Diastereomeric Thioureas Research Articles

INDIRECT HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ENANTIOSEPARATION OF RECEMIC AMINO ALCOHOLS WITH 1,3-DIACETOXY-1-(4-NITROPHENYL)-2-PROPYL ISOTHIOCYANATE AS DERIVATIZING AGENT

Synthetically and pharmaceutically interesting racemic amino alcohols with two adjacent chiral centres were analysed by means of indirect high performance liquid chromatography. For resolution of the enantiomers, the recently developed chiral derivatizing agent (1R,2R)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propyl isothiocyanate ((R,R)-DANI) was applied. The diastereomeric thioureas produced after derivatization were separated under reversed-phase conditions. Of the organic modifiers applied in the eluent, methanol proved much more effective than acetonitrile.

Reversed-phase high performance liquid chromatographic separation of the enantiomers of terbutaline by derivatization with 2,3,4,6-tetra-o-acetyl-beta-D-glucopyranosyl isothiocyanate.

The enantiomers of the bronchodilator terbutaline were separated by reversed-phase high performance liquid chromatography after derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate(GITC) reagent. The derivatization proceeded quantitatively within 1 h at room temperature. The corresponding diastereomeric thiourea derivatives were well resolved on an ODS column with acetonitrile-acetate buffer as a mobile phase. Elution orders of the diastereomers were confirmed by derivatization of R-(-)-terbutaline and S-(+)-terbutaline which were collected by semi-preparative chiral HPLC using Sumichiral OA-4700 column. The native fluorescence of terbutaline was quenched by derivatization with GITC. The detection limit was 25 ng when monitored at UV 278 nm.

Application of (1 S,2 S)- and (1 R,2 R)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate to the indirect enantioseparation of racemic proteinogenic amino acids

The application of (1 S,2 S)- or (1 R,2 R)-1,3-diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate as a new chiral derivatizing agent for the resolution of compounds possessing an amino group is described. The reagent is easily accessible in both enantiomeric forms after a simple two-step synthesis. Its applicability was demonstrated on the example of the resolution of a series of α-amino acids. The diastereomeric thiourea derivatives produced were separated by reversed-phase high-performance liquid chromatography. The effects of pH, temperature and reagent excess on the derivatization kinetics were investigated, as were the effects of pH and organic modifier on the separation.

Development of new isothiocyanate-based chiral derivatizing agent for amino acids

(1S,2S)-1,3-Diacetoxy-1-(4-nitrophenyl)-2-propylisothiocyanate [(S,S)-DANI] has been developed as a new chiral derivatizing agent for resolution of compounds containing an amino group. The reagent is readily available in both enantiomeric forms. Its applicability was demonstrated by the resolution of representative α-amino acids. The diastereomeric thiourea derivatives produced were separated by reversed-phase (C18) high-performance liquid chromatography, with mixtures of 0.1% aqueous trifluoroacetic acid (pH∼2) and methanol as eluents.

A Novel Linking-Protecting Group Strategy for Solid-Phase Organic Chemistry with Configurationally Stable α-[N-(Phenylfluorenyl)]amino Carbonyl Compounds: Synthesis of Enantiopure Norephedrines on Solid Support

A novel linking strategy has been developed for synthesizing configurationally stable α-amino aldehyde on polymeric supports. Alkylation of l-alanine methyl ester with 9-bromo-9-p-bromophenylfluorenene, followed by ester hydrolysis and coupling to isoxazolidine, provided N-(9-p-bromophenylfluoren-9-yl)alanine isoxazolidide (5), which was transformed into its corresponding boronate 2 by a palladium-catalyzed cross-coupling reaction with diboron pinacol ester. Boronate 2 was anchored to four different polymeric aryl halides 6−9 in 70−99% yields. Polymer-bound alaninal 1b was then synthesized on non-cross-linked polystyrene by hydride reduction of isoxazolidide 10b. Treatment of alaninal 1b with phenylmagnesium bromide, cleavage of the resulting amino alcohol in a 1:2:2 TFA/CH2Cl2/anisole cocktail, and acylation with di-tert-butyl dicarbonate furnished N-(BOC)norephedrines 14 that were demonstrated to be enantiopure by conversion to diastereomeric thioureas 15 and analysis by HPLC. In summary, we have developed a process by which the 9-phenylfluoren-9-yl protecting group has been converted into a new linker for the solid-phase synthesis and manipulation of α-amino carbonyl compounds.

