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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