Synthesis of a New Bioconjugate Steroid Pyridinium Salt Derived from Allopregnanolone Acetate

Abstract

Because allopregnanolone and derivatives represent biologically active molecules, in this letter, we present the synthesis of a new bioconjugate steroid pyridinium salt derived from allopregnanolone in three steps. The key steps involve the formation of the hydrazone intermediate, followed by condensation with bromoacetyl bromide and subsequent coupling with pyridine to generate the pyridinium bromide salt. The new bioconjugate steroid pyridinium salt, 4, was fully characterized by proton and carbon nuclear magnetic resonance (¹H and ¹³C NMR) spectroscopy, mass spectrometry (MS), and Fourier transform infrared spectroscopy (FTIR). ¹H-NMR analysis revealed the presence of a dynamic rotameric mixture in a 7:3 ratio of Z/E amide conformers, which were identified by a 2D NOESY experiment.

1. Introduction

Steroids’ bioconjugation with other active molecules have multiple applications, from enhancing a particular activity, combining two activities, to even facilitating administration [1]. In this sense, allopregnanolone, a neuroactive steroid, is the natural metabolite of progesterone, synthesized within nervous tissue by specific enzymes. Contrary to progesterone and its first metabolite dihydroprogesterone, allopregnanolone is able to interact as a potent positive allosteric modulator of the GABA-A receptor [2]. The therapeutic potential of allopregnanolone and its derivatives (Figure 1a, left) has been explored under different pathological conditions [3], demonstrating interesting beneficial effects on spinal cord trauma [4], prevention of neuronal death [5], reduction in cholesterol accumulation and stroke [6], epilepsy [7,8,9], diabetes mellitus [10,11], HIV [12], neuroinflammation [13,14], protective effects against neurodegenerative diseases such as Alzheimer’s [15,16] and Parkinson’s [17,18], as well as anxiolytic [19,20] and anti-stress [21] actions. Therefore, allopregnanolone bioconjugate derivatives may represent a new interesting therapeutic perspective on many diseases.

Pyridinium salts (Figure 1b, right) are familiar structures in many natural products [22,23] and bioactive pharmaceuticals, which exhibit several beneficial biological activities including antimicrobial [24,25], anti-cancer [26], anti-malarial [27], and anti-cholinesterase inhibitors [28]. Even though there are a few reports of steroid–pyridinium bioconjugates, one of them was synthesized by Ortoleva–King from 3-β-hydroxy-5-pregnene-20-one in 50% yield [29]. This methodology was applied in the synthesis of turosteride, a new 5α-reductase inhibitor [30]. Therefore, it becomes an attractive salt drug construction.

Consequently, developing new bioconjugates coming from allopregnanolone acetate constitutes an area for discovering novel pharmaceuticals with improved or novel biological properties that can expand functional diversity and enable new applications. This work focused on synthesizing a new biomolecule that combines these privileged scaffolds, pyridinium salt, and allopregnanolone acetate, using hydrazone as a tether because these compounds are considered structurally versatile and promising anti-infective agents [31].

2. Results and Discussion

Synthesis of Steroidal Pyridinium Salt 4

The steroidal pyridinium salt was synthesized in three stages (Scheme 1). In the first step, compound 1 was condensed with hydrazine hydrate using ethanol as the solvent. The mixture was refluxed until the reaction was complete, as indicated by TLC (4 h). In this step, hydrazone 2 was used without purification to avoid its undesirable hydrolysis. Analysis of the crude reaction mixture by NMR spectroscopy confirmed the formation of hydrazone 2. Subsequently, 2 was condensed with bromoacetyl bromide using triethylamine as the base and CH₂Cl₂ as the solvent, and the resulting mixture was stirred at 0 °C for 30 min to afford the desired compound 3, with an overall yield of 90% after two steps. Finally, to obtain the desired pyridinium salt 4, compound 3 was dissolved in pyridine at 25 °C. The resulting mixture was stirred for 5 min. Then, diethyl ether was added in excess, and the pyridinium salt 4 was precipitated as a white solid.

Pyridinium salt 4 undergoes slow conformational flipping in solution at ambient temperature, as revealed by the analysis of the crude reaction by ¹H NMR, specifically in the form of 4a and 4b conformers in a 7:3 ratio. Two-dimensional NOESY experiments in CDCl3 allowed for differentiation between the 4a and 4b conformers, which are favored in a ratio of approximately 7:3. Key signals (cross-peaks) of conformer 4a was observed at δ 11.06 and δ 2.03, indicating spatial correlations between the NH proton and 3H-21; observations at 11.06 and 2.38 indicated the interaction between NH proton and H-17; finally, a cross-peak was observed at δ 11.06 (NH) and 6.27 (CH2). All these signals are consistent with the conformer proposed. On the other hand, a characteristic signal of minor conformer 4b was observed at δ 9.77 and 2.03, which represents the corresponding correlation of NH/3H-21. The analysis of the cross-peaks at δ 9.77 (NH) and 2.38 (H-17) confirmed the interaction between these protons. This behavior could be explained by the E vs. Z amide bond rotation, favoring conformer 4a, while N—N single-bond rotation is restricted by an allylic 1,3-strain consequence as shown in Scheme 2.