HPLC Separation of Metyrosine Enantiomers as Methyl Esters Derivatized with 2, 3, 4, 6-Tetra-O-acetyl-β-D-glucopyranosyl Isothiocyanate

Enantiomeric resolution of racemic metyrosine as methyl esters was investigated following the formation of diastereomeric thioureas with 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (TAGIT). Baseline separation (Rs>1.5) was achieved for the derivatized enantiomers on an octadecylsilane column under isocratic conditions using a mobile phase consisting of 35:65 v/v acetonitrile-distilled water containing 0.1% triethylamine pH 4.0 (adjusted with trifluoroacetic acid) at a flow rate of 1.0 mL and detection at 250 nm. Retention times, peak resolution, and separation factors were tabulated and a possible mechanism of separation is discussed.

Indirect separation of chiral proteinogenic α-amino acids using the fluorescence active (1 R,2 R)-N-[(2-isothiocyanato)cyclohexyl]-6-methoxy-4-quinolinylamide) as chiral derivatizing agent : A comparison

Enantiomerically pure trans-1,2-diaminocyclohexane (DACH) based (1 R,2 R)- and (1 S,2 S)-N-[(2-isothiocyanato)cyclohexyl]-6-methoxy-4-quinolinylamide (( R, R)- and ( S, S)-CDITC) was designed as a new fluorescence tagging chiral derivatizing agent (CDA) for the separation of amino acids and chiral amines. The derivatization of amino acids with CDITC yielded highly fluorescent diastereomeric thioureas. These derivatives were separated on a non-chiral RP-HPLC system employing C 18 as stationary phase. The respective optimum separation condition were evaluated and the optimum pH was found to be 4.30. The α-value achieved ranged from 1.14 to 3.16 with peak resolutions ranging between 0.59 and 13.94. The fluorescent labeled amino acids were detected at λ ex=333 nm, λ em=430 nm. These fluorescence properties almost matched the nitrogen laser line of 335 nm making laser-induced fluorescence detection (LIF) possible. The HPLC separations were compared to quasi capillary electrochromatographic (CEC) measurements of the same diastereomers using polyvinyl pyrolidone (PVP) as pseudo-stationary phase. All 19 chiral proteinogenic amino acids were separated. The selectivity values ranged between 1.026 and 1.232 with R s values from 1.37 to 16.25. Detection was achieved by a diode array detector. The effect of PVP concentration, pH, temperature and organic modifier on the separations was evaluated. The optimum CEC separation conditions were found to be 20 m M sodium citrate (pH 3), 0.5% (w/v) PVP, 9% 2-propanol and 1% tert.-butylmethyl ether.

Synthesis of new stable aliphatic isothiocyanate-based chiral derivatizing agent and application to indirect separation of chiral amino and thiol compounds

A new chiral derivatizing agent (CDA) (1S, 2S) N-[(2-isothiocyanato)-cyclohexyl)-pivalinoyl amide ((S,S)-PDITC) is described. The CDA is available from 1,2-diamino cyclohexane (DACH) via a straightforward synthesis in both the (R,R) and (S,S)-configuration and can serve as a highly selective, stable reagent for the indirect resolution of chiral primary and secondary amines, amino acids and thiol compounds. The resulting diastereomeric thioureas and dithiocarbamates can be separated by simple RP-HPLC as demonstrated with a number of pharmaceutically important examples of amines and amino alcohol-type drugs. The latter diastereomers are compared with the well-established GITC derivatized compounds. The separation factors (α) of the diastereomeric thioureas range between 1.03 and 2.08 and were usually higher than those of the GITC derivatives. The chemical stability of the PDITC derivatives is excellent due to the absence of hydrolyzable ester groups— considered an advantage compared to GITC derivatives.

