Semipreparative HPLC enantioseparation, chiroptical properties, and absolute configuration of two novel cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1002/chir.20705

A direct semipreparative HPLC enantioseparation of two chiral thiazolidinone derivatives having cyclooxygenase-2 inhibition activity was performed on the Chiralpak IA chiral stationary phase. Semipreparative amounts of enantiopure forms were collected using acetonitrile-ethanol-trifluoroacetic acid mixtures as mobile phase. The absolute configuration of both compounds was unequivocally established by single-crystal X-ray diffraction method and correlated to the chiroptical properties of isolated enantiomers.

Full text of DOI:10.1002/chir.20705

CHIRALITY 22:56–62 (2010)
Semipreparative HPLC Enantioseparation, Chiroptical
Properties, and Absolute Configuration of Two Novel
Cyclooxygenase-2 Inhibitors
1
1
2
3
ROBERTO CIRILLI,1 STEFANO FIORE, FRANCESCO LA TORRE, ELIAS MACCIONI, DANIELA SECCI,
*
MARIA LUISA SANNA,2 AND CRISTINA FAGGI4
1Istituto Superiore di Sanita`, Dipartimento del Farmaco, Rome, Italy
2Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Cagliari, Cagliari, Italy
3Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive Universita`
degli Studi di Roma ‘‘La Sapienza,’’ Rome, Italy
4Universita` degli studi di Firenze, Dipartimento di Chimica Organica, Sesto Fiorentino, Firenze, Italy
ABSTRACT
A direct semipreparative HPLC enantioseparation of two chiral thiazoli-
dinone derivatives having cyclooxygenase-2 inhibition activity was performed on the
Chiralpak IA chiral stationary phase. Semipreparative amounts of enantiopure forms
were collected using acetonitrile-ethanol-trifluoroacetic acid mixtures as mobile phase.
The absolute configuration of both compounds was unequivocally established by single-
crystal X-ray diffraction method and correlated to the chiroptical properties of isolated
C
VV
enantiomers. Chirality 22:56–62, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: HPLC; chiral stationary phase; chiralpak IA; absolute configuration;
chiroptical properties; anti-inflammatory agents; sulfonamide derivatives;
thiazolidinone derivatives; COX-2-inhibitors
INTRODUCTION
tors immobilized onto macroporous silica.6,7 The stability
of the immobilized-type CSPs is not conditioned by polar-
ity or nature of mobile phases, unlike the coated-type
CSPs which are compatible with a limited range of sol-
vents (typically hydrocarbon/alcohol mixtures).8,9 It has
been demonstrated that 3,5-dimethylphenylcarbamate of
amylose fixed to the chromatographic matrix (Chiralpak
IA) has enantiomer resolving ability for a very broad range
of chiral analytes in normal-phase, polar organic and
reversed-phase modes.10–13 Its high efficiency and versatil-
ity makes IA packing material especially attractive for the
production of pure enantiomers from a racemate at the
semipreparative scale.14–18
The development of single-enantiomer drugs requires
sophisticated methodologies and materials to overcome
the additional problems relating to chirality, such as the
production and analysis of enantiomerically pure mole-
cules and the determination of their absolute configura-
tion. Approximately 10–50 mg of single enantiomers are
usually necessary for exhaustive biological and structural
characterizations of a chiral bioactive compound. Conse-
quently, the first part of this report is devoted to the devel-
opment of a suitable IA CSP/eluent system for the isola-
Non-steroidal anti-inflammatory drugs (NSAIDs) exhibit
their biological action by inhibiting cyclooxygenase (COX)
enzymes that are required for prostaglandin synthesis.
Three COX isozymes have been indentified so far: COX-1,
COX-2, and COX-3.1 The third physiological form of COX
(COX-3) has recently been supposed to be a COX-2 variant
appearing 48 h after the start of the inflammatory process,
which may activate the biosynthesis of endogenous media-
tors involved in the resolution of inflammation. COX-2 is
almost undetectable under physiological conditions, but it
is expressed in inflammatory cells, whereas COX-1 is
involved in cytoprotective physiological mechanisms and
expressed in many tissues such as the stomach and the
kidney. After this evidence there has been a growing inter-
est in the search for new COX-2 selective inhibitors with
anti-inflammatory activity and without the side effects
related to COX-1 inhibition such as ulcers and bleeding.
