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Synthesis of new compounds of the aryloxyaminopropanol type and their HPLC enantioseparation

Synthesis of new compounds of the aryloxyaminopropanol type and their HPLC enantioseparation Keywords Kúcové slová: INTRODUCTION In previous works, some new compounds of the aryloxyaminopropanol type derived from 4 hydroxypropiophenones with isopropyl and tert-butyl group in the hydrophilic part of the molecule were prepared (Cizmáriková et al., 1990a, 2003). Their anti-izoprenaline activity and anti-arrhythmic activity (Cizmáriková & Kozlovský, 1994a,b) depend on the substitution of the hydrophilic and lipophilic parts of the molecule. In addition, the prepared compounds with higher alkoxymethyl (octyloxymethyl and nonyloxymethyl) group in position 3 of the aromatic ring have shown antimicrobial and local anaesthetic activities (Cizmáriková et al., 1990b). In many papers, compounds with isopropyl and tert-butyl group in * cizmarikova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae the hydrophilic part of molecule are active beta-adrenolytics (Cizmáriková & Racanská, 1998; Bruchatá & Cizmáriková, 2010; Keckésová & Sedlárová, 2010). Lower beta-adrenolytic activity was observed in the compounds with other alkyl groups such as isobutyl and diethyl groups (Griffith, 2003). The large variety of different aromatic rings and substituents on nitrogen atom leads to compounds with combined pharmacological properties with affinity to both types of adrenergic receptor, and (Bruchatá & Cizmáriková, 2010). In the work (Mosti et al., 2000), compound with cyclohexylamino moiety gives antiarrhythmic, local anaesthetic and analgesic activity. Compounds of aryloxyaminopropanol type possess in their structure a single stereogenic centre and exist as stereoisomers. The most widely used technique for the separation of their enantiomers was high-performance liquid chromatography (HPLC) (Wang et al., 2008) on different chiral stationary phases such as cyclodextrin (Wang & Ching, 2002; Zhang et al., 2008; Hroboová et al., 2004), immobilised proteins (Fulde & Frahm, 1999; Fornstedt et al., 1999), Amberlite XAD-4 (Agrawal & Patel, 2005) and cellulose and amylase-based phases (AboulEnein & Ali, 2001; Valentova et al., 2003). Indirect enantioseparation using derivatisation agents is less suitable at these compounds (Bojarsky, 2002, Bojarsky et al., 2005). This paper was oriented on a preparation, characterisation of the new prepared derivatives of aryloxyaminopropanol type and its enantioseparation at the Chiralpak AD and Chirobiotic T columns. Chromatographic parameters such as separation, retention and resolution factors were calculated. HPLC studies were performed using a Hewlett-Packard (series 1 100) HPLC system consisting of a quaternary pump equipped with an injection valve (Rheodyne) and a diode array detector. The macrocyclic chiral stationary phase was Chirobiotic T (250 mm × 4 mm LD particle size 5-m Advanced Separation technologies, Inc., USA). The mobile phase was a mixture of methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v). The separation was carried out at a flow rate of 1 ml min­1 and column temperature was 23°C. The chromatograms were scanned using Hewlett Packard (series 1 100) at 270 nm. The injection volume was 20 l. The analyte was dissolved in methanol (concentration 1 mg ml­1). Secondary studies were carried out using HPLC system AGILENT 1200, consisting of a quaternary pump and a diode detector. Chromatographic characteristics The separation factor was expressed as = k1/k2, where k1, k2 are retention factors for the first and second eluting enantiomers. The retention factors k' were calculated as follows: k1 = (t1 ­ t0)/t0 and k2 = (t2 ­ t0)/t0, where t0, t1 and t2 are the dead elution time and elution times of enantiomers 1 and 2. The stereochemical resolution factor (Rs) of the first and second eluting enantiomers was calculated as the ratio of the difference between the retention times t1 and