Enantioselective HPLC separation of bioactive C5-chiral 2-pyrazolines on lux amylose-2 and lux cellulose-2: Comparative and mechanistic approaches

ABSTRACT Stereoselective analytical HPLC separations have been developed for a series of biologically active chiral 2-pyrazolines (1-22) to be used in monitoring their resolution reactions or to custom semipreparative HPLC separations prior to biological assessment of both enantiomers. Polysaccharide-based chiral stationary phases (CSPs), namely, Lux amylose-2 and cellulose-2, have been used. Both normal (n-hexane/ethanol) and polar organic (ethanol, methanol, acetonitrile, or mixtures thereof) elution modes were very beneficial for the achievement of baseline separations. The impact of various chemical moieties embedded in the structures of 2-pyrazolines 1-22 and the adopted stationary phases on chiral recognition has been investigated. A case of reversed order of elution following alterations in either stationary phase or elution mode has been observed. Our findings recommend that normal elution mode can be used for optimizing semipreparative HPLC methods whereas polar organic mobile phases (such as acetonitrile and ethanol) are more suited to stereoselective reactions monitoring, routine quality control work, or for pharmacological and toxicological assays. These results settle the implementation of polysaccharide-based CSPs using different elution modes and declare the practicality of such CSPs in stereoselective HPLC. GRAPHICAL ABSTRACT


Introduction
Enantioselective HPLC on chiral stationary phases (CSPs) has become a powerful tool in the drug discovery process, where it is used not only for chiral analyses but also for the fast attainment of enantiopure drug candidates for preliminary comparative biological testing of bioactive enantiomers. [1][2][3] The impetus for reliance on chromatography has been fueled by advances in CSPs that allowed for the reliable, robust, and efficient resolution of milligram to gram scale of chiral drug molecules in a timely fashion. [4] The prochiral pharmacophoric moiety 2-pyrazolines (4,5-dihydro-1H-pyrazole) is a crucial part of numerous drug structures with a wide variety of pharmacological activities such as anticancer, [5] analgesic, [6] hypoglycemic, [7] antitumor, [8] anticonvulsant, [9] anti-inflammatory, [10] antibacterial, [11] and antifungal activities. [12] It has been well established that the absolute configuration of the chiral center at C-5 of the 5-substituted 2-pyrazolines can be an important modulator of their biological activities. [13] In previous studies, we have reported on the synthesis of libraries of N-substituted 5-(1,3-benzodioxol-5-yl)-3-tert-butyl-4,5-dihydro-1H-pyrazole derivatives ( Figure 1) that showed potent activity in the racemic form. [9,11] The pharmacological and toxicological properties of pure enantiomers of these derivatives are therefore very interesting to be assessed. Pure enantiomers could be achieved through either asymmetric synthesis or stereoselective resolution of racemates. In both cases, chiral analytical assays are prerequisite either to evaluate the stereochemical purity of the stereoselective synthesis products or to carry out enantioselective resolution of racemates. Also, chiral bioanalytical assays may be required for further in vitro and in vivo investigations.
In continuation of our previous studies on 2-pyrazoline candidates 1-22, we discuss here the development of chiral analytical techniques of compounds  under a variety of conditions. The potential of two polysaccharide-based chiral columns (Lux amylose-2 and cellulose-2) has been investigated. Also, different mobile-phase compositions were used to explore the effect of solvents on the stationary phase's selectivity and recognition abilities.

