Tamás Sohajda

Separation and characterization of modified pregabalins in terms of cyclodextrin complexation, using capillary electrophoresis and nuclear magnetic resonance.

The (S)-(+)-isomer of 3-isobutyl-GABA (pregabalin), the blockbuster drug in the treatment of neuropathic pain has been separated from its R isomer by cyclodextrin modified capillary zone electrophoresis (CZE) using uncoated fused-silica capillary. Derivatization of the single isomer and the racemate with tosyl- and dansyl-chloride was carried out to introduce strong UV chromophores of different size. CE-pH titrations were performed to determine the dissociation constants for both derivatives. 30 cyclodextrin (CD) derivatives as chiral agents were used at four different pH values to study the enantioseparation of the differently protonated guest molecules. The separation was optimized as a function of CD concentration, buffer type and concentration, pH and applied voltage. For the tosylated derivate the best resolution (R(s)=2.76) was found with 6-monodeoxy-6-mono-(3-hydroxy)-propylamino-beta-cyclodextrin hydrochloride (PA-beta-CD) at pH 6.8, while with the same selector at pH 7.2 enantioseparation with an R(s) value of 4.32 could be achieved for the dansylated pregabalin. At pH 2.5 for the dansylated derivative trimethylated alpha- and beta-CD systems resulted the most significant separation (R(s)=7.38 and R(s)=7.74, respectively). Experiments with dual CD systems were carried out as well. The stoichiometry of the complexes was determined using the Job plot method and resulted in a 1:1 complex in both cases. The structures of the inclusion complexes were elucidated using 2D ROESY NMR experiments.

Chiral separation of asenapine enantiomers by capillary electrophoresis and characterization of cyclodextrin complexes by NMR spectroscopy, mass spectrometry and molecular modeling

The enantiomers of asenapine maleate (ASN), a novel antipsychotic against schizophrenia and mania with bipolar I disorder have been separated by cyclodextrin (CD) modified capillary zone electrophoresis for the first time. 15 different CDs were screened as complexing agents and chiral selectors, investigating the stability of the inclusion complexes and their enantiodiscriminating capacities. Although initially, none of the applied chiral selectors gave baseline separation, β-CD proved to be the most effective chiral selector. In order to improve resolution, an orthogonal experimental design was employed, altering the concentration of background electrolyte, organic modifier, pH, capillary temperature and applied voltage in a multivariate manner. The developed method (160 mM TRIS-acetate buffer pH 3.5, 7 mM β-CD, at 20 °C, applying 15 kV) was successful for baseline separation of ASN enantiomers (R(s)=2.40±0.04). Our method was validated according to ICH guidelines and proved to be sensitive, linear, accurate and precise for the chiral separation of ASN. Properties of the inclusion complexes, such as stoichiometry, atomic level intermolecular host-guest connections are proposed on the basis of ROESY NMR measurement, ESI-MS spectrometry and molecular modeling studies. It was found that the ASN-β-CD complex is of 1:1 composition, and either of the aromatic rings can be accommodated in the β-CD cavity.

Separation of vinca alkaloid enantiomers by capillary electrophoresis applying cyclodextrin derivatives and characterization of cyclodextrin complexes by nuclear magnetic resonance spectroscopy

In this work, the enantiomeric separation of three vinca alkaloid enantiomers (vincamine, vinpocetine and vincadifformine) has been investigated in an aqueous capillary electrophoresis (CE) system using cyclodextrins (CDs). The investigated CDs were the native alpha-, beta-, and gamma-CDs and their hydroxypropylated, randomly methylated, carboxymethylated and sulfobutylated derivatives. The first part of this study consisted of the determination of the apparent averaged complex stability constants with the selected CDs. Several parameters, such as the nature and the concentration of the CD, were studied and were found to have a significant effect on the enantiomeric resolution for all studied compounds. All three vinca alkaloids were successfully enantioseparated with CDs where different migration orders were observed in case of several CDs depending on the cavity size or substituent of the host. Chiral separation and determination of the stability constants were also performed with NMR spectroscopy which confirmed the CE results. Averaged stoichiometries of the complexes were determined using the Job plot method resulting in a 1:1 complex irrespective of the alkaloid enantiomers or cyclodextrin derivative. The structures of the inclusion complexes were elucidated using 2D ROESY NMR spectroscopy. On the basis of NMR results reversal of enantiomer migration order was clarified in various cases.

