INTRODUCTION:

Heterocyclic chemistry constitutes a vast and important domain in organic synthesis and Heterocyclic compounds exist naturally in essential molecules such as nucleic acids and alkaloids. The synthesis of heterocyclic compounds is extensively used in various applications (materials, stains, herbicides).

The 2-aryl-imino-N-(2-aryl)-thiazoline compounds have been developed and studied for a long time due to their biological activities.^1-3 The rigid, semi planar structures of such compounds provide sites for a number of important substitutions. As well, the presence of one or several heteroatoms permits interactions, of electrostatic type (hydrogen-bond, Vander Waals-bond.), with the biologic target, while the aromatic cycles allow other interactions of hydrophobic nature.

The separation of isomers with cyclodextrins has been studied intensively in the last decades.^4-8Natural cyclodextrins (CD) constitute a family of cyclic oligosaccharides comprising repetitive 6, 7, or 8 glucose units (a-, b-, g-CD, respectively).

The inside of the molecule forms a hydrophobic cavity, while the outer surface is hydrophilic, enabling it to act as a host for a wide variety of lipophilic drugs and components.⁹ Cyclodextrins are capable of forming molecular inclusion complexes with hydrophobic compounds, thus greatly enhancing their solubility in water.^10-14. Moreover, cyclodextrins can increase the stability, and the bioavailability of the guest molecule.¹⁵

In this study, the separation of the various atropisomers of racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine (Figure 1) has been carried out by application of various native cyclodextrin (alpha-, beta-, and gamma-CD) and cyclodextrin derivatives, such as hydroxypropyl-a-cyclodextrin, hydroxypropyl-b-cyclodextrin and hydroxypropyl-g-cyclodextrin. Native cyclodextrin (alpha-, beta-, and gamma-CD), hydroxypropyl-a-cyclodextrin and hydroxypropyl-b-cyclodextrin were found not feasible for the separation in reversed phase high-performance liquid chromatography (RP-HPLC). Only Hydroxypropyl-g-cyclodextrin (HP-g-CD) was found feasible for the separation as shown in (Table 1).

Figure 1: Structure of N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine

Table 1: Effect of Cyclodextrin

Experiment	Cyclodextrin	Rs
1	α	0.60
2	HP-α-CD	0.61
3	β	0.74
4	HP-β-CD	0.52
5	g	<0.5
6	HP-g-CD	1.38

EXPERIMENTAL:

Chemicals and reagents

The preparation and purification of racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine has been described by Bouchkara et al.¹⁶ . HP-g-CD, Hexane and propan-2-ol of HPLC grade were purchased from Sigma-Aldrich Co. All the solvents used for column chromatography were of HPLC grade and distilled prior to use. Water was purified by triple distillation.

Instrumentation

Chromatographic separations were carried out on a Shimadzu HPLC system (UFLC) consisting of a thermostated-column device, a degasser and a variable-wavelength UV detector. The column used for analytical HPLC was C-18 (150 mm × 4.6 mm). The mobile phase was a mixture of hexane and propan-2-ol with a flow rate of 0.5-1 ml/min. The wavelength of UV detector was set at 254 nm and the column was operated at room temperature. The injection volume was 5 µL.

Preparation of solutions

Racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine is accurately weighted, transferred to volumetric flasks and dissolved in solution of mobile phase 50:50 (v/v) hexane-propan-2-ol to make individual stock solutions of 1 mmol/L. The stock solution is stored at 4 ◦C and was later diluted with mobile phase to the recommended concentration of 0.01 µmol/L.

Preparation of inclusion complexes

10 µL of 0.25 µmol/L concentration of cyclodextrin and 10 µL of 0.01 µmol/L of solute were mixed and shaken at the temperature of 25°C to obtain a stable state of solubilization.

Mobile phase optimization and effect of incubation time

The influence of mobile phase composition was studied, whereby the experiment was carried out in the presence of different volumes of hexane ranging from 0% to 100% (v/v) while keeping the HP-g-CD (0.125 µmol/L) and substrate (0.005 µmol/L) concentrations constant.

