New Pyridinium Salt Bioconjugates of Cholesterol and Methylpyridine Derivatives: Synthesis and Characterization

Abstract

The synthesis of three novel, valuable bioconjugates obtained by coupling cholesterol bromoacetate with pyridine derivatives via an SN2 reaction was successfully carried out. Each of the products was fully characterized by magnetic nuclear resonance (¹H, ¹³C, APT, ¹H−¹H COSY, ¹H–¹³C HMBC, ¹H–¹³C HSQC), infrared spectroscopy (IR), and high-resolution mass spectrometry (HRMS).

1. Introduction

Pyridinium salts are quaternary nitrogen salts that have attracted considerable attention due to their multiple biological activities, which include anti-cancer, antimicrobial and anti-neurodegenerative, attributed in part to the positive charge on the nitrogen atom which facilitates interactions with negatively charged biological targets [1,2,3,4]. Similarly, steroids have a wide range of biological activities, including anti-cancer, hormonal regulation, and acting against cardiac, infectious, and neurodegenerative diseases [5,6,7,8,9,10]. Given their complementary bioactivity, the bioconjugation of steroids with positive-nitrogen-containing heterocycles has emerged as a promising strategy for the development of novel hybrid molecules with enhanced or synergistic pharmacological properties.

Specifically, bioconjugated steroidal quaternary nitrogen salts have mainly been obtained using nitrogen-containing aromatic systems (see Figure 1) such as pyridines (1–7), indoles, and indoxazoles as nitrogen sources, highlighting their potential anti-cancer and antimicrobial properties [11,12,13,14,15,16]. Some strategies for the introduction of quaternary nitrogen salts onto the steroidal framework involves the direct functionalization of the nitrogen-containing aromatic ring, generating an alkyl halide-bearing intermediate that subsequently undergoes N-alkylation with the steroidal substrate (6 and 7). The second and more widely applied strategy consists of introducing a halogen-bearing linker onto the steroidal scaffold (typically via esterification of a hydroxyl group with a haloacid or haloacyl halide) followed by intermolecular SN2 nucleophilic substitution (2 and 4) with a pyridine or pyridine derivative to yield the corresponding pyridinium salts; in steroids, this has been done in cholesterol (3 and 5) and with a hydrazone derived mainly from allopregnanolone (1) [17,18,19,20].

Specifically, the polarity of the 3-hydroxy group in the cholesterol structure constitutes an active site for hydrogen bond interactions or substituted derivative preparation with a myriad of biological molecules (e.g., phospholipids in membrane, etc.). Therefore, the biological activities of cholesterol bioconjugate derivatives have gained considerable importance in cholesterol 3-hydroxy modification structures [21,22,23,24,25,26]. In this regard, a pyridinium salt derived from cholesterol has been previously reported [27]; the synthesis of cholesterol-derived pyridinium salts by esterifying their 3-hydroxyl group with bromoacetyl bromide, forming an acetate linker, and subsequent N-alkylation with pyridine derivatives represents a remarkable approach to obtaining novel steroidal bioconjugates. Building on this precedent, this study outlines the synthesis and complete spectroscopic characterization of novel bioconjugate cholesterol-derived pyridinium salts.

2. Results and Discussion

The addition of the linker to cholesterol was carried out from 2-bromoacyl bromide to generate the esterification at the C-3 hydroxyl of cholesterol (Scheme 1), using K₂CO₃ as a base, according to what has been reported in the literature [18]. Then, condensation was performed in a biphasic system, since the steroid was dissolved in DCM and the base in water, giving the desired compound 9 in high chemical yield after its purification in a Combiflash apparatus (Teledyne ISCO, Lincoln, NE, USA).

With the linker properly added, the S_N2 reaction conditions were investigated to obtain the respective bioconjugate pyridinium salts from the selected substituted pyridines. This reaction proved to be sensitive to the substituted pyridine derivative, as when the optimized reaction conditions were employed [3,18,27], the desired salt was not obtained. Where the reported methodology for the case of the salt derived from pyridine and cholesterol was employed, the yield was low; the results of reproducing three modified methodologies according to literature reports [28,29,30,31] are shown in

Table 1. Yield of pyridinium salt of cholesterol (10a–d) formation for each methodology.

