Ultrasound‐Assisted Microcontinuous Process Facilitates the Selective Deuteration of Steroid Hormones

Dan Wang; Xin Lv; Fang Wei

doi:10.1002/jlcr.4146

. 2025 May 5;68(5-6):e4146. doi: 10.1002/jlcr.4146

Ultrasound‐Assisted Microcontinuous Process Facilitates the Selective Deuteration of Steroid Hormones

Dan Wang ^1,^✉, Xin Lv ¹, Fang Wei ^1,²

PMCID: PMC12051752 PMID: 40323697

ABSTRACT

Constructing deuterated molecules efficiently and practically has been a long‐standing challenge. Deuterated steroid hormones are essential for medical research and drug metabolism studies and are thus in high demand; mild and selective methods for the deuteration of steroid hormones have remained unexplored. Herein, we demonstrate a practical and efficient approach to synthesize 12 deuterated steroid hormones with up to 98% selectivity and 99% d‐incorporation under an ultrasound‐assisted microcontinuous process. Optical rotation experiments confirm that steroid hormones configurations are preserved during the H/D exchange reaction. Our protocol enables rapid, inexpensive, and sustainable gram‐scale synthesis, facilitated by the reuse of deuterated solvents via molecular distillation technology. Applying synthetic deuterated steroid hormones as mass spectrometry standards, six steroid hormones in metabolites are accurately analyzed from Frozen Human Plasma‐1950 sample. Overall, this work has successfully demonstrated the application of ultrasound assisted microcontinuous processing in enhancing H/D exchange reactions.

Keywords: gram‐scale synthesis, H/D exchange, microcontinuous, steroid hormones, ultrasound

Herein, we demonstrate a practical and efficient approach to synthesize 12 deuterated steroid hormones with up to 98% selectivity and 99% d‐incorporation under an ultrasound‐assisted microcontinuous process. Our protocol enables rapid, inexpensive, and sustainable gram‐scale synthesis, facilitated by the reuse of deuterated solvents via molecular distillation technology.

graphic file with name JLCR-68-0-g003.jpg

1. Introduction

As an important secretion of the human body, steroid hormones have significant medicinal value [1]. They have a clear role in maintaining life, regulating sexual function, immune regulation, and skin disease treatment [2, 3, 4]. For example, progesterone, 17α‐hydroxyprogesterone, hydrocortisone, androstenedione, testosterone, and dehydroepiandrosterone are important steroid hormones in the human body, and they are commonly used as indicators in medical diagnosis analysis. Their corresponding stable isotope compounds can be used as quantitative internal standards of mass spectrometry, or as characteristic markers for the study of drug mechanism and metabolism (Figure 1a) [5, 6, 7, 8, 9, 10]. Few protocols exist for preparing deuterated steroid hormones, due to the difficulty in combining efficiency and economy, which poses a significant obstacle to the accurate identification of steroid hormone types and contents in clinical diagnosis [11].

Direct deuteration of C—H bonds. (a) The importance of deuterium‐labelled steroid hormones. (b) Synthesis of deuterium‐labeled steroid hormones. (c) Continuous flow‐ultrasound synthesis of deuterated steroid hormones (this work).