Synthesis and application of a new isothiocyanate as a chiral derivatizing agent for the indirect resolution of chiral amino alcohols and amines

The synthesis of the optical pure trans-1,2-diaminocyclohexane (DACH)-based N-3,5-dinitrobenzoyl-DACH-isothiocyanate (DDITC) is straightforward and the compounds, available in R, R and S, S configurations, can serve as highly selective, stable and optically pure chiral derivatizing agents (CDA) for the indirect resolution of chiral primary and secondary amines and in particular of amino alcohols. The synthetic route to obtain DDITC is described and RP-HPLC separations of a number of pharmaceutically important amino alcohols including ß-blockers derivatized with DDITC are presented. The latter diastereomers were compared with the well established GITC-derivatized compounds by RP-HPLC. The separation factor (α) of the diastereomeric thioureas ranged between 1.05 and 2.00 and the peak resolution ( R) ranged from 0.67 to 6.67 for the DDITC derivatives, and were usually higher than the values for the GITC derivatives. However, derivatization of secondary amines containing a tertiary butyl group at the amino function (e.g., timolol) is impossible using DDITC, a limitation not observed using GITC as CDA. The advantage of DDITC over other chiral isothiocyanates is the higher chromatographic selectivity and the high detection sensitivity, including the option of electrochemical detection due to the redox potential (−0.62 V) of the nitro group. UV detection of the DDITC-derivatized amino compounds shows excellent sensitivity because of the higher molar absorptivity (ε) deduced from the nitroaryl chromophor (e.g., DDITC derivative of metamphetamine, ϵ 242 = 26 000). The chemical stability of the DDITC derivatives is much higher than that of GITC derivatives owing to the absence of easily hydrolysable ester groups, which is considered a further advantage.

2,3,4,6-Tetra- O-benzoyl-β- d-glucopyranosyl isothiocyanate: An efficient reagent for the determination of enantiomeric purities of amino acids, β-adrenergic blockers and alkyloxiranes by high-performance liquid chromatography using standard reversed-phase columns

The use of the chiral derivatization reagent 2,3,4,6-tetra- O-benzoyl-β- d-glucopyranosyl isothiocyanate (BGIT) is described for the conversion of a variety of amino acids and β-adrenergic blockers into diastereomeric thioureas, which can be separated on achiral RP-18 HPLC columns. In comparison with the established reagent 2,3,4,6-tetra- O-acetyl-β- d-glucopyranosyl isothiocyanate (AGIT), BGIT shows increased sensitivity owing to the higher molar absorptivity on the BGIT derivatives. Also a series of monosubstituted alkyloxiranes was transformed with 2-propylamine into the corresponding amino alcohols, which were then further reacted with BGIT, 2,3,4,6-tetra- O-pivaloyl-β- d-galactopyranosyl isothiocyanate (PGIT) or AGIT, leading to the corresponding thiourea derivatives. The diastereomers derived from BGIT could be separated with excellent resolution on a standard RP-18 column, whereas the PGIT and AGIT derivatives showed less or no resolution.

A high performance liquid chromatographic method for the determination of albuterol enantiomers in human serum using solid phase extraction and chemical derivatization.

A high performance liquid chromatographic method was developed for the simultaneous assay of R(-)- and S(+)-albuterol in human serum. The assay involves solid phase extraction as a sample clean-up step and derivatization of racemic albuterol to its diastereomeric thioureas with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl isothiocyanate. Chromatographic separation was accomplished under isocratic conditions using an octadecylsilane column and a mobile phase consisting of 29:71 acetonitrile:distilled water containing 0.1% triethylamine, pH 4.0 (adjusted with concentrated phosphoric acid) at a flow rate of 0.8 mL/min. The diastereomers were detected using a fluorescence detector set at 223 nm excitation and no emission filter. Racemic bamethane was used as internal standard. Drug to internal standard peak-height ratios were linear over a 2-20 ng/mL range for each enantiomer. The limit of detection of each analyte was 1.0 ng/mL (S/N = 3).

By-products in the derivatization of amines with the chiral reagent 2,3,4,6-tetra-O-acertyl-β- d-glucopyranosyl isothiocyanate and their elimination

The formation of the desired diastereomeric thiourea derivatives did not proceed quantitatively when β-adrenoceptor blocking drugs were allowed to react with 2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate (GITC). A competing reaction caused losses, which increased with increasing excess of the reagent (decreasing relative amounts of amine), amounting to as much as 50%. Measurements indicate that the by-products were due to an impurity in the commercial reagent, but also in GITC produced in-house from the corresponding bromide. The side-reaction could be completely eliminated by pretreatment of the reagent solution with a limited amount of another amine. Similar observations of a reactive impurity were made with the chiral reagent α-methylbenzyl isothiocyanate.