Pyridine	Methodology 1 w/o Solvent, rt, 5 min	Methodology 2 Et2O, rt, 30 min	Methodology 3 AcCN, Reflux, 60 min	Pyridinium Salt
Pyridine	92%	86%	89%	10a
2-methyl-Pyridine	52%	51%	87%	10b
3-methyl-Pyridine	80%	85%	83%	10c
4-methyl-Pyridine	77%	90%	82%	10d

.

¹H-NMR spectra confirmed the obtention of the desired bioconjugate pyridinium salt. Based on the previously reported and characterized cholesterol bromoacetate structure, new signals were assigned to the four obtained pyridinium compounds. The most notable shift, which confirmed the obtention of the pyridinium salt in the cholesterol bromoacetate, was the methylene protons attached to the carbon connected with the pyridine moiety, which resonated around 6.11 ppm and 6.40 ppm depending on the pyridinium salt derivative due to the positive charge on the nitrogen atom (Figure 2). The signals at positions 2 and 6 showed the same effect, shifting to values between 9.4 ppm and 9.7 ppm, due to the lack of protection generated around the positive charge of nitrogen. The shifts in the signals at positions 3, 4, and 5 on the pyridine ring correspond to free pyridine, assigned using the COSY experiment. For the methylpyridines, the salts exhibit a methyl signal between 2.5 ppm and 3.0 ppm, with those affected by resonance due to the positive charge being more pronounced. The carbon signals introduced by the new substituents were assigned by HSQC experiments (see Supplementary Materials), and the connectivity of adjacent positions was subsequently established through HMBC analysis.

The IR spectra show characteristic absorption bands; while the bands around 3600–3400 cm⁻¹ are characteristic of the steroid, the presence of bands around 1600 is diagnostic for the presence of C=N⁺, corroborating the structure. The HRMS data show, in the case of salt, only the presence of the protonated cation, indicative of the loss of the anion prior to detection by the equipment. The physical properties and the specific optical rotation do not change significantly since no new asymmetric centers are formed. At the melting point, the salts rise to 200 °C, consistent with the formation of an ionic compound.

3. Materials and Methods

3.1. Characterization Equipment, Solvents and Reagents

One-dimensional and two-dimensional NMR spectra were measured using a Bruker Ascend^TM 500 (¹H 500 MHz, ¹³C 125 MHz) spectrometer (Bruker, Billerica, MA, USA) at 298 K; chemical shifts are reported in ppm and the coupling constants in Hz with TMS (0.0 for ¹H) and residual solvent peak of CDCl₃ (δ = 7.26 ppm for ¹H, δ = 77.16 ppm for ¹³C) are used as references. Mass spectra (FABMS and EI) and high-resolution mass spectra (HRMS-FAB, HRMS-EI) were obtained on a JEOL mass spectrometer, the MStation JMS-700, at 70 eV (JEOL, Tokyo, Japan). Infrared spectra were obtained using a Bruker Tensor 27 spectrophotometer (Bruker Corporation, Billerica, MA, USA). Specific rotation was obtained using an Autopol III Polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA), with a 1 dm cell holding a total volume of 1.0 mL at 25 °C with CHCl₃ as the solvent. The melting point was obtained using a Guoming RY-2 Melting Point Tester (NANBEI, Zhengzhou, Henan, China) city, state abbrev. if USA, country) with the open capillary method. Chromatography column purification was performed on silica gel (60, 0.063-0.2 mm/70–230 mesh ASTM). Combiflash purification was performed on a Combiflash Rf (Teledyne ISCO, Lincoln, NE, USA) All reagents and solvents used in the reaction were Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) reagent-grade.

