16601580

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Short communication

Chiral high-performance liquid chromatographic separation and circular

dichroism spectra of the enantiomers of cytotoxic aristocularine alkaloids

Salvatore Caccamese

a,∗

, Giovanna Scivoli

a

, Salvatore Bianca

a

,

Juan Manuel L´opez-Romero

b

, Francisco Javier Ortiz-L´opez

b

a_{Dipartimento di Scienze Chimiche, Università di Catania, viale A. Doria 6, 95125 Catania, Italy} b_{Departamento de Quimica Orgánica, Universidad de Málaga, 29071 Málaga, Spain}

Received 9 May 2006; received in revised form 25 July 2006; accepted 28 July 2006 Available online 21 August 2006

Abstract

The HPLC enantiomeric separation of the racemic cularinoid alkaloidsN-p-methoxy-1,␣-dihydroaristoyagonine (1) and 4,5-demethoxy-1,␣ -dihydroaristoyagonine (2) was accomplished using five chiral stationary phases (CSPs), some of them polysaccharide-derived ones. The molecular size and conjugative effect strongly affect the different enantioselectivity of the compounds1and2on the various CSPs investigated. Single enantiomers of1were isolated by repeated injections on an analytical HPLC column, and their circular dichroism spectra and optical rotations were measured. The cytotoxicity of the isolated enantiomers was measured on a human colon carcinoma cell line and compared with that of the racemic compound.

Keywords: Cularine alkaloids; Polysaccharide and Pirkle-type chiral stationary phases; Enantioselective chromatography; Circular dichroism spectra; Cytotoxicity of enantiomers

1. Introduction

Cularinoids are a group of isoquinoline alkaloids consisting of about 60 members. They are characterized by a benzoxepine skeleton but they occur naturally in various oxidation states. Among them the aristocularines are oxidized cularinoids and a notable example is aristoyagonine, which occupies a special place as it is the only example till date of a natural cular-ine alkaloid incorporating a five-membered lactam ring [1]. From the Spanish group, it has been reported that aristocular-ines and the reduced dihydroaristoculararistocular-ines exhibit significant activity against some tumoral cell lines[1], and the IC50 val-ues (<5␮g/mL against HT 29 human colon adenocarcinoma) are similar to those reported for aporphine alkaloids studied by them[2]. Thus, these alkaloids are candidate drugs.

Extensive reviews have been published on the effect of chi-rality in several activities of drugs[3]and on the stereochemical aspects of drug action[4].

∗_{Corresponding author. Tel.: +39 095 738 5029; fax: +39 095 580138.}

E-mail address:[email protected](S. Caccamese).

Owing to the experience of the Italian group in the HPLC separation of the enantiomers of racemic alkaloids with a very different skeleton[5,6], we tried to resolve the enantiomers of the racemic1and2.

In this article we report the direct enantioseparation of1and

2using an isocratic normal-phase HPLC with five chiral station-ary phases (CSPs) and variousn-hexane/ethanol mobile phases. The good enantioselectivity and resolution factor obtained with a polysaccharide-derived CSP (Chiralpak AD) allowed us to iso-late the single enantiomers of compound1by repeated injections and to measure the circular dichroism (CD) spectra, specific rota-tion and cytotoxicity of the isolated enantiomers.

2. Experimental

2.1. Instrumentation

The HPLC system consisted of a Varian 5060 liquid chro-matograph equipped with a Knauer injector possessing 20 or 100␮L sample loops, a Jasco Uvidec 100-III UV spectrophoto-metric detector operating at 280 nm and a Hewlett-Packard 3396 integrator. CD spectra were recorded on a Jasco 810

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larimeter, using 1 mm cell. Optical rotation was measured with a Jasco DIP-370 digital polarimeter, using a 10-cm microcell. NMR spectra were obtained on a Bruker WP-200 SY instrument, at 200 MHz for1H and 50.3 MHz for13C. Chemical shifts are given relative to residual CHCl3(δH7.24 ppm) in deuterochlo-roform.Jvalues are in Hz.13C chemical shifts are given relative to CDCl3(77.0 ppm) in deuterochloroform. Mass spectrum (EI-MS, 70 eV,m/z) was obtained on an Hewlett-Packard 5988 A instrument. UV spectrum was recorded on a Hewlett-Packard 8452 A spectrophotometer.

