Enantioseparation of Substituted 1, 3-Diazaspiro [4.5]Decan-4-Ones: HPLC Comparative Study on Different Polysaccharide Type Chiral Stationary Phases

Enantioseparation of Substituted 1, 3-Diazaspiro [4.5]Decan-4-Ones: HPLC Comparative Study on... Abstract Enantioseparation of substituted 1,3-diazaspiro[4.5]decan-4-ones (1–14) was achieved using different polysaccharide type chiral stationary phases (CSPs), namely, Chiralcel OJ, Chiralcel OD and Lux-Amylose-2 using different mobile phases which were either n-hexane/2-propanol or n-hexane/ethanol mixtures of various ratios (v/v) at flow rate 1 mL min−1. UV detection was carried out at 254 nm and temperature of 20°C. The retention behavior and selectivity of these CSPs were examined in isocratic normal phase high-performance liquid chromatography mode. The results revealed that the amylose CSP (Lux-Amylose-2) could separate almost all the compounds under investigation in contrast to cellulose CSPs (Chiracel OJ and Chiracel OD) which resolved fewer compounds. Introduction It has been widely accepted that a pair of optically pure enantiomers may exhibit quite different bioactivities, pharmacological and toxicological behaviors, etc. Therefore, their preparation and analysis have become increasingly important in many fields of science dealing with drugs, natural products, intermediates and agrochemicals (1). Chromatographic techniques, such as gas chromatography (GC) (2, 3), supercritical fluid chromatography (SFC) (4, 5), capillary electrochromatography (CEC) (6) and high-performance liquid chromatography (HPLC) (7, 8) have been extensively developed for the separation of enantiomers. Enantioseparations by HPLC is an essential tool for the research and development of chiral drugs. This can be achieved through: (i) indirect methods of chiral separations which involve the synthesis of diastereoisomers by a derivatizing chiral agent followed by chromatography on an achiral column; or (ii) direct methods involving separation of the racemic drugs to their corresponding enantiomers using chiral stationary phases (CSPs). Direct methods based on CSPs are preferred since they are rapid and suitable to resolution of racemates on both analytical and preparative scales (9). Among the well known CSPs are the polysaccharides which exhibit unique chiral recognition ability for a broad range of chiral compounds and have been widely used for HPLC separation techniques (10). Imidazolidin-4-ones represent a class of compounds with interesting biological activity. Through manipulation of the substituents around the imidazolidin-4-one core, molecules with a variety of biological properties have been discovered such as antimalarials (11), anxiolytics (12), antivirals (13), antimicrobials (14) and anticonvulsants (15). Recently, we have prepared a series of racemic substituted 1,3-diazaspiro[4.5] decan-4-ones (1–14) (16) (Figure 1), which displayed remarkable anticonvulsant activity against subcutaneous pentylenetetrazole (scPTZ) screening test. The aim of the present study is the separation of these racemic compounds to their respective enantiomers using different polysaccharide type CSPs, namely, cellulose tris-(4-methylbenzoate) (Chiralcel OJ), cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD) and amylose tris-(5-chloro,2-methylphenylcarbamate) (Lux 3μ-Amylose-2). Based upon the best condition for the enantioseparation the most suitable column could be selected for future preparative scale enantioseparation of these derivatives for further pharmacological evaluation. Figure 1. View largeDownload slide Structure of the chiral substituted 1, 3-diazaspiron[4.5]decan-4-ones (1–14). Group 1: compound 1 and 2; Group 2: compounds 3–6; Group 3: compounds 7–12; Group 4: compound 13 and 14. Asterisk indicates the position of asymmetric center. Figure 1. View largeDownload slide Structure of the chiral substituted 1, 3-diazaspiron[4.5]decan-4-ones (1–14). Group 1: compound 1 and 2; Group 2: compounds 3–6; Group 3: compounds 7–12; Group 4: compound 13 and 14. Asterisk indicates the position of asymmetric center. Materials and Methods Chemicals and reagents The substituted 1, 3-diazaspiro [4.5] decan-4-ones (1–14) were synthesized according to the reported procedures (16) (Figure 1). n-Hexane, 2-propanol and ethanol were of HPLC grade and obtained from Merck (Darmastadt, Germany). Instrumentation and Analytical Conditions The HPLC unit was an Agilent 1100 series apparatus equipped with a quaternary pump, a vacuum degasser, a column oven, a diode array UV detector and HP Chemstation software. The separations were performed using cellulose tris-(4-methylbenzoate) (Chiralcel OJ) (250 × 4.6 mm2 i.d., 10 μm particle size), cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD) (250 × 4.6 mm2 i.d., 10 μm particle size) HPLC columns obtained from Daicel Chemical Ind. (Tokyo, Japan) and amylose tris-(5-chloro,2-methylphenylcarbamate) (Lux 