Liquid chromatographic enantioseparation of three beta‐adrenolytics using new derivatizing reagents synthesized from (S)‐ketoprofen and confirmation of configuration of diastereomers

Diastereomers of racemic β‐adrenolytic drugs [namely (RS)‐propranolol, (RS)‐metoprolol and (RS)‐atenolol] were synthesized under microwave irradiation with (S)‐ketoprofen based chiral derivatization reagents (CDRs) newly synthesized for this purpose. (S)‐Ketoprofen was chosen for its high molar absorptivity (ε_o ~ 40,000) and its availability as a pure (S)‐enantiomer. Its ‐COOH group was activated with N‐hydroxysuccinimide and N‐hydroxybenzotriazole; these were easily introduced and also acted as good leaving groups during nucleophilic substitution by the amino group of the racemic β‐adrenolytics. The CDRs were characterized by UV, IR, ¹H‐NMR, HRMS and CHNS. Separation of diastereomers was achieved by RP HPLC and open column chromatography. Absolute configuration of the diastereomers was established with the help of ¹HNMR supported by developing their optimized lowest energy structures using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6‐31G* basis set (based on density functional theory), and elution order was established. RP HPLC conditions for separation were optimized and the separation method was validated. The limit of detection values were 0.308 and 0.302 ng mL^?1. Copyright © 2016 John Wiley & Sons, Ltd.     

Methods and approaches for determination and enantioseparation of (RS)‐propranolol

Propranolol, a β‐adrenergic receptor antagonist, is a chiral compound that is marketed as a racemate, but only the (S)‐(?)‐enantiomer is responsible for the β‐adrenoceptor blocking activity. Different chromatographic methods have been applied for separation and determination of enantiomers of (RS)‐propranolol. In this article a review is presented on different liquid chromatographic methods used for enantioseparation of (RS)‐propranolol, using both HPLC and TLC. In addition, some aspects of enantioseparation under achiral phases of liquid chromatography have been briefly mentioned.     

High‐performance liquid chromatographic enantioseparation of (RS)‐bupropion using isothiocyanate‐based chiral derivatizing reagents

A high‐performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate‐based chiral derivatizing reagents, (S)‐1‐(1‐naphthyl) ethyl isothiocyanate, (S)‐NEIT, and (R)‐α‐methyl benzyl isothiocyanate, (R)‐MBIT. The diastereomers synthesized with (S)‐NEIT were enantioseparated by reversed‐phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)‐MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040–0.043 µg/mL for each of the diastereomers prepared with (S)‐NEIT. Copyright © 2013 John Wiley & Sons, Ltd.     

Indirect enantioresolution of (R,S)‐mexiletine by reversed‐phase high‐performance liquid chromatography via diastereomerization with [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride], (S)‐naproxen and nine chiral reagents synthesized as variants of Marfey's reagent

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)‐mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)‐O,O'‐di‐p‐toluoyl tartaric acid anhydride] was synthesized and (S)‐naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5‐difluoro‐2,4‐dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10–30 pmol. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatographic enantioseparation of (RS)‐etodolac using (S)‐levofloxacin and determination of absolute configuration of the diastereomeric derivatives

(RS)‐Etodolac was isolated from commercial tablets and was purified and characterized to be used as racemic standard. A pair of diastereomeric derivatives was synthesized using (S)‐levofloxacin as a chiral derivatizing reagent. The derivatization reaction was carried out under conditions of stirring at room temperature (30°C for 1.5 h) as well as under microwave irradiation; the derivatives obtained by the two methods were compared. Reaction conditions for derivatization were optimized with respect to mole ratio of chiral derivatizing reagent and (RS)‐etodolac. No racemization was observed throughout the study. Separation of diastereomeric derivatives was successful using C₁₈ column and a binary mixture of methanol and triethyl ammonium phosphate buffer of pH 4.5 (80:20, v/v) as mobile phase at a flow rate of 1 mL min^?1 and UV detection at 223 nm. An efficient approach for recognizing chirality and determining the absolute configuration of the diastereomeric derivatives of (RS)‐etodolac is described, which in turn is a measure of the enantiomeric purity of (RS)‐etodolac since the diastereomeric derivatives were separated and isolated using preparative thin‐layer chromatography.     

