tritiation is carried out in the presence of a palladium catalyst.

From another point of view, there is provided a compound represented by the above formula (I) and a compound represented by the above formula (IV). According to a preferred embodiment of these inventions, St has the following formula:

[Chemical 10] (Wherein, W ¹ and W ² each independently represent a hydroxyl group which may have a protecting group) provides a compound represented by the above formula (I) or the above formula (IV) which is a steroid skeleton. To be done.

Further, the following formula (V):

[Chemical 11] (In the formula, R represents a hydrogen atom or a lower alkyl group, X and Y each independently represent a hydrogen atom or a hydroxyl group which may have a protective group, and W ¹ and W ² each independently have a protective group. A compound represented by the formula (1) is also provided.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The first aspect of the method of the present invention is characterized by including the above steps (A) to (C). A second aspect of the present invention is characterized by including the steps (B) and (C), and a third aspect comprising only the step (C). In the above formulas (I) to (IV), St represents a steroid skeleton. As the steroid skeleton, it is known that various compounds such as those having various substituents in the basic skeleton of cyclopentanoperhydrophenanthrene and those having a double bond or a lactone group in the ring are known. . The steroid to which the method of the present invention is applicable is not particularly limited, and a labeled steroid of a compound having any steroid skeleton as St can be produced. R represents a hydrogen atom or a lower alkyl group, and as the lower alkyl group, for example, a linear or branched alkyl group having 1 to 6 carbon atoms can be used, and preferably a methyl group or an ethyl group is used. be able to.

X and Y each independently represent a hydrogen atom or a hydroxyl group which may have a protecting group, and the hydroxyl protecting group can act as a protecting group in steps (A) and (B), for example. If it can be deprotected accordingly,
The type is not particularly limited. Various protective groups for hydroxyl group are known to those skilled in the art, and an appropriate protective group can be selected. For example, lower alkanoyl group such as acetyl group, halogenated lower alkanoyl group such as trifluoroacetyl group, benzyl group, substituted or unsubstituted aralkyl group such as p-methoxybenzyl group, aroyl group such as benzoyl group, trimethylsilyl group, etc. An alkylsilyl group and the like can be used, but the present invention is not limited thereto. Of these, an acetyl group can be preferably used.

The method of the present invention will be specifically described with reference to the following scheme with respect to brassinolide, which is the most important compound among brassinosteroids which are plant hormones. It is not limited to the methods of isotopically labeling Nolide, nor is it limited to the details of these methods. As brassinolide is a highly functionalized steroid, it will be readily appreciated by those skilled in the art that the method of isotopically labeling this compound is applicable to a wide variety of steroids.

[0014]

[Chemical 12]

After protecting the four hydroxyl groups of commercially available brassinolide (4) with acetyl groups to lead to 2,3,22,23-tetra-O-acetylbrassinolide (5), Voigit et al. Method (Voig
t, B., et al., Tetrahedron, 52, pp.10653-10658, 199
According to 6), a hydroxyl group is introduced at the 25-position of (5) to convert it to 2,3,22,23-tetra-O-acetyl-25-hydroxybrassinolide (6). Subsequent dehydration of (6) with thionyl chloride in pyridine gives a mixture of regioisomers of double bonds (7) and (8). The mixture was treated with 90% hydrous methanol containing 5% KOH and the cation exchange resin [Dowex-50W (H ⁺ fo
rm)] to re-close the lactone ring, Δ ^{25 (26)} -
A brassinolide (9) is obtained.

In the above-mentioned step of introducing a protective group, the protective group for the hydroxyl group is not limited to the acetyl group, is substantially inactive in the oxidation step and the dehydration step, and can be easily deprotected by an appropriate means. Any material may be used as long as it is used. In the oxidation step, for example, in addition to methyl (trifluoromethyl) dioxirane used by Voigit et al., Dimethyldioxirane, light irradiation of peracetic acid, sodium periodate-ruthenium trichloride, or ozone-silica gel is used as an oxidizing agent. Can be used.

In the dehydration step, a mixture of positional isomers of the double bond is generally obtained, but it is efficient to carry out the deprotection step without separating these isomers. As the dehydrating agent, phosphorus oxychloride-pyridine, heating in dimethylsulfoxide or hexamethylphosphoric triamide, methanesulfonyl chloride-pyridine, or
A dehydrating agent such as N-bromoacetamide or N-bromosuccinimide-pyridine may be used. In the deprotection step, an appropriate reaction may be carried out depending on the type of protecting group used, but when the lactone ring is opened by alkali treatment or acid treatment, the lactone ring may be reclosed by an appropriate method. It is possible.

Next, Δ ^{25 (26)} -brassinolide (9) was deuterated at room temperature under deuterium (≧ 99.5 D-mol%) atmosphere using 5% palladium-carbon in a tetrahydrofuran solvent. By hydrogenation, [25,26,27-2H7] brassinolide (10) can be obtained. When tritiated, tritium gas may be used instead of deuterium gas in the same manner. The catalyst for catalytic deuteration or catalytic tritiation, for example, Pd-C, Pd-BaCO ₃ , Pd-BaSO ₄ , Pd-CaCO ₃ and other palladium-based catalysts, platinum oxide and other platinum-based catalysts, Raney nickel, rhodium. -A rhodium catalyst such as alumina can be used. Alternatively, a homogeneous catalytic hydrogenation catalyst such as chlorotris (triphenylphosphine) rhodium may be used. The type of solvent is not particularly limited, for example,
Ethyl ether, dioxane, ethyl acetate and the like can be used.