At present, there are two main synthesis methods of deuterated steroid hormones. Firstly, deuterated steroid hormones are synthesized from precursors, such as cholesterol or ketene, which is difficult to achieve large‐scale synthesis due to the many steps and complex operation [12]. Another method is the direct synthesis of deuterated steroid hormones by steroid hormones via H/D exchange reaction [13]. H/D exchange reaction is typically carried out at high temperatures and requires the addition of an equivalent amount of strong acids or bases (such as HCl, HCOOH, and NaOH) as catalysts, resulting in poor functional group tolerance and safety risk [14, 15]. The metal‐catalyzed H/D exchange reaction has been widely studied due to its high selectivity, including ruthenium [16, 17, 18, 19, 20, 21], iridium [22, 23], and iron [24, 25]. The addition of metal catalysts increases the cost of synthesis and the post‐processing procedure. In recent years, some organic bases, such as pyrrolidine [26], 1,8‐diazabicyclo[5.4.0]undecen‐7‐ene [27], imidazolium [28, 29], B(C₆F₅)₃ [30], and N‐heterocyclic carbene [31] have been used in the H/D exchange reaction. In order to achieve efficient synthesis of deuterated steroid hormones, the d‐incorporation of standard compounds generally requires at least greater than 95%, and the reaction under heating conditions may lead to poor functional group compatibility and changes of compound configuration. Therefore, it is necessary to develop milder and greener deuteration methods with as high d‐incorporation as possible (Figure 1b).

Continuous flow synthesis is an environmentally friendly and sustainable alternative compared with batch synthesis. It has been widely used for pharmaceutical synthesis and laboratory research in recent decades [32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42]. Many previously challenging or dangerous reactions can be performed safely and efficiently in a flow cell [43, 44, 45]. However, these reactors have limitations in dealing with solids, especially when the involvement of solid particles in chemical reactions can cause blockage of channels.

In chemical reactions, ultrasonic waves can play a role in two ways: First, they can directly act on reactants, causing molecular vibrations, shearing and collisions, which facilitate the formation and breaking of chemical bonds, thereby accelerating the reaction. Second, through their physical effects, they can alter the temperature, pressure and flow properties of the reaction system, thus influencing the thermodynamic and kinetic conditions of the reaction and improving the reaction rate and yield. The combination of ultrasound‐assisted continuous processes with microreactors appears to be an effective synthesis method that can bring significant benefits [46, 47, 48, 49, 50]. In recent years, sonochemistry has played an increasingly important role in sustainable development and green chemistry with its unique advantages [51, 52, 53]. When low‐frequency ultrasound (20–80 KHz) [54] passes through a microchannel, it can enhance mixing [55], improve interfacial mass transfer [47, 56], promote deagglomeration [57, 58] and displace the particles deposited on the surface to inhibit clogging [59]. Research results showed that the combination of ultrasound and flow chemistry could accelerate the reaction rate from hours to minutes and greatly increase the yield of products [46]. Ultrasound‐assisted microcontinuous procedures will provide an effective strategy for the synthesis of deuterated steroid hormones.

Based on our previous work in continuous flow synthesis [35, 36, 60, 61, 62, 63], here, we report a novel strategy for the synthesis of deuterated steroid hormones using continuous microflow‐ultrasound synergy in H/D exchange. The reaction performed at room temperature enabled the preparation of highly selective deuterated building blocks, representative deuterated steroid hormones with high and reliable d‐incorporation (Figure 1c). Furthermore, this continuous microflow‐ultrasound synergistic reaction supported scalable synthesis.

2. Results and Discussion

2.1. Optimization of the Reaction Conditions

We set out to investigate our proposed continuous microflow‐ultrasound synergistic synthesis of deuterated steroid hormones, with the entire experimental apparatus setup illustrated in Figure S1. Initially, progesterone (1a) was chosen as the model to optimize reaction conditions (Figure 2). After several efforts, the optimal reaction conditions were established using CD₃OD as both deuterium source and solvent, in the presence of HCOOH. The reaction was carried out in a 1 m long PEEK tube with an inner diameter of 100 μm, at a flow rate of 0.2 mL/min, and under 20 kHz ultrasound for 3 h. As shown in Figure 2a, we first investigated the effect of ultrasonic frequency on the reaction, and the results showed that the most suitable ultrasonic frequency for this reaction was 40 KHz. In the control experiment, stirring instead of continuous microflow‐ultrasound synergy resulted in low conversion. After comprehensive consideration of the reactivity, d‐incorporation, solubility and price, CD₃OD was selected as the deuterium source and solvent (Figure 2b). The amount of HCOOH used was crucial for achieving good reaction efficiency. Less product (yield < 5%) was obtained without the addition of acid, indicating that the acid acts as the key catalyst in this transformation (Figure 2c). Shortening the reaction time to 1 h resulted in incomplete reaction, while extending the reaction time to 4 h had no positive effect on the results (Figure 2d). Among different flow rates and tube diameters, it was found that a PEEK tube with an inner diameter of 100 μm and a flow rate of 0.2 mL/min had the best reaction effect (Figure 2e,f). The use of a continuous flow‐ultrasonic synergistic device allows the reaction to be performed safely at room temperature, with less acid used, and results in higher reaction efficiency and higher d‐incorporation.