Instrumentation and analytical conditions
The HPLC unit was Agilent 1100 series apparatus equipped with a quaternary pump, a vacuum degasser, autosampler, column compartment, and a diode array UV-detector. The signal was acquired and processed by HP Chemstation software. The columns used were Lux amylose-2 (amylose tris (5-chloro-2-methylphenylcarbamate) and Lux cellulose-2 (cellulose tris(3-chloro-4-methylphenylcarbamate)) (Phenomenex, Le Pecq, France). The dimensions of both columns are 250 mm � 4.6 mm, 3 µm. The flow rate was 1 mL min À 1 . All the samples were measured at wavelength 254 nm at 25°C. The optical rotation of compound 8 has been measured using KRUSS P8000 polarimeter, Optronic, Germany.
Normal elution mode, mixtures of alkane and lower alcohols as polar modifier (methanol, ethanol, and 2-propanol), is the recommended and most reported mobile phases for coated polysaccharide-based CSP. One elution mode is not adequate to achieve baseline separation of versatile analytes with diverse properties. Hence, the compatibility of the recently launched Lux columns (amylose-2 and cellulose-2) with all elution modes (normal, reverse, polar ionic, and polar organic) provided a wider horizon for chiral separation on coated polysaccharide-based CSPs.
The exact chiral recognition mechanisms of polysaccharide-based CSPs have not been fully revealed, although expectations about formation of solute-CSP complexes through inclusion of the enantiomers into the chiral cavities of the CSP were documented. [2,3] Nonspecific interactions control the retention of the enantiomer, and stereoselective interactions regulate the separation. [1,[13][14][15][16] Solute-CSP complexes are formed mainly through noncovalent molecular interactions such as hydrogen bonding, dipole-dipole, and π-π interactions between the compounds and the chiral selector groups attached to the polysaccharide. [1,[15][16][17] The supramolecular configuration of the polysaccharide shows dissimilar chiral cavities. The cavity of amylose is helical whereas cellulose is straight polymer chains. However, for both amylose and cellulose, the polar carbamate groups of the chiral selector are positioned inside whereas hydrophobic aromatic groups are located outside the polymer chain. [18] As well, the geometric characteristics (such as size, shape, and location of the functional groups) of the solute crucially contribute to the chiral recognition by defining the fitting extent of the solute into the chiral cavities and the strength of attractive interactions between various functional groups in the solute and the CSP. [1,16] At the inception of the present study, the recommended mixtures of n-hexane/2-propanol or ethanol have been used to investigate the elution and separation characters of compound 4 on amylose-2 column. Using a gradient of 20-50% 2-propanol as polar modifier in n-hexane failed to give baseline separation besides late elution. The replacement of 2-propanol with ethanol led to baseline separation at 8/2 v/v within 20 min R s ¼ 1.54. Furthermore, the use of 50% ethanol in n-hexane enhances the resolution within shorter run time 10 min R s ¼ 1.77 (Table 1 and Figure 2).

Enantioseparation using N-hexane/ethanol 1/1 V/V
Based on the efficiency of n-hexane/ethanol 1/1 v/v in separation of compound 4, this mobile phase has been selected for further investigations for separation of the 2-pyrazoline derivatives 1-22 on both amylose-2 and cellulose-2 columns. The results are summarized in Table 2.
Both amylose-2 and cellulose-2 have exhibited excellent chiral recognition abilities for all compounds except for the fourth group (compounds 19-22) on amylose-2.
It was of interest to discuss the resolution of compounds 4, 12, and 19-22. The resolution of compound 4, the most polar compound with nonsubstituted carboxamide side chain, has been tremendously enhanced from 1.71 on amylose-2 to 21.07 on cellulose-2 accompanied with delayed elution (35 min). On the other hand, compound 12, the most nonpolar with chloroacetyl side chain, showed the reverse situation being well separated on amylose-2 (R s ¼ 7.85) with late elution (25 min) but not separated at all on cellulose-2. Moreover, compounds 19-22 (fourth group) that have added methylene bridge compared with compounds 13-17 (third group) were fully separated on cellulose-2 with optimal R s 8-18 but completely unresolved on amylose-2. Further substitutions in the aryl or the alicyclic amine moieties were noncrucial for the chiral recognition using n-hexane/ethanol 1/1 v/v. However, the 4-OH group has enhanced the resolution of compounds 16 and 21 on cellulose-2 column when compared to the nonsubstituted piperidine derivative 15 and 20 (Supplementary Figure 1). It is worth mentioning that cellulose-2 is better for relatively polar compounds (like 4) and amylose-2 is the best for relatively nonpolar compounds (like 12) using n-hexane/ ethanol 1/1 v/v. This has been confirmed by superior resolutions of compounds 1-3 and 5-11 (less polar derivatives of 4) in comparison to 4 on amylose-2 that contrasts the obtained resolution when cellulose-2 was used. Regarding compounds 18-22, the occurrence of alicyclic amine moiety of compound 12 completely omits the recognition abilities of amylose-2 column but substantially improves the chiral recognition ability of cellulose-2 (Supplementary Figure 1). This could be explained by the decrement of lipophilicity of compound 12 -through addition of alicyclic amines-which is favored by cellulose-2 but not ideal for amylose-2 column. Concerning