Having described an example of the method of the present invention, the following examples describe the above steps in specific and detail. Therefore, those skilled in the art can appropriately select the starting compound, the protecting group, the reaction reagent, the reaction conditions and the like while referring to these descriptions disclosed in the present specification, and appropriately modify or modify the above method as necessary. As a result, the desired labeled steroid can be easily produced.

[0020]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. The compound numbers in the examples correspond to the compound numbers in the above scheme.

Example 1: [25,26,27-2H7] brassinolide (10)
Preparation of 2,3,22,23-tetra-O-acetyl-25-hydroxybrassinolide (6) (5.5 mg, 8.3 μmol) in anhydrous pyridine (500
μl) and thionyl chloride (15 μl, 21 μl
0 μmol) was added under ice cooling. The reaction solution was stirred at the same temperature for 30 minutes and further at room temperature for 30 minutes, and ethyl acetate was added. The reaction solution was saturated aqueous sodium hydrogen carbonate solution, water,
And washed successively with saturated aqueous potassium hydrogen sulfate solution,
It was dried over sodium sulfate. After distilling off the solvent, the residue was subjected to silica gel column chromatography, and 2,3,22,23-tetra-O-acetyl-Δ was collected from the elution part of dichloromethane.
A 65:35 mixture of ^{25 (26)} -brassinolide (7) and 2,3,22,23-tetra-O-acetyl-Δ ^{24 (25)} -brassinolide (8) (4.
6 mg, 86%) was obtained. This mixture was subjected to high performance liquid chromatography to obtain a colorless amorphous powder of each isomer.

2,3,22,23-Tetra-O-acetyl-Δ ^{25 (26)} -
Brassinolide (7) ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.73 (3H, s, 18-H3), 0.
98 (3H, s, 19-H3), 1.78 (3H, s, 27-H3), 1.98, 2.0
0, 2.02 and 2.11 (each 3H, each s, 4 × OAc), 2.99
(1H, dd, J = 12.2 and 4.5 Hz, 5-H), 3.98-4.18 (2H, 7
-H2), 4.74 and 4.84 (each 1H, each br s, 26-H2),
4.87 (1H, m, 2-H), 5.13 (1H, br d, J = 8.2Hz, 22- or
23-H), 5.27 (1H, dd, J = 8.2 and 3.5 Hz, 22- or 23-
H), 5.37 (1H, m, 3-H). 2,3,22,23-Tetra-O-acetyl-Δ ^{24 (25)} -Brassinolide (8) ¹ H-NMR (300 MHz, CDCl ₃ ): δ 0.66 (3H, s, 18-H3),
0.98 (3H, s, 19-H3), 1.98, 2.00, 2.05 and 2.11 (ea
ch 3H, each s, 4 × OAc), 2.99 (1H, dd, J = 12.2and 4.
3 Hz, 5-H), 3.98-4.18 (2H, 7-H2), 4.87 (1H, m, 2-
H), 5.28 (1H, brd, J = 9.7 Hz, 22-H), 5.37 (1H, m, 3-
H), 5.86 (1H, d, J = 9.7 Hz, 23-H).

2,3,22,23-tetra-O-acetyl-Δ ^{25 (26)} -
Brassinolide (7) and 2,3,22,23-tetra-O-acetyl-Δ
^{24 (25)} -65: 35 mixture of brassinolide (8) (5.3 mg)
Was dissolved in 90% water-containing methanol (500 μl) containing 5% KOH, and the mixture was stirred at room temperature for 1 hour and then heated under reflux for 2 hours. After adding methanol (2 ml) and water (1 ml), add cation exchange resin [Dowex-50W (H ⁺ form)] to make it acidic (pH 3-4) under ice-cooling stirring, and further at room temperature. Stir for 3 hours.
After the resin was filtered off, the solvent was distilled off and the obtained crystals were subjected to silica gel column chromatography. Chloroform-methanol, 30: 1, Δ25 ⁽²⁶⁾ -brassinolide (9) [2.3] mg, 90% yield from (7), colorless needles (CHCl _3-
CH ₃ OH): mp 232-233 ° C] was obtained.

¹ H-NMR (300 MHz, CDCl ₃ -CD ₃ OD, 10: 1):
δ 0.72 (3H, s, 18-H3), 0.91 (3H, s, 19-H3), 0.95
(3H, d, J = 6.4 Hz, 21- or 28-H3), 1.02 (3H, d, J = 7.
0 Hz, 21- or 28-H3), 1.78 (3H, s, 27-H3), 2.10 (1
H, ddd, J = 15.2, 12.1 and 2.2 Hz, H-4b), 2.23 (1H,
m, 24-H), 3.13 (1H, dd, J = 12.1 and 4.5 Hz, 5-H),
3.56 and 3.58 (each 1H, br ABq, J = 6.9 Hz, 22- and
23-H), 3.63 (1H, ddd, J = 12.2, 4.6 and 2.8 Hz, 2-
H), 3.97 (1H, m, 3-H), 4.10 (2H, d-like, J = 5.3 Hz,
7-H2), 4.81 and 4.91 (each 1H, each br s, 26-H2).

