Analytical and semipreparative high performance liquid chromatography separation of stereoisomers of...

J. Sep. Sci. 2006, 29, 1399 –1406 R. Cirilli et al. 1399

Roberto Cirilli1

Rosella Ferretti1

Bruno Gallinella1

Francesco La Torre1

Antonello Mai2

Dante Rotili2

1Dipartimento del Farmaco,Istituto Superiore di Sanit�,Roma, Italy

2Istituto Pasteur-FondazioneCenci Bolognetti, Dipartimentodi Studi Farmaceutici, Universit�degli studi di Roma “LaSapienza”, Roma, Italy

Original Paper

Analytical and semipreparative high performanceliquid chromatography separation of stereoisomersof novel 3,4-dihydropyrimidin-4(3H)-one derivativeson the immobilised amylose-based Chiralpak IAchiral stationary phase

Direct HPLC separation of stereoisomers of three novel 5-methyl-2-(alkylthio)-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones endowed with antiviral andpotential antiproliferative and morphological differentiation activity against mela-noma cells was performed by using the new immobilised amylose-based ChiralpakIA chiral stationary phase. Stereoselective conditions were achieved using normalphase eluents containing “non-standard” solvents such as ethyl acetate, methyl tert-butyl ether, or dichloromethane. In order to study the chiroptical properties of sin-gle stereoisomers, mg-scale separations were performed on analytical and semipre-parative size Chiralpak IA columns in combination with ethyl acetate-based eluents.

Keywords: Antiviral and antitumoral agents / Chiral separation / Chiralpak IA / Dihydropyrimidi-none derivatives / “Non-standard” eluents /

Received: January 12, 2006; revised: February 16, 2006; accepted: February 20, 2006

DOI 10.1002/jssc.200600019

1 Introduction

It has long been acknowledged that enantiomers ofchiral drugs may have distinct interactions with biologi-cal systems and consequently different pharmacokinetic,pharmacological, or toxicological activities, and thusmay be considered as different compounds. Develop-ment of a single enantiomer as drug candidate hasbecome standard practise in the pharmaceutical indus-try and research laboratories. Two approaches have gen-erally been adopted to obtain optically active com-pounds: the “racemic approach”, which involves the pro-duction of racemic compounds and successive enantio-meric separation by chromatographic techniques, andthe “enantiomeric approach”, which relies on stereose-lective or stereospecific synthesis. On a smaller scale, thefirst one offers the advantage of furnishing both enantio-mers, which are then available for comparative biologi-

cal testing at an early stage of drug development. In thelast two decades, direct enantioseparation by HPLC onCSPs has acquired a prominent role in analytical and pre-parative applications because of its simplicity, rapidity,and flexibility, and the growing availability of new effec-tive stationary phases. Polysaccharide-derived CSPs havebeen recognized as the most powerful packing materialfor analytical and preparative chromatographic chiralseparations due to their broad selectivity range and ver-satility [1–7]. Most commercially available “first genera-tion” polysaccharide-based CSPs are semi-synthetic poly-mers substantially obtained by conversion of hydroxylgroups of monosaccharide units into arylcarboxylate orarylcarbamate moieties [8] with the chiral polymers phy-sically coated on a silica matrix. Due to their coated nat-ure, these CSPs can only be used with a limited range ofsolvents such as polar solvents (e. g., alcohols) or non-polar solvents (e. g., alkanes) or their combination.Recently, by using a patent-protected immobilisationtechnology, a “second generation” of polysaccharide-based CSPs for HPLC with a high solvent versatility hasbeen designed and launched [9]. The new commerciallyavailable Chiralpak IA CSP [10] is based on the same poly-meric selector of the coated-type Chiralpak AD with thedistinctive difference that the 3,5-dimethylphenylcarba-mate of amylose is immobilised and not simply coatedonto a silica matrix. As reported in recent publications

Correspondence: Dr. Rosella Ferretti, Dipartimento del Farma-co, Istituto Superiore di Sanit�, Viale Regina Elena 299, 00161Rome, Italy.E-mail: [email protected]: +39-06-49387100

Abbreviations: CD, circular dichroism; CSP, chiral stationaryphase; e. e., enantiomeric excess; EA, ethyl acetate; MtBE, methyltert-butyl ether; S-DABO, thiodihydroalkoxybenzyloxopyrimi-dine

i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

1400 R. Cirilli et al. J. Sep. Sci. 2006, 29, 1399 –1406

[11–13], the immobilised-type IA CSP shows an excellentdegree of enantioselectivity towards many classes oforganic compounds and a total solvent compatibilityunder normal-phase and polar-mode conditions. Theadvantage of using this type of CSP was particularly evi-dent in preparative applications [14,15].

