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Chiral separation of bioactive cyclic
Mannich ketones by HPLC and CE using
cellulose derivatives and cyclodextrins
as chiral selectors
Nina Grobuschek a, Lawan Sriphong b, Martin G. Schmid a,Tamas Lorand c, Hassan Y. Aboul-Enein d, Gerald Gubitz a,*
aInstitute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens University,
Universitatsplatz 1, A-8010 Graz, AustriabDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Silpakorn University,
Nakorn Prathom 73000, ThailandcDepartment of Medical Chemistry, Faculty of Medicine, University Pecs, H-7601 Pecs,
Szigeti ut 12, HungarydBioanalytical and Drug Development Laboratory, Biological and Medical Research Department (MBC-03),
King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia
Received 1 July 2001; accepted 14 December 2001
Abstract
The chiral separation of cyclic Mannich ketones of potential pharmaceutical interest is
investigated using HPLC and CE. These Mannich ketones show a marked antibacterial and
antifungal activity. In HPLC, stationary phases containing cellulose derivatives or h-cyclodextrinwere used and in CE different cyclodextrins, such as h-CD, g-CD, carboxymethyl-h-CD and
succinyl-h-CD were added to the background electrolyte as chiral selectors.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: High performance liquid chromatography; Capillary electrophoresis; Enantiomer separation; Mannich
ketones; Cellulose derivative; Cyclodextrins
0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0165 -022X(02 )00089 -1
* Corresponding author. Tel.: +43-316-380-5371; fax: +43-316-380-9846.
E-mail address: gerald.guebitz@kfunigraz.ac.at (G. Gubitz).
www.elsevier.com/locate/jbbm
J. Biochem. Biophys. Methods 53 (2002) 25–36
1. Introduction
The family of a,h-unsaturated ketones is known to possess antimicrobial effects, as
they react as alkylating agents with the essential thiol groups of enzymes of micro-
organisms [1].
In order to receive more efficient water-soluble unsaturated ketones, several chiral
cyclic Mannich ketones were prepared either from arylidenecycloalkanones or from fused
bicyclic ketones as potential antibacterial and antifungal agents.
These substances showed more selective toxicity towards microorganisms than the
parent unsaturated ketones. Their breakdown can either afford reactive vinyl ketones in a
1,2-elimination or they can undergo reverse Mannich reaction [1]. These vinyl ketones
produced as intermediates have much higher affinity towards thiols than hydroxy- and
amino groups present in the nucleic acids, therefore they do not show the mutagenic side
effect of some alkylating agents used in therapy [2]. The Mannich ketones showed a
marked in vitro antibacterial effect against some Gram-positive strains as Staphylococcus
saprophyticus OKI 120008, Staphylococcus aureus OKI 118003, Micrococcus luteus
ATCC 9341 and Bacillus subtilis ATCC 6633, observed in low minimal inhibitory
concentration (MIC) values as 12.5–3.25 mg/l, while only some of the compounds were
active against the Escherichia coli strains [3]. In addition, they had in vitro antifungal
effects against Candida spp. and Aspergillus sp. [4] and they showed cytotoxic activity in
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tests on A431
(human epidermoid adenocarcinoma) cells, too [5].
Since the antibacterial effect might be restricted to one of the enantiomers, the
investigation of the individual enantiomers would be of interest. For that purpose, the
separation of the enantiomers on a micro-preparative scale is required.
The present paper deals with investigations for the analytical-scale chiral separation of
these cyclic Mannich ketones by HPLC and CE, which can be regarded as pilot
experiments for separation on preparative scale.
Among others, polysaccharides and cyclodextrins have been found to be powerful
selectors in HPLC and CE.
Cellulose derivative-based phases were developed by Ichida et al. [6] and Okamoto et
al. [7] and were found to show enantioselectivity for a broad spectrum of compounds [8–
12]. Especially the cellulose tris-(3,5-dimethylphenylcarbamate) phase (Chiralcel ODR)showed a marked chiral recognition ability for different classes of compounds. In addition
to the normal phase mode, this stationary phase was shown to be applicable also in the
reversed phase mode (Chiralcel OD-RHR) [13–15].Cyclodextrins have been used as chiral selectors both in HPLC [16,17] and in CE
[18–21] and represent the most frequently used chiral selectors for a broad application
range.