3.2. Cholesteryl 2′BrAcetate (9)

Cholesterol (1 mmol, 386 mg) was dissolved in 5.0 mL of DCM and 10.0 mL (2 eq) of a 1.0 N K₂CO₃ solution was added and placed under vigorous stirring. Subsequently, 160.0 µL of BrAcBr (1.8 eq) were added and stirred at room temperature for 10 min. Afterward, it was extracted with DCM (2 × 10.0 mL), and washes were performed with water (2 × 10.0 mL). The organic phases were combined and dried with anhydrous Na₂SO₄, reduced-pressure distillation was used to remove the solvent, and the mixture was subsequently purified by column chromatography, using silica as the stationary phase and hexane-ethyl acetate (10:0 to 8.5:1.5) as the mobile phase, to obtain 482 mg of a white solid (95%), m.p. 198–199 °C, [α_D] = −43.2° (c = 0.1 mg/mL). FTIR: 686, 802, 1000, 1172, 1285, 1403, 1746, 2935 ¹H NMR (500 MHz, CDCl₃): 5.39 (m, H-6, 1H), 4.67 (dddd, J_2β-3 = 12.2 Hz, J_4α−3 = 8.4 Hz, J_2α−3 = 8.3 Hz, J_4β−3 = 4.27 Hz, H-3, 1H), 3.80 (s, CH_2-Br, 2H), 2.36 (m, H-4, 2H), 2.04–1.93 (m, 2H), 1.92–1.75 (m, 2H), 1.70–1.04 (m, 16H), 1.02 (s, CH₃-19, 3H), 0.97 (m, 2H), 0.91 (d, J₂₁₋₂₀ = 6.5 Hz CH₃-21, 3H), 0.87 (d, J₂₆₋₂₅ = 2.4 Hz, CH₃-26, 3H), 0.85 (d, J = 2.2 Hz, CH₃-27, 3H), 0.68 (s, CH₃-18, 3H) ¹³C NMR: 166.82 (COO), 139.34 (C-5), 123.23 (C-6), 76.29 (C-3), 56.82 (C-14), 56.28 (C-17), 50.14 (C-9), 42.46 (C-13), 39.85, 39.66, 37.92 (C-4), 37.02, 36.71, 36.33, 35.93 (C-20), 32.04, 31.98, 28.37, 28.16, 27.65 26.53 (CH₂Br), 24.42, 23.97, 22.97 (C-26), 22.71 (C-27), 21.18, 19.44 (C-19), 18.86 (C-21), 12.00 (C-18); HRMS (EI+) m/z calcd for C₂₉H₄₇BrO₂⁺ [M⁺]^•+; 506.2759/508.2739, found 506.2761/508.2738.

3.3. Pyridinium Salt Procedures

3.3.1. Methodology 1

Cholesteryl 2′BrAcetate (0.1 mmol, 50.0 mg) was mixed with the corresponding pyridine (Py (6.2 mmol, 491 mg, 500 μL), 2-methyl-Py (5.07 mmol, 472 mg, 500 μL), 3-methylpyridine (5.14 mmol, 478 mg, 500 μL) and 4-methyl-Py (5.12 mmol, 477 mg, 500 μL)) and stirred for 5.0 min at room temperature; after this time, 5.0 mL of diethyl ether was added to precipitate the corresponding salt and dissolve the remaining unreacted starting material. The mixture was then filtered, and the solid obtained was washed with diethyl ether (5 × 5.0 mL) to obtain a white solid.

3.3.2. Methodology 2

Cholesteryl 2′BrAcetate (0.1 mmol, 50 mg) was dissolved in 1.0 mL of diethyl ether and R-pyridine was added (Py (1.241 mmol, 98.2 mg, 100 μL), 2-methyl-Py (1.014 mmol, 94.4 mg, 100 μL), 3-methyl-Py (1.028 mmol, 95.7 mg, 100 μL) and 4-methyl-Py (1.024 mmol, 95.4 mg, 100 μL)) and stirred at room temperature for 30.0 min. After this time, the formation of a white powder was observed, and solids were filtered and washed with diethyl ether (5 × 5.0 mL) to remove the remains of residual unreacted pyridine to obtain a white solid.

3.3.3. Methodology 3

Cholesteryl 2′BrAcetate (0.1 mmol, 50 mg) was dissolved in 5.0 mL of acetonitrile and R-pyridine was added (Py (1.241 mmol, 98.2 mg, 100 μL), 2-methyl-Py (1.014 mmol, 94.4 mg, 100 μL), 3-methyl-Py (1.028 mmol, 95.7 mg, 100 μL) and 4-methyl-Py (1.024 mmol, 95.4 mg, 100 μL)), and heated at reflux for 1.0 h. The solvent was removed under reduced pressure and resuspended in 15.0 mL of ethyl ether, and solids were filtered and washed with diethyl ether (5 × 5.0 mL) to remove the remains of residual unreacted pyridine to obtain a white solid.

