Method for preparing dehydroepiandrostene-D6 from androstenedione-D8
Dehydroepiandone-D6 was successfully prepared by esterification of androthenone-D8 through five-step reactions of enol esterification, upprotection, reduction, deprotection and deuterium hydrogen replacement, which solved the problem of lack of efficient synthesis routes in the prior art and achieved the preparation process under efficient and mild conditions.
Patent Information
- Application Number
- CN202510403628.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-01
- Publication Date
- 2025-06-03
AI Technical Summary
The lack of a clear synthetic route in the prior art has led to the efficient preparation of dehydroepiandrosterone-D6 not yet resolved.
Dehydroepiandine-D8 was used as raw material, and dehydroepiandine-D6 was prepared through five-step reactions of enol esterification, upprotection, reduction, deprotection and deuterium hydrogen replacement.
It realizes efficient preparation of dehydroepiandrosterone-D6, with mild reaction conditions, simple operation and wide application prospects.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of the preparation of organic compounds, and relates to a method for preparing dehydroepiandrosterone-D6 using androstenedione-D8. Background Art
[0002] Steroid hormones play an important role in maintaining the stability of the human internal environment, regulating metabolism, growth and development, and reproductive behavior, and have high reference value for the screening and diagnosis of related diseases. Since steroid hormones have low content, similar structures, and a wide variety, the preferred method for clinically detecting steroid hormone molecules is liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the commonly used standard substances for detection are stable isotope-labeled steroid compounds. Among them, deuterium-labeled steroid compounds are easier to synthesize and cheaper than 13 C-labeled substances, and their applications are more extensive.
[0003] Dehydroepiandrosterone and its metabolite dehydroepiandrosterone sulfate are important steroid hormones in the human body. Accurately quantifying their content in human biological matrices is of great significance for the diagnosis of clinical physiological and psychological diseases.
[0004] Lu Youli et al. (Laboratory Medicine, 2022, 2: 165-173) established an LC-MS / MS method for detecting steroid hormones, which can be used as a rapid and effective method for the auxiliary and differential diagnosis of polycystic ovarian syndrome (PCOS). Among them, deuterated dehydroepiandrosterone is used as a deuterated internal standard to correct the matrix effect.
[0005] The chemical name of dehydroepiandrosterone-D6 is 3β-hydroxy-4-en-17-one-androstane-2,2,3,4,4,6-D6, and its structure is shown in Formula a. The main deuteration positions are 2,2,3,4,4,6.
[0006]
[0007] There are few reports on the synthesis of dehydroepiandrosterone-D6, and there is no clearly reported synthesis route. Therefore, it is urgent to find a scheme for efficiently preparing dehydroepiandrosterone-D6. Summary of the Invention
[0008] In order to solve the deficiencies of the existing technology, the purpose of the present invention is to provide a method and application for preparing dehydroepiandrosterone-D6 using androstenedione-D8. In the present invention, purification and separation are easy, the reaction conditions are mild, the operation is simple and convenient, and the efficient preparation of dehydroepiandrosterone-D6 can be realized. In the preparation method of the present invention, androstenedione-D8 is used as a raw material, and dehydroepiandrosterone-D6 is prepared through five steps: enol esterification, protection, reduction, deprotection, and deuterium-hydrogen replacement.
[0009] The present invention provides a method for preparing dehydroepiandrosterone-D6 using androstenedione-D8. The method uses androstenedione-D8 as a raw material and prepares dehydroepiandrosterone-D6 through five steps: enol esterification, protection, reduction, deprotection, and deuterium-hydrogen substitution.
[0010] The reaction process of the method of the present invention is shown in Reaction Scheme (I):
[0011]
[0012] Among them, R is an acetyl group or a propionyl group.
[0013] The method for preparing dehydroepiandrosterone-D6 using androstenedione-D8 in the present invention includes the following steps:
[0014] Step (1) Enol esterification reaction: Dissolve the compound of formula (1), i.e., androstenedione-D8, in a first solvent, and carry out an enol esterification reaction under an organic acid condition to obtain the compound of formula (2); the reaction process is shown in Reaction Scheme (A):
[0015]
[0016] Among them, R is an acetyl group or a propionyl group.
[0017] Step (2) Protection reaction: Dissolve the compound of formula (2) obtained in step (1) in a second solvent, and carry out a protection reaction under the conditions of ethylene glycol, triethyl orthoformate, and an organic acid to obtain the compound of formula (3); the reaction process is shown in Reaction Scheme (B):
[0018]
[0019] Among them, R is an acetyl group or a propionyl group.
