Synthesis, antioxidant activity, and HPLC enantioseparation of aryloxyaminopropanols derived from naphthalen-2-ol

Synthesis of 2-[(naphthalen-2-yloxy)methyl]oxirane (Bruchatá et al., 2006)

Potassium hydroxide (0.169 mol) was added to a mixture of (±)-2-(chloromethyl)oxirane (0.153 mol) and 2-naphthol (0.148 mol) in 50 ml of water. The reaction proceeded for 48 h at room temperature and under constant stirring. Thereafter, the product of the reaction was extracted into ethyl acetate and properly washed with a 5% NaOH solution as well as with water. The solution of the resulting oxirane derivative was dried with magnesium sulfate. After evaporating of the solvent, the product was crystallized from hexane and used in the following synthetic step:

C₁₃H₁₂O₂, 200.23, yield 63%, 51 °C–53 °C (hexane), 55 °C (Srivastava et al., 2004) 50 °C–51 °C (Bruchatá et al., 2006) ¹H NMR (CD₃OD): δ 2.78–2.81 (m, 2H, CH(O)CH₂), 3.38–3.41 (m, 1H, CH), 3.94–4.00 (m, 2H, ArOCH₂), 7.13–7.18 (m, 2H, H^1,3naph), 7.24–7.33 (t, 1H, H⁷ naph), 7.34–7.42 (t, 1H, H⁶ naph), 7.73–7.77 (m, 3H, H^4,5,8 naph)

Synthesis of 2-naphthyloxyaminopropanols (Bruchatá et al., 2006)

Into a 250-ml round-bottom flask was subsequently added 0.08 mol oxirane derivative, 150 ml ethanol (EtOH), and 0.08 mol amine. The reaction mixture was kept at 30 °C for 4 h and then heated for 4 h under reflux. EtOH as well as the unreacted amine were removed under vacuum. The distillation residue was diluted with 50 ml of water and the base was extracted in an extraction funnel into diethyl ether. The extract was properly dried with K₂CO₃, and diethyl ether was removed in vacuum. The base can be crystallized from a suitable solvent.

The final products can be isolated as free bases or as salts with acids. The salts were prepared by adding an ether solution of the anhydrous acid (e.g., fumaric acid) to a dry ether solution of the base until complete precipitation. The salts were finally purified by crystallization from ethyl acetate. The free bases can be released from their salts by alkalizing their aqueous solutions with ammonia and subsequent extraction into diethyl ether.

Melting points of the products were determined using a Kofler block (HMK; Franz Küstner, Germany) and were uncorrected. The purity of the prepared substances was checked by thin-layer chromatography (TLC) using silica plates Silufol^® UV 254 (Merck) and ethyl acetate:diethylamine = 9:1 (v/v) as the mobile phase. Spectroline CM-10 (Sigma-Aldrich, St. Louis, MO, USA) was used for the detection under ultraviolet (UV)–visible (VIS) light.

Infrared spectra of the substances were recorded on Nicolet 6700 (Thermo Scientific, Waltham, MA, USA) spectrophotometer using an attenuated total reflexion (ATR) extension with ZnSe crystals. GENESYS 10S spectrophotometer was used for the measurement of UV–VIS spectra in the wavelength range 200–400 nm. Solutions of the prepared aryloxyamino-propanols in MeOH (as free bases or as salts) had the concentration of approximately 0.2 mol/m³.

¹H-NMR spectroscopy measurements were performed on a Varian Gemini 2000 spectrometer (Varian Inc., Palo Alto, CA, USA) with an operational frequency of 300 MHz for ¹H-NMR and 75 MHz for ¹³C-NMR. Tetramethylsilane was employed as the internal standard. Deuterated solvents (chloroform, MeOH, DMSO, and water) were used for the preparation of sample solutions. Chemical shifts were given in ppm (d). The multiplicity of the signals was denoted as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet.

The elemental analysis was performed on a FLASH 2000 Organic Elemental Analyzer (Thermo Scientific).