To determine the effect of time on resolution, the experiment was performed at different incubation times (Table 2) with a flow rate of 0.5 mL/min while keeping the conditions as described above.

Table 2: Effect of time on the Atropisomers Resolution

Time (hours)	A1 – RT (min)	A2 – RT (min)	SF	Rs
0	5.50	6.76	1.26	1.24
0.5	5.52	6.76	1.24	1.08
1	5.49	6.75	1.26	1.11
1.5	5.50	6.75	1.25	1.13
2	5.53	6.77	1.24	1.11
24	5.51	6.76	1.25	1.12

RT: Retention Time (t₁and t₂); Rs: Resolution; SF (α): Separation Factor

Partition coefficient determination

The lipophilicity of racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine was evaluated from their n-hexane–water partition coefficient, K_P, as follows: Equal volumes of freshly prepared solute/hexane solutions (0.25 mol/L) and of solute/water solutions were mixed and vigorously stirred at 37°C for 1 h. The two phases were separated by brief centrifugation (1000 g for 20 s). The solute concentration in either the hexanic or the aqueous phase was determined by a Schimadzu UV-VIS spectrophotometer (UV-1800). K_P was evaluated as the ratio of the solute concentration in n-hexane to that in water.

RESULTS:

Separation of atropisomers

The chromatograph of racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine showed a single peak (Figure 2) indicating the purity of the compound. In order to achieve the separation of the atropisomers, the influence of the concentration of HP-g-CD on the resolution were examined. Addition of HP-g-CD (0.05-250 µmol/L) to the analyte using a mobile phase composed of 50:50 (v/v) hexane-propan-2-ol was studied. The atropisomeric separation ability was evaluated by resolution. The best results were obtained using 0.125 µmol/L HP-g-CD while the higher concentration of 25 µmol/L and above showed a decrease in the efficiency of the column.

Figure 2: Chromatogram of racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine.

A typical chromatogram for the resolution of racemic-N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine using 0.125 µmol/L HP-g-CD and mobile phase of hexane-propan-2-ol (50:50, v/v) is shown in (Figure 3). The two atropisomers clearly appear at Rt 1.99 and 2.52 min.

Figure 3: Separation of atropisomers at 0.125µM concentration of HP-g-CD, 0.005µM concentration of analyte.

Effect of mobile phase parameter

Hexane-propan-2-ol proportion was found to be important for improvement of enantioselectivity. Different percentages (a range of 0-100%) of Hexane-propan-2-ol solutions were employed and evaluated. As shown in (Figure 4), the best results were obtained for a 50% hexane. Resolutions less than 0.5 were observed for hexane percentages below 20% and higher than 80%.

Figure 4: Effect of hexane proportion on the atropisomers resolution. 0.125µmol/L concentration of HP-gamma-CD, 0.005µmol/L concentration of mixture are used.

Effect of Time

The effect of time was also investigated and, as shown in (Table 2), there was no observed difference in resolutions. This indicates that that the formation of the complex was obtained in a very short time compared to periods usually needed (several seconds vs. 15 min)¹⁷. The extreme rapidity of the complex formation may be justified by the short hydrophobic chains of the analyte which can easily fill the hydrophobic cavities of CD molecules. Accordingly, the study was carried out employing a complex consisting of a molar ratio of 25:1 HP-g-CD: analyte and a flow rate of 0.5 mL/min.

Partition coefficient determination

The success of atropisomers separation greatly depends on the polarity of the analyte. In fact, the use of a lipophylic compound gives the best results due to the lipophylic cavity of the cyclodextrin. Therefore, the lipophylic characteristic will enhance the inclusion of the anlyte in the lipophylic cyclodextrin cavity.^{18, 19}

The partition coefficient Kp is the ratio of the concentrations of the analyte in an organic solvent (hexane) versus that in water. Since the capacity of inclusion of the anlyte in the cavity of the cyclodextrin is directly related to lipophilicity, the determination of this coefficient is essential to determine if the inclusion process is possible.

