Method for preparing testosterone D3 by using 17-carbonyl androstane-5-ene-3-ethylene ketone-16, 16-D2

Abstract

The invention discloses a method for preparing testosterone D3 by using 17-carbonyl androstane-5-ene-3-ethylene ketone-16 and 16-D2, which is characterized in that the 17-carbonyl androstane-5-ene-3-ethylene ketone-16 and 16-D2 is used as a raw material, and the testosterone D3 is prepared by two steps of reduction and deprotection. According to the preparation method, 17-carbonyl androstane-5-ene-3-ethylene ketone-16, 16-D2 is taken as a raw material, reduction is carried out through sodium boron deuteride, deuterium atoms are introduced to the 17 site, then deprotection is carried out, and high-abundance testosterone D3 (abundance gt; 96%). The invention also provides a novel compound of formula (2). The raw materials are easy to prepare, the deuteration efficiency is high, the reaction condition is mild, the operation is simple, the efficient preparation of testosterone D3 can be realized, and the testosterone D3 has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of organic compound preparation technology, and relates to a method for preparing testosterone-D3 using 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2. Background Technology

[0002] Steroid hormones play a crucial role in maintaining homeostasis, regulating metabolism, growth and development, and reproductive behavior in the human body, and are of high reference value for screening and diagnosing related diseases. Due to the low concentration, similar structures, and diverse types of steroid hormones in the body, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the preferred method for detecting steroid hormone molecules in clinical practice. Stable isotope-labeled steroid compounds are commonly used standards for detection, among which deuterium-labeled steroid compounds are relatively... 13 C-labeled compounds are easy to synthesize, cheaper, and have a wider range of applications.

[0003] Testosterone is the most abundant androgen in the human body, primarily synthesized by the interstitial cells of the testes, with the adrenal glands also secreting androgens. The vast majority of testosterone in the blood is bound to plasma proteins, with only a small amount existing in free form. Only free testosterone possesses biological activity. The main physiological functions of testosterone are promoting the development and growth of reproductive organs, stimulating libido, and simultaneously promoting and maintaining the development of male secondary sexual characteristics, maintaining the function of the prostate and seminal vesicles, and spermatogenesis. Furthermore, it can promote protein synthesis, bone growth, and erythrocyte production. This indicator can be used to assess testicular androgen secretion function, helping to screen for male sexual development abnormalities, testicular tumors, and infertility.

[0004] Lu Youli et al. (Laboratory Medicine, 2022, 2:165-173) established an LC-MS / MS method for detecting steroid hormones, which can serve as a rapid and effective method for the auxiliary and differential diagnosis of polycystic ovarian syndrome (PCOS). Testosterone-D3 is used as a deuterated internal standard to correct for matrix effects.

[0005] The chemical name of testosterone-D3 is 17β-hydroxyandrost-4-en-3-one-16,16,17-D3, and its structure is shown in Formula a. The main deuterated position is 16,16,17-trideuterium.

[0006]

[0007] There are few reports on the synthesis of testosterone-D3. In 1986, Raymond McCague et al. (J. Chem. Soc., Chem. Commun., 1986) reported a rearrangement reaction starting from androstenedione using octafluorotoluene, followed by deuteration and reduction under basic conditions, and finally removal of octafluorotoluene to obtain testosterone-D3. The main problem is that the rearrangement reaction using octafluorotoluene requires harsh conditions and high temperatures, resulting in a product abundance of 95% and a moderate yield. The reaction route is shown in Scheme 1.

[0008]

[0009] In 1995, patent CZ 287320B6 reported a method for preparing testosterone-D3 from raw material I. Starting from raw material I, testosterone-D3 was constructed through deuteration and reduction, followed by a rearrangement reaction. However, no specific isotopic abundance of the final product was reported, the yield was unsatisfactory at only 36%, and raw material I was difficult to prepare. The route is shown in Scheme 2.

[0010]

[0011] The main problems with all the publicly reported methods are low yield, difficulty in obtaining raw materials, low deuteration efficiency, and final isotope abundance not reaching 96% or higher. These methods are not suitable as standard materials for deuterated hormones. Therefore, it is urgent to find a highly efficient method for preparing testosterone-D3. Summary of the Invention

[0012] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing testosterone-D3 using 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2. In this method, 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2 is used as a raw material. A deuterium atom is introduced at the 17-position by reduction with sodium borodeuteride, followed by deprotection. This process involves two steps: reduction and deprotection, to obtain high-abundance testosterone-D3 (abundance > 96%). The compound of formula (2) in this invention is a novel structural intermediate that is easy to prepare. It exhibits high deuteration efficiency in the synthesis of testosterone-D3, is easy to purify and separate, has mild reaction conditions, and is simple and convenient to operate, thus enabling efficient preparation of testosterone-D3.

[0013] In the preparation method of the present invention, 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2 is used as raw material, and the target product testosterone-D3 is prepared through two steps of reduction and deprotection.