2.2. Enantioselective columns

Two polysaccharide-derived columns (250 mm×4.6 mm) were Chiralpak AD (amylose tris-3,5-dimethylphenylcarba-mate) and Chiralcel OD-H (cellulose tris-3,5-dimethylphe-nylcarbamate), both coated on silica gel, 10 and 5␮m, respec-tively. A third polysaccharide derived column was Chiral-pak IB (250 mm×4.6 mm), a new CSP based on cellu-lose tris-3,5-dimethylphenylcarbamate immobilized onto sil-ica gel. The three columns were supplied by Daicel (Tokyo). The Pirkle-type column (250 mm×4.6 mm) was 2R, 3R ␤ -GEM packed with undecanyl-N -3,5-dinitrobenzoyl-3-amino-3-phenyl-2-(1,1-dimethyl ethyl) propanoate covalently bonded to 5␮m 3-propylsilica from Regis (Morton Grove, IL, USA). The network polymeric column (250 mm×4.6 mm) was Kro-masil CHI-DMB (2R,3R-N,N-diallyl-l-tartardiamide based) from EKA Nobel AB (Bohus, Sweden). A column in-line filter with 0.5␮m stainless steel frit of 3 mm diameter from Rheo-dyne was used to protect the HPLC columns. Disposable PTFE filters of 0.2␮m pore size were used for filtration of sample solu-tions. Column void volume (t0) was measured by injection of tri

tert-butylbenzene as a non-retained marker[7]. Other HPLC

chromatographic parameters were those typically employed [8].

2.3. Chemicals

Compound 1 (N-p-methoxy-1,␣-dihydroaristoyagonine) is a new compound and its synthesis and characterization are described in the next section. The synthesis and full charac-terization of compound2have been described elsewhere[1].

2.4. Cytotoxicity assays

HT-29 and MDA-MB-231 cells were obtained from the American Type Culture Collection; P-388 and Schabel cells were kindly provided by Pharmamar (Madrid, Spain). The effects of the alkaloids on cellular proliferation were assayed by the MTI [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method [9]. Drug concentrations used in the test were in the range 0.3125–10␮g/mL. Details of the method have been described elsewhere[2].

3. Results and discussion

3.1. Chromatographic and chiroptical results

Table 1provides chromatographic data for the normal-phase separation of the enantiomers of the compounds1and2 (for-mulas shown inFig. 1) on five chiral stationary phases (CSP).

For compound1, using the three polysaccharide-derived CSP, the separation factor ␣ ranges from 1.18 to 1.29 but there is a marked difference in the resolution factorRs in experiments performed under the same polarity of the mobile phase, as shown in entries 3, 5, 10; 2, 9, where Chiralcel OD-H affords the minor

Rs. The Chiralpak IB gave much betterRs factor, as shown in Fig. 2a and c, and this is done for the immobilization of the chiral selector. Indeed, both OD-H and IB have the same chiral selector (cellulose tris-3,5-dimethylphenylcarbamate) but this is simply quoted in the support in the first one. The strong enhancement of the resolution factor and peak shape for the new commercially available Chiralpak IB was fully investigated[10].

A Pirkle-type CSP (␤-GEM) was also used for the enan-tioseparation of compound1. It gave good results, as shown in entries 11 and 12. However, the obtained capacity factors and elution times were too high, and this precludes a semiprepara-tive application, as also shown inFig. 2d. The third CSP type used for the enantioseparation of compound1 was a network polymeric CSP (Kromasil), but even by using very low polarity of the mobile phase, the separation and resolution factors were much less satisfactory than those obtained with the other CSP. As expected for the normal-phase behaviour of these CSPs, the resolution factorRsof compound1improves by decreasing the polarity of the mobile phase, as shown by comparison of the

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Table 1

Enantioselective HPLC resolution of alkaloids1and2on five chiral stationary phases