For the chiral separation of these Mannich ketones, we investigated a cellulose tris-
(3,5-dimethylphenylcarbamate), Chiralcel ODR, in normal and reversed phase mode
and a h-CD phase, Cyclobond IR, in normal phase, polar organic and reversed phase
mode.
For CE investigations native CDs and negatively charged CDs, such as succinyl-h-CDand carboxymethyl-h-CD were used.
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–3626
2. Material and methods
2.1. Instrumentation and separation conditions
HPLC was carried out with a HP 1090 Liquid Chromatograph (Hewlett Packard,
Palo Alto, CA, USA) equipped with a diode array detector (detection at 230 nm). A
Chiralcel ODR column 15� 0.46 cm I.D. (Daicel Chemical Industries, Tokyo, Japan)
(cellulose tris-(3,5-dimethylphenylcarbamate)) was used in the normal phase mode and
a Chiralcel OD-RHR column 25� 0.46 cm I.D. (Daicel Chemical Industries) was
operated in the reversed phase mode. Separations on the Cyclobond I 2000 seriesRcolumn 25� 0.46 cm I.D. (Astec, Whippany, NJ, USA) were carried out in the polar-
organic mode, the normal phase and the reversed phase mode. Temperature was 25 jC,samples were injected automatically by an autosampler (5–15 Al) and flow rate was
0.4–1 ml/min. The mobile phase was filtered if necessary through a cellulose nitrate
filter (Sartorius, Goettingen, Germany) with pore size of 0.2 Am and degassed with
helium for 20 min.
CE was performed using a PrinCE capillary electrophoresis system (PrinCE
Technologies, Emmen, Netherlands), equipped with a LAMBDA 1000 UV/VIS
detector (Bischoff Analysentechnik, Leonberg, Germany) and an external pressure
device (12 bar). The system was interfaced with a personal computer, DAX
software being used for data processing. The fused silica capillary (50 Am I.D.,
effective length 26 cm) was purchased from Microquarz (Munich, Germany). The
capillary was thermostated at 25 jC, detection was accomplished via on-column
measurement of UV absorption at 208 nm. The applied voltage was 10 or 15 kV,
respectively, and velocity of EOF was determined by injecting DMSO. Samples and
Table 1a
Compounds investigated: Mannich ketones of cycloalkanones
Sample n R Ar
1 1 1-Piperidyl Phenyl
2 1 1-Piperidyl 4V-OCH3-C4H6
3 1 1-Piperidyl 3V-OCH3-C4H6
4 1 4-Morpholinyl 3V-OCH3-C4H6
5 1 1-Piperidyl 2V-OCH3-C4H6
6 2 4-Morpholinyl Phenyl
7 2 Pyrrolidinyl Phenyl
8 2 4V-Methyl-piperidyl Phenyl
9 3 4-Morpholinyl Phenyl
10 3 1-Piperidyl Phenyl
11 4 4-Morpholinyl Phenyl
12 4 1-Piperidyl Phenyl
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–36 27
electrolytes were filtered through a 0.45-Am pore size filter (Schleich/Schuell,
Dassel, Germany).
2.2. Chemicals and solutions
All chemicals were of analytical grade. Hexane and isopropanol were purchased from
Roth (Karlsruhe, Germany), acetonitrile, methanol, diethylamine (DEA) and sodium
dihydrogenphosphate, boric acid, sodium hydroxide and dimethylsulfoxide (DMSO) from
E. Merck (Darmstadt, Germany). Sodium perchlorate, acetic acid, triethylamine and
perchloric acid and h-CD, carboxymethyl-h-CD and g-CD were purchased from
Fluka (Buchs, Switzerland), and succinyl-h-CD was obtained from Cyclolab R
Lab. (Budapest, Hungary). Water was deionized and doubly distilled.
2.3. Synthesis of Mannich ketones
The Mannich ketones (Table 1a and b) have been prepared either from the correspond-
ing bicyclic unsaturated ketones (2-arylidenecycloalkanones) [22] or from fused ketones
as tetralones, indanones, etc. (Fig. 1) [3]. The bases were liberated and precipitated by
Fig. 1. Synthesis of the Mannich ketones by the classical Mannich reaction.