A heterogeneous solution of 5% palladium-carbon (2.0 mg) in tetrahydrofuran (500 μl) was added to deuterium (≧ 99.
(5 D-mol%) After stirring for 15 minutes at room temperature under an atmosphere (1 atm), a solution of Δ ^{25 (26)} -brassinolide (9) (1.0 mg) in tetrahydrofuran (500 μl) was added, and further for 1 hour. It was stirred. After the catalyst was filtered off, the solvent was distilled off and the obtained crystal was subjected to high performance liquid chromatography [Senshu Pak ODS-1151D,
150 × 4.6 mm id, Senshu Scientific Co., Ltd .; Mobile phase:
Water-acetonitol = 55:45; flow rate: 1.0 ml / min] [25,
26,27-2H7] Brassinolide (10) (0.68 mg, 68%) was obtained as colorless crystals.

¹ H-NMR (300 MHz, CDCl ₃ -CD ₃ OD, 10: 1):
δ 0.72 (3H, s, 18-H3), 0.84 (3H, d, J = 6.9 Hz, 21-
or 28-H3), 0.89 (3H, d, J = 0.65 Hz, 21- or 28-H3),
0.92 (3H, s, 19-H3), 3.12 (1H, dd, J = 12.2 and 4.7
Hz, 5-H), 3.53 and 3.69 (each 1H, br ABq, J = 8.5 H
z, 22- and 23-H), 3.67 (1H, m, 2-H), 3.99 (1H, m,
3-H), 4.06-4.12 (2H, 7-H2); this spectrum is 26-H3
The spectrum was almost the same as that of the standard brassinolide (4) except that the signal based on 27-H3 was absent. FAB-MS: m / z 481 (2%), 482 (4), 483 (12), 484 (16),
485 (30), 486 (24), 487 (36), 488 ([M + 1] +, 100),
FAB-MS for 489 (34); brassinolide (4): m / z 479 (24
%), 480 (13), 481 ([M + 1] +, 100), 482 (33), it was found that this product had an isotopic purity of 60%.

[0027]

Industrial Applicability According to the method of the present invention, a labeled steroid can be efficiently produced, and a highly labeled steroid compound having 7 isotopes introduced in one molecule can be produced. is there.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4C091 AA06 BB01 CC03 DD03 DD13                       EE03 EE04 EE05 FF04 FF14                       GG01 HH01 JJ03 KK01 LL01                       MM03 NN01 PA02 PA05 PB05                       QQ01 SS07

Claims (9)

[Claims] 1. The following formula (I): (In the formula, St represents a steroid skeleton, R represents a hydrogen atom or a lower alkyl group, X and Y each independently represent a hydrogen atom or a hydroxyl group which may have a protecting group, Z
Is a deuterium atom or a tritium atom), and the method comprises the following steps: (A) the following formula (II): (In the formula, St, R, X, and Y are as described above.)
By oxidizing the compound represented by the following formula (III): (In the formula, St, R, X, and Y are as described above.)
A step of obtaining a compound represented by the following formula: (B) The compound represented by the above formula (III) obtained in the above step is dehydrated to obtain the following formula (IV): (In the formula, St, R, X, and Y are as described above.)
A step of obtaining a compound represented by the formula; and (C) a step of catalytic deuteration or catalytic tritiation of the compound represented by the above formula (IV) obtained in the above step. 2. A method for producing a labeled steroid represented by the formula (I) according to claim 1, which comprises the formula (I) according to claim 1.
A method comprising a step of catalytically deuterating or catalytically tritiating a compound represented by V). 3. A method for producing a labeled steroid represented by formula (I) according to claim 1, comprising the following steps: (D) a compound represented by formula (III) according to claim 1. Dehydration of the compound to obtain the compound represented by the formula (IV) according to claim 1; and (E) the compound represented by the formula (IV) obtained in the above step is subjected to deuteration or contact. A method comprising the step of tritiating. 4. The method according to claim 1, wherein the catalytic deuteration or catalytic tritiation is carried out in the presence of a palladium catalyst. 5. The method according to claim 1, wherein the compound of formula (IV) in which X and Y are hydroxyl groups is catalytically deuterated or catalytically tritiated. 6. The method according to claim 1, wherein R is a hydrogen atom, a methyl group, or an ethyl group. 7. A compound represented by the formula (IV) according to claim 1. 8. The following formula (V): (In the formula, R represents a hydrogen atom or a lower alkyl group, X and Y each independently represent a hydrogen atom or a hydroxyl group which may have a protective group, and W ¹ and W ² each independently have a protective group. A compound represented by the formula (1). 9. A compound represented by formula (I) according to claim 1.