Über Pterinchemie. 71. Mitteilung. Trennung der Diastereomeren (6R)- und (6S)-5,6,7,8-Tetrahydro-L-neopterin

Separation of the Diastereomers (6R) and (6S)-5,6,7,8-Tetrahydro-L -neopterin The mixture of the diastereomers of the pentaacetylderivative IV of (6RS)-5,6,7,8-tetrahydro-L -neopterins could be separated by fractional crystallisation in methanol into the diastereomers IV A and IV B. Hydrolysis with hydrochloric acid gives the pure, diastereomeric, (6R)- and (6S)-5,6,7,8-tetrahydro-L -neopterins.     

Über Pterinchemie. 72. Mitteilung. Trennung der Diastereomeren (6R)- und (6S)-5,6,7,8-Tetrahydro-L-biopterin

Separation of the Diastereomers (6R)- and (6S)-5,6,7,8-Tetrahydro-L -biopterin The mixture of the diastereomers of the tetraacetylderivative IV of (6RS)-5,6,7,8-tetrahydro-L -biopterin could be separated by fractional crystallisation in methanol into the diastereomers IV A and IV B. Hydrolysis with hydrochloric acid gives the pure, diastereomeric, (6R)- and (6S)-5,6,7,8-tetrahydro-L -biopterins.     

Application of optically pure amines as chiral auxiliaries to develop trichloro‐s‐triazine‐based new chiral derivatizing reagents for reversed‐phase high‐performance liquid chromatographic enantioseparation of dl‐selenomethionine

(R)‐(+)‐naphthylethyl amine and (S)‐(+)‐1‐benzyl‐3‐aminopyrrolidine were incorporated as chiral auxiliaries, by nucleophilic substitution of chlorine atoms, in cyanuric chloride (CC) or its 6‐butoxy derivative. There were obtained four new chiral derivatizing reagents (CDRs) as two dichloro and two monochloro triazine reagents. The CDRs so obtained were characterized and their optical purity was ascertained. Diastereomers of dl ‐selenomethionine were synthesized under microwave irradiation for 60 or 90 s (at 80% power of 800 W). Reversed‐phase high‐performance liquid chromatographic separation of diastereomers was carried out on a C₁₈ column using mixtures of acetonitrile with aqueous trifluoroacetic acid as mobile phase. The detection was made at 230 nm using a photodiode array detector. The separation behaviors in terms of retention times and resolutions were compared. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery. Copyright © 2013 John Wiley & Sons, Ltd.     

Stereoselective and Enantioselective Syntheses of the Four Stereoisomers of Muscol from (3RS)‐Muscone

Two trans stereoisomers of 3‐methylcyclopentadecanol (=muscol), (1R,3R)‐ 2 and (1S,3S)‐ 2 , were efficiently synthesized from (3RS)‐3‐methylcyclopentadecanone (=muscone; (3RS)‐ 1 ) by a highly stereoselective reduction (Scheme). L‐Selectride^® (=lithium tri(sec‐butyl)borohydride) was used, followed by the enantiomer resolution by lipase QLG (Alcaligenes sp.). The cis stereoisomers of muscol, (1S,3R)‐ 2 and (1R,3S)‐ 2 , were obtained by the Mitsunobu inversion of (1R,3R)‐ 2 and (1S,3S)‐ 2 , respectively (Scheme). The absolute configuration of (1R,3R)‐ 2 was determined by X‐ray crystal‐structure analysis of its 3‐nitrophthalic acid monoester, 2‐[(1R,3R)‐3‐methylcyclopentadecyl hydrogen benzene‐1,2‐dicarboxylate ((1R,3R)‐ 3b ), and by oxidation of (1R,3R)‐ 2 to (3R)‐muscone.     