t2 to the arithmetic sum of the two peaks' widths w1 and w2: Rs = 2(t2 ­ t1)/(w1 + w2). EXPERIMENTAL Chemicals All HPLC grade solvents were obtained from Merck (Germany). Synthesis (3-Chloromethyl-4-hydroxyphenyl)propan-1-one and (3-alkoxymethylphenyl-4-hydroxyphenyl)propan-1-one were prepared according to Cizmáriková et al. (2002). [4-(3-Alkylamino-2-hydroxypropoxy)phenyl]propan-1-one and [4-(3-cycloalkylamino-2-hydroxypropoxy)phenyl] propan1-one were prepared according to Cizmáriková et al. (2012). Instruments The melting points were determined using a Kofler Micro Hot Stage and were quoted uncorrected. The purity of the prepared compounds was assessed using Silufol® UV 254 (Merck) sheets in the solvent system ethyl acetate/diethylamine (9.5:0.5, v/v). UV spectra were run on spectrophotometer GENESYS 10s UV­Vis in methanol. Concentration of compounds was about 10-1 mol m-3. IR spectra were recorded using Nicolet 6700 (Thermo Scientific). 1H NMR and 13C NMR were recorded on the Varian Gemini 2000 Spectrometer operating at 300 MHz for protons and 75 MHz for carbons. Elementary analysis was carried out on FLESCH 2000 (Thermo Scientific) within 0.3 % of the theoretical values. HPLC analysis HPLC was carried out using the chiral stationary phases (Chiralpak AD) based on the amylase tris(3,5-dimethylphenylcarbamate) (0.46 × 25). The mobile phases consisted of hexane/ ethanol/methanol/diethylamine 85:7.5:11.25:0.1, v/v/v/v (A) and hexane/ethanol/methanol/diethylamine 85:7.5:7.5:0.1, v/v/v/v (B). Samples for analysis were prepared as approximately 1 mg ml­1 solution in methanol. Separations were carried out at a flow rate of 0.8 ml min­1 and the column temperature was maintained at 25°C. Chromatograms were scanned at a wavelength of 267 ± 8 nm. RESULTS AND DISCUSSION The aim of this study was to prepare four derivatives of aryloxyaminopropanol type with different groups in hydrophilic and lipophilic parts of a molecule. The compound without side alkoxymethyl chain was prepared by a two-step synthesis and those with alkoxymethyl group by a four-step synthesis from 4-hydroxyphenylpropan-1-one. Oxirane intermediates prepared by the reaction of 4-hydroxyphenylpropan-1-one with epichlorohydrin gave final products by the reaction with appropriate amine (phenylamine, cyclohexylamine and isobutylamine). These were isolated in the form of free bases or salts with fumaric acid and with oxalic acid (Table 1). Compound I was isolated only in the form of free base; salts with fumaric and oxalic acid could not be prepared due to increased basicity of its nitrogen atom. The purity of the final products was checked by thin-layer chromatography using ethyl acetate/diethylamine as the mobile phase (Table 1). The structures of prepared compounds were confirmed by IR, UV and 1H NMR, 13C NMR spectra (Tables 2­4). The stretching vibration of the characteristic group in the IR spectra were (OH) 3166­3400 cm­1, (NH) (base) 3073­3274 cm­1, (C=C) 1578­1604 cm­1, (C=O) 1668­1682 cm­1, (CAl OCAr) 1251­ 1276 cm­1 (Table 2). The UV spectra of bases display two bands corresponding to * transition at max = 202­270 nm, log = 3.86­4.80 (Table 3). Compound I with two aromatic rings gives four bands corresponding to * transition (Table 3). Cizmáriková, R. et al. Table 1. Physico-chemical parameters of prepared compounds. R2 O OH R1 H3C O Compound Form of compound I Base R1 R2 NH N H Empirical formula Mr C20H25O4N 251.33 M.p. (°C) Solvent 67­69 Hexane Yield (%) RF 62 0.73 CH2OCH3 II Base CH2OCH3 IIa Fumarate III Base IIIa Fumarate IIIb Oxalate IV Base IVa Fumarate M.p. melting point, RF retardation factor N H NH C20H31O4N 349.47 82­87 Hexane C40H62O8N2 · C4H4O4 646,79 (CH3)2CHCH2NH H C16H23O3N 279.02 C16H23O3N · C4H4O4 331.46 C16H23O3N · C2H2O2 (CH3)2CHCH2NH CH3CH2OCH2 C38H66O8N2 · C4H4O4 646.79 C19H31O4N 337.44 117­120 Ethylacetate 57­59 Hexane 164­167 Ethylacetate 159­160 Ethanol Viscous oil 154-156 propan-2-ol H NMR and 13C NMR spectra of free bases showed the proof of the final structure