3μ-Amylose-2) (250 × 4.6 mm2 i.d., 3 μm particle size)purchased from Phenomenex Company (Torrance, USA). The mobile phases were either n-hexane/2-propanol or n-hexane/ethanol mixtures of various ratios (v/v) at flow rate 1 mL min−1. UV detection was carried out at 254 nm and temperature of 20°C. The injection volume was 5 μL of concentration 1 mgmL−1 sample solution. Results and Discussion Figure 1 illustrated the chemical structure of the racemic substituted 1, 3-diazaspiro [4.5] decan-4-ones (1–14). The results of the separations using different polysaccharides CSPs are summarized in Tables I–III. The chiral compounds 1–14 of the present study were divided into four groups based on the N1 substitution. Group 1 contains the unsubstituted secondary amines 1 and 2. The second group consists of compounds 3–6 where they have aliphatic amines. Group 3 includes compounds 7–12 which were substituted with saturated heterocyclic amines. The last group contains compounds 13 and 14 which were substituted with unsaturated heterocyclic amine. Table I. Enantioseparation of Chiral Substituted 1, 3-Diazaspiro[4.5]Decan-4-Ones (1–14) on Lux-Amylose-2 Column Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Lux- Amylose  1  Isopropanol 20%  1.24  2.46  1.98  8.02  2  Isopropanol 20%  1.88  2.92  1.55  4.81  3  Isopropanol 25%  0.65  1.11  1.70  2.63  4  Isopropanol 25%  0.80  1.45  1.81  2.68  5  Isopropanol 10%  2.81  3.81  1.35  1.4  Isopropanol 20%  0.53  0.81  1.52  1.53  Isopropanol 25%  0.51  0.78  1.52  1.55  6  Isopropanol 10%  3.22  4.5  1.39  1.6  Isopropanol 20%  0.68  1.03  1.51  1.38  Isopropanol 25%  0.66  0.99  1.50  1.38  7  Isopropanol 20%  1.21  2.11  1.74  2.54  8  Isopropanol 20%  1.41  2.76  1.95  2.59  9  Isopropanol 20%  0.92  1.5  1.63  2.34  10  Isopropanol 20%  1.08  1.87  1.73  2.44  11  Isopropanol 20%  2.85  3.65  1.28  1.63  12  Isopropanol 20%  3.14  4.56  1.45  2.14  13  Isopropanol 25%  4.52  5.78  1.27  1.09  Isopropanol 30%  2.59  3.34  1.28  0.97  Isopropanol 40%  1.32  1.71  1.29  0.90  14  Isopropanol 20%  7.17  11.5  1.60  1.48  Isopropanol 25%  5.08  7.89  1.55  1.40  Isopropanol 30%  2.8  4.37  1.56  1.41  Isopropanol 40%  1.59  2.43  1.52  1.02  Ethanol 10%  6.52  8.06  1.23  1.55  Ethanol 20%  2.23  2.69  1.21  1.05  Ethanol 30%  1.12  1.33  1.18  0.78  Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Lux- Amylose  1  Isopropanol 20%  1.24  2.46  1.98  8.02  2  Isopropanol 20%  1.88  2.92  1.55  4.81  3  Isopropanol 25%  0.65  1.11  1.70  2.63  4  Isopropanol 25%  0.80  1.45  1.81  2.68  5  Isopropanol 10%  2.81  3.81  1.35  1.4  Isopropanol 20%  0.53  0.81  1.52  1.53  Isopropanol 25%  0.51  0.78  1.52  1.55  6  Isopropanol 10%  3.22  4.5  1.39  1.6  Isopropanol 20%  0.68  1.03  1.51  1.38  Isopropanol 25%  0.66  0.99  1.50  1.38  7  Isopropanol 20%  1.21  2.11  1.74  2.54  8  Isopropanol 20%  1.41  2.76  1.95  2.59  9  Isopropanol 20%  0.92  1.5  1.63  2.34  10  Isopropanol 20%  1.08  1.87  1.73  2.44  11  Isopropanol 20%  2.85  3.65  1.28  1.63  12  Isopropanol 20%  3.14  4.56  1.45  2.14  13  Isopropanol 25%  4.52  5.78  1.27  1.09  Isopropanol 30%  2.59  3.34  1.28  0.97  Isopropanol 40%  1.32  1.71  1.29  0.90  14  Isopropanol 20%  7.17  11.5  1.60  1.48  Isopropanol 25%  5.08  7.89  1.55  1.40  Isopropanol 30%  2.8  4.37  1.56  1.41  Isopropanol 40%  1.59  2.43  1.52  1.02  Ethanol 10%  6.52  8.06  1.23  1.55  Ethanol 20%  2.23  2.69  1.21  1.05  Ethanol 30%  1.12  1.33  1.18  0.78  Table II. Enantioseparation of Chiral Substituted 1, 3-Diazaspiro[4.5]Decan-4-Ones (1–14) on Chiracel OJ Column Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OJ  1  Isopropanol 10%  1.89  2.96  1.56  3.12  Isopropanol 20%  1.33  2.05  1.54  2.34  2  Isopropanol 10%  1.56  2.65  1.69  2.92  Isopropanol 20%  1.07  1.77  1.65  2.16  3  Isopropanol 10%  0.65  0.65  1  –  4  Isopropanol 5%  0.49  0.73  1.48  0.44  5  Isopropanol 10%  0.15  0.34  2.26  –  6  Isopropanol 20%  0.14  0.17  1.21  –  7  Isopropanol 10%  1.03  1.03  1  –  8  Isopropanol 10%  0.76  0.76  1  –  9  Isopropanol 10%  0.49  0.49  1  –  10  Isopropanol 10%  0.3  0.3  1  –  11  Isopropanol 10%  4.48  5.44  1.21  0.52  12  Isopropanol 10%  3.44  3.44  1  –  13  Ethanol 20%  3.31  7.29  2.20  2.52  14  Ethanol 20%  2.31  5.78  2.50  2.18  Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OJ  1  Isopropanol 10%  1.89  2.96  1.56  3.12  Isopropanol 20%  1.33  2.05  1.54  2.34  2  Isopropanol 10%  1.56  2.65  1.69  2.92  Isopropanol 20%  1.07  1.77  1.65  2.16  3  Isopropanol 10%  0.65  0.65  1  –  4  Isopropanol 5%  0.49  0.73  1.48  0.44  5  Isopropanol 10%  0.15  0.34  2.26  –  6  Isopropanol 20%  0.14  0.17  1.21  –  7  Isopropanol 10%  1.03  1.03  1  –  8  Isopropanol 10%  0.76  0.76  1  –  9  Isopropanol 10%  0.49  0.49  1  –  10  Isopropanol 10%  0.3  0.3  1  –  11  Isopropanol 10%  4.48  5.44  1.21  0.52  12  Isopropanol 10%  3.44  3.44  1  –  13  Ethanol 20%  3.31  7.29  