All new compounds were fully characterized by ¹H, ¹³C and 2D NMR, and IR spectroscopy. For the target compound, mass spectrometry (HRMS) was used (Supplementary Material).

3. Materials and Methods

3.1. General

NMR spectra 1D and 2D were recorded on Bruker-500 (500 MHz), with TMS (0.0 ppm for ¹H) and residual solvent peak of CDCl₃ (δ = 7.26 ppm for ¹H, δ = 77.16 ppm for ¹³C) used as reference. Optical rotations were determined at room temperature using an Autopol III polarimeter, with a 1 dm cell holding a total volume of 1 mL, with reference to the sodium D line. Infrared spectra were obtained using a Bruker Tensor 27 spectrophotometer (Bruker Corporation, Billerica, MA, USA). Mass spectra (FABMS) and high-resolution mass spectra (HRMS-FAB) were obtained on JEOL mass spectrometer, the MStation JMS-700, at 70 eV (JEOL, Tokyo, Japan). Column chromatography was performed on silica gel (60, 0.063–0.2 mm/70–230 mesh ASTM). All chemicals used were of reagent grade and employed without any additional purifications.

3.2. Synthesis Procedures

3.2.1. Allopregnanolone 17-Hydrazineylideneethyl Acetate (2)

To a stirred solution of the compound 1 (1 equiv, 0.83 mmol, 300 mg) in EtOH (15 mL) at room temperature, hydrazine hydrate (2 equiv, 150 μL) was added dropwise, and the mixture was refluxed. After 4 h, the solvent was removed under reduced pressure. The crude reaction was analyzed by NMR spectroscopy, confirming the formation of hydrazone 2, which was employed without purification for the next reaction. White solid. melting point 162–164 °C. ¹H NMR (500 MHz, CDCl₃), δ (ppm, J Hz): 4.64 (2H, s, NH₂), 4.37 (1H, tt, J = 10.9, 4.9 Hz, 3-H), 1.94–1.82 (2H, m), 1.70 (3H, s), 1.55–1.46 (2H, m), 1.46–1.33 (5H, m), 1.33–1.22 (4H, m), 1.18 (1H, dd, J = 12.5, 3.7 Hz), 1.09–0.99 (2H, m), 0.97 (4H, t, J = 6.1 Hz), 0.91–0.79 (3H, m), 0.72 (1H, td, J = 13.6, 3.9 Hz), 0.60 (1H, dd, J = 12.2, 5.3 Hz), 0.51 (3H, s), 0.38 (1H, q, J = 6.7 Hz), 0.25 (3H, s). ¹³C NMR (125 MHz, CDCl₃) δ 170.5 (C, OC(O)CH₃), 151.5 (C-20), 73.5 (C-3), 59.0, 55.9, 54.2, 44.6, 44.0, 39.0, 36.7, 35.6, 35.4, 33.9, 31.8, 28.4, 27.4, 24.1, 23.0, 21.3, 21.1, 15.5, 13.3, 12.2.

3.2.2. Allopregnanolone 17-Bromoacetyl Hydrazineylindene Ethyl Acetate (3)

To a stirred solution of hydrazone 2 (1 equiv., 0.26 mmol, 100 mg) in CH₂Cl₂ at 0 °C, triethylamine (2 equiv, 0.39 mmol, 40 mg) was added, followed by the slow addition of bromoacetyl bromide (1.2 equiv, 0.31 mmol, 640 mg). The resulting mixture was stirred for 30 min. Finally, the solvent was removed. The resulting mixture was purified through column chromatography (SiO_2, hexane: ethyl acetate, 90:10) to obtain the corresponding compound 3 (90% yield). White solid. melting point 158–160 °C. [α]_D²⁰ = +25.00 (c 0.733, CHCl₃). ¹H NMR (500 MHz, CDCl₃), δ (ppm, J Hz): 8.88 (1H, s), 4.62 (1H, tt, J = 11.0, 4.9 Hz, 3-H), 4.16 (2H, q, J = 11.3 Hz, CH₂Br), 2.26–2.11 (2H, m), 1.95 (3H, s), 1.78 (3H, s), 1.70–1.56 (5H, m), 1.56–1.47 (2H, m), 1.45–1.37 (1H, m), 1.33–1.16 (7H, m), 1.10 (3H, td, J = 9.6, 4.0 Hz), 0.96 (1H, td, J = 13.6, 3.9 Hz), 0.91–0.79 (1H, m), 0.75 (3H, s), 0.63 (1H, td, J = 11.2, 4.1 Hz), 0.51 (3H, s). ¹³C NMR (125 MHz, CDCl₃) δ 170.7 (C, OC(O)CH₃), 168.7 (C, NC(O)CH₂), 153.9 (C-20), 73.6 (C-3), 59.1, 56.2, 54.2, 44.6, 44.3, 39.1, 36.7, 35.6, 35.5, 33.9, 31.8, 28.4, 27.4, 26.8, 24.0, 23.3, 21.4, 21.2, 17.0, 13.5, 12.2. HRMS (FAB⁺) m/z calcd. for [C₂₅H₄₀BrN₂O₃]^•+: 495.2222; found: 495.2233. FTIR: 3190, 2927, 1727, 1674, 1365, 1242, 1026, 731 cm⁻¹.