proton and carbon signals of the aminopropanol chain (Tables 4 and 5). In this work, a direct HPLC method was used for enantioseparation of prepared racemic compounds using chiral stationary phases based on derivatised amylose (Chiralpak AD) and native teicoplanin (Chirobiotic T). Mobile phases A hexane/ethanol/methanol/diethylamine (85:3.75:11.25:0.1, v/v/v/v) and B hexane/ethanol/methanol/diethylamine (85:7.75:7.75:0.1, v/v/v/v) were used for the separations on the Chiralpak AD column. From Table 6 it is evident that good enantioseparations were achieved for all prepared compounds. The obtained separation factors were in the interval from 1.16 to 1.41 and resolution factors in the interval 2.36­6.35. In the case of compounds III, IIIa and IIIb in the B mobile phase, all forms of compound have similar values of selectivity factor and the values of resolution factors were in order oxalate>base>fumarate. Comparing the type of the mobile phase, greater value of Rs was obtained for the compound IIa in the form of base at the B mobile phase (Table 6). The results of the enantioresolution on the Chirobiotic T in polar organic separation mode (mobile phase methanol/acetonitrile/acetic acid/triethylamine 45:55:0.3:0.2, v/v/v/v) has shown that compounds with phenylamino and cyclohexylamino (I and II) are very well separated with resolution factor values in the range 1.93­2.36 and selectivity factors in the range 1.13­1.15. Comparing the enantioseparations of racemic compound with isobutyl substituent (compound III) on Chiralpak AD and on Chirobiotic T chiral stationary phases, lower enantioresolution was obtained ( = 1.07 and Rs = 0.92) on teicoplanin stationary phase (Table 7 and Figs 1 and 2). The mechanism of the separation on the both chiral phases is based on the interaction between chiral selector (derivatised amylase or native teicoplanin) and analyte by forming complexes between the enantiomeric analytes and chiral cavities. Detailed mechanism of enantioseparation was discussed in the papers Cizmáriková et al. (2012), Valentova et al. (2003) and Hroboová et al. (2001). Our study of pharmacological activities of the prepared compounds continues, and the results will be published in the next paper. CONCLUSION In this paper, four new compounds of the aryloxyaminopropanol type were prepared from 4-hydroxyphenylpropan1-one by two- and four-step syntheses. Chemical names of these compounds are: 1-[4-(2-hydroxy-3-phenylaminopropoxy)phenyl]propan-1-one, 1-[3-(methoxymethyl)-4-(2hydroxy-3-cyclohexylaminopropoxy)phenyl]propan-1-one, 1-[4-(2-hydroxy-3-isobutylaminopropoxy)phenyl]propan1-one and 1-[3-(ethoxymethyl)-4-(2-hydroxy-3-isobutylaminopropoxy)phenyl]propan-1-one. An enantioseparation of the prepared compounds was performed by using HPLC on an amylase tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and native teicoplanin (Chirobiotic T). The chromato- graphic results such as retention factor, separation factor = 1.16­1.41) and resolution factor (Rs = 2.36­6.35) have shown that Chiralpak AD is more suitable for enantioseparation of some of the prepared compounds than Chirobiotic T. ACKNOWLEDgEMENT This work was supported by the Slovak Research and Development Agency under the contract no. APVV-0516-12, grant UK/190/2013 and VEgA grant 1/0164/11. The authors thank Dr. Karlovska for recording 1H NMR and 13C NMR and Ms. Nedorostova for IR and UV spectra and Mr. Lehotsky for recording HPLC. Table 2. Values of stretching vibrations in IR spectra of prepared bases. Compounds I II III IIIa IIIb Iva , absorbtion maximum. * (OH, NH). (OH) (cm1) 3166 3283 3298* 3298 3400 3270 (NH) (cm1) 3274 3073 (C=C) (cm1) 1602 1600 1578 1604 1600 1600 1600 (C=O) (cm1) 1675 1670 1679 1679 1668 1682 (ArOalk) (cm1) 1265 1276 1257 1251 1254 1255 Table 3. Values of max and log in UV spectra, = m2 ·mol-1. Compounds I 205 203 202 203 203 max 1 (nm) 4.47 4.00 4.58 4.40 4.80 Log 1 220 216 II III IIIa IVa max 2 (nm) Log 2 max 3 (nm) Log 3 max 4 (nm) Log 4 max, wave length; , molar extinction coefficient. Compounds Solvent I CDCl3 Table 4. 