At the present time, the Coxib family is considered to be
the most promising class of COX-2 selective inhibitors.2–4
Recently, a novel series of chiral thiazolidin-4-one deriva-
tives showing a good inhibitory activity towards COX-2
and structurally related to Celecoxib and Rofecoxib (see
Figure 1), has been presented by some authors of this
work.5 In this article, we focus on the HPLC enantiosepa-
ration of two of them (compound 1 and 2, Fig. 1) using
the amylose-based Chiralpak IA as a chiral stationary
phase (CSP). The Chiralpak IA CSP is the first of a com-
mercially available series of polysaccharide-based selec-
C
*Correspondence to: R. Cirilli, Istituto Superiore di Sanita`, Dipartimento del
Farmaco, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: rcirilli@iss.it
Received for publication 23 October 2008; Accepted 12 January 2009
DOI: 10.1002/chir.20705
Published online 24 March 2009 in Wiley InterScience
(www.interscience.wiley.com).
VV
2009 Wiley-Liss, Inc.

HPLC ENANTIOSEPARATION OF COX-2 INHIBITORS
57
kin-Elmer polarimeter model 241 equipped with Hg/Na
lamps and a 40-ll flow-cell. The signal was acquired and
processed by Clarity software (DataApex, Prague, Czech
Republic).
The mobile phases were filtered and degassed by soni-
cation just before use. In analytical enantioseparations,
standard solutions were prepared by dissolving about 2
mg of sample into 10 ml of ethanol or methanol. The injec-
tion volume was 20–50 ll. In semipreparative enantiosepa-
ration a 1000 ll sample loop was used. After semiprepara-
tive separation, the collected fractions were analyzed by
chiral analytical columns to determine their enantiomeric
excess (e.e.).
The column hold-up time (t0 5 3.0 min for 250 3 4.6
mm I.D. column) was determined from the elution of an
unretained marker (toluene), using ethanol as eluent,
delivered at a flow-rate of 1.0 mL/min.
Specific rotations of stereoisomers of 1 and 2, dissolved
in acetonitrile, were measured at 589 nm by a Perkin-
Elmer polarimeter model 241 equipped with a Na lamp.
The volume of the cell was 1 ml and the optical path 10
cm. The system was set at a temperature of 208C using a
Neslab RTE 740 cryostat. The circular dichroism (CD)
spectra of enantiomers of 1 and 2, dissolved in acetonitrile
(concentration 0.2 mg/ml) in a quartz cell (0.1 cm-path
length) at 208C were measured using a Jasco Model J-700
spectropolarimeter. The spectra were average computed
over three instrumental scans and the intensities are pre-
sented in terms of ellipticity values (mdeg).
Fig. 1. Chemical structure of COX-2 inhibitors.
tion of enantiomerically pure forms of 1 and 2 at the semi-
preparative scale. The composition of the eluent was sys-
tematically investigated taking into account several param-
eters that affect the scaling-up procedure such as resolu-
tion, sample solubility and retention time.
Finally, the absolute configuration and chiroptical prop-
erties of collected enantiomers were exhaustively deter-
mined.
General Procedure for the Preparation of
4-(2-(4-chlorophenyl)-4-oxothiazolidin-3-yl)
benzenesulfonamide, 1, and 4-(2-(naphthalen-2-yl)-4-
oxothiazolidin-3-yl)benzenesulfonamide, 2
Benzenesulfonamide (4.00 g, 0.0232 mmol), 4-chloro-
benzaldehyde or 2-naphthaldehyde (0.0232 mmol), and
mercapthoacetic acid (3.3 mL, 0.0464 mmol), dissolved in
120 mL of toluene, were reacted under stirring at reflux in
a Dean-Stark apparatus for 48 h. The precipitated crude
solid was washed with ether and column chromato-
graphed on silica gel, using CHCl3/methanol as eluent.
The separated white solid was crystallized from ethanol.
MATERIALS AND METHODS
The chemicals, solvents for synthesis and spectral grade
solvents were purchased from Aldrich (Italy) and used
without further purification. Melting points (uncorrected)
were determined automatically on a FP62 apparatus (Met-
1
tler-Toledo). H-NMR spectra were recorded on a Bruker
AMX (300 MHz). Chemical shifts are expressed as d units
(parts per millions) using TMS as an internal standard.