Enantiomeric high‐performance liquid chromatography resolution and absolute configuration of 6β‐benzoyloxy‐3α‐tropanol

The absolute configuration of the naturally occurring isomers of 6β‐benzoyloxy‐3α‐tropanol ( 1 ) has been established by the combined use of chiral high‐performance liquid chromatography with electronic circular dichroism detection and optical rotation detection. For this purpose (±)‐ 1 , prepared in two steps from racemic 6‐hydroxytropinone ( 4 ), was subjected to chiral high‐performance liquid chromatography with electronic circular dichroism and optical rotation detection allowing the online measurement of both chiroptical properties for each enantiomer, which in turn were compared with the corresponding values obtained from density functional theory calculations. In an independent approach, preparative high‐performance liquid chromatography separation using an automatic fraction collector, yielded an enantiopure sample of _OR(+)‐ 1 whose vibrational circular dichroism spectrum allowed its absolute configuration assignment when the bands in the 1100–950 cm^‐1 region were compared with those of the enantiomers of esters derived from 3α,6β‐tropanediol. In addition, an enantiomerically enriched sample of 4 , instead of _OR(±)‐ 4 , was used for the same transformation sequence, whose high‐performance liquid chromatography follow‐up allowed their spectroscopic correlation. All evidences lead to the _OR(+)‐(1S,3R,5S,6R) and _OR(?)‐(1R,3S,5R,6S) absolute configurations, from where it follows that samples of 1 isolated from Knightia strobilina and Erythroxylum zambesiacum have the _OR(+)‐(1S,3R,5S,6R) absolute configuration, while the sample obtained from E. rotundifolium has the _OR(?)‐(1R,3S,5R,6S) absolute configuration.     

Resolution of (S,S)‐4‐(2,2,4‐tri­methyl­chroman‐4‐yl)­phenyl camphanate and its 4‐chromanyl epimer by crystallization

Dianin's compound (4‐p‐hydroxy­phenyl‐2,2,4‐tri­methyl­chroman) has been resolved by crystallization of the (S)‐(−)‐camphanic esters (S,S)‐ and (R,S)‐4‐(2,2,4‐tri­methyl­chroman‐4‐yl)­phenyl 4,7,7‐tri­methyl‐3‐oxo‐2‐oxabi­cyclo[2.2.1]heptane‐1‐carboxyl­ate, both C₂₈H₃₂O₅, from 2‐methoxy­ethanol, yielding the pure S,S diastereomer. The relative stereochemistry of both diastereomers has been determined by X‐ray crystallography, from which the absolute stereochemistry could be deduced from the known configuration of the camphanate moiety. The crystallographic conformations have been analysed, including the 1:1 disorder of the R,S diastereomer.     

Preparation and structural analysis of (±)‐cis‐ethyl 2‐sulfanylidenedecahydro‐1,6‐naphthyridine‐6‐carboxylate and (±)‐trans‐ethyl 2‐oxooctahydro‐1H‐pyrrolo[3,2‐c]pyridine‐5‐carboxylate

Bicycle ring closure on a mixture of (4aS,8aR)‐ and (4aR,8aS)‐ethyl 2‐oxodecahydro‐1,6‐naphthyridine‐6‐carboxylate, followed by conversion of the separated cis and trans isomers to the corresponding thioamide derivatives, gave (4aSR,8aRS)‐ethyl 2‐sulfanylidenedecahydro‐1,6‐naphthyridine‐6‐carboxylate, C₁₁H₁₈N₂O₂S. Structural analysis of this thioamide revealed a structure with two crystallographically independent conformers per asymmetric unit (Z′ = 2). The reciprocal bicycle ring closure on (3aRS,7aRS)‐ethyl 2‐oxooctahydro‐1H‐pyrrolo[3,2‐c]pyridine‐5‐carboxylate, C₁₀H₁₆N₂O₃, was also accomplished in good overall yield. Here the five‐membered ring is disordered over two positions, so that both enantiomers are represented in the asymmetric unit. The compounds act as key intermediates towards the synthesis of potential new polycyclic medicinal chemical structures.     

Resolution and isolation of enantiomers of (±)‐isoxsuprine using thin silica gel layers impregnated with l‐glutamic acid,comparison of separation of its diastereomers prepared with chiral derivatizing reagents having l‐amino acids as chiral auxiliaries

Thin silica gel layers impregnated with optically pure l ‐glutamic acid were used for direct resolution of enantiomers of (±)‐isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l ‐alanine, l ‐valine and S‐benzyl‐l ‐cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed‐phase high‐performance liquid chromatography on a C₁₈ column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin‐layer chromatography (TLC) on reversed phase (RP) C₁₈ plates. Diastereomers prepared with enantiomerically pure (+)‐isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)‐isoxsuprine. The elution order in the experimental study of RP‐TLC and RP‐HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1–0.09 µg/mL in TLC while it was in the range of 22–23 pg/mL in HPLC and 11–13 ng/mL in RP‐TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.     