1H NMR spectral data of compounds (ppm) (TMS). 1.19­1.24 (t, 3H, CH3CH2), 2.94­3.00 (q, 2H, CH3CH2), 3.26­3.32 (m, 2H, CH2NH), 3.40 (s, 3H, OCH3), 4.11­4.13 (m, 2H, CH2CH), 4.24­4.28 (m, 1H, CH2CH), 4.48­4.59 (m, 2H, CH2OCH3), 6.66­6.69 (d, 2H, C2ANLH, C6ANLH), 6.71­6.77 (t, 1H, C4ANLH), 6.91­6.94 (d, 1H, C5ARH), 7.17­7.22 (t, 2H, C3ANLH, C5ANLH), 7.93­7.96 (m, 2H, C2ARH, C6ARH) 1.04­1.13 (t, 3H, C=OCH2CH3), 1.16­1.25 (m, 6H, C3,4,5-CHexH2), 1.60­1.75 (m, 4H, C2,6-CHexH2), 2.40­2.46 (m, 1H, C1-CHexH), 2.70­2.77 (q, 2H, C=OCH2), 2.91­2.96 (m, 2H, CH2NH), 2.99 (s, 3H, OCH3), 3.55­3.62 (m, 1H, CHOH), 3.97­4.02 (m, 2H, OCH2CH), 4.07­4.10 (m, 2H, CH2OCH3), 6.93­6.96 (d, 1H, C5-ARH), 7.92­7.95 (d, 2H, C2,6-ARH) 0.91 (d, 6H, CH-(CH3)2), 1.75 (m, 1H, CH-(CH3)2), 2.45 (m, 2H, NHCH2CH), 2.76 (m, 2H, CHOH-CH2), 2.85 (m, 2H, CO-CH2), 4.05 (m, 3H, O-CH2CH). 1.02­1.05 (d, 6H, CH(CH3)2), 1.13­1.18 (t, 3H, C=OCH2CH3), 1.22­1.26 (t, 3H, OCH2CH3), 2.05­2.14 (m, 1H, CH(CH3)2), 3.00­3.02 (m, 2H, CH2CH(CH3)2), 3.05­3.09 (m, 2H, CHOHCH2NH), 3.27­3.34 (m, 2H, C=OCH2), 3.64­3.71 (q, 2H, OCH2CH3), 4.16­4.27 (m, 2H, OCH2CH), 4.38­4.45 (m, 1H, CHOH), 4.83 (s, 2H, CARCH2), 6.08 (s, 1H, CfumHCOO), 6.52 (s, 1H, CfumHCOOH), 7.07­7.10 (d, 1H, C3ARH), 7.98­8.01 (d, H, C2ARH, C6ARH) (ppm) (multiplicity and number of protons) II CDCl3 III CDCl3 IVa D2O , chemical shift. Cizmáriková, R. et al. Compounds I CDCl3 II CDCl3 III CDCl3 Table 5. 13C NMR spectral data of compounds (ppm) (TMS). (ppm) 8.39 (CH3CH2), 31.52 (CH3CH2), 46.30 (CH2CH), 58.23 (OCH3), 68.62 (CH2CH), 70.55 (OCH2CH), 71.74 (CH2OCH3), 112.22 (C5AR), 113.21 (C2,6ANL), 114.19 (C3AR), 118.02 (C4ANL), 126.68 (C1AR), 129.34 (C3,5ANL), 130.28 (C2,6AR), 148.07 (C1ANL), 160.80 (C4AR), 199.49 (C=O) 8.42 (C=OCH2CH3), 25.00 (C3,5-CHex), 26.05 (C4-CHex), 31.43 (C=OCH2CH3), 33.72 (C6-CHex), 34.02 (C2-CHex), 48.55 (CH2NH), 56.72 (C1-CHex), 68.21 (OCH3), 68.94 (CHOH), 70.62 (OCH2CH), 76.59 (CH2OCH3), 114.21 (C3,5-AR), 130.21 (C2,6-AR), 162.43 (C1-AR), 183.18 (C4-AR), 199.49 (C=O) 20.54 (CH(CH3)2), 26.38 (CH(CH3)2), 28.45 (COCH2), 51.53 (CHOH-CH2), 57.75 (CH2CH(CH3)2), 67.78 (CHOH), 70.63 (O-CH2), 114.22 (CAr3,5), 130.60 (CAr2,6), 162.62 (CAr4), 196.81 (CO) 10.93 (C=OCH2CH3), 21.86, 21.98 (CH(CH3)2), 28.15 (CH(CH3)2), 34.45 (C=OCH2CH3), 52.59 (CHOHCH2NH), 57.73 (CH2CH(CH3)2), 67.96 (OCH2CH), 72.37 (CHOH), 117.32 (C5AR), 132.58 (C1AR), 133.69 (C2,6AR), 138.21 (C3AR), 165.26 (C4AR), 183.55 (CfumOO), 208.31 (C=O) , chemical shift. Compounds I I II IIa III IIIa IIIb IVa *Mobile phase: IVa D2O Table 6. Chromatographic data for enantioseparation of prepared compounds on amylase tris(3,5-dimethylphenylcarbamate) bonded chiral stationary phase (Chiralpak AD). t1 41.10 58.58 38.07 33.07 27.98 27.91 27.91 29.40 k1 10.14 14.46 8.97 7.91 6.93 6.51 6.84 6.74 1.37 1.36 1.30 1.36 1.33 1.41 1.32 1.16 Rs 5.70 2.39 3.14 6.35 4.90 4.28 5.67 2.36 Mobile phase A B A B B B B B A hexane/ethanol/methanol/diethylamine (85:3.75:11.25:0.1, v/v/v/v) and B hexane/ethanol/methanol/diethylamine (85:7.75:7.75:0.1, v/v/v/v). Rs stereochemical resolution factor; t1, elution time for enantiomer 1; k1, retention factor for enantiomer 1; , separation factor. Table 7. Chromatographic data for the enantioseparation on teicoplanin-bonded chiral stationary phase (Chirobiotic T). Compound I IIa IIIa t1 21.43 17.84 19.65 k1 3.87 3.05 3.47 1.15 1.13 1.07 Rs 2.36 1.93 0.92 Mobile phase: methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v); Rs, stereochemical resolution factor; t1, elution time for enantiomer 1; k1, retention factor for enantiomer 1; , separation factor. Fig. 1. Separation of enantiomers of compound IIIa. Column: Chiralpac AD; Mobile phase: hexane/ethanol/methanol/ dietylamine (85:7.5:7.5:0.1, v/v/v/v). Fig. 2. Separation of enantiomers of compound IIIa.Column: Chirobiotic T; Mobile phase: methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

Synthesis of new compounds of the aryloxyaminopropanol type and their HPLC enantioseparation