Entry Compound CSP A(%)a _k

1 t1 t2 α Rs

1 1 OD-H 10 16.44 52.2 61.3 1.18 1.5

2 1 OD-H 20 7.92 26.7 31.1 1.18 1.3

3 1 OD-H 30 4.34 15.9 18.4 1.18 1.0

4 1 AD 10 11.35 41.4 48.4 1.20 –c

5 1 AD 30 8.73 32.6 40.5 1.27 2.5

6 1 AD 40 5.91 23.1 28.8 1.28 2.4

7 1 AD 40d 5.71 18.2 22.7 1.29 2.2

8 1 AD 40e 5.40 15.5 19.4 1.29 2.0

9 1 IB 20 7.73 26.2 30.9 1.20 2.3

10 1 IB 30 4.32 16.0 18.7 1.20 2.2

11 1 ␤-GEM 30 16.46 44.9 57.6 1.30 3.1

12 1 ␤-GEM 50 8.92 25.5 32.4 1.30 2.4

13 1 Kromasil 2 6.28 19.2 21.0 1.10 1.1

14 1 Kromasil 5 3.49 11.9 12.7 1.09 0.8

15 2 OD-H 10 3.76 14.2 18.5 1.38 2.6

16 2 OD-H 20 1.99 8.9 11.0 1.35 2.0

17 2 OD-H 30 1.32 6.9 8.3 1.34 1.0

18 2 AD 10 5.94 23.3 29.8 1.33 1.7

19 2 AD 20 2.74 12.5 14.9 1.26 1.2

20 2 AD 30 1.72 9.1 10.2 1.19 1.0

21 2 IB 10 4.56 16.7 18.7 1.14 2.3

22 2 IB 20 2.20 9.6 10.5 1.13 2.0

23 2 ␤-GEM 10 11.39 31.8 1.00

24 2 Kromasil 2 4.16 13.6 1.00

a_{Percentage of ethanol doped with 0.5% of TFA in}_n_{-hexane at a flow rate of 1 mL/min unless otherwise specified;}_t

0, min = 3.0 (OD-H), 3.4 (AD), 3.0 (IB), 2.6 (␤-GEM), 2.6 (Kromasil).

b_{Retention factor of the first eluted enantiomer.} c_{Second peak too large and tailed.}

d _{Flow rate at 1.3 mL/min,}_t

0= 2.7. Experimental conditions used for semipreparative isolation. e_{Flow rate at 1.5 mL/min,}_t

0= 2.4.

entries 1, 2, 3; 5, 6; 9, 10; 11, 12; 13, 14, although the separation factorαis almost unaffected by the percentage of doped ethanol in the mobile phase. This demonstrates that there is no compe-tition between the analyte and the alcohol for the active sites of the CSP. Also, the different flow rates of the mobile phase do not affect the separation factor on the Chiralpak AD column although the Rs increases strongly as the flow rate decreases (entries 6, 7, 8).

A similar behaviour in the enantioseparation of compound2

on the three investigated polysaccharide-derived CSP is found as illustrated inTable 1. In general, the Chiralcel OD-H and Chi-ralpak AD columns gave much better separation factor for2with respect to1in the same experimental conditions. In particular, compounds1and2show very differentk₁values under the same polarity of the mobile phase and CSP, as shown by comparison of entries 2, 16; 4, 18; 5, 20; 9, 22. Okamoto and Yashima[11] demonstrated that steric factors and hydrogen bonding play an important role in the fitting of the solute into the chiral cavity of the polysaccharide-derived CSP. Compared with compound

2, compound 1 has an increased bulkiness and a conjugative electron donating effect from theN-p-methoxyphenyl group up the carbonyl group. This, in turn, results in a stronger hydrogen bonding between the C O of the compound1and the NH of the carbamate moiety of the CSP. The combination of these two fac-tors could be the cause of the extended retention of compound

1. The lack of the electron donatingN-p-methoxyphenyl group

in compound2explains also the lack of complementary interac-tion with the 3,5-dinitrobenzoyl electron-attracting group of the ␤-GEM CSP[12]. This results in the lack of enantioseparation as shown inFig. 2h and by comparison of entries 23 (compound

2) and 11 (compound1). The lack or presence of this group also explains the different polarity of the same mobile phase that must be used to get good enantioseparation for1and2. As an example,Fig. 2shows the behaviour of these compounds with four CSPs using an-hexane/ethanol-doped mobile phase of dif-ferent polarity (70:30 compound1, 90:10 compound2). Thus, a single chromatographic system for both compounds cannot be used.