The solubility of a sample in the mobile phase is consid-ered to be one of the most critical factors for preparativechromatography. In order to render a separation practi-cally feasible and/or economically attractive on a pre-parative scale, the racemic compound must show anappreciable solubility in the eluent. This requirementcannot be satisfied in practice with the first generationof polysaccharide-derived chiral stationary phases,because of their coated nature; solvents of intermediatepolarity, such as methyl tert-butyl ether (MtBE), ethyl acet-ate (EA), tetrahydrofuran, acetone, 1,4-dioxane, andchlorinated solvents, can dissolve or swell the chiralpolymers, and their use must be excluded in the prepara-tion of eluent and sample [16].

In continuation of our work on the enantioseparation ofbiologically active chiral compounds [17–21], we reporthere on the direct HPLC separation of stereoisomers ofthree novel 5-methyl-2-(alkylthio)-6-(2,6-difluorophe-nylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (S-DABO deriv-atives) [22] (compounds 1–3, Fig. 1), endowed with anti-viral and potential antiproliferative and morphologicaldifferentiation activity against melanoma cells, on thenew immobilised amylose-based Chiralpak IA CSP. Theadvantages gained by use of “non-standard” mobilephases with respect to alcohol-based eluents in the analy-tical and mg-scale enantioseparation of compounds 1–3on the Chiralpak IA CSP are illustrated. The work is com-pleted with the determination of chiroptical proprieties(specific rotations and circular dichroism spectra) of theisolated stereoisomers.

2 Experimental

2.1 Chemicals

Stereoselective HPLC analysis of compounds has beenperformed by using stainless-steel Chiralpak IA columns(25064.6 and 250610 mm id, 5 lm) (Daicel ChemicalIndustries, Tokyo, Japan). Mobile phases for chromato-graphy were prepared from HPLC grade solvents. Ethanoland ethyl acetate (EA) were obtained from Baker (Deven-ter, Holland); n-hexane, 2-propanol, CH3CN, MtBE, THF,and dichloromethane (CH2Cl2) were purchased fromMerck (Darmstadt, Germany). The hold-up time marker1,3,5-tri-tert-butylbenzene was purchased from Sigma(St. Louis, MO, USA).

Compounds 1–3 (Fig. 1) were synthesised by a chemicalpathway developed by Mai et al. (Mai, A., Artico, M., Sbar-della, G., unpublished data).

2.2 Instrumentation

Analytical and semipreparative HPLC enantioseparationswere performed by using a Perkin Elmer (Norwalk, CT,USA) 200 LC pump equipped with a Rheodyne (Cotati,CA, USA) injector with a 20-lL, 200-lL, or 1-mL sampleloop, a Perkin Elmer column oven, and a Perkin Elmer290 UV detector. The sign of the optical rotations of enan-tiomers of the compounds tested was measured on-lineat a wavelength of 365 nm by a Perkin Elmer model 241polarimeter equipped with Hg/Na lamps and a 40-lLflow-cell. The system was kept at a constant temperatureof 238C. The signal was acquired and processed by CSW1,7 software (Data Apex, Prague, Czech Republic).

Specific rotations of the single enantiomers of analytes1–3 dissolved in ethanol (about 0.2 g/100 mL) were meas-ured on a Perkin Elmer 241 polarimeter equipped with aNa lamp operating at 589 nm. The volume of the cell was1 mL and the length of the optical path 10 cm. The sys-tem was thermostated at 238C.


Figure 1. Structures of the chiral analytes 1–3.