Stereospecific High-Performance Liquid Chromatographic Assay for the Enantiomers of Phenylpropanolamine in Human Plasma

A stereospecific high-performance liquid chromatographic assay for the enantiomers of phenylpropanolamine (PPA) in human plasma has been developed. The method is based on reaction of extracted PPA with the chiral derivatizing agent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate to form diastereomeric thiourea derivatives. These thiourea derivatives are stable for at least 24 h, readily separable by reversed-phase chromatography, and amenable to ultraviolet detection at 254 nm. Standard curves for each of the PPA enantiomers are linear over the concentration range 10-250 micrograms/L. Over the approximate range of the standard curve, precision (intra- and interday) ranged from 2.2-7.0% while accuracy ranged from 99.2-107.3%. The method has been shown to be free from interference from endogenous plasma constituents and from a range of drugs containing a primary or secondary amine function. Individual PPA enantiomer plasma concentration-time profiles measured for a subject administered racemic PPA are included to demonstrate the utility of the assay.

A high-performance liquid chromatographic assay for the enantiomers of bevantolol in human plasma.

A method was developed and validated for the simultaneous analysis of (+)- and (-)-bevantolol in human plasma. The assay involves plasma protein precipitation, derivatization of racemic bevantolol to its diastereomeric thioureas with 2,3,4,5-tetra-o-acetyl-alpha-D-glucopyranosyl isothiocyanate, and solid-phase extraction of the diastereomers from 0.5 ml human plasma. Chromatographic separation was accomplished under isocratic conditions using a reversed-phase C-18 analytical column and mobile phase consisting of equal parts of 75 mM dibasic ammonium phosphate buffer (adjusted to pH 3.5 with phosphoric acid) and acetonitrile, with a detection wavelength of 220 nm. The absolute peak-height method was employed for quantitation. Retention times for the diastereomers of (+)- and (-)-bevantolol were 7.4 and 6.4 min, respectively. The method is suitable for the quantification of the enantiomers over a concentration range of 40 to 800 ng/ml per enantiomer.

Reversed-phase high-performance liquid chromatographic separation of the enantiomers of trimetoquinol hydrochloride by derivatization with 2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate and application to the optical purity testing of drugs

The enantiomers of the bronchodilator trimetoquinol hydrochloride were separated by reversed phase high-performance liquid chromatography after derivatization with 2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate (TAGIT) reagent. The corresponding diastereomeric thiourea derivatives were baseline resolved on an ODS column with a resolution of more than 2 within 10 rain. The derivatization proceeded smoothly and quantitatively within 15 min at room temperature even in aqueous solution. The favourable UV adsorption of the derivatized enantiomers permitted the detection of the ( R)-(+)-isomer in ( S)-(-)-trimetoquinol hydrochloride down to the 0.2% level. The determination of the optical purity of trimetoquinol hydrochloride drug substance and in tablets and injections was succesfully achieved. The optimization of the derivatization procedures and separation conditions is described.

Reversed-phase HPLC separation of the enantiomers of denopamine after derivatization with GITC chiral reagent

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed to determine the optical purity of denopamine, which is a new cardiotonic agent having an asymmetric carbon in a molecule. The enantiomers were converted to diastereomeric thiourea derivatives using 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) reagent. Separation of the enantiomers of denopamine as diastereomers was successfully achieved by reversed-phase HPLC within 10 min using an ODS column and UV detection. Derivatization of denopamine proceeded rapidly under the alkaline conditions and the ambient temperature. This method was applied to the determination of the optical purity of denopamine drug substances and those in tablets. The favorable UV absorption of the derivatives enabled the optical antipode to be determined down to the 0.2% level.