C₁₆H₁₃O₂N base yield 59%, R_f: 0.62, m.p. 135 °C–6 °C (cyclohexane); 132.8–135.7 (Fagerstroem et al., 2006), ¹H-NMR (CDCl₃): δ 1.27 (d, 6H, CH(CH₃)₂), 2.97 (s, 1H, CH(CH₃)₂), 4.04–4.16 (m, 2H, CH₂O), 4.19–4.42 (m, 4H, Ar-O-CH₂-CH(OH)), 7.11–7.15 (d, 1H, H³naph), 7.23 (s, 1H, H¹naph), 7.32 (t, 2H, H^6,7naph), 7.41–7.44 (m, 2H, H^4,8naph), 7.71–7.76 (m, 1H, H⁵naph)

¹³C-NMR (DMSO): δ 20.79 (CH(CH₃)₂), 48.93 (CH(CH₃)₂), 49.66 (CH₂NH), 66.43 (CHOH), 70.26 (OCH₂), 106.66 (C³naph), 118.70 (C¹naph), 123.63 (C⁶naph), 126.67 (C^7,8 naph), 127.47 (C⁵ naph), 128.83, 129.23 (C–C_cond naph), 134.30 (C⁴ naph), 156.39 (C² naph)

C₁₇H₂₃O₂N base yield 69%, R_f: 0.66, m.p. 112 °C–4 °C (cyclohexane); 113 °C–14 °C (Bruchatá et al., 2006) fumarate 213 °C–215 °C, 215 °C–216 °C (Bruchatá et al., 2006), ¹H-NMR (CDCl₃): δ 1.14 (s, 9H, C(CH₃)₃) 2.40 (dd, 1H, CH₂NH), 3.99–4.03 (m, 3H, OCH₂CH(OH)), 4.16 (m, 1H, OCH₂), 7.18 (d, 1H, H³ naph), 7.24 (s, 1H, H¹ naph), 7.33 (t, 1H, H⁶ naph), 7.43 (t, 1H, H⁷ naph), 7.69–7.77 (m, 3H, H^4,5,8 naph)

¹³C-NMR (DMSO): δ 29.07 (C(CH₃)₃), 44.67 (CH₂NH), 50.56 (C(CH₃)₃), 68.53 (CHOH), 70.49 (OCH₂), 106.73 (C³ naph), 118.79 (C¹ naph), 123.68 (C⁶ naph), 126.36, 126.36 (C^7,8 naph), 127.60 (C⁵ naph), 129.02, 129.37 (C–C_cond naph), 134.44 (C⁴ naph), 156.62 (C² naph)

C₁₅H₁₉O₂N base yield 62%, R_f: 0.67, m.p. 77 °C–79 °C (hexane); fumarate 139 °C–141 °C hydrochloride 170 °C–2 °C, IR (cm⁻¹): base 3350 (νOH), 1629, 1600 (νC=C), 1258 (νArOalk); UV fumarate (CH₃OH, ɛ in m²/mol): λ₁ 261 log ɛ₁ 3.99, λ₂ 271 log ɛ₂ 4.01, λ₃ 313 log ɛ₃ 3.45

¹H-NMR (CD₃OD): δ 2.85 (s, 6H, N(CH₃)₂), 3.24–3.35 (m, 2H, CHCH₂N), 4.05–4.17 (m, 2H, ArOCH₂), 4.35–4.40 (m, 1H, CH₂CHOH), 6.37 (s, 2H, CH fumar), 7.13 (d, 1H, H³ naph), 7.23 (s, 1H, H¹ naph), 7.32 (t, 2H, H^6,7 naph), 7.41–7.44 (m 2H, H^4,8 naph), 7.71–7.76 (m, 1H, H⁵ naph)

C₁₉H₂₅O₂N base yield 65%, R_f: 0.48, m.p. 62 °C–64 °C (cyclohexane), fumarate m.p. 161 °C–163 °C (ethyl acetate); IR (cm⁻¹): 3326 (νOH, νNH), 1629, 1600 (νC=C), 1216 (νArOalk); UV (CH₃OH, ɛ in m²/mol): λ₁ 229 log ɛ₁ 4,02, λ₂ 272 log ɛ₂ 2,85