[0014] The reaction process of the preparation method described above in this invention is shown in reaction formula (I):

[0015]

[0016] The method of the present invention includes the following steps:

[0017] Step (1) Reduction: The compound of formula (1) is dissolved in the first solvent and reacted in the presence of a reducing agent to obtain the compound of formula (2); the reaction process is shown in reaction formula (A):

[0018]

[0019] Step (2) Deprotection: The compound of formula (2) obtained in step (1) is dissolved in a second solvent and deprotected under acidic conditions to obtain the target product testosterone-D3; the reaction process is shown in reaction formula (B):

[0020]

[0021] In step (1) of the present invention, the reduction reaction specifically involves reacting the compound of formula (1) with a reducing agent in the first solvent to obtain the compound of formula (2).

[0022] In step (1), the first solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, etc.; preferably, it is ethanol.

[0023] In step (1), the reducing agent is selected from one or more of sodium borodeuteride, lithium tetradeuterium aluminum, etc.; preferably, it is sodium borodeuteride.

[0024] In step (1), the molar ratio of the compound of formula (1) and the reducing agent is 1:(1-2); preferably, it is 1:1.

[0025] In step (1), the temperature of the reduction reaction is 0℃-50℃; preferably, it is 20℃.

[0026] In step (1), the reduction reaction takes 1-12 hours; preferably, it takes 1 hour.

[0027] In step (2), the deprotection reaction specifically involves the compound of formula (2) undergoing a deprotection reaction in the second solvent under the action of acid to obtain testosterone-D3.

[0028] The acid is selected from one or more of hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, etc.; preferably, it is p-toluenesulfonic acid.

[0029] Wherein, the weight ratio of the compound of formula (2) to the acid is 1:(0.01~0.1); preferably, it is 1:0.05.

[0030] The second solvent is selected from one or more of water, acetone, methanol, etc.; preferably, it is acetone.

[0031] The temperature of the deprotection reaction is 10–75°C; preferably, it is 20°C.

[0032] The deprotection reaction takes 0.5 to 12 hours; preferably, it takes 12 hours.

[0033] In one specific embodiment, 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2 is used as a raw material. A deuterium atom is directly introduced at the 17 position by reduction with sodium borodeuteride, followed by deprotection under mild conditions, to prepare high-abundance testosterone-D3. Preferably, the isotopic abundance of the testosterone-D3 is >96%.

[0034] This invention also proposes that the above-mentioned method for preparing testosterone-D3 using 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2 has important applications in liquid chromatography-mass spectrometry for the medical detection of hormones, using testosterone-D3 as a deuterated internal standard, in measuring the residues of multiple anabolic hormones in animal-derived foods and in the differential diagnosis of polycystic ovary syndrome.

[0035] The present invention also proposes a novel structural intermediate, the structure of which is shown in formula (2):

[0036]

[0037] The preparation method of the compound of formula (2) includes:

[0038] Compound (1), namely 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2, was dissolved in a first solvent and reacted in the presence of a reducing agent to obtain compound (2); the reaction process is shown in reaction formula (A):

[0039]

[0040] The first solvent is selected from one or more of methanol, ethanol, and tetrahydrofuran; and / or the reducing agent is selected from one or more of sodium borodeuteride and lithium tetradeuterium; and / or the molar ratio of the compound of formula (1) to the reducing agent is 1:(1-2); and / or the temperature of the reduction reaction is 0℃-50℃; and / or the time of the reduction reaction is 1-12h.

[0041] The high-abundance testosterone-D3 synthesized in this invention can be applied to liquid chromatography-mass spectrometry (LC-MS / MS) for the medical detection of hormones. For example, national standards stipulate that testosterone-D3 is used as an internal standard to confirm and quantify the residues of multiple anabolic hormones in animal-derived foods. Furthermore, by using testosterone-D3 as a deuterated internal standard to correct for matrix effects, an LC-MS / MS method for detecting steroid hormones has been established, which can serve as a rapid and effective method for the auxiliary and differential diagnosis of polycystic ovary syndrome (PCOS).

[0042] The beneficial effects of this invention include: the compound of formula (2) is a novel intermediate, the structure of which has not been reported in any literature. Furthermore, no relevant literature has been found on the method for synthesizing testosterone-D3 from this intermediate. At the same time, the raw materials are simple and easy to prepare, the deuteration efficiency is high, the purification and separation are easy, the reaction conditions are mild, and the operation is simple and convenient. It can achieve efficient preparation of testosterone-D3, and the isotopic abundance of the obtained testosterone-D3 is greater than 96%. It can be applied to the liquid chromatography-mass spectrometry method for medical detection of hormones and has broad application prospects. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a high-resolution mass spectrum of high-purity testosterone-D3 in Example 3 of the present invention. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0046] Example 1: Reduction reaction using ethanol as solvent

[0047]

[0048] 200 mg of compound (1) was mixed with 10 ml of ethanol and cooled to 15 °C. 33 mg of sodium borodeuteride solid was added in portions, and the reaction temperature was maintained at 20 °C. The mixture was stirred for 1 h, and water-soluble ammonium chloride was added to quench the polarity. The mixture was extracted with ethyl acetate, separated, washed once with water, and the organic phase was concentrated to obtain compound (2), which was a white solid of 180 mg, with a yield of 90%.