2.20  2.52  14  Ethanol 20%  2.31  5.78  2.50  2.18  Table III. Enantioseparation of Chiral Substituted 1, 3-Diazaspiro[4.5]Decan-4-Ones (1–14) on Chiracel OD Column Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OD  1  Isopropanol 2%  5.23  6.14  1.17  0.88  2  Isopropanol 10%  0.38  0.59  1.55  1.11  3  Isopropanol 10%  NR        4  Isopropanol 10%  NR        5  Isopropanol 10%  NR        6  Isopropanol 2%  1.17  1.17  1  –  7  Isopropanol 20%  0.17  0.17  1  –  8  Isopropanol 10%  NR        9  Isopropanol 2%  1.69  1.69  1  –  10  Isopropanol 20%  0.02  0.02  1  –  11  Isopropanol 20%  0.53  0.66  1.24  0.83  12  Isopropanol 20%  0.52  0.71  1.36  1.12  13  Isopropanol 20%  2.65  9.47  3.57  6.97  14  Isopropanol 20%  2.75  4.12  1.49  1.90  Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OD  1  Isopropanol 2%  5.23  6.14  1.17  0.88  2  Isopropanol 10%  0.38  0.59  1.55  1.11  3  Isopropanol 10%  NR        4  Isopropanol 10%  NR        5  Isopropanol 10%  NR        6  Isopropanol 2%  1.17  1.17  1  –  7  Isopropanol 20%  0.17  0.17  1  –  8  Isopropanol 10%  NR        9  Isopropanol 2%  1.69  1.69  1  –  10  Isopropanol 20%  0.02  0.02  1  –  11  Isopropanol 20%  0.53  0.66  1.24  0.83  12  Isopropanol 20%  0.52  0.71  1.36  1.12  13  Isopropanol 20%  2.65  9.47  3.57  6.97  14  Isopropanol 20%  2.75  4.12  1.49  1.90  NR = no resolution. Enantioseparation on Lux-Amylose-2 Successful enantioseparations for all analyts under investigation were achieved using Lux-Amylose-2 column in combination with n-hexane/2-propanol or n-hexane/ethanol. Lux-Amylose-2 column exhibited high chiral recognition ability for the compounds of group 1using n-hexane/2-propanol 80:20 v/v as mobile phase with Rs 8.02 and 4 for compounds 1 and 2, respectively (Figure 2A). It could be observed that in group 1 the methyl substitution in the aromatic ring increased the retention in compound 2 than unsubstituted derivative 1. Concerning group 2 enantioselectivity was observed for all compounds of this group however, a baseline enantioseparation could be observed only for the diethyl derivatives 3 and 4 rather than the dipropyl derivatives 5 and 6. Also, as in group 1 the retention was higher in presence of methyl group in the distal aromatic moiety. The compounds of the third group substituted with heterocyclic amines 7–12 were well resolved on Lux-Amylose-2 column with n-hexane/2-propanol 80:20 v/v as a mobile phase and have Rs > 2 except the morpholine derivative 11 which showed relatively lower resolution (Figure 2B). It was obvious from the results of the third group that compounds bearing heterocyclic amine with an additional heteroatom, i.e, morpholine moiety have higher retention than that of the other compounds and generally as in groups 1 and 2 the aromatic substitution with methyl group increased the retention. On the other hand, the substitutions in group 4 with unsaturated heterocyclic amine and with an additional basic center, i.e, imidazole ring worsen the interaction between this CSP and the analyte. For compound 13 the separation factor was rather high but due to low peak efficiency a baseline enantioseparation could not be observed under any tested conditions. Moreover, compound 14 could barely achieve baseline separation using n-hexane/ethanol 90:10 v/v as a mobile phase after trying various ratios of n-hexane/2-propanol as well as n-hexane/ethanol as shown in Table I. Lux-Amylose-2 column has used in this study has a particle size of 3 μm as compared to the other two columns namely Chiralcel OJ and Chiralcel OD which had a larger particle size of 10 μm. This could be a likely explanation of the improved chiral resolution on Lux-Amylose-2 column for many of the solutes. Figure 2. View largeDownload slide (A) and (B) Chromatograms of enantiomeric separation of compounds 1 and 11, respectively, on Lux- amylose-2 using n-hexane/2-propanol 80:20 v/v. Figure 2. View largeDownload slide (A) and (B) Chromatograms of enantiomeric separation of compounds 1 and 11, respectively, on Lux- amylose-2 using n-hexane/2-propanol 80:20 v/v. Enantioseparation on Chiracel OJ It was found that Chiracel OJ exhibited less enantioselectivity than Lux-Amylose-2 column towards the diazaspirodecanones 1–14. The secondary amines 1 and 2 of the group 1 were the best resolved compounds with baseline separation on the Chiracel OJ column although the resolution was not as good as Lux-Amylose-2. In contrast to Lux-Amylose-2 column the retention of this group decreased upon methyl substitution of the aromatic ring. The derivatives of the second group containing aliphatic amine side chain 3–6 were less retained and were not as well resolved by Chiracel OJ as compared to the results obtained by Lux-Amylose-2. Also, this CSP did not display chiral recognition ability towards the compounds of group 3 having saturated heterocyclic moiety 7–12 as all of them eluted as unresolved single peak with a