[0020] Step (3) Reduction reaction: Dissolve the compound of formula (3) obtained in step (2) in a third solvent, and use a reducing agent to carry out a reduction reaction to obtain the compound of formula (4); the reaction process is shown in Reaction Scheme (C):
[0021]
[0022] Among them, R is an acetyl group or a propionyl group.
[0023] Step (4) Deprotection reaction: Dissolve the compound of formula (4) obtained in step (3) in a fourth solvent, and carry out a deprotection reaction under an acidic condition to remove ethylene glycol to obtain the compound of formula (5); the reaction process is shown in Reaction Scheme (D):
[0024]
[0025] Step (5) Deuterium-hydrogen exchange reaction: Dissolve the compound of formula (5) obtained in step (4) in a fifth solvent, and under acidic conditions, carry out a deuterium-hydrogen exchange reaction to obtain the target compound dehydroepiandrosterone-D6; the reaction process is shown in reaction formula (E):
[0026]
[0027] In step (1) of the present invention, the enol esterification reaction is specifically: Dissolve the compound of formula (1) in a first solvent, and under the condition of an organic acid, carry out an enol esterification reaction to obtain the compound of formula (2).
[0028] Among them, the first solvent is selected from one or more of acetic anhydride, propionic anhydride, etc.; preferably, it is acetic anhydride.
[0029] Among them, the organic acid is selected from one or more of p-toluenesulfonic acid, methanesulfonic acid, etc.; preferably, it is p-toluenesulfonic acid.
[0030] Among them, the weight ratio of the compound of formula (1), the first solvent, and the organic acid is 1:(1-4):(0.1-1); preferably, it is 1:2:0.15.
[0031] Among them, the temperature of the enol esterification reaction is 0°C to 50°C; preferably, it is 25°C.
[0032] Among them, the time of the enol esterification reaction is 1 to 12 h; preferably, it is 6 h.
[0033] In step (2) of the present invention, the protection reaction is specifically: Dissolve the compound of formula (2) in a second solvent, and under the conditions of ethylene glycol, triethyl orthoformate, and an organic acid, carry out a protection reaction to obtain the compound of formula (3).
[0034] Among them, the second solvent is selected from one or more of dichloromethane, tetrahydrofuran, ethyl acetate, etc.; preferably, it is dichloromethane.
[0035] Among them, the organic acid is selected from one or more of p-toluenesulfonic acid, methanesulfonic acid, etc.; preferably, it is p-toluenesulfonic acid.
[0036] Among them, the weight ratio of the compound of formula (2), the second solvent, ethylene glycol, triethyl orthoformate, and the organic acid is 1:(5-20):(1-4):(1-4):(0.05-0.10); preferably, it is 1:20:2:2:0.05.
[0037] Among them, the temperature of the protection reaction is 0°C to 50°C; preferably, it is 25°C.
[0038] Among them, the time of the upper protection reaction is 1 to 12 h; preferably, it is 12 h.
[0039] In step (3) of the present invention, the reduction reaction is specifically as follows: the compound of formula (3) is dissolved in a third solvent, and a deuterated reducing agent is used to carry out a reduction reaction to obtain the compound of formula (4).
[0040] Among them, the third solvent is selected from one or more of deuterated methanol, deuterated ethanol, tetrahydrofuran, etc.; preferably, it is deuterated methanol.
[0041] Among them, the reducing agent is selected from one or more of sodium borodeuteride, potassium borodeuteride, etc.; preferably, it is sodium borodeuteride.
[0042] Among them, the weight ratio of the compound of formula (3), the third solvent, and the reducing agent is 1:(5 - 10):(0.5 - 2.0); preferably, it is 1:10:0.5.
[0043] Among them, the temperature of the reduction reaction is 0 °C to 50 °C; preferably, it is 25 °C.
[0044] Among them, the time of the reduction reaction is 1 to 12 h; preferably, it is 1 h.
[0045] In step (4) of the present invention, the deprotection reaction is specifically as follows: the compound of formula (4) undergoes a deprotection reaction in a fourth solvent under the action of an acid to remove ethylene glycol and obtain the compound of formula (5).
[0046] Among them, the fourth solvent is selected from one or two of water, acetone, etc.; preferably, it is acetone.
[0047] Among them, the acid is selected from one or more of hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, etc.; preferably, it is p-toluenesulfonic acid.
[0048] Among them, the weight ratio of the compound of formula (4), the fourth solvent, and the acid is 1:(5 - 10):(0.01 - 0.1); preferably, it is 1:10:0.1.