Optimizing conditions: single factor variables of optimized reaction condition: (a) ultrasonic frequency; (b) deuterium source; (c) additives; (d) reaction time; (e) flow rate; (f) the inner diameter of the peek tube. Optimized reaction conditions: 0.3 mmol progesterone (**1a)**, 2 mL CD₃OD, 5 mol% HCOOH, in 1 m long peek tube with inner diameter of 100 μm at 0.2 mL/min flow rate, 40 KHz ultrasonic frequency, room temperature, ambient air atmosphere, 3 h, isolated yield of progesterone‐d ₉ (1b).

2.2. Substrate Scope of Steroid Hormones

Steroid hormones can accurately reflect the health status of the human body, which participate in many physiological and pathological pathways [2, 3, 4, 64, 65]. As shown in Figure 3, with the optimized reaction conditions in hand, the scope of steroid hormones was then explored. This protocol was found to be compatible with steroid hormones that contain carbonyl group, double bond group and hydroxyl group. The conversion of 12 steroid hormones to the corresponding deuterated steroid hormones was synergistically synthesized using continuous microflow‐ultrasound. Progesterone‐d ₉ (1b, 98% yield, d‐incorporation > 95%), dehydroepiandrosterone‐d ₂ (2b, 98% yield, d‐incorporation > 99%), pregnenolone‐d ₄ (3b, 98% yield, d‐incorporation > 99%), hydrocortisone‐d ₅ (4b, 98% yield, d‐incorporation > 93%), androsterone‐d ₂ (5b, 98% yield, d‐incorporation > 95%), androstenedione‐d ₇ (6b, 98% yield, d‐incorporation > 90%), 17α‐hydroxyprogesterone‐d ₈ (7b, 98% yield, d‐incorporation > 75%), cortisone‐d ₇ (8b, 98% yield, d‐incorporation > 99%), desoxycortisone‐d ₇ (9b, 98% yield, d‐incorporation > 80%), testosterone‐d ₅ (10b, 98% yield, d‐incorporation > 82%), corticosterone‐d ₅ (11b, 98% yield, d‐incorporation > 68%), and 11α‐hydroxyprogesterone‐d ₈ (12b, 98% yield, d‐incorporation > 89%) gave outstanding yields and excellent d‐incorporation. In addition, to verify that the configuration of each compound remained unchanged during the transformation, we analyzed the specific rotation of 5a, 5b, 6a, and 6b, respectively. The results showed that 5a and 6a and their corresponding products 5b and 6b had the same specific rotation (+) and were dextrorotatory compounds.

Substrate scope of steroid hormones. Reaction conditions: 0.3 mmol steroid hormones (a), 2 mL CD₃OD, 5 mol% HCOOH, in 1 m long peek tube with inner diameter of 100 μm at 0.2 mL/min flow rate, 40 KHz ultrasonic frequency, room temperature, ambient air atmosphere, 3 h, isolated yield of deuterated steroid hormones‐d _x (b).