Coupling constants J are valued in Hertz (Hz). Elemental
1
analysis for C, H, and N were performed on a Perkin- 1. Yield 45%; mp: 201–2038C. H-NMR (DMSO) d 4.05 (d,
Elmer 240 B microanalyzer and the analytical results were
within 60.4% of the theoretical values for all compounds.
All reactions were monitored by TLC performed on silica
gel plates 0.2-mm thick (60 F254 Merck); spots were
visualized by UV light.
1H, CH2, J 5 16.0), 4.18 (d, 1H, CH2, J 5 15.9), 6.73 (s,
1H, CH), 7.43 (s, 2H, NH2, D2O-exch.), 7.47 (d, 2H, Ar,
J 5 8.4); 7.57 (d, 2H, Ar, J 5 8.4); 7.67 (d, 2H, 4-Ar, J 5
8.7), 7.86 (d, 2H, Ar, J 5 8.7). Anal. Calcd for
C15H13ClN2O3S2: C, 48.84; H, 3.55; N, 7.59. Found: C,
HPLC enantioseparations were performed using stain-
less-steel Chiralpak IA (250 3 4.6 mm I.D. and 250 3 10 2. Yield 52%; mp: 213–2158C. H-NMR (DMSO) d 4.09 (d,
48.86; H, 3.56; N, 7.60.
1
mm I.D.) columns (Chiral Technologies Europe, Illkirch,
France). HPLC-grade solvents were supplied by Carlo
Erba (Milan, Italy). The HPLC apparatus consisted of a
Perkin-Elmer (Norwalk, CT) 200 lc pump equipped with a
Rheodyne (Cotati, CA) injector, a 50-ll sample loop, a
HPLC Dionex TCC-100 oven (Sunnyvale, CA) and a Per-
1H, CH2 J 5 16.2); 4.24 (d, 1H, CH2 J 5 16.2); 6.91
(s,1H, CH); 7.33 (s, 2H, NH2, D2O-exch.), 7.59-7.63 (m,
2H, naft.); 7.68–7.73 (m, 3H, naft. and phenyl); 7.82 (d,
2H, phenyl, J 5 8.7); 7.96-8.01 (m, 4H, naft). Anal.
Calcd for C19H16N2O3S2: C, 59.36; H, 4.19; N, 7.29.
Found: C, 59.36; H, 4.18; N, 7.30.
Chirality DOI 10.1002/chir

58
CIRILLI ET AL.
X-ray Crystal Structure Analysis
As it can be noticed from the molecular weight, the
asymmetric unit contains an acetonitrile molecule cocrys-
tallized with two molecules of compound (2)-1.
The non-hydrogen atoms were refined anisotropically,
whereas the hydrogen atoms were refined as isotropic.
Hydrogens were assigned in calculated positions except
for those on sulfonamidic nitrogens that were located in
the Fourier difference map.
An important observation on the crystal packing is the
presence of two intramolecular and three intermolecular
hydrogen bonds.
X-ray diffraction analysis of (2)-1 and (1)-2. In
both cases Cu/Ka radiation (40mA/-40KV), monochro-
mated by an Oxford Diffraction Enhance ULTRA assembly
and an Oxford Diffraction Excalibur PX Ultra CCD were
used for cell parameters determination and data collection.
The integrated intensities, measured using the x scan
mode, were corrected for Lorentz and polarization
effects.19
Direct methods of SIR200420 were used to resolve the
structure and it was refined using the full-matrix least
squares on F2 provided by SHELXL97.21
Multi-scan symmetry-related measurement was used as
the experimental absorption correction type.
Compound (1)-2. C19H16N2O3S2, cell content
C38H32N4O6S4, PM 5 768.92, Monoclinic, space group P
˚
21, a 5 10.252(1), b 5 10.204(1), c 5 17.259(1) A, b 5
3
˚
97.449(1), V 5 1790.3(3) A , Z 5 2 Dc 5 1.426, l 5 2.886
mm21, F(000) 5 800.
Compound (2)-1. C15H13N2O3S2Cl, cell content
Colorless, needle shaped crystals were obtained from
C32H29N5O6S4Cl2, PM 5 778.74, Triclinic, space group P acetonitrile/ethanol and RX-analysis was performed with a
˚
1, a 5 8.053(1), b 5 8.286(1), c 5 13.737(1)A, a 5 Goniometer Oxford Diffraction KM4 Xcalibur2 at low tem-
3
˚
92.451(3) b 5 102.290(3) g 5 97.267(4), V 5 886.1(2) A , perature (100 K).