relatively low retention factor except the morpholine derivative 11 which was separated but with very poor resolution. However, this column showed better enantioseparation of the imidazole derivatives 13 and 14 (Figure 3) with higher resolution compared to that on the Lux-Amylose-2. The aforementioned trend of a decreasing retention time with methyl substitution of the aromatic ring was also observed for those imidazole derivatives as the compounds of group 1 as shown in Table II. Figure 3. View largeDownload slide Chromatogram of enantiomeric separation of compound 14 on Chiracel OJ using n-hexane/ethanol 80:20 v/v. Figure 3. View largeDownload slide Chromatogram of enantiomeric separation of compound 14 on Chiracel OJ using n-hexane/ethanol 80:20 v/v. Enantioseparation on Chiracel OD Chiracel OD column which contains phenylcarbamate moiety as Lux-Amylose-2 column but attached to the cellulose backbone instead of amylose exhibited lower enantioselectivity towards most of the compounds under investigation compared to the amylose derivative. Unfortunately, partial resolution only was observed for the secondary amines 1 and 2 (Figure 4A) of the group 1 after trying various ratios of n-hexane/2-propanol as a mobile phase. Also, as on the Chiracel OJ column the retention time of those secondary amines 1 and 2 decreased upon methyl substitution of the aromatic ring. This column was the worst one for the separation of group 2 compounds having aliphatic amine side chain 3–6 as they were not strongly retained on this CSP as can be derived from low retention time. As in Chiracel OJ column most of the compounds of the group 3 bearing saturated heterocyclic amines 7–12 did not resolve on Chiracel OD column and almost not retained on this column. However, as in the Lux-Amylose-2 column the morpholine derivatives 11 and 12 were more retained than the other compounds of group 3 on this stationary phase and could be resolved but without baseline separation. On the contrary to Lux-Amylose-2 column the substitution with unsaturated heterocyclic amine as in compounds 13 and 14 significantly improved the chiral recognition ability of this column. Both of the imidazole derivatives 13 (Figure 4B) and 14 could achieve baseline separation as on Chiracel OJ with a comparable or higher resolution factor. Also, it should be noticed that the retention time was decreased by methyl substitution of the aromatic group (Table III). Figure 4. View largeDownload slide (A) Chromatogram of enantiomeric separation of compound 2 using n-hexane/isopropanol 90:10 v/v and (B) Chromatogram of enantiomeric separation of compound 13 using n-hexane/ isopropanol 80:20 v/v on Chiracel OD. Figure 4. View largeDownload slide (A) Chromatogram of enantiomeric separation of compound 2 using n-hexane/isopropanol 90:10 v/v and (B) Chromatogram of enantiomeric separation of compound 13 using n-hexane/ isopropanol 80:20 v/v on Chiracel OD. It is well known that the polar carbamate residues are probably the most important adsorption sites for the enantio-recognition process of phenylcarbamate derivatives of cellulose and amylose. The NH and C = O groups interact with the functional groups of the racemate through hydrogen bonding in addition to dipole–dipole interaction of the C = O group. On the contrary, only acceptor hydrogen bonding with chiral analyte are provided by the docking ester sites on benzoate derivative of cellulose. Besides, the π– π interaction between the phenyl groups of the phenylcarbamates and an aromatic group of a racemate may also play some role in the chiral discrimination (17, 18). This could justify the separation of the unsubstituted secondary amines 1 and 2 on the three CSPs used for this investigation where they displayed a lower number of hydrogen bond interaction with the CSPs. In addition, the higher retention of the morpholine derivatives 11 and 12 on the three columns compared to the other congeners of group 2 may be due to presence of an additional oxygen atom which leads to increase the number of hydrogen bonds. Also, this case occurred with the imidazole derivatives 13 and 14 due to the presence of an additional basic center. Moreover, the π–π interaction between the phenyl groups of the phenylcarbamates of the CSPs and the imidazole ring may further increase the retention of these derivatives. Conclusion This investigation showed that the amylose CSP (Lux-Amylose-2) could separate most of the compounds under investigation in contrast to cellulose CSPs (Chiracel OJ and Chiracel OD) which could resolve fewer compounds. This difference in the chiral recognition ability between amylose and cellulose might be attributed to their difference in configuration where amylose possesses a helical structure while the cellulose forms a linear and rigid structure. 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Enantioseparation of Substituted 1, 3-Diazaspiro [4.5]Decan-4-Ones: HPLC Comparative Study on Different Polysaccharide Type Chiral Stationary Phases