2.3. Scale‐Up Synthesis of Progesterone‐d ₉ and the Recirculation Times of CD₃OD

To further explore the practicality of our approach, we expanded the reaction of 1a to a 4 mmol scale and obtained the desired product (1b) in 90% isolated yield and 96% d‐incorporation under continuous microflow‐ultrasound synergy (Figure 4a). Due to the amount of CD₃OD in the reaction process, it was recycled through molecular distillation. As shown in Figure 4b, CD₃OD was recycled four times, and the yield of 1b remained at 98%. These results demonstrate that this method has the potential for large‐scale synthesis of deuterated steroid hormones.

Application experiments: Scale‐up synthesis (a) and the recirculation times of CD₃OD (b).

3. Mechanistic Studies

The results of mass spectrometry monitoring of continuous microflow‐ultrasound synergistically synthesized progesterone‐d ₉ are shown in Figure 5. Progesterone ([M + H]⁺ = 315.2348) was rapidly converted into progesterone‐d ₈ within 30 min of reaction initiation and gradually converted to progesterone‐d ₉ ([M + H]⁺ = 324.2871) from 30 min to 3 h. The positions of progesterone molecule that participate in the H/D exchange reaction are at the ortho‐position of carbonyl and alkenyl, which are active C—H bonds. However, there is a C—H on the tertiary carbon of the reaction sites, which is slightly less reactive due to the influence of steric hindrance, resulting in a slower reaction rate.

Mass spectrometry monitoring of reaction process.

Keto‐enol tautomerism is common in carbonyl‐containing compounds, and acid catalysis contributes to the reaction to enol. In addition, as the enol structure contains a hydroxyl group, it contributes to the stabilization of the enol structure and the conversion of the ketone structure to the enol structure in a solvent containing a hydrogen bond acceptor [66]. Therefore, HCOOH catalyst took advantage of converting ketone to enol, which is conducive to the smooth progress of H/D exchange reaction.

The active site of the compound is associated with the pKa of the C—H bond. The acidity hierarchy of C—H bonds within the hormone structure is as follows: C —H (acyl ortho‐site) is more acidic than C—H (allyl), which is more acidic than C—H (alkenyl), which in turn is more acidic than C—H (alkyl). Consequently, the reactions described in this paper can selectively target active C—H bonds (C—H (acyl ortho‐site), C—H (allyl), and C—H (alkenyl)).

The possible mechanism of the microflow‐ultrasound synergistically H/D exchange reaction is shown in Figure 6. First, under the help of acid, progesterone is converted from a ketone structure to an enol structure to obtain intermediate A. Then, under the combined help of CD₃OD and acid, H/D exchange occurs at the α‐position of alkenyl group, and it is converted to keto substrate to obtain intermediate B. Next, under the help of acid, the keto‐enol structure is converted again to yield intermediate C. Finally, under the help of CD₃OD and acid, H/D exchange occurs again to produce progesterone‐d ₉.

4. Application of Deuterated Steroid Hormones

Isotopic compounds can serve as internal standards for mass spectrometry analysis [67, 68, 69, 70, 71]. To demonstrate the applicability of synthetic deuterated steroid hormones, these compounds were employed as internal standards in the analysis of Metabolites in Frozen Human Plasma‐1950 (NIST SRM 1950). The analysis of steroid hormones was performed using UPLC‐MS, with the detailed instrument information provided in the Supporting Information. The gradient conditions for the liquid chromatography are shown in Table S1 of the supplementary information. Six steroid hormones were quantified in NIST SRM 1950, including progesterone, 17α‐hydroxyprogesterone, hydrocortisone, cortisone, corticosterone, and testosterone. The concentrations of these compounds, their limits of detection (LODs), limits of quantitation (LOQs), and comparisons to reported values [72, 73, 74] for NIST SRM 1950 are summarized in Tables 1. While LODs define the concentrations below which reported data may vary by more than 100%, low‐precision data can still be generated at lower concentrations, indicating the presence of metabolites with an instrumental signal‐to‐noise ratio greater than 3:1 [75]. The LODs of the six steroid hormones and their isotopic compounds are lower than the corresponding steroid hormone levels in NIST SRM 1950. Therefore, the use of synthetic deuterated steroid hormones as internal standards in mass spectrometry analysis provides a reliable method for analyzing steroid hormones in samples.