Z 5 1 Dc 5 1.459, l 5 4.282 mm21, F(000) 5 402.
Six thousand eight hundred and sixty eight reflections
Colorless, prismatic shaped crystals suitable for collec- were collected with a 4.35<y<70.58 range with a 93.8%
tion were obtained from acetonitrile/ethanol and RX-analy- completeness to theta; 4691 were independent, the param-
sis was performed with a Goniometer Oxford Diffraction eters were 485 and the final R index was 0.0414 for reflec-
KM4 Xcalibur2 at room temperature.
tions having I>2rI, and 0.0478 for all data. Absolute Flack
Six thousand seven hundred and six reflections were structure parameter 0.0109 with esd 0.0180.
collected with a 5.40<y<61.91 range with a 96.9% com-
As it can be noticed from the molecular weight, the
pleteness to theta; 4024 were independent, the parameters asymmetric unit contains two molecules of compound (1)-
were 460 and the final R index was 0.0361 for reflections 2 but there is no symmetry plane (and a space group
having I>2rI, and 0.0492 for all data. Absolute Flack struc- P 21/m), due to the fact that we have a pure enantiomer
ture parameter 0.0388 with esd 0.0138.
and not a racemic mixture.
TABLE 1. Retention factor (k1) for the first eluting enantiomer, enantioseparation (a), and resolution (Rs) factors of
1 and 2 in polar organic conditions
Compound
Mobile phase (v:v)
k1
a
Rs
1
Methanola
0.37 (S)-(2)d
0.51 (S)-(2)
0.73 (S)-(2)
0.57 (S)-(2)
0.27 (S)-(2)
0.31 (S)-(2)
0.46 (S)-(2)
0.34 (S)-(2)
0.21 (S)-(2)
0.14 (S)-(2)
0.44 (S)-(2)
0.67 (S)-(2)
1.02
3.86
4.43
1.38
2.19
2.37
1.29
3.93
3.87
3.14
2.92
2.00
1.87
1.00
1.13
1.23
1.00
1.61
1.40
8.08
13.47
2.72
6.58
3.92
1.22
13.92
11.86
7.19
6.04
4.48
5.98
–
Ethanolb
2-Propanolc
1-Propanolc
Acetonitrilea
Ethyl acetatea
Ethanol-Acetonitrile-TFA 95:5:0.1b
Ethanol-Acetonitrile-TFA 90:10:0.1b
Ethanol-Acetonitrile-TFA 75:25:0.1b
Ethanol-Acetonitrile-TFA 50:50:0.1a
Methanol
2
Ethanol
2-Propanol
1-Propanol
Acetonitrile
Ethyl acetate
Ethanol-Acetonitrile-TFA 95:5:0.1
Ethanol-Acetonitrile-TFA 90:10:0.1
0.82 (S)-(2)
0.34 (S)-(2)
0.33
0.61 (S)-(2)
0.55 (S)-(2)
1.10
1.08
–
4.51
3.01
Column, Chiralpak IA (250 3 4.6 mm I.D.); flow rate, temperature, 258C; detection, UV at 254 and 280 (for ethyl acetate) nm and polarimetric at 365 nm.
a1 ml min21
b0.5 ml min21
c0.35 ml min21
.
.
.
dAbsolute configuration and on-line optical rotation of the first-eluted enantiomer.
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF COX-2 INHIBITORS
59
RESULTS AND DISCUSSION
The polar organic mode is an attractive condition for
semipreparative HPLC enantioseparation of analytes with
medium polarity and poor solubility or insolubility in mix-
tures containing hydrocarbon.23 This is the case of com-
pounds 1 and 2 which are highly soluble in acetonitrile,
sparingly soluble in alcohol and ethyl acetate and insolu-
ble in n-hexane. Successful enantioseparations using Chi-
ralpak IA CSP in combination with polar organic eluents
have been previously reported.11,24 Thus, two analytes
were initially screened on Chiralpak IA using net metha-
nol, ethanol, 2-propanol, acetonitrile and ethyl acetate as a
mobile phase.
The obtained chromatographic results are summarized
in Table 1. Among alcohol-based mobile phases, 2-propa-
nol and 1-propanol gave the lowest selectivity. In practical
experience, these two alcohols are rarely used as a mobile
phase for semipreparative enantioseparation, because of
their high viscosity and difficulty of evaporation from col-
lected fractions.25
The best chiral discrimination for compound 1 was
obtained with ethanol, as demonstrated in Figure 2. Mar-
ginal (compound 1) or null (compound 2) enantioselectiv-
ity was observed with ethyl acetate.