¹H-NMR (CDCl₃): 1.16–1.25 (m, 6H, H^3,4,5 cyclohex), 1.60–1.75 (m, 4H, H^2,6 cyclohex), 2.40–2.46 (m, 1H, H¹cyclohex), 2.79–2.95 (m, 2H, CH₂N), 3.60–3.88 (m, 4H, (CH₂)₂ cyclohex), 4.09–4.42 (m, 4H, Ar-O-CH₂-CH(OH)), 7.14 (d, 1H, H³ naph), 7.25 (s, 1H, H¹ naph), 7.33 (t, 2H, H^6,7 naph), 7.42–7.45 (m, 2H, H^4,8 naph), 7.70–7.75 (m, 1H, H⁵ naph)

C₁₉H₁₉ NO₂ base yield 65%, R_f: 0.31, m.p. 89 °C–92°C (cyclohexane); IR (cm⁻¹): 3270 (νOH, NH), 3055 (νNH), 1628, 1600 (νC=C), 1219 (νArOalk); UV (CH₃OH, ɛ in m²/mol): λ₁ 225 log ɛ₁ 3.81, λ₂ 271 log ɛ₂ 3.31

¹H-NMR (CDCl₃): δ 3.58–3.71 (m, 2H, CH₂N), 4.15–4.18 (m, 3H, 4H, Ar-O-CH₂-CH(OH)), 7.30–7.35 (m, 1H, H⁴ anil), 7.39–7.45 (m, 1H, H³ naph), 7.51–7.63 (m, 5H, H^1,6,7 naph, H^3,5 anil), 7.73–7.77 (m, 3H, H^4,5,7 naph)

MW 293.36, calc. %C 77.79 %H 6.53 %N 4.77, found %C 77.58 %H 6.32 %N 4.49

C₂₃H₂₇O₄N base yield 75%, R_f: 0.57, m.p. 93.5 °C–95 °C (cyclohexane); 138 °C–142 °C (ethyl acetate), IR (cm⁻¹) (fumarate): 2760, 3155 (νOH, NH), 1625, 1597 (νC=C), 1256 (νArOalk); UV (CH₃OH, ɛ in m²/mol): λ₁ 203 log ɛ₁ 3.52, λ₂ 226 log ɛ₂ 3.75, λ₃ 272 log ɛ₂ 3.67

¹H-NMR (CDCl₃): δ 2.62–2.68 (m, 4H, CH₂CH₂), 2.72–2.76 (m, 2H, CHCH₂N), 3.68 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.96–4.06 (m, 3H, ArOCH₂CH), 6.69–6.70 (m, 3H, H¹ naph, H^2′,6′arom), 6.79–6.82 (m, 1H, H³ naph), 7.13–7.17 (m, 1H, H⁵ benz), 7.29–7.36 (t, 2H, H^6,7 naph), 7.45–7.46 (m, 2H, H^4,8 naph), 7.78–7.82 (m, 1H, H⁵ naph)

MW 349.48, calc. %C 79.05 %H 7.79 %N 4.01, found %C 79.24 %H 7.53 %N 4.22

C₁₇H₂₁O₂N base yield 73%, R_f: 0.67, m.p. 64 °C–65 °C (heptane); 65 °C–66 °C (Bruchatá et al., 2006), fumarate m.p. 98 °C–99 °C (cyclohexane); 93 °C–97 °C (Bruchatá et al., 2006)

¹H-NMR (CDCl₃): δ 1.79–1.83 (m, 4H, 2H^3,4 pyrrol, 2.54–2.90 (m, 6H, CH₂N, 2H^2,5 pyr) 4.09–4.15 (m, 4H, Ar-O-CH₂-CH(OH)), 7.15–7.77 (m, 7H, CH naph)

¹³C-NMR (DMSO): δ 26.84 (C^2,5 pyr), 56.08 (C^3,4 pyr), 61.53 (CH₂N pyr), 65.22 (CHOH), 66.95 (C^3,5 morph), 73.87 (OCH₂), 106.83 (C³ naph), 118.86 (C¹ naph), 123.68 (C⁶ naph), 126.46, 126.32 (C^7,8 naph), 127.61 (C⁵ naph), 129.05, 129.42 (C–C_cond naph), 134.30 (C⁴ naph), 156.24 (C² naph)