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© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Enantioseparation of substituted 1,3-diazaspiro[4.5]decan-4-ones (1–14) was achieved using different polysaccharide type chiral stationary phases (CSPs), namely, Chiralcel OJ, Chiralcel OD and Lux-Amylose-2 using different mobile phases which were either n-hexane/2-propanol or n-hexane/ethanol mixtures of various ratios (v/v) at flow rate 1 mL min−1. UV detection was carried out at 254 nm and temperature of 20°C. The retention behavior and selectivity of these CSPs were examined in isocratic normal phase high-performance liquid chromatography mode. The results revealed that the amylose CSP (Lux-Amylose-2) could separate almost all the compounds under investigation in contrast to cellulose CSPs (Chiracel OJ and Chiracel OD) which resolved fewer compounds. Introduction It has been widely accepted that a pair of optically pure enantiomers may exhibit quite different bioactivities, pharmacological and toxicological behaviors, etc. Therefore, their preparation and analysis have become increasingly important in many fields of science dealing with drugs, natural products, intermediates and agrochemicals (1). Chromatographic techniques, such as gas chromatography (GC) (2, 3), supercritical fluid chromatography (SFC) (4, 5), capillary electrochromatography (CEC) (6) and high-performance liquid chromatography (HPLC) (7, 8) have been extensively developed for the separation of enantiomers. Enantioseparations by HPLC is an essential tool for the research and development of chiral drugs. This can be achieved through: (i) indirect methods of chiral separations which involve the synthesis of diastereoisomers by a derivatizing chiral agent followed by chromatography on an achiral column; or (ii) direct methods involving separation of the racemic drugs to their corresponding enantiomers using chiral stationary phases (CSPs). Direct methods based on CSPs are preferred since they are rapid and suitable to resolution of racemates on both analytical and preparative scales (9). Among the well known CSPs are the polysaccharides which exhibit unique chiral recognition ability for a broad range of chiral compounds and have been widely used for HPLC separation techniques (10). Imidazolidin-4-ones represent a class of compounds with interesting biological activity. Through manipulation of the substituents around the imidazolidin-4-one core, molecules with a variety of biological properties have been discovered such as antimalarials (11), anxiolytics (12), antivirals (13), antimicrobials (14) and anticonvulsants (15). Recently, we have prepared a series of racemic substituted 1,3-diazaspiro[4.5] decan-4-ones (1–14) (16) (Figure 1), which displayed remarkable anticonvulsant activity against subcutaneous pentylenetetrazole (scPTZ) screening test. The aim of the present study is the separation of these racemic compounds to their respective enantiomers using different polysaccharide type CSPs, namely, cellulose tris-(4-methylbenzoate) (Chiralcel OJ), cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD) and amylose tris-(5-chloro,2-methylphenylcarbamate) (Lux 3μ-Amylose-2). Based upon the best condition for the enantioseparation the most suitable column could be selected for future preparative scale enantioseparation of these derivatives for further pharmacological evaluation. Figure 1. View largeDownload slide Structure of the chiral substituted 1, 3-diazaspiron[4.5]decan-4-ones (1–14). Group 1: compound 1 and 2; Group 2: compounds 3–6; Group 3: compounds 7–12; Group 4: compound 13 and 14. Asterisk indicates the position of asymmetric center. Figure 1. View largeDownload slide Structure of the chiral substituted 1, 3-diazaspiron[4.5]decan-4-ones (1–14). Group 1: compound 1 and 2; Group 2: compounds 3–6; Group 3: compounds 7–12; Group 4: compound 13 and 14. Asterisk indicates the position of asymmetric center. Materials and Methods Chemicals and reagents The substituted 1, 3-diazaspiro [4.5] decan-4-ones (1–14) were synthesized according to the reported procedures (16) (Figure 1). n-Hexane, 2-propanol and ethanol were of HPLC grade and obtained from Merck (Darmastadt, Germany). Instrumentation and Analytical Conditions The HPLC unit was an Agilent 1100 series apparatus equipped with a quaternary pump, a vacuum degasser, a column oven, a diode array UV detector and HP Chemstation software. The separations were performed using cellulose tris-(4-methylbenzoate) (Chiralcel OJ) (250 × 4.6 mm2 i.d., 10 μm particle size), cellulose tris-(3,5-dimethylphenylcarbamate) (Chiralcel OD) (250 × 4.6 mm2 i.d., 10 μm particle size) HPLC columns obtained from Daicel Chemical Ind. (Tokyo, Japan) and amylose tris-(5-chloro,2-methylphenylcarbamate) (Lux 3μ-Amylose-2) (250 × 4.6 mm2 i.d., 3 μm particle size)purchased from Phenomenex