In the case of compound 2, enantioselectivity differ-
ences between methanol and ethanol were not large. How-
ever, ethanol yielded a higher resolution than methanol
due to better efficiency. Although from the analysis of
chromatographic data net ethanol proved to be the best
performing mobile phase, it was not considered conven-
ient for scaling-up enantioseparation at the semiprepara-
tive level. This for two valid reasons: (i) the sample solubil-
Fig. 2. Chromatograms of 1 obtained by simultaneous on-line polari-
metric (gray line) and UV (black line) detection. Column, Chiralpak IA
(250 3 4.6 mm I.D.); eluent, from top to bottom: ethyl acetate, acetonitrile
and ethanol; see Table 1 for other conditions.
An important observation on the crystal packing is the ity of 1 and 2 in ethanol was not high enough to allow
presence of four intermolecular hydrogen bonds. appreciable amounts of racemate to be separated for single
The non-hydrogen atoms were refined anisotropically injection and (ii) retention times were too long. In practice,
whereas hydrogen atoms were refined as isotropic; they owing to the high viscosity of ethanol and in accordance
were assigned in calculated positions.
with the column back-pressure limit indicated for the Chi-
The X-ray CIF file for these structures have been depos- ralpak IA column (about 1500 psi), the retention times can-
ited at the Cambridge Crystallographic Data Centre and not be appreciably shortened by increasing the flow rate.
allocated with deposition numbers CCDC 710777 (Com-
It can be seen from the results resumed in Table 1, the
pound 1) and CCDC 710778 (Compound 2). (Copies of progressive addition of acetonitrile to ethanol, in the pres-
the crystallographic data for this article can be obtained, ence of a constant amount (0.1%) of trifluoroacetic acid,
free of charge, from CCDC, 12 Union Road, Cambridge, lead to a reduction in retention, enantioselectivity and re-
CB2 1EZ UK [e-mail: deposit@ccdc.cam.ac.uk; internet:// solution. The effects on enantioselectivity and resolution
www.ccdc.cam.ac.uk]).
were more pronounced for compound 2 which was only
TABLE 2. Chromatographic and polarimetric analysis of the pooled fractions containing the first (F1) and second (F2)
eluted enantiomers of 1 and 2
F1b
F2b
Compound
Mobile phase
Aa
20
e.e.(%)
[a]2D0
e.e.(%)
[a]2D0
1
Ethanol/acetonitrile/TFA 90:10:0.1
(v/v/v)
Ethanol/acetonitrile/TFA 95:5:0.1
(v/v/v)
>99.0
2136 (c 5 0.1, CH3CN)
298 (c 5 0.1, CH3CN)
>99.0
1140 (c 5 0.1, CH3CN)
197 (c 5 0.1, CH3CN)
2
10
>99.0
>99.0
Column: Chiralpak IA (250 3 10 mm I.D.); flow-rate, 3.0 ml min21; detector, UV at 280 nm; temperature, 258C.
aAmount of sample (in mg) resolved in a single semipreparative run.
bEnantiomeric purity and polarimetric data for the pooled fractions containing the first (F1) and second (F2) eluted enantiomers.
Chirality DOI 10.1002/chir

60
CIRILLI ET AL.
On-line (Table 1) and off-line (Table 2) polarimetric anal-
ysis indicated that the first eluting enantiomer of both
compounds was levorotatory under all investigated condi-
tions.
At the best of our knowledge, no stereochemical charac-
terization for molecules structurally related to 1 or 2 has
been reported. We therefore used single-crystal X-ray dif-
fraction to unambiguously assign26 the absolute configura-
tion to the collected enantiomers. As illustrated in Figure
4, (2)-1 and (1)-2 were shown to have (S) and (R) abso-
lute configuration at the stereogenic center located on the
thiazolidinone ring.
HPLC analysis of non racemic samples enriched by
enantiomers of known chirality made it possible to assign
the enantiomer elution order on the Chiralpak IA CSP: the
(S)-(2)-enantiomers of both compounds 1 and 2 were
eluted before their (R)-(1) counterparts (Table 1).