Table 1b
Compounds investigated: fused Mannich ketones
Sample n R1 R2
13 1 H 1-Piperidyl
14 2 H 2-(1,2,3,4-Tetrahydro)-
isoquinolyl
15 3 H 1-Piperidyl
16 1 5-OCH3 1-Piperidyl
17 2 5-OCH3 1-Piperidyl
18 2 6-OCH3 1-Piperidyl
19 2 7-OCH3 1-Piperidyl
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–3628
methanolic HCl after recrystallisation. The structure of these Mannich ketones was
confirmed by NMR measurements.
3. Results and discussion
3.1. Cellulose derivatives used as CSP in HPLC
Cellulose derivatives possess still the helical structure of cellulose and due to the
derivatization of the hydroxy groups a tertiary structure is built, forming chiral cavities
Table 2
Separation data for Mannich ketones by HPLC using Chiralcel-ODR column in the normal phase mode are shown
Sample k1 k2 a Rs
1 0.77 0.84 1.10 0.91
4 3.20 3.56 1.11 1.46
5 3.06 3.96 1.30 4.23
8 0.52 0.62 1.19 0.90
9 1.16 1.41 1.22 2.23
11 0.90 1.54 0.58 3.90
16 0.78 0.91 1.17 1.02
Only data of resolved compounds are given under optimized conditions. Mobile phase: hexane/isopropanol
(90:10) + 0.2% DEA.
Table 3
Separation data for Mannich ketones by HPLC using a Chiralcel-OD-RHR column in the reversed phase mode
Sample k1 k2 a Rs Mobile phase *
1 25.46 28.53 1.12 1.77 1
2 4.61 4.99 1.08 0.64 3
3 13.24 13.71 1.04 0.62 2
4 2.62 2.91 1.12 0.83 3
5 8.48 9.40 1.11 0.87 3
6 1.61 3.32 2.06 4.32 4
7 2.70 8.06 2.98 7.25 4
8 5.98 12.36 2.07 3.19 4
9 1.75 3.34 1.91 4.26 4
10 3.91 9.88 2.53 3.96 4
11 3.50 – 1.00 0.00 3
12 4.10 4.75 1.16 0.83 4
13 1.62 – 1.00 0.00 2
14 9.12 11.07 1.21 0.81 3
15 4.24 4.40 1.04 0.33 2
16 2.20 2.31 1.05 0.84 2
17 2.80 3.56 1.27 1.57 3
18 5.24 5.48 1.05 0.40 2
19 2.22 – 1.00 0.00 3
Only results under optimized conditions are shown.
*Mobile phase 1: 0.5 M NaClO4, pH:2/ACN (75:25). Mobile phase 2: 0.5 M NaClO4, pH:2/ACN (70:30).
Mobile phase 3: 0.5 M NaClO4, pH:2/ACN (65:35). Mobile phase 4: 0.5 M NaClO4, pH:2/ACN (60:40).
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–36 29
capable for stereoselective inclusion of molecules. Chiralcel OD and Chiralcel OD-RH
phases are based on macroporous silica gel coated with the semisynthetic polymers. These
polymers consist of D-glucose units in h-1,4-linkage derivatized with 3,5-dimethylphe-
nylcarbamate groups [23]. For the chiral recognition hydrogen bondings, dipole–dipole
and k–k interactions [11,24], which take place between the diphenylmethylcarbamate
residue and aromatic moieties of the analytes, are responsible. We investigated the chiral
separation of cyclic Mannich ketones using cellulose tris-(3,5-dimethylphenylcarbamate)
as chiral stationary phase in the normal phase mode (Chiralcel ODR column) and the
reversed phase mode (Chiralcel OD-RHR column). In the normal phase mode, we used
hexane/isopropanol (90:10) containing 0.2% DEA as mobile phase, whereby out of 19
compounds investigated 7 were resolved, 2 of them baseline separated (Table 2). On a
column containing the same selector suitable for operating in the reversed phase mode, 10
Fig. 2. Influence of ACN content on the selectivity (a) using an OD-RHR column in the reversed phase mode.
Mobile phase: 0.5 M NaClO4, pH2 containing different amounts of ACN.
Fig. 3. Chromatogram of compound 17 using a Chiralcel OD-RHR column in the reversed phase mode. Mobile
phase: 0.5 M NaClO4, pH2/ACN (60:40), flow: 0.4 ml/min, Inj.: 15 Al, sample concentration: 1 mg/ml in water.
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–3630
compounds were baseline resolved and 6 showed partial resolution (Table 3). As mobile
phase 0.5 M NaClO4, pH 2 containing different amounts of ACN (20–40%) was used.