HPLC enantioseparation of racemic bupropion,baclofen and etodolac: modification of conventional ligand exchange approach by pre‐column formation of chiral ligand exchange complexes

Separation of racemic mixture of (RS)‐bupropion, (RS)‐baclofen and (RS)‐etodolac, commonly marketed racemic drugs, has been achieved by modifying the conventional ligand exchange approach. The Cu(II) complexes were first prepared with a few l ‐amino acids, namely, l ‐proline, l ‐histidine, l ‐phenylalanine and l ‐tryptophan, and to these was introduced a mixture of the enantiomer pair of (RS)‐bupropion, or (RS)‐baclofen or (RS)‐etodolac. As a result, formation of a pair of diastereomeric complexes occurred by ‘chiral ligand exchange’ via the competition between the chelating l ‐amino acid and each of the two enantiomers from a given pair. The diastereomeric mixture formed in the pre‐column process was loaded onto HPLC column. Thus, both the phases during chromatographic separation process were achiral (i.e. neither the stationary phase had any chiral structural feature of its own nor did the mobile phase have any chiral additive). Separation of diastereomers was successful using a C₁₈ column and a binary mixture of MeCN and TEAP buffer of pH 4.0 (60:40, v/v) as mobile phase at a flow rate of 1 mL/min and UV detection at 230 nm for (RS)‐Bup, 220 nm for (RS)‐Bac and 223 nm for (RS)‐Etd. Baseline separation of the two enantiomers was obtained with a resolution of 6.63 in <15 min. Copyright © 2016 John Wiley & Sons, Ltd.     

A novel approach for enantioseparation as applied to (RS)‐etodolac from pharmaceutical formulations: LC MS and density functional theory support for confirmation of diastereomers so separated

In the present studies formation of diastereomers of (RS)‐etodolac was confirmed using LC‐MS when [M + H]⁺ or [M]⁺ were recorded for the diastereomers. The lowest energy optimized structures of two diastereomers were drawn, which confirmed the three‐dimensional geometry of the diastereomers. This supports the optimized analytical separation conditions. In addition, separation of diastereomers was successful using a C₁₈ column and a binary mixture of methanol and triethyl ammonium phosphate buffer of pH 4.5 (80:20, v/v) as mobile phase at a flow rate of 1 mL min^?1 and UV detection at 223 nm. The separation method was validated as per International Conference on Harmonization guidelines. (RS)‐Etodolac was isolated from commercial tablets and purified and characterized to be used as racemic standard. Three pairs of diastereomers were synthesized using enantiomerically pure amines, namely, (R)‐(+)‐α‐methyl benzyl amine, (S)‐(?)‐α,4‐dimethylbenzylamine and (R)‐(?)‐1‐cyclohexylethylamine. Derivatization reactions were carried out under conditions of stirring at room temperature (30 °C for 2 h) as well as under microwave irradiation (MWI), and the two types of diastereomers were compared. Reaction conditions for derivatization were optimized with respect to mole ratio of chiral derivatizing agent and (RS)‐etodolac and MWI time. No racemization was observed throughout the study. Copyright © 2015 John Wiley & Sons, Ltd.     

(2S)‐2‐[1‐(2,4‐Di­fluoro‐5‐iodo­phenyl)‐2‐deoxy‐β‐d‐ribo­furanos‐5‐yloxy]‐8‐methyl‐4H‐1,3,2‐benzodioxa­phosphole 2‐oxide

The title compound, C₁₉H₁₈F₂IO₆P, prepared as a potential antiviral and anticancer agent from 3‐methyl­salicyl­chloro­phosphane and 1‐(2,4‐di­fluoro‐5‐iodo­phenyl)‐2‐deoxy‐β‐d ‐ribo­furan­ose, is one of a 1:1 mixture of two diastereomers. The diastereomers differ in their configuration, S or R, at the asymmetric phosphorus center. X‐Ray crystallographic analysis of the title compound has determined the absolute configuration at the asymmetric P center to be S.     