J. Sep. Sci. 2006, 29, 1399 –1406 Stereoselective HPLC on immobilised Chiralpak IA CSP 1401

The circular dichroism (CD) spectra of enantiomers ofthe chiral analytes 1–3, dissolved in ethanol (concentra-tion about 0.4 mg/mL) in a quartz cell (0.1 cm pathlength), were recorded by a Jasco J-710 spectropolari-meter (Japan Spectroscopic, Tokyo, Japan). The systemwas thermostated at 258C. The spectra are averaged overthree instrumental scans and the intensities are pre-sented in terms of De values.

2.3 Chromatographic conditions

Analytical separations were performed with standardsolutions prepared by dissolving each analyte in chroma-tographic eluent (concentration about 0.5 mg/mL). Theinjection volume was 20 lL. Mobile phases used in thisstudy were filtered through a Millipore (Yonezawa,Japan) membrane filter (0.45 lm) and degassed beforeuse. The flow rate was 1 mL/min and the column tem-perature was set at 258C.

Semipreparative enantioseparations of the analytes 1–3were carried out using 250610 mm id and 25064.6 mmid Chiralpak IA columns, in combination with mixturesof MtBE–EA (75 :25, v/v) and pure EA. The flow rate wasset at 4 mL/min, or 1 mL/min, whilst the column tem-perature was maintained at 258C. After stereoselectivesemipreparative separation, the collected fractions were

analysed on a chiral analytical Chiralpak IA column todetermine their enantiomeric excess (e. e.).

3 Results and discussion

3.1 Chromatographic enantioseparation

2-(sec-Butylthio)-6-[1-(2,6-difluorophenyl)propyl]-3,4-dihy-dro-5-methylpyrimidin-4(3H)-one (1) has two stereogeniccentres, so four stereoisomers are possible. In order toachieve the diastereo- and enantioselective separation ofall four stereoisomers in a single run on the immobi-lised-type Chiralpak IA CSP, various eluents based on“standard” and “non-standard” solvents were investi-gated. Figure 2 shows typical chromatograms obtainedduring optimisation of the chromatographic method. Asa general trend, on going from “standard” eluents to“non-standard” eluents the resolution dramaticallyimproved. Stereoisomers of compound 1 were notresolved at all when a conventional mobile phase consist-ing of n-hexane–2-propanol (85 :15, v/v) was used, whiletwo weakly separated peaks were achieved when ethanolwas used as alcoholic modifier. It is interesting to notethat the combination of various coated-type polysacchar-ide-based CSPs such as Chiralpak AD, Chiralcel OD, andChiralcel OJ with standard mobile phases containing


Figure 2. Influence of the mobile phasecomposition on diastereo- and enantio-separation of compound 1. Column: Chi-ralpak IA (25064.6 mm id); eluent: asindicated; flow rate:1 mL/min; detection:UV at 280 nm; column temperature:258C.


ethanol or 2-propanol did not produce any improvementin resolution. The immobilization of polysaccharidederivatives on a silica matrix broadens the range of possi-ble solvents, permitting the use of alternative mobilephases and exploration of new selectivity profiles, andtheoretically enhancing the chiral separations. Asreported in Fig. 2, with a mixture of n-hexane–THF–ethanol (75 :25 :1, v/v/v) compound 1 was partiallyresolved and three eluted peaks were obtained. By usinga mobile phase consisting of n-hexane–CH2Cl2 –2-propa-nol (85 :15 :1, v/v/v), the chiral resolving ability of IA CSPsubstantially remained constant. When pure EA wasused as mobile phase, the second peak and the thirdpeak were baseline separated with a and Rs values of 1.19and 2.42, respectively, and with greatly increased a andRs values for the second and the first peak (2.45 and 8.24,respectively). With a mixture consisting of MtBE andCH3CN (85 :15, v/v) the first eluted peak split and fourpeaks, corresponding to all four stereoisomers of 1,appeared. Nevertheless, the first pair of stereoisomerswas still narrowly separated (a = 1.06 and Rs = 0.63) whilethe resolution between other eluted peaks was complete(Rs A 2.05). Separation of the first eluted pair of stereoi-somers was improved when EA-based binary mixtureswere used as eluents. Better results in terms of stereose-lectivity were observed on using the IA CSP in combina-tion with a mixture of MtBE–EA (75 :25, v/v). Under theseconditions the separation between the first and secondstereoisomer improved to a maximum value (a = 1.12and Rs = 1.46) and good resolution between other peakswas achieved (Rs A3.49). Since the absolute configurationof the stereoisomers of 1 is unknown, to identify thepeaks in enantiomeric relationship the optical rotation