Characterization of aspartame-cyclodextrin complexation

The inclusion complex formation of aspartame (guest) and various cyclodextrins (host) were examined using 1H NMR titration and capillary electrophoresis. Initially the protonation constants of aspartame were determined by NMR-pH titration with in situ pH measurement to yield log K1=7.83 and log K2=2.96. Based on these values the stability of the complexes formed by aspartame and 21 different cyclodextrins (CDs) were studied at pH 2.5, pH 5.2 and pH 9.0 values where aspartame exists predominantly in monocationic, zwitterionic and monoanionic form, respectively. The host cyclodextrin derivatives differed in various sidechains, degree of substitution, charge and purity so that the effect of these properties could be examined systematically. Concerning size, the seven-membered beta-cyclodextrin and its derivatives have been found to be the most suitable host molecules for complexation. Highest stability was observed for the acetylated derivative with a degree of substitution of 7. The purity of the CD enhanced the complexation while the degree of substitution did not provide obvious consequences. Finally, geometric aspects of the inclusion complex were assessed by 2D ROESY NMR and molecular modelling which proved that the guests aromatic ring enters the wider end of the host cavity.

Efficacy and ototoxicity of different cyclodextrins in Niemann–Pick C disease

Niemann–Pick type C (NPC) disease is a fatal, neurodegenerative, lysosomal storage disorder characterized by intracellular accumulation of unesterified cholesterol (UC) and other lipids. While its mechanism of action remains unresolved, administration of 2‐hydroxypropyl‐β‐cyclodextrin (HPβCD) has provided the greatest disease amelioration in animal models but is ototoxic. We evaluated other cyclodextrins (CDs) for treatment outcome and chemical interaction with disease‐relevant substrates that could pertain to mechanism.

Chiral recognition of imperanene enantiomers by various cyclodextrins: A capillary electrophoresis and NMR spectroscopy study

The enantiomers of imperanene, a novel polyphenolic compound of Imperata cylindrica (L.), were separated via cyclodextrin‐modified capillary electrophoresis. The anionic form of the analyte at pH 9.0 was subject to complexation and enantioseparation CE studies with neutral and charged cyclodextrins. As chiral selectors 27 CDs were applied differing in cavity size, sidechain, degree of substitution (DS) and charge. Three hydroxypropylated and three sulfoalkylated CD preparations provided enantioseparation and the migration order was successfully interpreted in each case in terms of complex mobilities and stability constants. The best enantioresolution (RS = 1.26) was achieved using sulfobutyl‐ether‐γ‐CD (DS ∼4), but it could be enhanced by extensive investigations on dual selector systems. After optimization (CD concentrations and pH) RS = 4.47 was achieved using a 12.5 mM sulfobutyl‐ether‐γ‐CD and 10 mM 6‐monodeoxy‐6‐mono‐(3‐hydroxy)‐propylamino‐β‐cyclodextrin dual system. The average stoichiometry of the complex was determined with Jobs method using NMR‐titration and resulted in a 1:1 complex for both (2‐hydroxy)propyl‐β‐ and sulfobutyl‐ether‐γ‐CD. Further NMR experiments suggest that the coniferyl moiety of imperanene is involved in the host‐guest interaction.

Evaluation of the interaction between sitagliptin and cyclodextrin derivatives by capillary electrophoresis and nuclear magnetic resonance spectroscopy

An aqueous capillary electrophoretic method was developed for chiral analysis of the novel anti‐diabetic drug, sitagliptin. The acid–base profiling of the analyte was carried out using both capillary electrophoresis and nuclear magnetic resonance pH titrations. The apparent complex stability and chiral separation properties were investigated with 30 different cyclodextrins under acidic conditions. The effect of concentration and pH of the BGE, temperature of the capillary, and the type and concentration of the chiral selector on the enantiomer resolution were thoroughly investigated. The effects of dual cyclodextrin systems on separation were also extensively studied. Complete separation of racemic sitagliptin with good resolution (RS=2.24) was achieved within a short time (15 min) with optimized parameters (10°C, pH=4.4, 40 mM phosphate buffer) of a sulfobutylether‐β‐cyclodextrin (averaged degree of substitution ∼4) and native β‐cyclodextrin dual system. The averaged stoichiometry of the inclusion complex was determined using the Job plot method with both 1H and 19F NMR experiments and resulted in a 1:1 complex. The structure of the inclusion complex was elucidated using 2‐D ROESY NMR experiments.