On the bases of the chromatographic results inTable 1, we resorted to the experimental conditions in entry 7 to perform the isolation of the single enantiomers of1. This was accom-plished by 100␮L repeated injections (0.3–0.4 mg) of a solution of1 (25 mg in 7 mL ethanol). This injected amount is, in our experience, good enough to avoid overloading problems using a column of analytical size, as this one, and to maintain a zero baseline in the 4.5 min interval between the eluting peaks with-out a crossing point between the elution of the two enantiomers. Collection of the eluates corresponding to the two major chro-matographic peaks gave, after filtration and rotoevaporation, 8 and 7 mg of the first and the second eluate, respectively. The quantitative circular dichroism (CD) spectra of both compounds were measured in ClCH2CH2Cl, and they are reported after

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Fig. 2. Typical HPLC enantioseparation of compounds1and2as a function of CSP, using the same mobile phase for both compounds but with different polarity for1and2. Conditions: compound1(upper traces) OD-H (a), AD (b), IB (c),␤-GEM (d); compound2OD-H (e), AD (f), IB (g),␤-GEM (h). Mobile phase

n-hexane/ethanol doped with 0.5% TFA, 70:30 (compound1), 90:10 (compound2), at 1 mL/min in all cases.

subtraction of the baseline of the ClCH2CH2Cl solvent. The CD spectra of the two eluted peaks were almost mirror images of each other, as shown inFig. 3indicating their enantiomeric nature.

Specific rotation [α]22_D +116.5 (c 0.4, ClCH2CH2Cl) was measured for the first-eluted sample of1, whereas the second afforded an experimental [α]22_D −118 (c 0.4, ClCH2CH2Cl). The slight difference in the values is due to the paucity of the weighted material.

Fig. 3. CD spectra (1,2-dichloroethane, 22◦C) of the enantiomers of compound 1obtained from the first (1) and the second (2) eluted peaks.

After chiroptical measurement, the solutions of the individual enantiomers were taken to dryness in a drying pistol in vacuum and in the presence of P2O5as desiccant and the power residues used for cytotoxicity assays, as reported in Section3.3.

3.2. Synthesis and NMR spectral data of compound1

Compound 1 was prepared by the reaction of 2-(2 -methoxyphenoxy)-4,5-dimethoxy-N-(p -methoxyphenyl)-phe-nylacetamide with oxalyl chloride in the presence of the Lewis acid stannyl chloride, following reported procedure. The exper-imental details were also reported[1]. Product1was isolated in 37% yield, m.p. 169.3–170.2◦C, by column chromatography on silica gel, using dichlorometane:MeOH, 9:1, as eluting mixture. 1H NMR (CDCl3) 7.54 (1H, d, J= 8.0, Ar-H), 7.40 (2H, d,J= 9.0, Ar-H), 7.05−6.90 (3H, m, Ar-H), 6.81 (1H, s, Ar-H), 6.45 (1H, s, Ar-H), 5.14 (1H, dd,J= 11.1 and 3.1, H-1), 3.96, 3.86, 3.80, 3.77 (4×3H, 4×s, 4×OMe), 3.16 (1H, dd,

J= 14.0 and 3.1, HCH), 2.80 (1H, dd,J= 14.0 and 11.1, HCH); 13_{C NMR (CDCl}

3) 166.4, 157.4, 151.9, 148.6, 147.5, 144.5, 139.2, 132.4, 129.5, 125.1 (2C), 124.1, 118.6, 114.5 (2C), 114.9, 113.7, 112.2, 105.1, 58.7, 56.4, 56.2, 56.0, 55.4, 37.5;

m/z(%) 434 (M+_{+ H, 26), 433 (M}+_{, 92), 418 (45), 402 (100),} 92 (55); UV (EtOH) (logε) 228 (4.09), 280 (3.98) nm; Anal. Calcd. for C25H23NO6: C 69.37, H 5.35, N 3.23, found C 69.24,