Enantioseparation using ethanol 100%
Motivated by the role of ethanol in the enhancement of chiral recognition of amylose-2 and cellulose-2 columns and the compatibility of such columns with highly polar solvents, it was interesting to investigate the ingenuity of pure ethanol in the separation of tested compounds. Results are summarized in Table 3. It was noticeable that the chiral recognition by both amylose-2 and cellulose-2 using 100% ethanol is very close to that of using n-hexane/ethanol 1/1 v/v. All compounds, except the fourth group (18-22) on amylose-2, have been separated within shorter time with lower resolutions but still baseline-separated. Meanwhile, cellulose-2 showed better baseline separations with extremely sharp, fast eluted peaks in comparison to amylose-2 using ethanol 100% or cellulose-2 using n-hexane/ Figure 2). It is noteworthy that the maximum retention time for all compounds in cellulose-2 column using 100% ethanol was 12 min. Compound 4 has been eluted within 12 min and R s ¼ 8.17 ( Supplementary  Figure 2), which is so much better if compared to the elution on cellulose-2 using n-hexane/ethanol 1/1 v/v (35 min and R s ¼ 21.07). As well, compound 12, which was not separated on cellulose-2 using n-hexane/ethanol 1/1 v/v (Supplementary   Figure 1), has been resolved within 10 min and R s ¼ 5.44, which is highly superior over amylose-2 that separates compound 12 after 19 min and R s ¼ 3.94 using 100% ethanol (Supplementary Figure 2). The ability of cellulose-2 to resolve compound 12 using 100% ethanol could be justified by the absence of n-hexane that mainly interferes with the nonpolar interactions, which are the major interaction forces between compound 12 and CSP. The polarity effect is also working while using 100% ethanol; thus, cellulose-2 still shows better resolution for relatively polar compounds (compound 4 showed the best resolution using 100% ethanol).

Enantioseparation using methanol 100%
The ingenuity of ethanol 100% in chiral separation of compounds 1-22 on polysaccharide-based CSP acted as an impetus to explore the cleverness of further polar solvents in chiral resolution of this group of compounds on cellulose-2 and amylose-2 columns. 100% methanol has been investigated where results are summarized in Table 4. High polarity of methanol led to weakly retained compounds translated in short runs with bad resolution. The only remarkable point using absolute methanol is complementary resolving abilities displayed by amylose-2 and cellulose-2 columns. Thus, compounds that have not been separated on amylose-2 (first, third and fourth groups) have been separated on cellulose-2 and vice versa (Supplementary Figure 3). In general, methanol 100% as a mobile phase is not the best choice for chiral resolution of such group of compounds on the used CSP. With the exception of compounds 1 and 4 on cellulose-2 and 11 on amylose-2, the resolution values did not exceed 2, and almost 50% of the compounds did not achieve baseline separation. With respect to such results n-hexane/ethanol 1/1 v/v and ethanol 100% look highly superior over methanol 100% for compounds 1-22.

Enantioseparation using acetonitrile 100%
The recent research reporting the significance of using acetonitrile on cellulose and amylose-based CSP [19][20][21] acted as an impetus to investigate acetonitrile efficacy in separation of compounds 1-22. Indeed, acetonitrile showed greater advantage over methanol and comparable efficacy to ethanol and n-hexane/ethanol mobile phases. In terms of peak sharpness and retention time, acetonitrile is the best mobile phase for this group of compounds using these CSPs. Using cellulose-2, baseline separations have been achieved for all compounds except 18 (Table 5 and Supplementary Figure 4).
Cellulose-2 is still sensitive to the polarity of the solute showing better recognition abilities for relatively polar compounds as seen by the resolution of compounds 4 and 12 compared to compounds 5-11 and 18-22 respectively (Supplementary Figure 4). Also, compounds 1-3 (lacking polar NH moiety) were inferiorly separated as their relatively polar analogues (compounds 5, 10, and 11 having NH group) on cellulose-2. Similarly, the third group (compounds 13-17 lacking lipophilic CH 2 moiety) was better separated on cellulose-2 than the fourth group (compounds 18-22 having the lipophilic CH 2 moiety). This was precisely the opposite when amylose-2 column was used. Further clue has been introduced by the OH group at C-4 of compounds 16 and 21, which has changed the feebly resolved (16) or unresolved (21) on amylose-2 to be fully resolved on cellulose-2 (Supplementary Figure 4). In contrary, compound 17 (with ethyl substitute in C-4) exhibited baseline separation on amylose-2, but only partial separation on cellulose-2 column has been observed (Supplementary Figure 4). Besides having very short runs, the ability of acetonitrile to resolve three members of the fourth group (compounds 18-22) on amylose-2 column represents a very important advantage over ethanol and n-hexane/ethanol systems.