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de Gruyter
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Abstract

Keywords Kúcové slová: INTRODUCTION In previous works, some new compounds of the aryloxyaminopropanol type derived from 4 hydroxypropiophenones with isopropyl and tert-butyl group in the hydrophilic part of the molecule were prepared (Cizmáriková et al., 1990a, 2003). Their anti-izoprenaline activity and anti-arrhythmic activity (Cizmáriková & Kozlovský, 1994a,b) depend on the substitution of the hydrophilic and lipophilic parts of the molecule. In addition, the prepared compounds with higher alkoxymethyl (octyloxymethyl and nonyloxymethyl) group in position 3 of the aromatic ring have shown antimicrobial and local anaesthetic activities (Cizmáriková et al., 1990b). In many papers, compounds with isopropyl and tert-butyl group in * cizmarikova@fpharm.uniba.sk © Acta Facultatis Pharmaceuticae Universitatis Comenianae the hydrophilic part of molecule are active beta-adrenolytics (Cizmáriková & Racanská, 1998; Bruchatá & Cizmáriková, 2010; Keckésová & Sedlárová, 2010). Lower beta-adrenolytic activity was observed in the compounds with other alkyl groups such as isobutyl and diethyl groups (Griffith, 2003). The large variety of different aromatic rings and substituents on nitrogen atom leads to compounds with combined pharmacological properties with affinity to both types of adrenergic receptor, and (Bruchatá & Cizmáriková, 2010). In the work (Mosti et al., 2000), compound with cyclohexylamino moiety gives antiarrhythmic, local anaesthetic and analgesic activity. Compounds of aryloxyaminopropanol type possess in their structure a single stereogenic centre and exist as stereoisomers. The most widely used technique for the separation of their enantiomers was high-performance liquid chromatography (HPLC) (Wang et al., 2008) on different chiral stationary phases such as cyclodextrin (Wang & Ching, 2002; Zhang et al., 2008; Hroboová et al., 2004), immobilised proteins (Fulde & Frahm, 1999; Fornstedt et al., 1999), Amberlite XAD-4 (Agrawal & Patel, 2005) and cellulose and amylase-based phases (AboulEnein & Ali, 2001; Valentova et al., 2003). Indirect enantioseparation using derivatisation agents is less suitable at these compounds (Bojarsky, 2002, Bojarsky et al., 2005). This paper was oriented on a preparation, characterisation of the new prepared derivatives of aryloxyaminopropanol type and its enantioseparation at the Chiralpak AD and Chirobiotic T columns. Chromatographic parameters such as separation, retention and resolution factors were calculated. HPLC studies were performed using a Hewlett-Packard (series 1 100) HPLC system consisting of a quaternary pump equipped with an injection valve (Rheodyne) and a diode array detector. The macrocyclic chiral stationary phase was Chirobiotic T (250 mm × 4 mm LD particle size 5-m Advanced Separation technologies, Inc., USA). The mobile phase was a mixture of methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v). The separation was carried out at a flow rate of 1 ml min­1 and column temperature was 23°C. The chromatograms were scanned using Hewlett Packard (series 1 100) at 270 nm. The injection volume was 20 l. The analyte was dissolved in methanol (concentration 1 mg ml­1). Secondary studies were carried out using HPLC system AGILENT 1200, consisting of a quaternary pump and a diode detector. Chromatographic characteristics The separation factor was expressed as = k1/k2, where k1, k2 are retention factors for the first and second eluting enantiomers. The retention factors k' were calculated as follows: k1 = (t1 ­ t0)/t0 and k2 = (t2 ­ t0)/t0, where t0, t1 and t2 are the dead elution time and elution times of enantiomers 1 and 2. The stereochemical resolution factor (Rs) of the first and second eluting enantiomers was calculated as the ratio of the difference between the retention times t1 and t2 to the arithmetic sum of the two peaks' widths w1 and w2: Rs = 