TABLE 1.

Steroids in NIST SRM 1950. ^a

Analyte	LOD	LOQ	t_R (min)	Q1 (Da)	Reported NIST	Measured NIST
Progesterone	0.477	1.59	8.22	315.23	5.21 ± 0.35	5.15 ± 0.62
Progesterone‐d ₉	0.398	1.33	8.20	324.29	—	—
17α‐hydroxyprogesterone	0.302	1.01	7.06	331.23	2.26 ± 0.70	2.28 ± 0.91
17α‐hydroxyprogesterone‐d ₈	0.295	0.982	7.03	339.28	—	—
Hydrocortisone	0.275	0.916	6.99	363.22	244 ± 15	246 ± 23
Hydrocortisone‐d ₅	0.272	0.906	6.94	368.28	—	—
Cortisone	0.277	0.922	7.76	361.20	46.3 ± 6.1	47.1 ± 5.7
Cortisone‐d ₆	0.281	0.936	7.70	368.24	—	—
Corticosterone	0.288	0.959	7.59	347.22	6.01 ± 1.41	5.95 ± 1.55
Corticosterone‐d ₅	0.284	0.946	7.56	352.25	—	—
Testosterone	0.346	1.15	7.13	289.22	8.31 ± 0.721	8.44 ± 1.20
Testosterone‐d ₅	0.337	1.12	7.11	294.25	—	—

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Note: Measured values are means ± SD (n = 3). Reference values of NIST are medians of laboratory means.

Abbreviations: LOD, limit of detection; LOQ, limit of quantitation.

^{^a}

Values are in nM.

5. Conclusions

In summary, we have developed a practical and efficient method for the synthesis of deuterated steroid hormones under ultrasound‐assisted microcontinuous process. Twelve deuterated steroid hormones were synthesized at room temperature with a selectivity of up to 95% and a d‐incorporation of up to 99%. This transformation enabled fast and efficient gram‐scale synthesis. In addition, deuterated solvents can be reused several times by molecular distillation technology to save experimental costs. The H/D exchange reaction was monitored by mass spectrometry, and a reasonable reaction mechanism was proposed. Using synthetic deuterated steroid hormones as internal standards in NIST SRM 1950 sample could accurately identify and quantify six kinds of steroid hormones. Further studies on the synthesis applications of isotopic compounds are currently underway in our group.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1. Continuous flow‐ultrasound synergy apparatus setup in its entirety.

Table S1. Gradient elution conditions.

JLCR-68-0-s001.docx^{(19.6MB, docx)}

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 22301313 and U21A20274). We also gratefully acknowledge the support of National Key R&D Program Key Special Project (Grant No. 2021YFD1600103), Hubei Province Technology Innovation Special Project (Grant No. 2021BEC021), and Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS‐ASTIP‐2013‐OCRI). We thank Prof. Aiwen Lei for his help with the NMR experiments.

Funding: This work was supported by National Natural Science Foundation of China (22301313 and U21A20274), National Key Research and Development Program of China (2021YFD1600103), Hubei Province Technology Innovation Special Project (2021BEC021), and Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS‐ASTIP‐2013‐OCRI).

Contributor Information

Dan Wang, Email: wangdan06@caas.cn.

Fang Wei, Email: willasa@163.com.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Continuous flow‐ultrasound synergy apparatus setup in its entirety.

Table S1. Gradient elution conditions.

JLCR-68-0-s001.docx^{(19.6MB, docx)}

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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PERMALINK

Ultrasound‐Assisted Microcontinuous Process Facilitates the Selective Deuteration of Steroid Hormones

Dan Wang

Xin Lv

Fang Wei

ABSTRACT

1. Introduction

FIGURE 1.

2. Results and Discussion