Using photometry,²⁰ Kp was found to be 29, which indicates a preference for N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine towards the lipid phase thus proving the possibility of penetrating the cyclodextrin cavity.

DISCUSSION:

The optimal separation conditions may differ based on three factors: the chemical structure of the compound, the experimental parameters (concentration and mobile phase) and the structure of the cyclodextrin selector. In this study we compared the effectivity of cyclodextrin selectors (a-, HP-a-CD, b-, HP-b-CD, g- and HP-g-CD). The derivatives of cyclodextrin were chosen on the basis of their selective properties, the previous usage in other studies, and availability.

Among the three cyclodextrins and the three cyclodextrin derivatives applied, the analyte showed stereoselective interaction only with HP-g-CD. The reason could be related to the capacity and the polarity of HP-g-CD cavity.

Thus, a-, HP-a-CD, b-, and HP-b-CD seem to have a small hydrophobic cavity preventing the analyte inclusion phenomenon from taking place. g- and HP-g-CD have a large cavity that allows inclusion. The large size of the cavity in the case of g-CD encourages the exit of the analyte after inclusion. Only HP-g-CD shows a persistent inclusion of the analyte, due to the presence of the hydroxypropyle groups. These results prove that the predominating separation mechanism of CD for 2-aryl-imino-N-(2-aryl)-thiazoline compounds was based on the phenomenon of analyte-CD inclusion, where a transient diastereomeric complex is formed between the CD and the analyte.^21-22

CONCLUSION:

The objective of this study was the atropisomers separation of N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine. The method used consists in adding the cyclodextrin with the analyte. A complex analyte-cyclodextrin forms, and has passed on a stationary phase in HPLC.

Several types of cyclodextrins have been tried. Only the HP-g-CD showed a capacity to form the complex and to achieve the separation. The use of the hydroxypropylated cyclodextrins is giving new opportunities for the study of 2-aryl-imino-N-(2-aryl)-thiazoline compounds.

The method involves direct separation of the atropisomers on a reverse stationary phase with cyclodextrin as chiral selector at specific mobile phase. The chromatographic conditions described herein provide a novel, rapid and reliable approach for separation and analysis of N-[(2Z)-4-methyl-3-(2-methylphenyl)-1,3-thiazol-2(3H)-ylidene]-N-(2-chlorophenyl)amine atropisomers from synthesized sample. Supplementary studies in NMR must be made to confirm the inclusion of the analyte inside the cavity of cyclodextrin.

ACKNOWLEDGEMENTS:

This research is supported by the management committee of scientific research at the Lebanese University.

REFERENCES:

1. Roussel C, Vanthuyne N, Shineva N, Bouchekara M. and Djafrie A. Atropisomerism in the 2-arylimino-N-(2-aryl)-thiazoline series. Arkivoc. 2008 (Viii): 28-41.

2. Roussel C, Vanthuyne N, Shineva N, Bouchekara M, Djafri A, Elguero J, Alkorta I. Atropisomerism in the 2-Arylimino-N-(2-hydroxyphenyl)thiazoline Series: Influence of Hydrogen Bonding on the Racemization Process. Journal of Organic Chemistry. 73(2), 2008: 403-411.

3. Loftsson T. and Brewster M.E., Pharmaceutical applications of cyclodextrins, Journal of Pharmaceutical Sciences. 85(10); 1996: 1017–1025.

4. Szejtli J. Introduction and general overview of cyclodextrin chemistry. Chemical Review. 98(5); 1998: 1743-1753.

5. Szemán J, Ganzler K, Salgó A. and Szejtli J. Effect of the degree of substitution of cyclodextrin derivatives on chiral separations by high-performance liquid chromatography and capillary electrophoresis. Journal of. Chromatography A. 728 (1-2); 1996: 423-431.

6. Weseloh G, Bartsch H. and König W.A. Separation of basic drugs by capillary electrophoresis using selectively modified cyclodextrins as chiral selectors. Journal of Microcolumn Separations. 7(4), 1995: 355-363.

7. Arnaud S, Varesio E, Dreux M, and Veuthey J.L. Binding constant dependency of amphetamines with various commercial methylated-beta-cyclodextrins. Electrophoresis., 20 (13); 1999: 2670-2679.