Company (Torrance, USA). The mobile phases were either n-hexane/2-propanol or n-hexane/ethanol mixtures of various ratios (v/v) at flow rate 1 mL min−1. UV detection was carried out at 254 nm and temperature of 20°C. The injection volume was 5 μL of concentration 1 mgmL−1 sample solution. Results and Discussion Figure 1 illustrated the chemical structure of the racemic substituted 1, 3-diazaspiro [4.5] decan-4-ones (1–14). The results of the separations using different polysaccharides CSPs are summarized in Tables I–III. The chiral compounds 1–14 of the present study were divided into four groups based on the N1 substitution. Group 1 contains the unsubstituted secondary amines 1 and 2. The second group consists of compounds 3–6 where they have aliphatic amines. Group 3 includes compounds 7–12 which were substituted with saturated heterocyclic amines. The last group contains compounds 13 and 14 which were substituted with unsaturated heterocyclic amine. Table I. Enantioseparation of Chiral Substituted 1, 3-Diazaspiro[4.5]Decan-4-Ones (1–14) on Lux-Amylose-2 Column Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Lux- Amylose  1  Isopropanol 20%  1.24  2.46  1.98  8.02  2  Isopropanol 20%  1.88  2.92  1.55  4.81  3  Isopropanol 25%  0.65  1.11  1.70  2.63  4  Isopropanol 25%  0.80  1.45  1.81  2.68  5  Isopropanol 10%  2.81  3.81  1.35  1.4  Isopropanol 20%  0.53  0.81  1.52  1.53  Isopropanol 25%  0.51  0.78  1.52  1.55  6  Isopropanol 10%  3.22  4.5  1.39  1.6  Isopropanol 20%  0.68  1.03  1.51  1.38  Isopropanol 25%  0.66  0.99  1.50  1.38  7  Isopropanol 20%  1.21  2.11  1.74  2.54  8  Isopropanol 20%  1.41  2.76  1.95  2.59  9  Isopropanol 20%  0.92  1.5  1.63  2.34  10  Isopropanol 20%  1.08  1.87  1.73  2.44  11  Isopropanol 20%  2.85  3.65  1.28  1.63  12  Isopropanol 20%  3.14  4.56  1.45  2.14  13  Isopropanol 25%  4.52  5.78  1.27  1.09  Isopropanol 30%  2.59  3.34  1.28  0.97  Isopropanol 40%  1.32  1.71  1.29  0.90  14  Isopropanol 20%  7.17  11.5  1.60  1.48  Isopropanol 25%  5.08  7.89  1.55  1.40  Isopropanol 30%  2.8  4.37  1.56  1.41  Isopropanol 40%  1.59  2.43  1.52  1.02  Ethanol 10%  6.52  8.06  1.23  1.55  Ethanol 20%  2.23  2.69  1.21  1.05  Ethanol 30%  1.12  1.33  1.18  0.78  Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Lux- Amylose  1  Isopropanol 20%  1.24  2.46  1.98  8.02  2  Isopropanol 20%  1.88  2.92  1.55  4.81  3  Isopropanol 25%  0.65  1.11  1.70  2.63  4  Isopropanol 25%  0.80  1.45  1.81  2.68  5  Isopropanol 10%  2.81  3.81  1.35  1.4  Isopropanol 20%  0.53  0.81  1.52  1.53  Isopropanol 25%  0.51  0.78  1.52  1.55  6  Isopropanol 10%  3.22  4.5  1.39  1.6  Isopropanol 20%  0.68  1.03  1.51  1.38  Isopropanol 25%  0.66  0.99  1.50  1.38  7  Isopropanol 20%  1.21  2.11  1.74  2.54  8  Isopropanol 20%  1.41  2.76  1.95  2.59  9  Isopropanol 20%  0.92  1.5  1.63  2.34  10  Isopropanol 20%  1.08  1.87  1.73  2.44  11  Isopropanol 20%  2.85  3.65  1.28  1.63  12  Isopropanol 20%  3.14  4.56  1.45  2.14  13  Isopropanol 25%  4.52  5.78  1.27  1.09  Isopropanol 30%  2.59  3.34  1.28  0.97  Isopropanol 40%  1.32  1.71  1.29  0.90  14  Isopropanol 20%  7.17  11.5  1.60  1.48  Isopropanol 25%  5.08  7.89  1.55  1.40  Isopropanol 30%  2.8  4.37  1.56  1.41  Isopropanol 40%  1.59  2.43  1.52  1.02  Ethanol 10%  6.52  8.06  1.23  1.55  Ethanol 20%  2.23  2.69  1.21  1.05  Ethanol 30%  1.12  1.33  1.18  0.78  Table II. Enantioseparation of Chiral Substituted 1, 3-Diazaspiro[4.5]Decan-4-Ones (1–14) on Chiracel OJ Column Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OJ  1  Isopropanol 10%  1.89  2.96  1.56  3.12  Isopropanol 20%  1.33  2.05  1.54  2.34  2  Isopropanol 10%  1.56  2.65  1.69  2.92  Isopropanol 20%  1.07  1.77  1.65  2.16  3  Isopropanol 10%  0.65  0.65  1  –  4  Isopropanol 5%  0.49  0.73  1.48  0.44  5  Isopropanol 10%  0.15  0.34  2.26  –  6  Isopropanol 20%  0.14  0.17  1.21  –  7  Isopropanol 10%  1.03  1.03  1  –  8  Isopropanol 10%  0.76  0.76  1  –  9  Isopropanol 10%  0.49  0.49  1  –  10  Isopropanol 10%  0.3  0.3  1  –  11  Isopropanol 10%  4.48  5.44  1.21  0.52  12  Isopropanol 10%  3.44  3.44  1  –  13  Ethanol 20%  3.31  7.29  2.20  2.52  14  Ethanol 20%  2.31  5.78  2.50  2.18  Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OJ  1  Isopropanol 10%  1.89  2.96  1.56  3.12  Isopropanol 20%  1.33  2.05  1.54  2.34  2  Isopropanol 10%  1.56  2.65  1.69  2.92  Isopropanol 20%  1.07  1.77  1.65  2.16  3  Isopropanol 10%  0.65  0.65  1  –  4  Isopropanol 5%  0.49  0.73  1.48  0.44  5  Isopropanol 10%  0.15  0.34  2.26  –  6  Isopropanol 20%  0.14  0.17  1.21  –  7  Isopropanol 10%  1.03  1.03  1  –  8  Isopropanol 10%  0.76  0.76  1  –  9  Isopropanol 10%  0.49  0.49  1  –  10  Isopropanol 10%  0.3  0.3  1  –  11  Isopropanol 10%  4.48  5.44  1.21  0.52  12  Isopropanol 10%  3.44  3.44  1  –  13  Ethanol 20%  3.31  7.29  2.20  2.52  14  Ethanol 20%  2.31  5.78  2.50  2.18  Table III. Enantioseparation