[0049] Among them, the temperature of the deprotection reaction is 0 °C to 50 °C; preferably, it is 25 °C.
[0050] Among them, the time of the deprotection reaction is 1 to 12 h; preferably, it is 12 h.
[0051] In step (5) of the present invention, the deuterium-hydrogen exchange reaction is specifically as follows: the compound of formula (5) is dissolved in a fifth solvent, and under acidic conditions, a deuterium-hydrogen exchange reaction is carried out to obtain the target compound dehydroepiandrosterone-D6.
[0052] Among them, the fifth solvent is selected from one or more of methanol, ethanol, water, etc.; preferably, it is ethanol.
[0053] Among them, the acid is selected from one or more of hydrochloric acid with a mass concentration of 5% and sulfuric acid with a mass concentration of 5%, etc.; preferably, it is hydrochloric acid with a concentration of 5%.
[0054] Among them, the weight ratio of the compound of formula (5), the fifth solvent, and the acid is 1:(5 - 10):(1 - 10); preferably, it is 1:10:5.
[0055] Among them, the temperature of the deuterium-hydrogen substitution reaction is 50 - 75 °C; preferably, it is 70 °C.
[0056] Among them, the time of the deuterium-hydrogen substitution reaction is 1 - 24 h; preferably, it is 12 h.
[0057] In the preparation method of the present invention, using commercially available androstenedione-D8 as the raw material, enol esterification is first carried out, then the 17-carbonyl group is protected with ethylene glycol. Under the condition of a deuterated solvent, the 3-carbonyl group is reduced by sodium borodeuteride to introduce deuterium atoms at the 3-position and 4-position respectively. Then, the ethylene glycol protection is removed, and finally, deuterium-hydrogen substitution is carried out to replace the deuterium atom at the 16-position with a hydrogen atom, thereby preparing dehydroepiandrosterone-D6. The reaction conditions of the present invention are mild, the operation is simple, and the efficient preparation of dehydroepiandrosterone-D6 can be achieved, having broad application prospects.
[0058] The beneficial effects of the present invention include: The present invention provides a brand-new method for preparing dehydroepiandrosterone-D6. In the present invention, the raw materials are easy to prepare, purification and separation are easy, the reaction conditions are mild, the operation is simple and convenient, and the efficient preparation of dehydroepiandrosterone-D6 can be achieved. Detailed Description of the Invention
[0059] Combined with the following specific examples, the present invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content.
[0060] The following description of at least one exemplary embodiment is actually only illustrative and in no way restricts the present invention and its application or use. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0061] In all the examples shown and discussed here, any specific value should be interpreted as merely exemplary and not as a limitation. Therefore, other examples of the exemplary embodiments may have different values.
[0062] The present invention discloses a method for preparing dehydroepiandrosterone-D6 using androstenedione-D8. Using androstenedione-D8 as a raw material, dehydroepiandrosterone-D6 is prepared through five steps: enol esterification, protection, reduction, deprotection, and deuterium-hydrogen substitution. In the preparation method of the present invention, commercially available androstenedione-D8 is used as a raw material. First, enol esterification is carried out, then the 17-carbonyl group is protected with ethylene glycol. Under the condition of a deuterated solvent, the 3-carbonyl group is reduced with sodium borodeuteride to introduce deuterium atoms at the 3-position and 4-position respectively. Then, the ethylene glycol protection is removed. Finally, deuterium-hydrogen substitution is carried out to replace the deuterium atom at the 16-position with a hydrogen atom, and dehydroepiandrosterone-D6 is prepared. The reaction conditions of the present invention are mild and the operation is simple, and the efficient preparation of dehydroepiandrosterone-D6 can be achieved.
[0063] The test materials used in the examples of the present invention are all conventional biochemical reagents without special instructions.
[0064] Example 1 3-Enol esterification reaction (acetyl group)
[0065]
[0066] Add androstenedione-D8 (1 g, 3.41 mmol) to a three-necked flask, then add 2 g of acetic anhydride, stir until clear, and then add 0.15 g of p-toluenesulfonic acid. The system is controlled at 25 °C for reaction for 6 h. After TCL detection shows that the reaction is complete, the temperature of the system is lowered to 0-5 °C, and the liquid of the system is slowly poured into 100 ml of aqueous sodium bicarbonate solution, stirred for 1 h, a solid precipitates, filtered, and the filter cake is washed with ice water and dried to obtain 0.91 g of a white compound of formula (2), with a molar yield of 80%.