at the single wavelength of 365 nm was monitored dur-ing chromatography. Because of the low optical activityof the analytes investigated, an amount of about 2 mg ofcompound 1 dissolved in 100 mL of mobile phase consist-ing of MtBE–EA 75 :25, v/v was injected onto a 1-cm idChiralpak IA column. The resulting chromatogramsobtained by simultaneous on-line polarimetric and UVdetection are illustrated in Fig. 3. By analysing the signof optical rotation and relative peak areas, it was possibleto identify the first and the fourth eluted stereoisomer asappertaining to the same enantiomeric pair (compound1a) and the second and third one to the diastereomericpair of enantiomers (compound 1b). Under the experi-mental conditions employed, the first eluting peak ofboth diastereomeric pairs was levorotatory. Assignmentof the enantiomeric nature of the chromatographicpeaks by a chiroptical method based on on-line circulardichroism measurements was precluded by the high cut-off of EA (280 nm) present in the eluent. The best chroma-tographic results relative to simultaneous separation of1a and 1b on the IA CSP are summarised in Table 1.

So far, the above-mentioned optimised chromatographicmethod is the only effective way for the separation of thediastereoisomers of 1. In fact, all previous attempts toseparate 1a and 1b on achiral stationary phases such asSilica, Si-NH2, Si-Diol, and Si-CN in combination with nor-mal-phase eluents failed.

The structures of pyrimidinone derivatives 2 and 3 arestrictly correlated with compound 1 (Fig. 1). The difluoro-phenylpropyl group of 1 is replaced by the difluoroben-zyl moiety in compound 2, while substitution of the sec-butyl group linked to the sulphur atom by a propyl


Figure 3. Polarimetric (365 nm)(top) and UV (330 nm) (bottom)chromatograms of compound 1.Column: Chiralpak IA(250610 mm id); eluent: MtBE–EA (75 :25, v/v); flow rate:1 mL/min; column temperature: 258C.


group generates compound 3. Thus, in contrast to com-pound 1, analytes 2 and 3 have a single stereogenic cen-ter located in two different alkyl side chains of the dihy-dropyrimidinone structure.

Compounds 2 and 3 exhibited a chromatographic beha-viour similar to compound 1 on the IA CSP. Figure 4shows the effect of mobile phase composition on theenantioselectivity factors of 2 and 3. The enantiomers ofthese two S-DABO derivatives were not resolved at allwhen eluting mixtures with “standard” solvents such asn-hexane–2-propanol or n-hexane–ethanol were used.Compound 2 was also not resolved when the eluting mix-ture contained a “non-standard” solvent, such as THF,while a narrow separation was observed for compound 3(a = 1.16). The separation improved for both compoundswhen the eluting mixture contained CH2Cl2 and a valuesof 1.12 and 1.32, respectively, were achieved. The best

results in terms of enantioselectivity were obtained byusing the mixture MtBE–EA (75 :25, v/v) for compound 2(a = 1.36) and pure EA for compound 3 (a = 2.21). Table 1reports the chromatographic parameters obtained in theoptimised separation of analytes 2 and 3 on Chiralpak IACSP. Although the recognition mechanisms for the poly-saccharide-based chiral stationary phases have not beenfully understood at a molecular level, some hypothesesbased on chromatographic, computational, and spectro-scopic approaches have been formulated [3, 8, 23–26]. Itis generally thought that chiral selectivity is based on acombination of different factors that control the degreeof fitting of enantiomers into the chiral cavities of thepolymeric selector. This fitting is affected by attractiveand/or repulsive interactions of different magnitude andorigin.

In our case, the analytes investigated have multiplepotential points of interaction with the active sites of theCSP and it is difficult to identify a clear structure-enan-tioselectivity dependence.