C₁₆H₁₆O₂N₂ base yield 62%, m.p. 122 °C–125 °C (cyclohexane), R_f: 0,31, IR (cm⁻¹) (fumarate): 3113 (νOH), 1628, 1601 (νC=C), 1217 (νArOalk); UV (CH₃OH, ɛ in m²/mol): λ₁ 226 log ɛ₁ 4.02, λ₂ 272 log ɛ₂ 2.85, ¹H-NMR (CDCl₃): 3.94–3.97 (m, 2H, CH₂ CH₂CH), 4.09–4.00 (m, 3H, ArCH₂CH), 4.25–4.12 (m, 1H, CH₂CHOH), 6.97 (d, 2H, H⁴, H⁵ imi), 7.61 (s, 1H, H² imi), 7.85–7.17 (m, 7H, CH naph)

MW 268.32, calc. %C 71.62 %H 6.01 %N 10.44, found %C 71.45 %H 6.22 %N 10.23

C₁₇H₁₈O₂N₂ base yield 64%, R_f: 0.49, m.p. 127 °C–130 °C (cyclohexane); fumarate m.p. 136 °C–138 °C, IR (cm⁻¹) (base): 3057 (νOH), 1629, 1600 (νC=C), 1258 (νArOalk); UV fumarate (CH₃OH, ɛ in m²/mol): λ₁ 261 log ɛ₁ 3.96, λ₂ 271 log ɛ₂ 3.96, λ₃ 321 log ɛ₃ 3.40, ¹H-NMR (CDCl₃): δ 2.39 (s, 3H, CH₃), 4.29–4.39 (m, 2H, CH₂N), 4.42–4.85 (m, 3H, CH₂CHOH), 7.21 (d, 1H, H⁴ imi), 7.22 (d, 1H, H¹ naph), 7.29–7.31 (m, 2H, H^7,8 naph), 7.33–7.34 (m, 1H, H⁵ imi), 7.38 (d, 1H, H³ naph), 7.73–7.74 (m, 2H, H^4,6 naph), 7.76 (d, 1H, H⁵ naph)

MW 282.35, calc. %C 72.32 %H 6.45 %N 9.92, found %C 72.41 %H 6.65 %N 9.73

C₁₈H₂₃O₂N base yield 61%, R_f: 0.69, m.p. 82 °C–84 °C (hexane), 83 °C–84 °C (Bruchatá et al., 2006) fumarate 156 °C–159 °C (ethyl acetate), 157 °C–158 °C (Bruchatá et al., 2006)

¹H-NMR (CDCl₃): δ 1.48–1.59 (m, 6H, 2H^3,4,5 piper), 2.41 (d, 2H, CH₂N), 2.53 (t, 4H, 2H^2,6 piper), 4.09–4.15 (m, 5H, Ar-O-CH₂-CH(OH)), 7.25 (s, 1H, H¹ naph), 7.29 (d, 1H, H³ naph), 7.34 (t, 1H, H⁶ naph), 7.42 (t, 1H, H⁷naph), 7.70–7.76 (m, 3H, H^4,5,8 naph)

¹³C-NMR (DMSO) δ 24.05 (C⁴ piper), 25.85 (C^3,5 piper), 54.75 (C^2,6 piper), 61.26 (CH₂N piper), 65.17 (CHOH), 66.95 (C^3,5 morph), 70.33 (OCH₂), 106.69 (C³ naph), 118.84 (C¹ naph), 123.66 (C⁶ naph), 126.74, 126.33 (C^7,8 naph), 127.59 (C⁵ naph), 129.01 (C⁴ naph), 129.35 (C–C_cond naph), 134.30 (C⁴ naph), 156.65 (C² naph)

C₁₇H₂₁O₃N base yield 58%, R_f: 0.40, m.p. 71 °C–73 °C (hexane); 70 °C–72 °C (Bruchatá et al., 2006), fumarate 132 °C–134 °C (ethyl acetate), 134 °C–136 °C (Bruchatá et al., 2006)

¹H-NMR (CDCl₃): δ 2.49–2.72 (m, 6H, H^2,6 morph, CH₂-N), 3.73–3.76 (m, 6H, H^3,5 morph, OCH(OH)), 4.10 (m, 2H, Ar-O-CH₂), 3.38 (m, 1H, CH-OH), 2.68 (m, 2H, CH₂-N morph), 7.16–7.21 (m, 2H, H^1,3 naph), 7.33–7.36 (t, 2H, H^6,7naph), 7.43–7.45 (m, 2H, H^4,8 naph), 7.71–7.77 (m, 1H, H⁵ naph)