[0067] Example 2 17-Ketal protection
[0068]
[0069] Add the compound of formula (2) (0.8 g, 2.39 mmol) and 16 g of dichloromethane to a three-necked flask, stir until the system is clear, then add 1.6 g of ethylene glycol, 1.6 g of triethyl orthoformate, and 0.04 g of p-toluenesulfonic acid, and stir at 25 °C for 12 h. After TCL detection, the raw materials have reacted completely. Wash with aqueous sodium bicarbonate solution three times, separate the aqueous phase, and concentrate the organic phase to obtain 0.82 g of a white solid compound of formula (3), with a molar yield of 91%.
[0070] Example 3 Reduction reaction (using deuterated methanol as a solvent)
[0071]
[0072] In a three-necked flask, add the compound of formula (3) (0.8 g, 2.11 mmol) and 8 g of deuterated methanol, stir until the system becomes clear, then add 0.4 g of sodium borodeuteride solid, stir at 25 °C for 1 h, detect by TLC. After the raw materials have reacted completely, add dichloromethane solution, wash twice with aqueous ammonium chloride solution, concentrate the organic phase to obtain the crude product of the compound of formula (4), and then carry out silica gel column chromatography separation. The eluent is petroleum ether:ethyl acetate = 5:1, to obtain 0.75 g of white solid (4) compound, with a molar yield of 93%.
[0073] Example 4 Reduction reaction (in tetrahydrofuran solvent)
[0074]
[0075] In a three-necked flask, add the compound of formula (3) (0.8 g, 2.11 mmol) and 8 g of tetrahydrofuran, stir until the system becomes clear, then add 0.4 g of sodium borodeuteride solid, stir at 25 °C for 1 h, detect by TLC. After the raw materials have reacted completely, quench with aqueous ammonium chloride solution, add ethyl acetate for extraction, concentrate the organic phase to obtain the crude product of the compound of formula (4), and then carry out silica gel column chromatography separation. The eluent is petroleum ether:ethyl acetate = 5:1, to obtain 0.67 g of white solid (4) compound, with a molar yield of 83%.
[0076] Example 5 17-Deprotection
[0077]
[0078] In a three-necked flask, add the compound of formula (4) (0.7 g, 2.35 mmol) and 7 g of acetone, add 0.07 g of p-toluenesulfonic acid, stir at 25 °C for 12 h, detect by TLC. After the raw materials have reacted completely, add dichloromethane solution, wash once with aqueous sodium bicarbonate solution, concentrate the organic phase to obtain 0.58 g of the compound of formula (5). Molar yield: 96%.
[0079] Example 6 Deuterium-hydrogen exchange reaction (in methanol solvent)
[0080]
[0081] In a three-necked flask, add the compound of formula (5) (0.5 g, 1.69 mmol) and 5 g of methanol, add 2.5 g of 5% hydrochloric acid, heat to 60 °C, keep the temperature for reaction for 12 h, cool to 25 °C, pour the reaction solution into ice water, solid precipitates, filter, wash with water, dry the filter cake, carry out silica gel column chromatography separation. The eluent is petroleum ether:ethyl acetate = 5:1, to obtain 0.40 g of white solid dehydroepiandrosterone-D6. Molar yield: 80%.
[0082] Example 7 Deuterium-hydrogen exchange reaction (in ethanol solvent)
[0083]
[0084] In a three-necked flask, add the compound of formula (5) (0.5 g, 1.69 mmol) and 5 g of ethanol, then add 2.5 g of 5% hydrochloric acid. Heat the mixture to 70 °C and keep the reaction for 12 h. Cool it to room temperature, pour the reaction solution into ice water, and a solid will precipitate. Filter it, wash it with water, dry it, and perform silica gel column chromatography separation. The eluent is petroleum ether:ethyl acetate = 5:1, and 0.44 g of white solid dehydroepiandrosterone-D6 is obtained. Molar yield: 88%. 1 H NMR(400MHz,CDCl 3 )δ2.43-2.50(1H,m),2.04-2.14(2H,m),1.92-1.99(1H,m),1.83-1.87(2H,m),1.03(3H,s,H-19),0.89(3H,s,H-18)。HRMS(ESI):calcd for C 19 H 22 D 6 O 2 [M+Na] + 317.2466;found 317.2371.
[0085] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, all changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.
[0086] As used in the present invention, the term "comprising" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.
[0087] As used in the present invention, the term "and / or" includes any one and all combinations of one or more of the related listed items. Although the embodiments of the present specification have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present specification, and the scope of the present specification is defined by the appended claims and their equivalents.