The CD spectra of the enantiomers of 1 and 2 are
shown in Figure 5. Solutions of (R)-1 exhibited two strong
Cotton effects of opposite sign at 209 and 229 nm followed
by two weaker CD bands at 250 (negative) and 280 nm
(positive). It should be noted that, in the case of the (R)-2
enantiomer, the intense negative CD band located at a
Fig. 3. (a): Semipreparative resolution of 20 mg of 1; (b) Analytical
HPLC enantioseparation of 1; (c) and (d): Chromatographic analysis of
isolated enantiomers. Column, (a): Chiralpak IA (250 3 10 mm I.D.),
(b), (c), and (d): Chiralpak IA (250 3 4.6 mm I.D); eluent, ethanol-acetoni-
trile-TFA 90-10-0.1 (v/v/v); flow-rate, (a): 3.0 ml min21, (b), (c), and (d):
0.5 ml min21; detector, UV at 280 (a) and 254 nm (b, c and d); tempera-
ture, 258C.
weakly resolved with net acetonitrile. However, the addi-
tion of 10% of the non-alcoholic cosolvent produced a
reduction in the elution time of the more retained enan-
tiomer of 1 by about 40% without significatively impairing
enantioselectivity or resolution (the enantioselectivity and
resolution factor values changed from 4.43 and 13.47 to
3.87 and 11.86, respectively).
As was expected from the acid nature of the analytes,
the presence of 0.1% of trifluoroacetic acid caused an
enhancement in peak symmetry. A further benefit which
was particularly useful for semipreparative applications
consisted in improved sample solubility.
So, based on the chromatographic results of analytical
screening, the acetonitrile-ethanol-TFA 90-10-0.1 (v/v/v)
and acetonitrile-ethanol-TFA 95-5-0.1 (v/v/v) mixtures
were identified, respectively, as the most suitable eluents
for the enantioseparation of 1 and 2 on the 1-cm I.D. IA
column.
Table 2 shows the enantiomeric excess and rotation spe-
cific for pooled fractions containing the first eluted (F1)
and second eluted (F2) enantiomer, after injection of 20
mg of 1 and 10 mg of 2. The racemic forms were dis-
solved in 1000 lL of the acetonitrile-ethanol-TFA 50-50-0.1
(v/v/v) mixture. As shown in Figure 3, the isolation of
enantiopure forms of 1 was performed in short also in
presence of unknown impurities.
Fig. 4. An ORTEP view of the molecular structure of the (S)-(2)-1
enantiomer (top) and (R)-(1)-2 enantiomer (bottom).
Chirality DOI 10.1002/chir

HPLC ENANTIOSEPARATION OF COX-2 INHIBITORS
61
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Fig. 5. CD spectra of enantiomers of 1 and 2 in acetonitrile.
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shorter wavelength was red shifted at 227 nm. The slight
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To sum up, the enantioselective HPLC method based on
Chiralpak IA CSP in the presence of acetonitrile-ethanol-
TFA mixtures provided rapid access to mg amounts of
enantiopure forms of compounds 1 and 2. The application
of the optimized CSP/eluent system supports a first com-
parative biological evaluation of the influence of stereo-
chemistry on cyclooxygenase-2 inhibition activity. Further,
the crystallographic and CD data spectra presented in this
study may be used to empirically assign the absolute con-
figuration of compounds structurally related to 1 and 2.
14. Cirilli R, Ferretti R, Gallinella B, De Santis E, Zanitti L, La Torre F.
High-performance liquid chromatography enantioseparation of proton
pump inhibitors using the immobilized amylose-based Chiralpak IA
chiral stationary phase in normal-phase, polar organic and reversed-
phase conditions. J Chromatogr A 2008;1177:105–113.
15. Cirilli R, Simonelli A, Ferretti R, Bolasco A, Chimenti P, Secci D, Mac-
cioni E, La Torre F. Analytical and semipreparative high performance
liquid chromatography enantioseparation of new substituted 1-thiocar-
bamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazoles on polysaccharide-based
chiral stationary phases in normal-phase, polar organic and reversed-
phase conditions. J Chromatogr A 2006;1101:198–203.
16. Weng W, Guo H, Zhan F, Fang H, Wang Q, Yao B, Li S. Chromato-
graphic enantioseparations of binaphthyl compounds on an immobi-
lized polysaccharide-based chiral stationary phase. J Chromatogr A
2008;1210:178–184.
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