Ethylation of the benzylic carbon of compound 2 gener-ates the more conformationally restricted compound 3with a stereogenic center directly linked to the 2,6-difluorophenyl group. This structural modification leadsto an enhancement in selectivity under each chromato-graphic condition tested. The experimental results are inaccordance with a favourable contribution to the mole-cular recognition process exerted by putative charge-transfer interaction between complementary aromaticmoieties of the polymeric selector and analyte.

3.2 mg-Scale separation of stereoisomers of 1–3and their chiroptical characterisation

During the last two decades the number of chiral station-ary phases available for the separation of enantiomershas grown rapidly, making it possible to develop an ana-lytical separation of almost any racemic mixture; thusselectivity is generally not a problem. Besides, immobili-sation of the polysaccharide derivatives on the chromato-graphic matrix offers the possibility of developing sep-aration methods with no constraint on the choice ofmobile phase and sample solvent [11]. This last point haspractical relevance for enantioseparations at preparativelevels, where sample solubility in the mobile phase is aprerequisite for successful resolution.

The dihydropyrimidinones investigated revealed a verylow solubility (a1 mg/mL) in hexane–alcohol mixtures,an intermediate solubility in methanol (a5 mg/mL), anda good solubility in EA (A30 mg/mL).

Thus, to achieve mg-scale separation of the stereoisomersof 1–3 on the IA CSP the optimised EA-based eluents indi-cated in Table 1 were selected.


Table 1. Retention factors (k1) for the first eluting enantio-mer, enantioseparation (a), and resolution (Rs) factors of 1–3 in the optimised stereoselective separation on Chiralpak IACSP (25064.6 mm id).

Compound Eluent (v/v) k1 a Rs

1a MtBE–EA (75 :25) 1.68 1.96 9.631b MtBE–EA (75 :25) 1.88 1.40 4.722 EA 100% 2.16 1.36 3.943 MtBE–EA (75 :25) 0.75 2.21 8.38

Flow rate: 1 mL/min; column temperature: 258C; detection:UV at 280 nm.

Figure 4. Enantioselectivity factor (a) of compounds 2 and 3as a function of the mobile phase composition. Eluent: A: n-hexane–2-propanol (85 :15, v/v); B: n-hexane–ethanol(95 :5, v/v); C: n-hexane–EA (50 :50, v/v); D: MtBE–EA(75 :25, v/v); E: EA 100%; F: MtBE–CH3CN (85 :15, v/v); G:n-hexane–THF–ethanol (75 :25 :1, v/v/v); H: n-hexane–CH2Cl2 –ethanol (85 :15 :1, v/v/v); I: CH3CN 100%. For otherexperimental conditions see Fig. 2.


Table 2 summarises the chromatographic operating con-ditions employed and the chromatographic and polari-metric data pertinent to each enantiomer that was col-lected in a single fraction.

Semipreparative enantioseparations of compounds 1(1a+1b) and 2 were achieved on a 1-cm id Chiralpak IAcolumn. The yields were greater than 60% and the enan-tiomeric excess (e. e.) of the collected fractions rangedfrom 96% to 99%. Figure 5 shows the chromatogramsobtained on injecting 10 mg of compound 1 and the ana-lytical checks on all four single stereoisomers obtained.The importance of sample solubility for the productivityof a mg-scale resolution was clear in the loading study ofcompound 3 on an analytical size Chiralpak IA column.The polarimetric HPLC traces obtained by using pure EAas eluent and solvating agent are depicted in Fig. 6. Thehigh chiral resolving ability of the Chiralpak IA/EA sys-tem and the very high sample solubility (about 80 mg/

mL) permitted complete separation of the two enantio-mers at a load as high as 15 mg of racemate.

Polarimetric analysis revealed that the first eluted enan-tiomer of each enantiomeric pair investigated was levo-rotatory in ethanol solution, at a wavelength of 589 nm.Furthermore, the optical activity of the enantiomers ofthe two diastereomeric pairs 1a and 1b was opposite(Fig. 3). This result agrees with the enantiomeric natureof the stereoisomers of 1a and 1b, as previouslydescribed.