3.4. 3-(Cholest-5-en-3β-yloxy)-2-oxoethyl-pyridin-1-ium bromide (10a)

The best yield was obtained by methodology 1, obtaining 54 mg of a white solid (92%), m.p. 215–217 °C, [α_D] = −129.6° (c = 0.1 mg/mL). FTIR: 716, 1068, 1237, 1375, 1492, 1624, 1746, 2936, 3094, 3562. ¹H NMR (500 MHz, CDCl₃): 9.42 (d, J_{Py-H2−Py-H3} = 5.9 Hz, Py-H2, Py-H-6, 2H), 8.55 (t, J_{Py-H4−Py-H3} = J_{Py-H4−Py-H5} = 7.8 Hz, Py-H4, 1H), 8.10 (d, J_{Py-H2−Py-H3} = 6.9 Hz, Py-H3, Py-H5, 2H), 6.21 (s, CH₂-N⁺, 2H), 5.33 (m, H-6, 1H), 4.67 (m, H-3, 1H), 2.35 (m, H₂-4, 2H), 2.01–0.98 (m, 27H), 0.97 (s, CH₃-19, 3H), 0.88 (d, J₂₁₋₂₀ = 6.4 Hz, CH₃-21, 3H), 0.83 (m, CH₃-26, CH₃-27, 6H), 0.64 (s, CH₃-18, 3H). ¹³C NMR: 165.32 (COO), 146.78 (2-Py, 6-Py), 146.25 (4-Py), 138.99 (C-5), 127.86 (3-Py, 5-Py), 123.46 (C-6), 77.84 (C-3), 61.35 (CH₂-N⁺), 56.75 (C-14), 56.25 (C-17), 50.04 (C-9), 42.38 (C-13), 39.78, 39.58, 37.99 (C-4), 36.93, 36.59, 36.26, 35.87 (C-20), 31.97, 31.86, 28.29, 28.07, 27.75, 24.34, 23.94, 22.89 (C-26), 22.63 (C-27), 21.11, 19.38 (C-19), 18.80 (C-21), 11.94 (C-18); HRMS (FAB) m/z calcd for C₃₄H₅₃NO₂ [M + H⁺] ⁺; 507.4065, found 507.4069.

3.5. 1-[3-(Cholest-5-en-3β-yloxy)-2-oxoethyl]-2-methylpyridin-1-ium bromide (10b)

The best yield was obtained by methodology 3, obtaining 45 mg of a white solid (87%), m.p. 214–215 °C, [α_D] = −86.4° (c = 0.1 mg/mL). FTIR: 571, 728, 996, 1222, 1375, 1466, 1633, 1745, 2413, 2934, 3416. ¹H NMR: 9.67 (m, Py-H6, 1H), 8.39 (dd, J_{Py-H3−Py-H4} = J_{Py-H3−Py-H2} = 7.84 Hz, 1H, 7.90 (m, Py-H5, Py-H3), 6.04 (m, CH₂-N⁺, 2H) 5.21 (m, H-6, 1H), 4.64 (m, H-3, 1H), 2.85 (s, CH₃-Py, 3H), 2.31 (m, H₂-4, 2H), 2.20–0.98 (m, 27H), 0.95 (s, CH₃-19, 3H), 0.84 (d, J = 6.5 Hz, CH₃-21, 3H), 0.79 (m, CH₃-26, CH₃-27, 6H), 0.61 (s, CH₃-18, 3H) ¹³C NMR: 165.00 (COO), 156.24 (2-Py), 148.05 (6-Py), 146.23 (4-Py), 138.89 (C-5), 129.93 (3-Py), 125.98 (5-Py), 123.52 (C-6), 77.76 (C-3), 59.48 (CH₂-N⁺), 56.74 (C-14), 56.19 (C-17), 50.05 (C-9), 42.38 (C-13), 39.76, 39.57, 37.94 (C-4), 36.88, 36.60, 36.25, 35.85 (C-20), 31.96, 31.86, 28.28, 28.07, 27.72, 24.34, 23.90, 22.89 (C-26), 22.63 (C-27), 21.27 (Me-Py), 21.09, 19.36 (C-19), 18.79 (C-21), 11.93 (C-18); HRMS (FAB) m/z calcd for C₃₅H₅₅NO₂ [M + H⁺] ⁺; 521.4222, found 521.4227.