2(t2 ­ t1)/(w1 + w2). EXPERIMENTAL Chemicals All HPLC grade solvents were obtained from Merck (Germany). Synthesis (3-Chloromethyl-4-hydroxyphenyl)propan-1-one and (3-alkoxymethylphenyl-4-hydroxyphenyl)propan-1-one were prepared according to Cizmáriková et al. (2002). [4-(3-Alkylamino-2-hydroxypropoxy)phenyl]propan-1-one and [4-(3-cycloalkylamino-2-hydroxypropoxy)phenyl] propan1-one were prepared according to Cizmáriková et al. (2012). Instruments The melting points were determined using a Kofler Micro Hot Stage and were quoted uncorrected. The purity of the prepared compounds was assessed using Silufol® UV 254 (Merck) sheets in the solvent system ethyl acetate/diethylamine (9.5:0.5, v/v). UV spectra were run on spectrophotometer GENESYS 10s UV­Vis in methanol. Concentration of compounds was about 10-1 mol m-3. IR spectra were recorded using Nicolet 6700 (Thermo Scientific). 1H NMR and 13C NMR were recorded on the Varian Gemini 2000 Spectrometer operating at 300 MHz for protons and 75 MHz for carbons. Elementary analysis was carried out on FLESCH 2000 (Thermo Scientific) within 0.3 % of the theoretical values. HPLC analysis HPLC was carried out using the chiral stationary phases (Chiralpak AD) based on the amylase tris(3,5-dimethylphenylcarbamate) (0.46 × 25). The mobile phases consisted of hexane/ ethanol/methanol/diethylamine 85:7.5:11.25:0.1, v/v/v/v (A) and hexane/ethanol/methanol/diethylamine 85:7.5:7.5:0.1, v/v/v/v (B). Samples for analysis were prepared as approximately 1 mg ml­1 solution in methanol. Separations were carried out at a flow rate of 0.8 ml min­1 and the column temperature was maintained at 25°C. Chromatograms were scanned at a wavelength of 267 ± 8 nm. RESULTS AND DISCUSSION The aim of this study was to prepare four derivatives of aryloxyaminopropanol type with different groups in hydrophilic and lipophilic parts of a molecule. The compound without side alkoxymethyl chain was prepared by a two-step synthesis and those with alkoxymethyl group by a four-step synthesis from 4-hydroxyphenylpropan-1-one. Oxirane intermediates prepared by the reaction of 4-hydroxyphenylpropan-1-one with epichlorohydrin gave final products by the reaction with appropriate amine (phenylamine, cyclohexylamine and isobutylamine). These were isolated in the form of free bases or salts with fumaric acid and with oxalic acid (Table 1). Compound I was isolated only in the form of free base; salts with fumaric and oxalic acid could not be prepared due to increased basicity of its nitrogen atom. The purity of the final products was checked by thin-layer chromatography using ethyl acetate/diethylamine as the mobile phase (Table 1). The structures of prepared compounds were confirmed by IR, UV and 1H NMR, 13C NMR spectra (Tables 2­4). The stretching vibration of the characteristic group in the IR spectra were (OH) 3166­3400 cm­1, (NH) (base) 3073­3274 cm­1, (C=C) 1578­1604 cm­1, (C=O) 1668­1682 cm­1, (CAl OCAr) 1251­ 1276 cm­1 (Table 2). The UV spectra of bases display two bands corresponding to * transition at max = 202­270 nm, log = 3.86­4.80 (Table 3). Compound I with two aromatic rings gives four bands corresponding to * transition (Table 3). Cizmáriková, R. et al. Table 1. Physico-chemical parameters of prepared compounds. R2 O OH R1 H3C O Compound Form of compound I Base R1 R2 NH N H Empirical formula Mr C20H25O4N 251.33 M.p. (°C) Solvent 67­69 Hexane Yield (%) RF 62 0.73 CH2OCH3 II Base CH2OCH3 IIa Fumarate III Base IIIa Fumarate IIIb Oxalate IV Base IVa Fumarate M.p. melting point, RF retardation factor N H NH C20H31O4N 349.47 82­87 Hexane C40H62O8N2 · C4H4O4 646,79 (CH3)2CHCH2NH H C16H23O3N 279.02 C16H23O3N · C4H4O4 331.46 C16H23O3N · C2H2O2 (CH3)2CHCH2NH CH3CH2OCH2 C38H66O8N2 · C4H4O4 646.79 C19H31O4N 337.44 117­120 Ethylacetate 57­59 Hexane 164­167 Ethylacetate 159­160 Ethanol Viscous oil 154-156 propan-2-ol H NMR and 13C NMR spectra of free bases showed the proof of the final structure proton and carbon signals of the aminopropanol chain (Tables 4 and 5). In