Fig. 2 shows the influence of the ACN content on a. With increasing amounts of ACN, the
Table 4
Separation data for Mannich ketones by HPLC using a Cyclobond I 2000R column in the polar organic and the
reversed phase mode are shown
Sample k1 k2 a Rs Mobile
phase *
1 2.52 – 1.00 0.00 1
3.50 – 1.00 0.00 2
2 2.56 – 1.00 0.00 1
3.63 – 1.00 0.00 2
3 1.96 – 1.00 0.00 1
1.58 – 1.00 0.00 4
4 0.27 – 1.00 0.00 1
3.26 – 1.00 0.00 2
5 1.98 – 1.00 0.00 1
6.78 8.41 1.24 2.45 4
6 0.39 0.42 1.07 0.56 1
5.66 5.87 1.04 0.64 2
7 0.01 – 1.00 0.00 1
6.15 6.39 1.04 0.62 2
8 0.01 – 1.00 0.00 1
7.42 – 1.00 0.00 2
9 0.24 0.40 1.65 2.45 1
1.38 2.05 1.49 4.67 3
10 3.50 – 1.00 0.00 1
1.44 2.18 1.51 4.58 3
11 0.17 0.25 1.44 1.61 1
1.24 1.75 1.41 3.68 3
12 1.83 2.44 1.33 3.80 1
1.19 1.65 1.39 3.35 3
13 1.32 – 1.00 0.00 1
0.71 – 1.00 0.00 4
14 0.24 – 1.00 0.00 1
3.12 3.30 1.06 0.78 4
15 1.69 1.74 1.03 0.60 1
2.12 2.57 1.21 1.93 4
16 1.57 – 1.00 0.00 1
1.31 – 1.00 0.00 4
17 2.04 – 1.00 0.00 1
7.40 8.05 1.09 0.90 4
18 2.69 2.76 1.03 0.63 1
4.11 4.36 1.06 0.72 4
19 2.41 – 1.00 0.00 1
4.71 4.92 1.05 1.44 4
Only data under optimized conditions are given.
Polar organic mode: *Mobile phase 1: ACN/MeOH/Hac/TEA (95:5:0.3:0.2). Reversed phase mode: *Mobile
phase 2: ACN/1%TEAA (15:85); 3: ACN/1%TEAA (25:75); 4: ACN/1%TEAA (7:93).
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–36 31
retention time decreased, while a did not change significantly. In Fig. 3, the chromatogram
of compound 17 is shown.
3.2. Cyclodextrins used as CSP in HPLC
Cyclodextrins represent one of the most frequently used chiral selectors. These cyclic
oligosaccharides consist of 6 (a-CD), 7 (h-CD), or 8 (g-CD) glucopyranose units with a
truncated cone providing a hydrophobic cavity with a hydrophilic surface. The hydroxy
groups in positions 2, 3 and 6 are available for derivatization modifying the depth of the
cavity and the solubility. The chiral recognition is based on inclusion of the hydrophobic
group of the analyte into the hydrophobic cavity of the CD and additional lateral
interactions with the hydroxyl groups at the C-2 and C-3 at the upper rim of the CD,
such as hydrogen bonds and dipole–dipole interactions. Armstrong and DeMond [16]
developed the first HPLC-phase based on cyclodextrin chemically bonded to silica gel. We
used a h-CD column (Cyclobond I series 2000R) and the separations were performed in
Fig. 4. Chromatogram of compound 9 using a Cyclobond IR column. (a) In the polar organic mode. Mobile
phase: ACN/MeOH/Hac/TEA (95:5:0.3:0.2), flow: 1.0 ml/min, Inj.: 5 Al, sample concentration: 1 mg/ml in
mobile phase. (b) In the reversed phase mode. Mobile phase: ACN/1% TEAA, pH 4 (25:75), flow: 0.5 ml/min,
Inj.: 5 Al, sample concentration: 1 mg/ml in mobile phase.
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–3632
the normal phase, the reversed phase and the polar organic mode. In the normal phase
mode we used as mobile phase hexane/isopropanol (90:10), but no satisfying separations
were achieved. Using the polar organic mode with a mobile phase consisting of ACN/
MeOH/HAc/TEA (95:5:0.3:0.2), 3 compounds were baseline and 3 partially resolved. The
separation data of all Mannich ketones under these conditions are presented in Table 4.