Enantioseparation using acetonitrile/ethanol or methanol 1/1 V/V
To further explore the chiral recognition abilities of amylose-2 and cellulose-2 columns, a highly polar system (acetonitrile/ ethanol 1/1 v/v) has been investigated for the resolution of the tested compounds. Results are summarized in Supplementary Table 1 and Figure 5. The use of acetonitrile/ethanol as mobile phase did not introduce any added benefit over absolute ethanol or acetonitrile. It was advantageous for particular examples like compound 4, which has been baseline-separated within 6 min with R s ¼ 7.16 on cellulose-2. With regards to run time, acetonitrile/ethanol 1/1 v/v has achieved great improvement over 100% acetonitrile (36 min), 100% ethanol (12 min), and n-hexane/ethanol (35 min) for compound 4. The preference of cellulose-2 column toward more polar compounds has been changed with respect to third and fourth groups. Thus, using this solvent combination, compounds (18)(19)(20)(21)(22) were more efficiently resolved on cellulose-2 column than in compounds 13-17. However, in the case of amylose-2 column the situation was still similar to absolute ethanol or acetonitrile.
With respect to the number of compounds that were successfully baseline-separated, acetonitrile/methanol system showed superiority to acetonitrile/ethanol. Also, amylose-2 displayed greater chiral recognition abilities than cellulose-2 column using such highly polar combinations (Supplementary Table 2 and Figure 6).

Elution order in polysaccharides-based CSP
It has been previously documented that the elution order is reversible while altering between amylose and cellulose-based CSP. [22][23][24][25] More recently, other factors like additives to the mobile phase [19,26] or temperature [27] were found to alter the elution order in polysaccharide-based chiral selectors. In the present study further evidence has been introduced by compound 8, which was available in enantio-enriched form (ee 80% by HPLC n-hexane/ethanol 1/1 v/v on amylose-2) a ½ � 25 D ¼ þ 23° (c 1.0, CHCl 3 ). Using polar organic mode (ethanol, methanol, acetonitrile, acetonitrile/ethanol, or acetonitrile/methanol) the minor enantiomer was eluted first on amylose-2 and second on cellulose-2, but when using normal mode (n-hexane/ethanol) the minor enantiomer was first eluted on both columns (Figure 3). In our case, the probability of false reversal of the elution order [28] was neglected since the detection was not based on optical rotation. The only reported justification for reversed elution order was the change in supramolecular configuration of the CSPs that could result from varying the elution mode. Occasionally, reverse order of elution happens when changing the mobile phase on the same CSP [29] as well as using the same mobile phase but changing the CSP. [30] In the current study, both cases have been reported since the elution order of compound 8 was reversed when changing between amylose-2 and cellulose-2 column under all polar organic mode mobile phases. As well, when changing the elution mode from polar organic to normal on cellulose-2 the elution order has been reversed ( Figure 3).

Conclusions
The polysaccharide-phenylcarbamate-based CSPs, Lux amylose-2 and cellulose-2, have exhibited outstanding chiral recognition abilities toward different bioactive chiral 2-pyrazolines 1-22 in both polar organic and normal elution modes. Very high resolutions were realized for most compounds under different elution modes. Mechanistic approach based on different interactions between the CSP and compounds 1-22 and its effects on the chiral recognition abilities of Lux columns have been discussed. It has been confirmed that the intermolecular forces involved in analyte retention and enantioseparation are diverse. Thus, both the mobile and stationary phases, as well as the nature of the analyte, are contributing to the chiral recognition. Several chiral analytical methods for compounds 1-22 have been introduced and they can be utilized for monitoring asymmetric reactions or optimizing preparative chiral HPLC resolutions. Polar organic mode (acetonitrile 100%) was suggested for reaction monitoring since it showed optimum resolution within short analysis times and favorable peak shape. Fast elution is very crucial when having several samples to be analyzed as in quality control work, reactions follow-up, or pharmacological and toxicological screening. Normal mode (n-hexane/ethanol) was instructed for the development of preparative HPLC methods due to the big difference in retention between two enantiomers that will support column loadability. Altering the mobile or the stationary phases led to varied affinity patterns of compound 8. Table 5. Separation parameters of compounds 1-22 on Lux 3 μ amylose-2 and cellulose-2 using acetonitrile 100%. Column