¹³C-NMR (DMSO): δ 53.75 (C^2,6 morph), 61.08 (CH₂N morph), 65.33 (CHOH), 66.95 (C^3,5 morph), 70.11 (OCH₂), 106.71 (C³ naph), 118.75 (C¹ naph), 123.73 (C⁶ naph), 126.38, 126.75 (C^7,8 naph), 127.61 (C⁵ naph), 129.05, 129.42 (C–C_cond naph), 134.30 (C⁴ naph), 156.56 (C² naph)

C₁₈H₂₄O₂N₂ base yield 62 %, R_f: 0.37, m.p. 131 °C–133 °C (cyclohexane); 132 °C–134 °C (Bruchatá et al., 2006), fumarate 215 °C–217 °C (ethyl acetate), 215 °C–217 °C (Bruchatá et al., 2006)

¹H-NMR (CDCl₃): δ 2.30 (s, 3H, N-CH₃), 2.49–2.73 (m, 10H, CH₂ piper, CH₂N), 4.09–4.17 (m, 4H, Ar-O-CH₂-CH(OH)), 7.14–7.77 (m, 7H, CH naph)

¹³C-NMR (DMSO): δ 45.96 (N-CH₃), 53.14 (C^3,5 N-methylpiper), 55.10 (C^2,6 N-methylpiper), 60.39 (CH₂N-methylpiper), 65.07 (CHOH), 71.34 (OCH₂), 106.68 (C³ naph), 118.70 (C¹ naph), 123.67 (C⁶ naph), 126.52, 126.33 (C^7,8 naph), 127.59 (C⁵ naph), 129.01 (C⁴ naph), 129.35 (C–C_cond naph), 134.30 (C⁴ naph), 156.65 (C² naph)

C₂₄H₂₈ O₂N₂ base yield 64%, R_f: 0.84, m.p. 108 °C–109 °C (cyclohexane), fumarate 179 °C–180 °C (ethyl acetate), IR (cm⁻¹) fumarate 2915–3407 (νOH), 1629, 1600 (νC=C), 1261 (νArOalk); UV base (CH₃OH, ɛ in m²/mol): λ₁ 226 log ɛ₁ 4.59, λ₂ 272 log ɛ₂ 4.40, λ₃ 327 log ɛ₃ 2.82

¹H-NMR (CDCl₃): base δ 2.65–2.69 (m, 4H, H^2,6 piper), 2.89–2.91 (m, 2H, CHCH₂N), 3.10–3.14 (m, 4H, H^3,5 piper), 3.87 (s, 3H, OCH₃), 4.13–4.18 (m, CH₂CHOH), 6.85–6.98 (m, 4H, H^{3′,4′,5′,6′} arom), 7.16–7.21 (m, 2H, H^1,3 naph), 7.33–7.36 (t, 2H, H^6,7 naph), 7.43–7.45 (m, 2H, H^4,8 naph), 7.71–7.77 (m, 1H, H⁵ naph)

MW 376.50, calc. %C 76.56 %H 7.50 %N 7.44, found %C 76.35 %H 7.28 %N 7.23

Aqueous solutions of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) with a concentration of 7.7 μg/ml (14 mM) and K₂S₂O₈ with a concentration of 1.32 mg/ml (4.9 mM) were prepared. These solutions were combined in 1:1 ratio and left for 24 h in a refrigerator in the dark. The wells were filled with 60 μl of sample solution at a concentration of 10⁻² or 10⁻³ mol/dm³, respectively, and then 240 μl of ABTS solution was added. Thereafter, the absorbance was determined spectrophotometrically at a wavelength of 734 nm for 30 min at 1-min interval. The measurement was carried out on a 96-well plate. Three parallel measurements were performed for each sample. During measurement, the colorless ABTS undergoes oxidation by potassium peroxodisulfate, yielding the stable blue-green ABTS^•+ radical. Addition of antioxidants causes reduction of the ABTS^•+ radical and discoloration of the solution. The antioxidant activity relates to the activity of the standard substance trolox, whose activity was determined simultaneously with the samples measured. The method is dependent on pH, hence buffers containing disodium hydrogen phosphate and potassium dihydrogen phosphate were used for the measurement (Re et al., 1999; Malík et al., 2017).