this work, a direct HPLC method was used for enantioseparation of prepared racemic compounds using chiral stationary phases based on derivatised amylose (Chiralpak AD) and native teicoplanin (Chirobiotic T). Mobile phases A hexane/ethanol/methanol/diethylamine (85:3.75:11.25:0.1, v/v/v/v) and B hexane/ethanol/methanol/diethylamine (85:7.75:7.75:0.1, v/v/v/v) were used for the separations on the Chiralpak AD column. From Table 6 it is evident that good enantioseparations were achieved for all prepared compounds. The obtained separation factors were in the interval from 1.16 to 1.41 and resolution factors in the interval 2.36­6.35. In the case of compounds III, IIIa and IIIb in the B mobile phase, all forms of compound have similar values of selectivity factor and the values of resolution factors were in order oxalate>base>fumarate. Comparing the type of the mobile phase, greater value of Rs was obtained for the compound IIa in the form of base at the B mobile phase (Table 6). The results of the enantioresolution on the Chirobiotic T in polar organic separation mode (mobile phase methanol/acetonitrile/acetic acid/triethylamine 45:55:0.3:0.2, v/v/v/v) has shown that compounds with phenylamino and cyclohexylamino (I and II) are very well separated with resolution factor values in the range 1.93­2.36 and selectivity factors in the range 1.13­1.15. Comparing the enantioseparations of racemic compound with isobutyl substituent (compound III) on Chiralpak AD and on Chirobiotic T chiral stationary phases, lower enantioresolution was obtained ( = 1.07 and Rs = 0.92) on teicoplanin stationary phase (Table 7 and Figs 1 and 2). The mechanism of the separation on the both chiral phases is based on the interaction between chiral selector (derivatised amylase or native teicoplanin) and analyte by forming complexes between the enantiomeric analytes and chiral cavities. Detailed mechanism of enantioseparation was discussed in the papers Cizmáriková et al. (2012), Valentova et al. (2003) and Hroboová et al. (2001). Our study of pharmacological activities of the prepared compounds continues, and the results will be published in the next paper. CONCLUSION In this paper, four new compounds of the aryloxyaminopropanol type were prepared from 4-hydroxyphenylpropan1-one by two- and four-step syntheses. Chemical names of these compounds are: 1-[4-(2-hydroxy-3-phenylaminopropoxy)phenyl]propan-1-one, 1-[3-(methoxymethyl)-4-(2hydroxy-3-cyclohexylaminopropoxy)phenyl]propan-1-one, 1-[4-(2-hydroxy-3-isobutylaminopropoxy)phenyl]propan1-one and 1-[3-(ethoxymethyl)-4-(2-hydroxy-3-isobutylaminopropoxy)phenyl]propan-1-one. An enantioseparation of the prepared compounds was performed by using HPLC on an amylase tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and native teicoplanin (Chirobiotic T). The chromato- graphic results such as retention factor, separation factor = 1.16­1.41) and resolution factor (Rs = 2.36­6.35) have shown that Chiralpak AD is more suitable for enantioseparation of some of the prepared compounds than Chirobiotic T. ACKNOWLEDgEMENT This work was supported by the Slovak Research and Development Agency under the contract no. APVV-0516-12, grant UK/190/2013 and VEgA grant 1/0164/11. The authors thank Dr. Karlovska for recording 1H NMR and 13C NMR and Ms. Nedorostova for IR and UV spectra and Mr. Lehotsky for recording HPLC. Table 2. Values of stretching vibrations in IR spectra of prepared bases. Compounds I II III IIIa IIIb Iva , absorbtion maximum. * (OH, NH). (OH) (cm1) 3166 3283 3298* 3298 3400 3270 (NH) (cm1) 3274 3073 (C=C) (cm1) 1602 1600 1578 1604 1600 1600 1600 (C=O) (cm1) 1675 1670 1679 1679 1668 1682 (ArOalk) (cm1) 1265 1276 1257 1251 1254 1255 Table 3. Values of max and log in UV spectra, = m2 ·mol-1. Compounds I 205 203 202 203 203 max 1 (nm) 4.47 4.00 4.58 4.40 4.80 Log 1 220 216 II III IIIa IVa max 2 (nm) Log 2 max 3 (nm) Log 3 max 4 (nm) Log 4 max, wave length; , molar extinction coefficient. Compounds Solvent I CDCl3 Table 4. 