Synthesis and Optical Resolution of the Floral Odorant (±)‐2,3‐Dihydro‐2,5‐dimethyl‐1H‐indene‐2‐methanol,and Preparation of Analogues

The title compound (±)‐ 1 , a recently discovered, valuable, floral‐type odorant, has been synthesized by a straightforward procedure (Scheme 1). To determine the properties of the enantiomers of 1 , their separation by preparative HPLC and the determination of their absolute configuration by X‐ray crystallography were carried out (Figure). Furthermore, the analogues 2 – 6 were synthesized, either from differently methylated 2‐methylindan‐1‐ones (Schemes 2 and 3) or, in the case of the 2,4,6‐trimethylated homologue 6 , by a completely different synthetic approach (Scheme 4). An evaluation of (+)‐(S)‐ 1 , (−)‐(R)‐ 1 , and (±)‐ 1 showed only minor differences in terms of odor (Table).     

Analytical and semi‐preparative enantioresolution of (RS)‐ketorolac from pharmaceutical formulation and in human plasma by HPLC

An efficient, simple, validated, analytical and semi‐preparative HPLC method has been developed for direct enantioresolution of (RS)‐Ketorolac (Ket) using monochloro‐methylated derivatives of cellulose and amylose, i.e. cellulose (tris‐3‐chloro‐4‐methylphenylcarbamate) and amylose (tris‐5‐chloro‐2‐methylphenylcarbamate) as chiral stationary phases (CSPs) with photo diode array detection at 320 nm. Enantioresolution was carried out in samples of human plasma spiked with (RS)‐Ket under normal and reversed‐phase elution modes with suitable mobile phase compositions. The effect of nature of alcohols (MeOH, EtOH, PrOH and n‐BuOH) and other solvents (MeCN and MeOH) as organic modifiers in the mobile phase was investigated on the separation performance of two CSPs in terms of retention and separation of enantiomers. The best resolution was observed on cellulose‐based CSP using EtOH, while using 2‐PrOH (15%) and amylose‐based CSP obtained the highest retention. Under reversed‐phase elution mode the best enantioseparation was observed using 30% MeCN with ammonium formate buffer. The elution order of enantiomers was ascertained by determining specific rotations. The limit of detection and quantitation values were 5 and 15.5 ng/mL for each enantiomer of (RS)‐Ket, respectively. Copyright © 2016 John Wiley & Sons, Ltd.     

Reversed‐phase liquid chromatographic determination of enantiomers of atenolol in rat plasma using derivatization with Marfey's reagent

An HPLC method was established for enantioseparation of (R,S)‐atenolol (ATE) and determination of enantiomers in rat plasma. Marfey's reagent (1‐fluoro‐2,4‐dinitrophenyl‐5‐L‐alanine amide, FDNP‐L‐Ala‐NH₂, MR) was used as chiral derivatizing reagent with detection of diastereomers at 340 nm. It was shown that the R‐isomer eluted before the S‐isomer. The method was validated for linearity, repeatability, limits of detection and limit of quantification (LOQ). Recovery of ATE at LOQ was 92.8% for (R)‐ATE and 92.6% for (S)‐ATE. Copyright © 2009 John Wiley & Sons, Ltd.     

The Absolute Configuration of (+)‐Ethyl cis‐1‐Benzyl‐3‐hydroxypiperidine‐4‐carboxylate and (+)‐4‐Ethyl 1‐Methyl cis‐3‐Hydroxypiperidine‐1,4‐dicarboxylate; a Revision

Discrepancies between chiroptical data from the literature and our determination of the structure of the title compounds (+)‐ 5 and (+)‐ 9a were resolved by an unambiguous assignment of their absolute configuration. Accordingly, the dextrorotatory cis‐3‐hydroxy esters have (3R,4R)‐ and the laevorotatory enantiomers (3S,4S)‐configuration. The final evidences were demonstrated on both enantiomers (+)‐ and (?)‐ 5 by biological reduction of 4 by bakers' yeast and stereoselective [Ru^II(binap)]‐catalyzed hydrogenations of 4 (Scheme 2), by the application of the NMR Mosher method on (+)‐ and (?)‐ 5 (Scheme 3), as well as by the transformation of (+)‐ 5 into a common derivative and chiroptical correlation (Scheme 4).