The enantiomer elution order of 1–3 was determined byspiking a racemic sample with single enantiomersobtained by semipreparative separations. Under all theenantioselective conditions employed, the elution orderof the enantiomers of 1a, 1b, 2, and 3 was unchanged,with the (– )-enantiomer eluting before the (+)-enantio-mer.


Table 2. Chromatographic, quantitative, and polarimetric analysis of the first (F1) and second (F2) fraction recovered during thesemipreparative enantioseparations of 2 and 3

Compound Column(mm)

Eluent SR/V/FRa) ee (%) F1yield (%)

[a]D23 ee (%) F2yield (%)

[a]D23

2 IA (250610) MtBE–EA(75 :25, v/v)

20/300/4 98 80 –23(c = 0.30, EtOH)

A99.0 70 +27(c = 0.28, EtOH)

3 IA (25064.6) EA 100% 15/200/1 97 75 –32(c = 0.21, EtOH)

A99.0 70 +36(c = 0.22, EtOH)

a) SR: semipreparative run (mg), V: sample volume injection (lL), FR: flow rate (mL/min).

Figure 5. Trace (a): semipreparative separation of 10 mg of 1 (1a+1b) dissolved in 400 mL of the mobile phase. Column: Chi-ralpak IA (250610 mm id); eluent: MtBE–EA (75 :25, v/v); flow rate: 4 mL/min; temperature: 258C; detection: UV at 330 nm.Traces (b)– (e): purity control and specific rotations of the single fractions F1 –F4 collected at semipreparative scale. Column Chi-ralpak IA column (25064.6 mm id); eluent: MtBE–EA (75 :25, v/v); flow rate: 1 mL/min; temperature: 258C. Detection wave-length: 280 nm.


The CD spectra of enantiomers obtained by mg-scale sep-arations are depicted in Fig. 7. Each pair of enantiomersof 1–3 showed mirror-image CD spectra, confirmingtheir enantiomeric relationship. The CD spectrum of the(– )-1a and (– )-1b enantiomers exhibited an intense andnegative Cotton effect located at 240 nm. This band (1Lb)is probably due to the p–p* electronic transition of thearomatic system directly linked to the stereogenic cen-ter. An additional broad and weak positive band wasobserved at around 300 nm. Compound 3 shows thesame CD spectral profile as compound 1. This analogyseems be correlated with the fact that compound 3 con-tains the same chirophore moiety (Fig. 1). In the CD pro-file of compound 2 the intense band at 240 nm is

replaced by two weak bands located at around 220 and250 nm. These bands are probably ascribed to electronictransitions involving the non-bonding electron pair ofthe sulphur atom linked to the sec-butyl group.

4 Concluding remarksThe analytical and semipreparative separation of stereo-isomers of three novel pyrimidinone derivatives on theChiralpak IA CSP was demonstrated. Owing to the univer-sal solvent compatibility of the immobilised-type amy-lose-derived IA CSP, effective enantioselective conditionswere achieved using EA-based eluents. In the case of com-pound 1, having two stereogenic centers, the use of “non-


Figure 6. Loading study of compound 3 onan analytical size Chiralpak IA column.Amount injected in mg: 1 in 100 lL (dottedline), 8 in 200 mL (dashed line); 15 in200 lL of EA (solid line). Eluent: EA 100%;flow rate: 1 mL/min; temperature: 258C.Detection wavelength: 330 nm.

Figure 7. CD spectra of the enantio-mers of the compounds 1–3 in ethanolat 258C. Solid traces correspond to thesecond eluted enantiomer on the IACSP.


standard” solvents permitted simultaneous realisationof diastereo- and enantioselective conditions and separa-tion of all four stereoisomers in a single run. The advan-tages accruing from the use of the immobilised-type IACSP were evident also in preparative applications. Anamount of 15 mg of compound 3 was resolved in a singlestep on using an analytical size IA column and pure EA aseluent.

The findings of this study open the way for determina-tion of the absolute configuration of the single enantio-mer of S-DABO derivatives as well as for the determina-tion of their biological activity.

The authors are grateful to Mr C. De Sena and Mr L. Zanitti fortheir technical assistance.

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