of Chiral Substituted 1, 3-Diazaspiro[4.5]Decan-4-Ones (1–14) on Chiracel OD Column Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OD  1  Isopropanol 2%  5.23  6.14  1.17  0.88  2  Isopropanol 10%  0.38  0.59  1.55  1.11  3  Isopropanol 10%  NR        4  Isopropanol 10%  NR        5  Isopropanol 10%  NR        6  Isopropanol 2%  1.17  1.17  1  –  7  Isopropanol 20%  0.17  0.17  1  –  8  Isopropanol 10%  NR        9  Isopropanol 2%  1.69  1.69  1  –  10  Isopropanol 20%  0.02  0.02  1  –  11  Isopropanol 20%  0.53  0.66  1.24  0.83  12  Isopropanol 20%  0.52  0.71  1.36  1.12  13  Isopropanol 20%  2.65  9.47  3.57  6.97  14  Isopropanol 20%  2.75  4.12  1.49  1.90  Column  Compound  Eluent (% of alcohol)  k1  k2  α  RS  Chiracel OD  1  Isopropanol 2%  5.23  6.14  1.17  0.88  2  Isopropanol 10%  0.38  0.59  1.55  1.11  3  Isopropanol 10%  NR        4  Isopropanol 10%  NR        5  Isopropanol 10%  NR        6  Isopropanol 2%  1.17  1.17  1  –  7  Isopropanol 20%  0.17  0.17  1  –  8  Isopropanol 10%  NR        9  Isopropanol 2%  1.69  1.69  1  –  10  Isopropanol 20%  0.02  0.02  1  –  11  Isopropanol 20%  0.53  0.66  1.24  0.83  12  Isopropanol 20%  0.52  0.71  1.36  1.12  13  Isopropanol 20%  2.65  9.47  3.57  6.97  14  Isopropanol 20%  2.75  4.12  1.49  1.90  NR = no resolution. Enantioseparation on Lux-Amylose-2 Successful enantioseparations for all analyts under investigation were achieved using Lux-Amylose-2 column in combination with n-hexane/2-propanol or n-hexane/ethanol. Lux-Amylose-2 column exhibited high chiral recognition ability for the compounds of group 1using n-hexane/2-propanol 80:20 v/v as mobile phase with Rs 8.02 and 4 for compounds 1 and 2, respectively (Figure 2A). It could be observed that in group 1 the methyl substitution in the aromatic ring increased the retention in compound 2 than unsubstituted derivative 1. Concerning group 2 enantioselectivity was observed for all compounds of this group however, a baseline enantioseparation could be observed only for the diethyl derivatives 3 and 4 rather than the dipropyl derivatives 5 and 6. Also, as in group 1 the retention was higher in presence of methyl group in the distal aromatic moiety. The compounds of the third group substituted with heterocyclic amines 7–12 were well resolved on Lux-Amylose-2 column with n-hexane/2-propanol 80:20 v/v as a mobile phase and have Rs > 2 except the morpholine derivative 11 which showed relatively lower resolution (Figure 2B). It was obvious from the results of the third group that compounds bearing heterocyclic amine with an additional heteroatom, i.e, morpholine moiety have higher retention than that of the other compounds and generally as in groups 1 and 2 the aromatic substitution with methyl group increased the retention. On the other hand, the substitutions in group 4 with unsaturated heterocyclic amine and with an additional basic center, i.e, imidazole ring worsen the interaction between this CSP and the analyte. For compound 13 the separation factor was rather high but due to low peak efficiency a baseline enantioseparation could not be observed under any tested conditions. Moreover, compound 14 could barely achieve baseline separation using n-hexane/ethanol 90:10 v/v as a mobile phase after trying various ratios of n-hexane/2-propanol as well as n-hexane/ethanol as shown in Table I. Lux-Amylose-2 column has used in this study has a particle size of 3 μm as compared to the other two columns namely Chiralcel OJ and Chiralcel OD which had a larger particle size of 10 μm. This could be a likely explanation of the improved chiral resolution on Lux-Amylose-2 column for many of the solutes. Figure 2. View largeDownload slide (A) and (B) Chromatograms of enantiomeric separation of compounds 1 and 11, respectively, on Lux- amylose-2 using n-hexane/2-propanol 80:20 v/v. Figure 2. View largeDownload slide (A) and (B) Chromatograms of enantiomeric separation of compounds 1 and 11, respectively, on Lux- amylose-2 using n-hexane/2-propanol 80:20 v/v. Enantioseparation on Chiracel OJ It was found that Chiracel OJ exhibited less enantioselectivity than Lux-Amylose-2 column towards the diazaspirodecanones 1–14. The secondary amines 1 and 2 of the group 1 were the best resolved compounds with baseline separation on the Chiracel OJ column although the resolution was not as good as Lux-Amylose-2. In contrast to Lux-Amylose-2 column the retention of this group decreased upon methyl substitution of the aromatic ring. The derivatives of the second group containing aliphatic amine side chain 3–6 were less retained and were not as well resolved by Chiracel OJ as compared to the results obtained by Lux-Amylose-2. Also, this CSP did not display chiral recognition ability towards the compounds of group 3 having saturated heterocyclic moiety 7–12 as all of them eluted as unresolved single peak with a relatively low retention factor except the morpholine