[0049] Example 2 Reduction reaction using methanol as solvent

[0050]

[0051] 200 mg of compound (1) was mixed with 10 ml of methanol and cooled to 15 °C. 33 mg of sodium borodeuteride solid was added in portions, and the reaction temperature was maintained at 20 °C. The mixture was stirred for 1 h, and water-soluble ammonium chloride was added to quench the polarity. The mixture was extracted with ethyl acetate, separated, washed once with water, and the organic phase was concentrated to obtain compound (2), which was a white solid of 175 mg, with a yield of 88%.

[0052] Example 3 uses deprotection of p-toluenesulfonic acid

[0053]

[0054] 100 mg of compound (2) and 10 ml of acetone were mixed, and 5 mg of p-toluenesulfonic acid was added. The mixture was stirred at 20 °C for 12 h. After the reaction was completed, a sample was taken to check the reaction. The acetone was removed by vacuum distillation, ethyl acetate was added, and the mixture was washed once with water. The organic phase was concentrated and separated by column chromatography to obtain 80 mg of the product, with a yield of 93%. The isotopic abundance was 96.7%. 1 H NMR(400MHz, CDCl3)δ5.73(s,1H),2.47-2.26(m,4H),2.05-2.02(m,1H),1.87-1.84(m,2H),1.74-1.66(m,1H) ),1.59-1.54(m,1H),1.48-1.26(m,5H),1.20(s,3H),1.13-0.90(m,4H),0.79(s,3H).ESI-HRMS(m / z)[M+Na] + :calcd for C 19 H 25 D3NaO2314.2175; found 314.2175.

[0055] Example 4 uses hydrochloric acid for deprotection.

[0056]

[0057] 100 mg of compound (2) and 10 ml of methanol were mixed, and 10 mg of (5%) hydrochloric acid was added. The mixture was stirred at 20 °C for 12 h. After the reaction of the raw materials was completed, the methanol was removed by vacuum distillation, ethyl acetate was added, and the mixture was washed once with water. The organic phase was concentrated and separated by column chromatography to obtain 78 mg of product with a yield of 90% and an isotopic abundance of 96.4%.

[0058] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing testosterone-D3 using 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2, characterized in that, The method uses compound 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2 of formula (1) as a raw material, and prepares the target product testosterone-D3 through a two-step reaction of reduction and deprotection. The reaction process is shown in reaction formula (I):

2. The method as described in claim 1, characterized in that, The method includes the following steps: Step (1) Reduction: The compound of formula (1), namely 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2, is dissolved in the first solvent and reacted in the presence of a reducing agent to obtain the compound of formula (2); the reaction process is shown in reaction formula (A): Step (2) Deprotection: The compound of formula (2) obtained in step (1) is dissolved in a second solvent and deprotected under acidic conditions to obtain the target product testosterone-D3; the reaction process is shown in reaction formula (B):

3. The method as described in claim 2, characterized in that, In step (1), the first solvent is selected from one or more of methanol, ethanol, and tetrahydrofuran; and / or, the reducing agent is selected from one or more of sodium borodeuteride and lithium tetradeuterium; and / or, the molar ratio of the compound of formula (1) to the reducing agent is 1:(1-2); and / or, the temperature of the reduction reaction is 0℃-50℃; and / or, the time of the reduction reaction is 1-12h.

4. The method as described in claim 2, characterized in that, In step (2), the acid is selected from one or more of hydrochloric acid, sulfuric acid, and p-toluenesulfonic acid; and / or, the weight ratio of the compound of formula (2) to the acid is 1:(0.01 to 0.1); and / or, the second solvent is selected from one or more of water, acetone, and methanol; and / or, the temperature of the deprotection reaction is 10 to 75°C; and / or, the time of the deprotection reaction is 0.5 to 12 h.

5. The method as described in any one of claims 1, characterized in that, The isotopic abundance of testosterone-D3 prepared by the method is >96%.

6. The high-abundance testosterone-D3 prepared by the method according to any one of claims 1-4.

7. The method according to any one of claims 1-4, or the application of high-abundance testosterone-D3 as described in claim 6 in liquid chromatography-mass spectrometry for the medical detection of hormones.

8. An intermediate, characterized in that, The structure of the intermediate is shown in equation (2):

9. The method for preparing the compound of formula (2) as described in claim 8, characterized in that, The preparation method includes: Compound (1), namely 17-carbonylandrost-5-ene-3-ethylene glycol ketal-16,16-D2, was dissolved in a first solvent and reacted in the presence of a reducing agent to obtain compound (2); the reaction process is shown in reaction formula (A):

10. The preparation method according to claim 9, characterized in that, The first solvent is selected from one or more of methanol, ethanol, and tetrahydrofuran; and / or the reducing agent is selected from one or more of sodium borodeuteride and lithium tetradeuterium aluminum; and / or the molar ratio of the compound of formula (1) to the reducing agent is 1:(1-2); and / or the temperature of the reduction reaction is 0℃-50℃; and / or the time of the reduction reaction is 1-12h.

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