Claims
1. A method for preparing dehydroepiandrosterone-D6 using androstenedione-D8, characterized in that: The method uses androstenedione-D8 as a raw material, and prepares dehydroepiandrosterone-D6 through five steps of enol esterification, upper protection, reduction, deprotection, and deuterium hydrogen replacement; the reaction process of the method is shown in reaction formula (I): Reaction formula (I); Wherein, R is acetyl or propionyl.
2. The method according to claim 1, characterized in that The method comprises the following steps: Step (1) Enol esterification reaction: The compound of formula (1), i.e. androstenedione-D8, is dissolved in a first solvent and subjected to enol esterification reaction under organic acid conditions to obtain a compound of formula (2); the reaction process is shown in reaction formula (A): Reaction formula (A); Step (2) upper protection reaction: the compound of formula (2) obtained in step (1) is dissolved in a second solvent, and an upper protection reaction is carried out in the presence of ethylene glycol, triethyl orthoformate and an organic acid to obtain a compound of formula (3); the reaction process is shown in reaction formula (B): Reaction formula (B); Step (3) Reduction reaction: The compound of formula (3) obtained in step (2) is dissolved in a third solvent, and a reducing agent is used to perform a reduction reaction to obtain a compound of formula (4); the reaction process is shown in reaction formula (C): Reaction formula (C); Step (4) deprotection reaction: dissolving the compound of formula (4) obtained in step (3) in a fourth solvent, and performing a deprotection reaction under acidic conditions to remove ethylene glycol to obtain a compound of formula (5); the reaction process is shown in reaction formula (D): Reaction formula (D); Step (5) deuterium hydrogen replacement reaction: the compound of formula (5) obtained in step (4) is dissolved in a fifth solvent, and a deuterium hydrogen replacement reaction is carried out under acidic conditions to obtain the target compound dehydroepiandrosterone-D6; the reaction process is shown in reaction formula (E): Reaction formula (E); Wherein, R is acetyl or propionyl.
3. The method according to claim 2, characterized in that In the step (1), the first solvent is selected from one or both of acetic anhydride and propionic anhydride; and / or the organic acid is selected from one or both of p-toluenesulfonic acid and methanesulfonic acid; and / or the weight ratio of the compound of formula (1), the first solvent and the organic acid is 1:(1-4):(0.1-1).
4. The method according to claim 2, characterized in that In the step (1), the temperature of the enol esterification reaction is 0° C. to 50° C.; and / or the time of the enol esterification reaction is 1 to 12 hours.
5. The method according to claim 2, characterized in that In the step (2), the second solvent is selected from one or more of dichloromethane, tetrahydrofuran, and ethyl acetate; and / or the organic acid is selected from one or both of p-toluenesulfonic acid and methanesulfonic acid; and / or the weight ratio of the compound of formula (2), the second solvent, ethylene glycol, triethyl orthoformate, and the organic acid is 1:(5-20):(1-4):(1-4):(0.05-0.10); and / or the temperature of the upper protection reaction is 0°C to 50°C; and / or the time of the upper protection reaction is 1 to 12 hours.
6. The method according to claim 2, characterized in that In the step (3), the third solvent is selected from one or more of deuterated methanol, deuterated ethanol, and tetrahydrofuran; and / or the reducing agent is selected from one or two of sodium borodeuteride and potassium borodeuteride; and / or the weight ratio of the compound of formula (3), the third solvent, and the reducing agent is 1:(5-10):(0.5-2.0); and / or the temperature of the reduction reaction is 0°C to 50°C; and / or the time of the reduction reaction is 1 to 12 hours.
7. The method according to claim 2, characterized in that In the step (4), the fourth solvent is selected from one or both of water and acetone; and / or the acid is selected from one or more of hydrochloric acid, sulfuric acid, and p-toluenesulfonic acid.
8. The method according to claim 2, characterized in that In the step (4), the weight ratio of the compound of formula (4), the fourth solvent and the acid is 1:(5-10):(0.01-0.1); and / or the temperature of the deprotection reaction is 0°C-50°C; and / or the time of the deprotection reaction is 1-12 hours.
9. The method according to claim 2, characterized in that In the step (5), the fifth solvent is selected from one or more of methanol, ethanol, and water; and / or the acid is selected from one or both of 5% by mass hydrochloric acid and 5% by mass sulfuric acid.
10. The method according to claim 2, characterized in that In the step (5), the weight ratio of the compound of formula (5), the fifth solvent and the acid is 1:(5-10):(1-10); and / or the temperature of the deuterium hydrogen replacement reaction is 50 to 75° C.; and / or the time of the deuterium hydrogen replacement reaction is 1 to 24 hours.