ChemInform Abstract: Reversed‐Phase Liquid Chromatographic Separation of Enantiomeric and Diastereomeric Bases Related to Chloramphenicol and Thiamphenicol

The important antimicrobial agents Chloramphenicol and thiamphenicol are N-acylated amines whose chemical structures include two chiral centers. Each drug is the single enantiomer of R,Rconfiguration. The N-deacylated bases of the drugs are important intermediates in their synthesis and optical resolution. In this report, reversed-phase HPLC methods are described for the separation of enantiomeric and diastereomeric bases of the two drugs and of two closely related bases used in some syntheses of the drugs. The stereoisomeric bases were derivatized with a homochiral isothiocyanate and the resulting diastereomeric thioureas were separated on C18 columns with methanol:water mixtures as mobile phases and detection at 254 nm. The four stereoisomeric bases of Chloramphenicol and those of its unnitrated analogue were thus separable after derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate. This reagent also allowed the separation of the D-threoisomer of the p-mercaptomethyl analogue of thiamphenicol base from its stereoisomers. The stereoisomers of thiamphenicol base were similarly separated with (R)-α-methylbenzyl isothiocyanate as the derivatizing agent. The diastereomers of chloramphenicol base and of thiamphenicol base were chromatographically separable after derivatization with the nonchiral reagent benzyl isothiocyanate. The procedures developed may be useful in the determination of the stereoisomeric composition of the drugs in research and in quality control, and may be applicable to other similar agents whose chemistry and pharmacology are receiving considerable attention.

Reversed-Phase Liquid Chromatographic Separation of Enantiomeric and Diastereomeric Bases Related to Chloramphenicol and Thiamphenicol

The important antimicrobial agents Chloramphenicol and thiamphenicol are N-acylated amines whose chemical structures include two chiral centers. Each drug is the single enantiomer of R,Rconfiguration. The N-deacylated bases of the drugs are important intermediates in their synthesis and optical resolution. In this report, reversed-phase HPLC methods are described for the separation of enantiomeric and diastereomeric bases of the two drugs and of two closely related bases used in some syntheses of the drugs. The stereoisomeric bases were derivatized with a homochiral isothiocyanate and the resulting diastereomeric thioureas were separated on C18 columns with methanol:water mixtures as mobile phases and detection at 254 nm. The four stereoisomeric bases of Chloramphenicol and those of its unnitrated analogue were thus separable after derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate. This reagent also allowed the separation of the D-threoisomer of the p-mercaptomethyl analogue of thiamphenicol base from its stereoisomers. The stereoisomers of thiamphenicol base were similarly separated with (R)-α-methylbenzyl isothiocyanate as the derivatizing agent. The diastereomers of chloramphenicol base and of thiamphenicol base were chromatographically separable after derivatization with the nonchiral reagent benzyl isothiocyanate. The procedures developed may be useful in the determination of the stereoisomeric composition of the drugs in research and in quality control, and may be applicable to other similar agents whose chemistry and pharmacology are receiving considerable attention.

Determination of the enantiomeric composition of chiral epoxides using chiral derivatization and liquid chromatography

There appears to be a need for convenient chromatographic methods for the analysis of the enantiomeric composition of chiral oxiranes. In this report a new procedure for such purposes is described. Racemic mono-, 2,2-disubstituted and trans-2,3-disubstituted oxiranes were reacted with simple volatile alkylamines. The aminoalcohol products were derivatized with 2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate, and the resulting diastereomeric thioureas were resolved on C 18 liquid chromatographic columns using methanol-water mobile phases and detection at 254 nm. In several cases, the order of elution of the derivatives could be related to the configuration of the starting epoxide. The procedure is simple and convenient to carry out despite the two-reaction derivatization sequence, and may have broad utility in the enantiomeric analysis of chiral epoxides.

Optical resolution of amino acid enantiomers by high-performance liquid chromatography

Simultaneous analysis of common protein amino acid enentiomers was achieved by a chiral derivatization and a chiral mobile phase method. In the chiral derivatization method, 2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate was chosen as the reagent for the derivatization of enantiomeric amino acids to give diastereomeric thiourea derivatives. These derivatives were efficiently separated on a octadecylsilyl silica gel column by gradient elution and were detected by their absorbance at 250 nm. Derivatives of all common protein amino acid racemates, except cysteine, were resolved within about 2 h, although a few peaks were slightly overlapped. In the chiral mobile phase method, the optically active binary copper complex with N( ptoluenesulphonyl)- d-phenylglycine was used as a chiral additive in the mobile phase for the ligand-exchange chromatographic resolution of underivatized d, l-amino acids. Simultaneous resolution of common protein amino acid enantiomers on a resversed phase was achieved by a column-switching technique, utilizing two ODS columns of different lengths, and by gradient elution with acetonitrile. The column eluate was monitored fluorometrically after reaction with o-phthalaldehyde.