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A New Alkaloid from Two Coccinellid Beetles Harmonia axyridis and Aiolocaria hexaspilota
Naseer Alam, In Soo Choi, Kyung-Sik Song, Jongki Hong, Chong O. Lee, and Jee H. Jung* College of Pharmacy, Pusan National University, Pusan 609-735, Korea Department of Biochemistry, Kyungpook National University, Taegu 702-701, Korea Korea Basic Science Institute, Seoul 136-701, Korea Pharmaceutical Screening Center, Korea Research Institute of Chemical Technology, Daejeon 305-343, Korea Received August 4, 2001

Keywords : Coccinellid beetles, Harmonia axyridis, Aiolocaria hexaspilota, Cytotoxicity, Alzheimers disease.

Coccinellid beetles are known to secrete droplets of blood from the joints when they are molested.1 A number of alkaloids are present in this reflex bleeding which is considered as a mode of their chemical defense. These alkaloids are also responsible for their aposematic coloration. About fifty alkaloids like azaphenalenes, azabicyclononanes, harmonine, pyrrolidines, piperidines, aromatic amines, azamacrolides, and some dimmeric alkaloids have so far been isolated from about thirty five species of the Coccinellidae.2-4 The deterrent and toxic properties of these defensive alkaloids prompted

us to investigate Harmonia axyridis and Aiolocaria hexaspilota for their alkaloidal constituents. We here report the isolation of a new natural product 3-hydroxypiperidin-2-one (1) and harmonine (2) from these species of Coccinellidae. Compound 1 was isolated as a light yellow solid. The [M + H]+ peak was observed at m/z 116.0-712 in the HRFABMS which corresponded to the molecular formula C5H9NO2. The formula showed two degrees of unsaturation. A broad band at 3398 cm1 in the IR spectrum suggested the presence of OH and NH groups. A strong band at 1619 cm1 indicated an intra-molecular hydrogen bonded carbonyl group. The 1H NMR of 1 displayed a proton signal at 3.94 that was correlated to a carbon signal at 62.5. This signal showed correlations with the methylene protons at 2.28 and 2.09. The methylene proton signals at 3.36 and 3.20 showed correlation with a carbon signal at 46.5 indicating that they are vicinal to a nitrogen function. These proton
*

signals showed strong correlation with the methylene proton signal at 1.96 that showed further correlations with the methylene proton signals at 2.28 and 2.09. Thus the entire proton signals in the COSY spectrum comprised a single spin system. The 13C NMR spectrum featured a carbonyl carbon at 174.5 (C-2), an oxygenated carbon at 62.5 (C3), and a carbon attached to nitrogen at 46.5 (C-6). The signals at 31.0 and 24.5 were assigned to C-4 and C-5, respectively, and 1H-13C connectivities were confirmed by an HMQC experiment. The COSY and HMBC correlations were also in accordance with the proposed structure (Figure 1). Thus the gross structure was determined as 3-hydroxypiperidin-2-one. The compound showed an optical rotation of 53o which was the same in sign to that of the earlier 21 synthesized (S)-3-hydroxypiperidin-2-one ( [ ] D 6o)5,6 but the degree of rotation was different. The isolated compound was quite pure and the structure was well established by 2D NMR techniques so we deduce that 1 also has (3S)-stereochemistry. This compound has previously been synthesized 6 but to the best of our knowledge it is the first report of its occurrence as a natural product. The reported mp of the synthetic compound varied from 133 to 171 oC, depending on its optical purity.5,7-9 Compound 1 has melted at 122-124 o C and decomposed at 190-192 oC. Compound 2 was a brownish oil. Its 1H NMR featured the presence of a terminal methyl ( 1.04, 3H, d, J = 7.0 Hz, H-18), a methine ( 2.82-2.90, 1H, m) and methylene protons ( 2.67, 2H, t, J = 7.0 Hz) vicinal to a nitrogen atom, and a mono-unsaturated alkyl chain. The assignments were confirmed by COSY correlations and were supported by 13C

To whom correspondence should be addressed. Tel.: +82-51510-2803; Fax: +82-51-510-2803; E-mail: [email protected]

Figure 1. Key COSY and HMBC correlations for compound 1.

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Table 1. In Vitro Cytotoxicities (ED50 g/mL) of 1 and 2 against Human Solid Tumor Cellsa
compound 1 cisplatin doxorubicin 2 cisplatin doxorubicin A549 SK-OV-3 SK-MEL-2 >30 0.72 0.02 3.04 0.40 0.01 >30 1.23 0.11 2.86 0.77 0.02 >30 2.26 0.02 2.87 0.98 0.02 XF498 >30 1.03 0.08 1.03 0.48 0.03 HCT15 >30 1.10 0.04 1.10 0.41 0.02

a A549: human lung cancer; SK-OV-3: human ovarian cancer; SK-MEL2: human skin cancer; XF498: human CNS cancer; HCT15: human colon cancer. The compounds were assayed in two separate batches.