8. Szemán J, Roos N, and Csabai K. Ruggedness of enantiomeric separation by capillary electrophoresis and high-performance liquid chromatography with methylated cyclodextrins as chiral selectors. Journal of Chromatography A. 763 (1); 1997: 139-147.

9. Wainer IW. and Drayer D. Drug stereochemistry.Analytical methods and pharmacology. Marcel Dekker, New York, 1998.

10. Hermansson J. and von Bahr C. Simultaneous determination of d- and l-Propranolol in human plasma by high-performance liquid chromatography. Journal of Chromatography B. 221 (1); 1980: 109–117.

11. Hermansson J. Separation and quantification of (R)- and (S)-propranolol as their diastereomeric derivatives in human plasma by reversed-phase ion-pair chromatography. Acta Pharmceutica Suecica. 19 (1), 1982: 11–24.

12. Thompson JA, Holtzman JL, Tsuru M. and Lerman CL. Procedure for the chiral derivatization and chromatographyc resolution of R-(+)- and S-(−)-propranolol. Journal of Chromatography A. 238(2); 1982: 470–475.

13. Matsuo N. and Ohno N. Preparation of optically active 1-acetoxy-2-aryloxypropionitriles and its application to a facile synthesis of (S)-(−)-propranolol. Tetrahedron Letters. 26 (45); 1985: 5533–5534.

14. Terao Y, Murata M, Achiwa K, Nishino T, Akamatsu M. and Kamimura M. Highly efficient lipase-catalyzed asymmetric synthesis of chiral glicerol derivatives leading to practical synthesis of S-propranolol. Tetrahedron Letters. 29 (40); 1988: 5173–5176.

15. Del Valle EMM. Cyclodextrins and their uses, a review. Process Biochemistry. 39 (9); 2004: 1033–1046.

16. Bouchekara M, Djafrie A, Vanthuyne N. and Roussel C. Atropisomerism in some N,N’-diaryl-2-iminothiazoline derivatives: chiral separation and configurational stability. Arkivoc. 2002(x): 72-79.

17. Jordheim LP, Degobert G, Diab R, Peyrottes S, Perigaud C, Dumontet C. and Fessi H. Inclusion complexes of a nucleotide analogue with hydroxypropyl-beta-cyclodextrin. Journal of Inclusion Phenomena and Macrocyclic Chemistry. 63 (1-2); 2009: 11–16.

18. Butkus E, Martins JC, and Berg U. 1H NMR spectroscopic study of the interaction between cyclodextrins and bicyclo[3.3.1]nonanes. Journal of Inclusion Phenomena and Macrocyclic Chemistry. 26 (4), 1996: 209–218.

19. Grillo R, de Melo NF, Moraes CM, de Lima R, Menezes CM, and Ferreira EI. Study of the interaction between hydroxymethylnitrofurazone and 2-hydroxypropyl-betacyclodextrin, Journal of Pharmaceutical and Biomedical Analysis. 47 (2); 2008: 295–302.

20. Petracek FJ, and Zekhmeister L. Determination of partion coefficients of carotenoids as a tool in pigment analysis. Analytical Chemistry. 28 (9), 1956: 1484–1485

21. Lamparczyk H, and Zarzycki PK. The influence of temperature on the multiple separation of estrogenic steroids using mobile phases modified with β-cyclodextrin in high-performance liquid chromatography. Journal of Chromatography A. 668 (2), 1994: 413–417.

22. Tarkanyi G. Quantitative approach for the screening of cyclodextrins by nuclear magnetic resonance spectroscopy in support of chiral separations in liquid chromatography and capillary electrophoresis enantioseparation of norgestrel with alpha-, beta- and gamma-cyclodextrins. Journal of Chromatography A. 961 (2), 2002: 257–276.Esmolol hydrochloride (ESM, methyl 3-{4-[2-hydroxy-3-(isopropylamino) propoxy] phenyl} propionate hydrochloride.

Received on 27.09.2011 Modified on 11.10.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1800-1803