Chiral recognition of dapoxetine enantiomers with methylated-gamma-cyclodextrin: a validated capillary electrophoresis method.

The enantiomers of dapoxetine, a serotonin transporter inhibitor for the treatment of premature ejaculation have been separated by cyclodextrin modified capillary zone electrophoresis using uncoated fused-silica capillary. Over 20 cyclodextrins were screened as chiral selectors, investigating the stability of the inclusion complexes and enantioseparating properties. According to the preliminary experiments as chiral selector randomly methylated-γ-cyclodextrin was chosen. The basic chemical and instrumental parameters of enantioseparation as concentration of buffer, chiral selector and organic additive, pH, temperature and applied voltage were optimized afterwards using an orthogonal experimental design. Using this methodology not only the optimal parameter values for chiral separation (15 °C, +15 kV, 70 mM acetate, 20 v/v% MeOH, pH*=4.5, 3 mM methylated-γ-CyD) but also the significance order of factors on resolution was determined. Applying these parameters an optimal resolution of 7.01 was achieved. The optimized method was then validated according to the ICH guideline Q2 (R1) with regard to repeatability, linearity range, LOD, LOQ, accuracy and robustness.

Interactions of non-charged tadalafil stereoisomers with cyclodextrins: Capillary electrophoresis and nuclear magnetic resonance studies

The single isomer drug R,R-tadalafil (Cialis) contains two chiral centers thus four stereoisomers (R,R-, S,S-, S,R- and R,S-tadalafil) exist, however, only the most potent inhibitor, the R,R-tadalafil is in clinical use. In our study, over 20 charged cyclodextrin (CD) derivatives were studied for enantiospecific host-guest type interactions in CD-modified capillary electrophoresis. Tadalafil stereoisomers are non-charged; therefore, their electrophoretic separation poses a challenge. Several candidates of both positively and negatively charged hosts were found to be effective for the enantioseparation. Eight out of the beta derivatives and three of alpha derivatives (including sulfated, sulfoalkylated, carboxyalkylated and amino derivatives) resolved all four stereoisomers partially or completely. Cavity size-dependent absolute enantiomer migration order (EMO) reversals were observed in the case of sulfopropyl-alpha (EMO: R,S; S,R; R,R; S,S) and sulfopropyl-beta (S,S; R,R; S,R; R,S) derivatives, while substituent-dependent partial EMO reversals were detected for sulfobutyl-ether-alpha (R,S; S,R; S,S; R,R) and sulfated-alpha-CD (R,R; S,S; R,S; S,R) selectors. Complexation-induced (1)H NMR chemical shift changes reflected that the benzodioxole moiety plays a major role in cavity size-dependent EMO reversal. Sulfobutyl-ether-alpha-CD was the only selector that provided the desired EMO in which the clinically applied eutomer R,R-tadalafil migrates last. Finally, an electrophoretic method applying a background electrolyte (BGE) containing 75 mM Tris-acetic acid buffer (pH 4.75) and 7 mM sulfobutyl-ether-alpha-CD was developed for the baseline resolution of all isomers at 25 °C and +25 kV applied voltage.

Tapentadol enantiomers: Synthesis, physico-chemical characterization and cyclodextrin interactions.

The complete physico-chemical characterization of the single enantiomer analgesic drug R,R-tapentadol was quantitated in terms of protonation macro- and microconstants and octanol-water partition coefficient using pH-potentiometry, UV-pH and (1)H NMR-pH titrations. The protonation macroconstants were found to be logK1=10.59±0.01 and logK2=9.44±0.01, while the individual basicity of each protonation site was found to be logk(O)=9.94 and logk(N)=10.48 for the phenolate and tertiary amine functions, respectively. As a consequence, the zwitterionic form of tapentadol predominates in aqueous solutions. The potential optical impurity (S,S-tapentadol) was synthesized for the first time in a seven-step chiral synthetic procedure. The enantiomers of tapentadol were separated by cyclodextrin modified capillary zone electrophoresis. Over 15 cyclodextrins were investigated in terms of apparent complex stability and screened as chiral selectors, and the sulfated alpha-cyclodextrin was found to resolve the enantiomers with excellent resolution (Rs=16.2 and 9.1) at pH 4.75 and pH 9.0, respectively. The system containing 12mM selector in a 50mM TRIS-acetate buffer was amenable to detect S,S-tapentadol potential optical impurity at 0.1% concentration level.