3.2.3. Allopregnanolone 17-Hydrazineyl-2-oxoethyl pyridine-1-ium Bromide 4

To bromoacetate 3 (1 equiv, 0.036 mmol, 18 mg), pyridine (4 equiv, 0.14 mmol, 11 mg) was added and stirred for 5 min, then the mixture was purified by recrystallization and adding Et₂O (15 mL), and a white precipitate was observed. The solvent was removed by filtration on cotton, the organic residue taken to dry, and the pyridinium salt was obtained with 80% yield. White solid. melting point 193–195 °C. [α]_D²⁵ (c = 0.37, CHCl₃). Major conformer, 4a. ¹H NMR (500 MHz, CDCl₃), δ (ppm, J Hz): 11.06 (1H, s), 9.48 (2H, d, J = 6.0 Hz), 8.49 (1H, t, J = 7.8Hz), 8.07 (2H, t, J = 7.0 Hz), 6.27–6.18 (2H, m, CH₂Py), 4.69 (1H, td, J = 11.2, 5.5 Hz, 3-H), 2.38–2.23(2H, m), 2.20 (3H, s), 2.03 (3H, s), 1.95–1.79 (3H, m), 1.77–1.53 (7H, m), 1.41–1.23 (6H, m), 1.21–1.11 (3H, m), 1.02(1H, td, J = 13.9, 4.2 Hz), 0.91(1H, tt, J = 11.7, 5.5 Hz), 0.82 (3H, d, J = 6.9 Hz), 0.58 (3H, s). Minor conformer, 4b. 9.77 (s), 9.31 (d, J = 6.0 Hz), 8.54 (t, J = 7.7 Hz), 8.13 (t, J = 7.0 Hz), 6.16–6.11 (m, CH₂Py) ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 170.7(C, OC(O)CH₃), 163.8(C, NC(O)CH₂), 160.4(C-20), 145.9, 145.4, 127.8, 73.6(C-3), 61.1 (C, CH₂Py), 59.4, 56.2, 54.1, 44.6, 44.5, 38.9, 36.7, 35.5, 33.9, 31.8, 28.4, 27.4, 24.2, 23.2, 21.5, 21.1, 20.5, 13.5, 12.2. HRMS (FAB⁺) m/z calcd. [C₃₀H₄₄N₃O₃]^•+: 494.3383; found: 494.3371. FTIR: 3176, 2918, 2847, 1727,1702, 1260, 1030, 683, 592 cm⁻¹.

4. Conclusions

A new pyridinium salt derived from allopregnanolone is reported, with an overall yield of 72% after 3 steps. All physical, NMR, IR spectroscopy, and mass spectrometry characteristics were determined. The presence of two conformers in solution in a 7:3 ratio is the result of an allylic 1, 3-strain effect and Z vs. E amide bond rotation.

Author Contributions

Conceptualization, A.C.-C. and J.L.T.; Methodology, J.L.S.-J., V.C.-T. and H.R.-M.; investigation, J.S.-R. and H.R.-M.; resources A.C.-C. and J.S.-R.; writing—original draft preparation, H.R.-M. and J.L.S.-J.; writing—review and editing, A.C.-C., J.L.T. and V.C.-T. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data presented in this study are included in the Supplementary Materials.

Acknowledgments

Ramiro Caso for IR spectroscopy and Patricia Ruiz-Gutierrez for technical support on mass spectrometry analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figure and Schemes

Figure 1 (a) Allopregnanolone and derivatives with biological activities [3,8]. (b) Pyridinium salts with biological activities reported [18,19,20,21,22,23,24].

Supplementary Materials

NMR 1D/2D and FTIR spectra of compounds.

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