3.6. 1-[3-(Cholest-5-en-3β-yloxy)-2-oxoethyl]-3-methylpyridin-1-ium bromide (10c)

The best yield was obtained by methodology 2, obtaining 44 mg of a white solid (85%), m.p. 204–206 °C, [α_D] = −87.1° (c = 0.1 mg/mL). FTIR: 500, 720, 995, 1216, 1341, 1465, 1640, 1745, 2934, 3388. ¹H NMR: 9.24 (m, 2H-Py, 1H), 9.20 (d, J = 6.2 Hz, 6H-Py, 1H), 8.24 (d, J = 8.1 Hz, 4H-Py, 1H), 7.93 (dd, J₁ = 8.0, J₂ = 6.0 Hz, 5H-Py, 1H), 6.11 (s, CH₂-N⁺, 2H), 5.30 (m, H-6, 1H), 4.62 (m, H-3, 1H), 2.55 (s, CH₃-Py, 3H), 2.05–0.95 (m, 27H), 0.94 (s, CH₃-18, 3H), 0.84 (d, J = 6.6 Hz, CH₃-21, 3H), 0.79 (m, CH₃-26, CH₃-27, 6H), 0.61 (s, CH₃-19, 3H) ¹³C NMR: 165.43 (COO), 146.56 (4-Py), 146.34 (2-Py), 143.97 (6-Py), 139.02 (3-Py), 138.95 (C-5), 127.16 (5-Py), 123.42 (C-6), 77.77 (C-3), 61.04 (CH₂-N⁺), 56.75 (C-14), 56.22 (C-17), 50.04 (C-9), 42.37 (C-13), 39.76, 39.57, 37.99 (C-4), 36.92, 36.59, 36.25, 35.85 (C-20), 31.96, 31.86, 28.28, 28.07, 27.74, 24.33, 23.91, 22.88 (C-26), 22.63 (C-27), 21.09, 19.37 (C-19), 18.78 (C-21, Me-Py), 11.93 (C-18); HRMS (FAB) m/z calcd for C₃₅H₅₅NO₂ [M + H⁺] ⁺; 521.4222, found 521.4216.

3.7. 1-[3-(Cholest-5-en-3β-yloxy)-2-oxoethyl]-4-methylpyridin-1-ium bromide (10d)

The best yield was obtained by methodology 2, obtaining 47 mg of a white solid (90%), m.p. 204–205 °C, [α_D] = −74.2° (c = 0.1 mg/mL). FTIR: 829, 995, 1199, 1375, 1468, 1643, 1742, 2076, 2935, 3388. ¹H NMR: 9.17 (d, J = 6.2 Hz, 6H-Py, 2H-Py), 7.83 (d, J = 6.2 Hz, 5H-Py, 3H-Py, 2H), 6.14 (J_AB = 13Hz, CH₂-N⁺, 2H), 5.39 ppm (m, H-6, 1H), 4.72 (m, H-3, 1H), 2.71 (s, CH₃-Py, 3H), 2.39 (m, H₂-4, 2H), 2.06–1.05 (m, 27H), 1.03 (s, CH₃-19, 3H), 0.93 (d, J = 6.5 Hz, CH₃-21, 3H), 0.88 (d, J₂₆₋₂₅ = 2.2 Hz, CH₃-26, 3H), 0.87 (d, J₂₇₋₂₅ = 2.2 Hz, CH₃-27, 3H), 0.69 (s, CH₃-18, 3H) ¹³C: 165.59 (COO), 160.17 (4-Py), 145.76 (2-Py, 6-Py), 139.04 (C-5), 128.22 (3-Py, 5-Py), 123.54 (C-6), 77.91 (C-3), 60.77 (CH₂-N⁺), 56.80 (C-14), 56.24 (C-17), 50.08 (C-9), 42.43 (C-13), 39.81, 39.63, 38.03 (C-4), 36.96, 36.65, 36.30, 35.91 (C-20), 28.35, 28.15, 27.78, 24.40, 23.95, 22.96 (C-26), 22.70 (C-27), 22.66 (Me-Py), 21.14, 19.42 (C-19), 18.84 (C-21), 11.99 (C-18); HRMS (FAB) m/z calcd for C₃₅H₅₅NO₂ [M + H⁺] ⁺; 521.4222, found 521.4217.

4. Conclusions

Four cholesterol-derived pyridinium salts were synthesized and characterized, demonstrating high-yield reactions depending on the position of the methyl group in the pyridine ring, highlighting NMR as the main method of characterization. In particular, for ¹H, the signals at positions 2 and 6 of the pyridine ring appear up to 9.5 ppm and the signal of the methylene hydrogens attached to the positive nitrogen up to 6.3 ppm as an AB signal.