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H 5.28, N 3.22. The structure delucidation was obtained by the analysis of the spectral data. In fact, compound1 exhibits the characteristic 1H NMR spectrum for reduced cularine alkaloids, with an ABX system at 5.14 ppm (dd, J= 11.1, 3.1 Hz), 3.16 ppm (dd,J= 14.0 and 3.1 Hz) and 2.80 ppm (dd,

J= 14.0, 11.1 Hz) corresponding to the protons at positions 1 andα. The presence of two signals doubled at 7.54 and around 7.0 ppm in the1H NMR spectrum; the first one at low magnetic field because of anisotropic effect of the carbonyl group 4 in the molecule, confirm the existence of ao-tetrasubstituted ring A characteristic for the aristocularine skeleton. The13_{C NMR} spectrum is in agreement with the structure of1, showing 25 signals corresponding to the 25 carbon atoms in the molecule. One signal appears in the spectrum at 166.4 ppm which can be assigned to the amide carbonyl group, confirming the aristolactam ring. The six aliphatic signals between 37.5 and 58.7 ppm present in this spectrum can be attributed to the six aliphatic carbons in the molecule, four methoxy groups and the carbons 1 andα, indicating a reducing C. Also, the M+ atm/z

433 and the elemental analysis confirm the structure.

3.3. Cytotoxicity results on (±), (+), (−)1

The analytical isolation of minor amounts of the single enan-tiomers of1 afforded to perform a cytotoxicity assay against HT-29 human colon carcinoma cell line to investigate, if any, an enantioselectivity with respect to (±)1.

There is no significant difference in the antiproliferative effect among the single enantiomers and the racemic compound. The percentage of growth of the cells, referred to the control (no alka-loid), was 110–100 and 35–33 for concentrations of 0.3125 and 10␮g/mL, respectively. In particular, the IC50values obtained were 6.8, 6.9 and 6.7␮g/mL for (±)1, (+)1, and (−)1, respec-tively.

Thus, although these values are similar to that obtained for (±)2(5.0␮g/mL)[1]and for aporphine alkaloids[2], a phar-macological enantioselectivity is absent. The (±) 1 exhibits significant activity also against MDA-MB-231 human breast carcinoma, wild type P-388 and adriamycin resistant P-388 Schabel cell lines, the IC50 values being 3.8, 3.2, 2.7␮g/mL, respectively but, because of the paucity of the material, the indi-vidual enantiomers of1were not tested against these cell lines.

Acknowledgements

Financial support from MIUR-COFIN 2003 (Roma), project “Sostanze naturali ed analoghi sintetici con attivit`a antitu-morale” to S.C. is gratefully acknowledged. We also thank Dr. F.J. Alonso, Biochemistry and Molecular Biology Department, University of M´alaga for the cytoxicity assays.

References

[1] R. Suau, R. Rico, J.M. L´opez-Romero, A. Ruiz, F.J. Ortiz-L´opez, Arkivoc (Gainesville, Florida) 5 (2002) 62.

[2] R. Suau, J.M. L´opez-Romero, R. Rico, F.J. Alonso, C. Lobo, Tetrahedron 52 (1996) 11307.

[3] R. Crossley, Tetrahedron 48 (1992) 8155. [4] J. Caldwell, J. Chromatogr. A 719 (1996) 3.

[5] S. Caccamese, G. Principato, R. Jokela, A. Tolvanen, D. Din Belle, Chirality 13 (2001) 691.

[6] T. Putkonen, A. Tolvanen, R. Jokela, S. Caccamese, N. Parrinello, Tetra-hedron 59 (2003) 8589.

[7] H. Koller, K.-E. Rimb¨ock, A. Mannschreck, J. Chromatogr. 282 (1983) 89. [8] S. Allenmark, Chromatographic Enantioseparation: Methods and

Applica-tions, second ed., Ellis Horwood, Chichester, 1991, p. 89. [9] T. Mossmann, J. Immunol. Methods 65 (1983) 55.

[10] T. Zhang, D. Nguyen, P. Franco, T. Murakami, A. Ohnishi, H. Kurosawa, Anal. Chim. Acta 557 (2006) 221.

[11] Y. Okamoto, E. Yashima, Angew. Chem. Int. Ed. Engl. 37 (1998) 1020. [12] W.H. Pirkle, J.E. McCune, J. Chromatogr. 441 (1988) 311.