derivative 11 which was separated but with very poor resolution. However, this column showed better enantioseparation of the imidazole derivatives 13 and 14 (Figure 3) with higher resolution compared to that on the Lux-Amylose-2. The aforementioned trend of a decreasing retention time with methyl substitution of the aromatic ring was also observed for those imidazole derivatives as the compounds of group 1 as shown in Table II. Figure 3. View largeDownload slide Chromatogram of enantiomeric separation of compound 14 on Chiracel OJ using n-hexane/ethanol 80:20 v/v. Figure 3. View largeDownload slide Chromatogram of enantiomeric separation of compound 14 on Chiracel OJ using n-hexane/ethanol 80:20 v/v. Enantioseparation on Chiracel OD Chiracel OD column which contains phenylcarbamate moiety as Lux-Amylose-2 column but attached to the cellulose backbone instead of amylose exhibited lower enantioselectivity towards most of the compounds under investigation compared to the amylose derivative. Unfortunately, partial resolution only was observed for the secondary amines 1 and 2 (Figure 4A) of the group 1 after trying various ratios of n-hexane/2-propanol as a mobile phase. Also, as on the Chiracel OJ column the retention time of those secondary amines 1 and 2 decreased upon methyl substitution of the aromatic ring. This column was the worst one for the separation of group 2 compounds having aliphatic amine side chain 3–6 as they were not strongly retained on this CSP as can be derived from low retention time. As in Chiracel OJ column most of the compounds of the group 3 bearing saturated heterocyclic amines 7–12 did not resolve on Chiracel OD column and almost not retained on this column. However, as in the Lux-Amylose-2 column the morpholine derivatives 11 and 12 were more retained than the other compounds of group 3 on this stationary phase and could be resolved but without baseline separation. On the contrary to Lux-Amylose-2 column the substitution with unsaturated heterocyclic amine as in compounds 13 and 14 significantly improved the chiral recognition ability of this column. Both of the imidazole derivatives 13 (Figure 4B) and 14 could achieve baseline separation as on Chiracel OJ with a comparable or higher resolution factor. Also, it should be noticed that the retention time was decreased by methyl substitution of the aromatic group (Table III). Figure 4. View largeDownload slide (A) Chromatogram of enantiomeric separation of compound 2 using n-hexane/isopropanol 90:10 v/v and (B) Chromatogram of enantiomeric separation of compound 13 using n-hexane/ isopropanol 80:20 v/v on Chiracel OD. Figure 4. View largeDownload slide (A) Chromatogram of enantiomeric separation of compound 2 using n-hexane/isopropanol 90:10 v/v and (B) Chromatogram of enantiomeric separation of compound 13 using n-hexane/ isopropanol 80:20 v/v on Chiracel OD. It is well known that the polar carbamate residues are probably the most important adsorption sites for the enantio-recognition process of phenylcarbamate derivatives of cellulose and amylose. The NH and C = O groups interact with the functional groups of the racemate through hydrogen bonding in addition to dipole–dipole interaction of the C = O group. On the contrary, only acceptor hydrogen bonding with chiral analyte are provided by the docking ester sites on benzoate derivative of cellulose. Besides, the π– π interaction between the phenyl groups of the phenylcarbamates and an aromatic group of a racemate may also play some role in the chiral discrimination (17, 18). This could justify the separation of the unsubstituted secondary amines 1 and 2 on the three CSPs used for this investigation where they displayed a lower number of hydrogen bond interaction with the CSPs. In addition, the higher retention of the morpholine derivatives 11 and 12 on the three columns compared to the other congeners of group 2 may be due to presence of an additional oxygen atom which leads to increase the number of hydrogen bonds. Also, this case occurred with the imidazole derivatives 13 and 14 due to the presence of an additional basic center. Moreover, the π–π interaction between the phenyl groups of the phenylcarbamates of the CSPs and the imidazole ring may further increase the retention of these derivatives. Conclusion This investigation showed that the amylose CSP (Lux-Amylose-2) could separate most of the compounds under investigation in contrast to cellulose CSPs (Chiracel OJ and Chiracel OD) which could resolve fewer compounds. This difference in the chiral recognition ability between amylose and cellulose might be attributed to their difference in configuration where amylose possesses a helical structure while the cellulose forms a linear and rigid structure. 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Journal of Chromatographic ScienceOxford University Press

Published: Feb 1, 2018

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