I. The compounds were separated on a column with a polysaccharide chiral stationary phase. HPLC instrument AGILENT 1200 (Agilent Technologies, Santa Clara, CA, USA) was used, which comprised an automatic dosing system, quaternary high-pressure pump, degasser of the mobile phase, and a diode array detector. The collection and processing of chromatographic data was carried out by the software program Agilent ChemStation for LC system (Agilent Technologies). The separation of enantiomers was achieved on a chiral stationary phase Chiralpak AD (0.46 × 25cm; 5 μm ID) with tris(3,5-dimethylphenylcarbamate)amylose as the chiral selector. The mobile phase was an 80:10:10:0.1 (v/v/v/v) mixture of hexane, EtOH, MeOH, and N-ethylenamine, respectively. HPLC solvents were acquired from Merck (Darmstadt, Germany). The mobile phase flow rate was 0.8 ml/min, injection volume was 20 μl, and the column temperature was set to 25 °C. Chromatograms were recorded at a wavelength of 265 ± 8 nm.

II. The compounds were separated on a column with a macrocyclic chiral stationary phase using an HPLC instrument (Series 1100), containing a binary high-pressure pump, automatic dosing system, column thermostat, and diode array detector. Separation of enantiomers was carried out on a chiral stationary phase with the chiral selector teicoplanin (Chirobiotic T [0.45 × 25 cm, 5 μm ID]). The mobile phase consisted of a mixture of MeOH, acetonitrile, acetic acid, and triethylamine in a 45:55:0.3:0.2 (v/v/v/v) ratio. HPLC-quality solvents were purchased from Merck. Dead time was estimated as the elution time of MeOH. Sample solutions with 0.1 mg/ml concentration were prepared by dissolution of an exact amount of substance in MeOH.

The enantioseparation was evaluated setting the following chromatographic criteria: retention factork:k1=t1−t0/t0,k2=t2−t0/t0selectivity factorα:α=k2/k1resolution factorRs:Rs=2t2−t1/w1+w2 \matrix{ {{\rm{retention\, factor }}\left( {\rm{k}} \right):{{\rm{k}}_1} = \left( {{{\rm{t}}_1} - {{\rm{t}}_0}} \right)/{{\rm{t}}_0},{{\rm{k}}_2} = \left( {{{\rm{t}}_2} - {{\rm{t}}_0}} \right)/{{\rm{t}}_0}} \cr {{\rm{selectivity\, factor }}\left( \alpha \right):\alpha = {{\rm{k}}_2}/{{\rm{k}}_1}} \cr {{\rm{resolution\, factor }}\left( {{{\rm{R}}_{\rm{s}}}} \right):{{\rm{R}}_{\rm{s}}} = 2\left( {{{\rm{t}}_2} - {{\rm{t}}_1}} \right)/\left( {{{\rm{w}}_1} + {{\rm{w}}_2}} \right)} \cr } where t₁ and t₂ are the retention times (min) and w₁, w₂ are the peak widths at the bases of the peaks (min) for the respective enantiomers.

Mobile phase: hexane/EtOH/MeOH/ethylethanamine with the following composition: A 85:7.5:7.5:0.1v/v/v/v,B 80:10:10:0.1v/v/v/v,C 75:12.5:12.5:0.1v/v/v/v,D 70:15:15:0.1v/v/v/v \matrix{ {{\rm{A }}\,85:7.5:7.5:0.1\left( {{\rm{v}}/{\rm{v}}/{\rm{v}}/{\rm{v}}} \right),} \cr {{\rm{B }}\,80:10:10:0.1\left( {{\rm{v}}/{\rm{v}}/{\rm{v}}/{\rm{v}}} \right),} \cr {{\rm{C }}\,75:12.5:12.5:0.1\left( {{\rm{v}}/{\rm{v}}/{\rm{v}}/{\rm{v}}} \right),} \cr {{\rm{D }}\,70:15:15:0.1\left( {{\rm{v}}/{\rm{v}}/{\rm{v}}/{\rm{v}}} \right)} \cr }