Regarding the separations achieved in the reversed phase mode using h-CD, it can be seen
that 5 compounds were baseline and 8 partially resolved (Table 4). In this case ACN/
Table 5
Separation data for Mannich ketones by CE using different selectors added to the background electrolyte (BGE)
Sample t1 t2 a Rs BGE*
1 17.76 18.15 1.02 0.92 1
21.47 22.32 1.04 0.86 2
2 16.89 – 1 0 1
26.13 27.12 1.04 1.04 2
3 18.34 – 1 0 1
21.56 22.02 1.02 0.51 2
4 17.35 – 1 0 1
25.96 – 1 0 2
5 14.51 14.84 1.02 0.90 1
20.39 – 1 0 2
6 18.29 – 1 0 1
21.11 21.90 1.04 0.86 2
7 14.48 – 1 0 1
18.60 19.20 1.03 0.83 2
8 14.70 – 1 0 1
18.61 19.26 1.04 0.90 2
9 14.50 14.77 1.02 1.27 1
24.56 25.53 1.04 0.98 2
10 14.58 14.92 1.02 1.31 1
22.60 23.59 1.04 1.34 2
11 14.69 15.01 1.02 0.96 1
25.94 27.14 1.05 1.23 2
12 14.66 15.41 1.05 1.65 1
22.20 – 1 0 2
13 11.06 – 1 0 1
11.84 12.43 1.05 1.12 2
14 15.60 16.24 1.04 1.31 1
17.24 – 1 0 2
15 13.51 – 1 0 1
15.82 – 1 0 2
16 11.50 – 1 0 1
13.59 14.13 1.04 0.84 2
17 14.66 14.81 1.01 0.47 1
21.29 – 1 0 2
18 15.95 16.37 1.03 0.99 1
22.26 – 1 0 2
19 14.29 14.52 1.02 0.66 1
20.57 – 1 0 2
*BGE 1: 0.2 M H3BO3, pH5, 4% succinyl-h-CD. BGE 2: 0.2 M H3BO3, pH 5, 5% carboxymethyl-h-CD.
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–36 33
1%TEAA, pH 4 containing different amounts of ACN (7–25%) was used as mobile
phase. Compared to the reversed phase mode, retention times were shorter in the polar
organic mode (Fig. 4a,b; Table 4).
3.3. Cyclodextrins used as chiral selectors in CE
Native CDs, such as h-CD and g-CD were checked as additives to the background
electrolyte (BGE) to perform chiral separations of the Mannich ketones, however no
satisfying results were achieved. When negatively charged CDs, such as succinyl-h-CDand carboxymethyl-h-CD were used, some of the compounds investigated were
resolved.
This group of CDs showed higher selectivity attributed to the countercurrent mobility.
The negatively charged CDs tend to move to the anode while the positively charged
analyte moves to the cathode. Using 4% succinyl-h-CD as additive to the BGE (0.2 M
H3BO3 pH 5), out of 19 compounds investigated 2 were baseline and 7 partially resolved
(Table 5). The pH was checked in a range from 3 to 9 and the optimum was found to be 5.
With 5% carboxymethyl-h-CD as chiral selector, 3 compounds were baseline and 10
partially resolved (see Table 5). Fig. 5 shows the electropherogram of compound 18 using
succinyl-h-CD as chiral selector.
4. Conclusion
A cellulose tris-(3,5-dimethylphenylcarbamate) phase and a cyclodextrin phase were
compared for their ability to resolve cyclic Mannich ketones by HPLC. In several cases,
these two columns were found to be complementary, whereby the best results were
obtained with the Chiralcel OD-RHR column operated in the reversed phase mode.
Fig. 5. Electropherogram of compound 18 using 4% succinyl-h-CD as chiral selector. BGE: 0.2 M H3BO3, pH 5.
Capillary: 50 Am I.D., 70/26-cm effective length. 10 kV, 7 AA; Inj.: electrokinetic (3 kV, 0.1 min).
N. Grobuschek et al. / J. Biochem. Biophys. Methods 53 (2002) 25–3634
Furthermore, CE was also checked for its ability to resolve the Mannich ketones using
different CDs as chiral selectors. Whereas with native CDs no separations were observed,
with negatively charged CD derivatives some compounds were resolved.
Acknowledgements
This work was supported by the grant OTKAT 030261 and by a grant from the Fonds
zur Forderung der Wissenschaflichen Forschung (FWF, P 13815CHE and P 12767CHE).
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