1H NMR spectral data of compounds (ppm) (TMS). 1.19­1.24 (t, 3H, CH3CH2), 2.94­3.00 (q, 2H, CH3CH2), 3.26­3.32 (m, 2H, CH2NH), 3.40 (s, 3H, OCH3), 4.11­4.13 (m, 2H, CH2CH), 4.24­4.28 (m, 1H, CH2CH), 4.48­4.59 (m, 2H, CH2OCH3), 6.66­6.69 (d, 2H, C2ANLH, C6ANLH), 6.71­6.77 (t, 1H, C4ANLH), 6.91­6.94 (d, 1H, C5ARH), 7.17­7.22 (t, 2H, C3ANLH, C5ANLH), 7.93­7.96 (m, 2H, C2ARH, C6ARH) 1.04­1.13 (t, 3H, C=OCH2CH3), 1.16­1.25 (m, 6H, C3,4,5-CHexH2), 1.60­1.75 (m, 4H, C2,6-CHexH2), 2.40­2.46 (m, 1H, C1-CHexH), 2.70­2.77 (q, 2H, C=OCH2), 2.91­2.96 (m, 2H, CH2NH), 2.99 (s, 3H, OCH3), 3.55­3.62 (m, 1H, CHOH), 3.97­4.02 (m, 2H, OCH2CH), 4.07­4.10 (m, 2H, CH2OCH3), 6.93­6.96 (d, 1H, C5-ARH), 7.92­7.95 (d, 2H, C2,6-ARH) 0.91 (d, 6H, CH-(CH3)2), 1.75 (m, 1H, CH-(CH3)2), 2.45 (m, 2H, NHCH2CH), 2.76 (m, 2H, CHOH-CH2), 2.85 (m, 2H, CO-CH2), 4.05 (m, 3H, O-CH2CH). 1.02­1.05 (d, 6H, CH(CH3)2), 1.13­1.18 (t, 3H, C=OCH2CH3), 1.22­1.26 (t, 3H, OCH2CH3), 2.05­2.14 (m, 1H, CH(CH3)2), 3.00­3.02 (m, 2H, CH2CH(CH3)2), 3.05­3.09 (m, 2H, CHOHCH2NH), 3.27­3.34 (m, 2H, C=OCH2), 3.64­3.71 (q, 2H, OCH2CH3), 4.16­4.27 (m, 2H, OCH2CH), 4.38­4.45 (m, 1H, CHOH), 4.83 (s, 2H, CARCH2), 6.08 (s, 1H, CfumHCOO), 6.52 (s, 1H, CfumHCOOH), 7.07­7.10 (d, 1H, C3ARH), 7.98­8.01 (d, H, C2ARH, C6ARH) (ppm) (multiplicity and number of protons) II CDCl3 III CDCl3 IVa D2O , chemical shift. Cizmáriková, R. et al. Compounds I CDCl3 II CDCl3 III CDCl3 Table 5. 13C NMR spectral data of compounds (ppm) (TMS). (ppm) 8.39 (CH3CH2), 31.52 (CH3CH2), 46.30 (CH2CH), 58.23 (OCH3), 68.62 (CH2CH), 70.55 (OCH2CH), 71.74 (CH2OCH3), 112.22 (C5AR), 113.21 (C2,6ANL), 114.19 (C3AR), 118.02 (C4ANL), 126.68 (C1AR), 129.34 (C3,5ANL), 130.28 (C2,6AR), 148.07 (C1ANL), 160.80 (C4AR), 199.49 (C=O) 8.42 (C=OCH2CH3), 25.00 (C3,5-CHex), 26.05 (C4-CHex), 31.43 (C=OCH2CH3), 33.72 (C6-CHex), 34.02 (C2-CHex), 48.55 (CH2NH), 56.72 (C1-CHex), 68.21 (OCH3), 68.94 (CHOH), 70.62 (OCH2CH), 76.59 (CH2OCH3), 114.21 (C3,5-AR), 130.21 (C2,6-AR), 162.43 (C1-AR), 183.18 (C4-AR), 199.49 (C=O) 20.54 (CH(CH3)2), 26.38 (CH(CH3)2), 28.45 (COCH2), 51.53 (CHOH-CH2), 57.75 (CH2CH(CH3)2), 67.78 (CHOH), 70.63 (O-CH2), 114.22 (CAr3,5), 130.60 (CAr2,6), 162.62 (CAr4), 196.81 (CO) 10.93 (C=OCH2CH3), 21.86, 21.98 (CH(CH3)2), 28.15 (CH(CH3)2), 34.45 (C=OCH2CH3), 52.59 (CHOHCH2NH), 57.73 (CH2CH(CH3)2), 67.96 (OCH2CH), 72.37 (CHOH), 117.32 (C5AR), 132.58 (C1AR), 133.69 (C2,6AR), 138.21 (C3AR), 165.26 (C4AR), 183.55 (CfumOO), 208.31 (C=O) , chemical shift. Compounds I I II IIa III IIIa IIIb IVa *Mobile phase: IVa D2O Table 6. Chromatographic data for enantioseparation of prepared compounds on amylase tris(3,5-dimethylphenylcarbamate) bonded chiral stationary phase (Chiralpak AD). t1 41.10 58.58 38.07 33.07 27.98 27.91 27.91 29.40 k1 10.14 14.46 8.97 7.91 6.93 6.51 6.84 6.74 1.37 1.36 1.30 1.36 1.33 1.41 1.32 1.16 Rs 5.70 2.39 3.14 6.35 4.90 4.28 5.67 2.36 Mobile phase A B A B B B B B A hexane/ethanol/methanol/diethylamine (85:3.75:11.25:0.1, v/v/v/v) and B hexane/ethanol/methanol/diethylamine (85:7.75:7.75:0.1, v/v/v/v). Rs stereochemical resolution factor; t1, elution time for enantiomer 1; k1, retention factor for enantiomer 1; , separation factor. Table 7. Chromatographic data for the enantioseparation on teicoplanin-bonded chiral stationary phase (Chirobiotic T). Compound I IIa IIIa t1 21.43 17.84 19.65 k1 3.87 3.05 3.47 1.15 1.13 1.07 Rs 2.36 1.93 0.92 Mobile phase: methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v); Rs, stereochemical resolution factor; t1, elution time for enantiomer 1; k1, retention factor for enantiomer 1; , separation factor. Fig. 1. Separation of enantiomers of compound IIIa. Column: Chiralpac AD; Mobile phase: hexane/ethanol/methanol/ dietylamine (85:7.5:7.5:0.1, v/v/v/v). Fig. 2. Separation of enantiomers of compound IIIa.Column: Chirobiotic T; Mobile phase: methanol/acetonitrile/acetic acid/triethylamine (45:55:0.3:0.2, v/v/v/v).

Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Dec 1, 2013

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