Table 2. Enzyme Inhibitory Activity of 2a

Acetycholinesterase 12 47

Prolylendopeptidase 53 64

Neuraminidase 5 12

Expressed as % inhibition.

NMR spectrum. The LRFABMS showed an [M + H]+ ion at m/z 283 which gave a molecular formula C18H38N2 in combination with the NMR data. The location of the double bond was determined based on the significant allylic cleavages at m/z 168 and 114 in the FAB-CID MS/MS. These findings suggested the gross structure of compound 2 as harmonine, previously isolated from Harmonia leis conformis and Hippodamia convergens.10 The geometry of the double bond was determined as cis on the basis of the chemical shift value of the allylic carbons (see Experimental) while the absolute stereochemistry was presumed to be the same as that of harmonine.11 Compounds 1 and 2 were evaluated for their cytotoxicity against five human solid tumor cell lines, and 2 was found to posses significant cytotoxicity against these cell lines (Table 1). Compound 2 was also evaluated for its inhibitory activity on several enzymes that are considered as therapeutic targets of Alzheimers disease. It showed a week inhibitory activity against acetylcholinestrase (AChE), prolylendopeptidase (PEP), and neuraminidase (Table 2). Experimental Section General Experimental Procedures. Melting point was measured on an Electrothermal digital melting point apparatus and was uncorrected. Optical rotations were measured on a JASCO DIP-370 digital polarimeter. IR spectra were obtained using a JASCO FT/IR-410 spectrometer. 1H and 13 C NMR spectra were recorded on a Varian Unity Plus 300, and Varian INOVA 500 spectrometers. Chemical shifts were reported in reference to the respective residual solvent peaks (H 3.3 and C 49.0 for CD3OD). COSY spectra were recorded on a Varian INOVA 500 spectrometer and Bruker DMX 600 spectrometer for compound 1 and 2, respectively. HRFABMS data were obtained using a JEOL JMS-HX110/

110A. HPLC was performed on a Gilson 370 pump with a YMC amino 12S05 (250 10 mm i.d., S-5 m, 120 ) and YMC ODS-H80 (250 10 mm i.d., S-4 m, 80 ) column using a Shodex RI-71 detector. Animal Material. About 1000 adult ladybird beetles were collected and were kept at 20 oC until extraction. Isolation of Compounds. The frozen beetles were extracted with MeOH at room temp. and the combined extract was filtered and vacuum dried on a rotary evaporator to give a thick paste (9 g). Reversed-phase flash chromatographic separation of this extract with 20 0% H2O/MeOH and finally with acetone gave 5 fractions. Fraction 2 showed high cytotoxicity in the brine shrimp assay12 and was further separated into 16 fractions on reversed-phase MPLC (ODS, MeOH : H2O, 7 : 3). Fraction 2-2 was chromatographed on a reversed-phase HPLC (ODS, MeOH : H2O, 3 : 7) into 8 fractions and repeated HPLC purification (amino, CH3CN : H2O, 7 : 3) of fraction 2-2-1 yielded compound 1 (94 mg). Fractions 2-42-16 were combined into a single fraction (fraction 2-4) on the basis of TLC and brine shrimp assay and was further separated on chromatotron (Si gel, 2 mm) eluting with a gradient of CH2Cl2 : MeOH : NH3 (9 : 1 : 0.1 1 : 1 : 0.1) to give 7 fractions. Fraction 2-4-4 yielded compound 2 (13.5 mg) on further purification by HPLC (amino, CH3CN : H2O, 1 : 1). Compound 1: Yellowish brown solid; mp 122-124 oC (decomp. 190-192 oC, lit. 171.5 oC5, 143-145 oC7, 135-137 18 o 8 C , 133-135 oC9); [ ] D 53o (c 0.8, MeOH); IR max (film) 1 1 3398, 1619, 1405 cm ; H NMR (500 MHz, CD3OD) 3.94 (1H, dd, J = 7.9, 6.4 Hz, H-3), 3.36 (1H, dt, J = 11.6, 5.8 Hz, H-6), 3.20 (1H, dt, J = 11.6, 7.3 Hz, H-6), 2.28 (1H, ddd, J = 12.8, 7.9, 7.9 Hz, H-4), 2.09 (1H, ddd, J = 12.8, 6.4, 6.4 Hz, H-4), 1.94-1.99 (2H, m, H-5); 13C NMR (125 MHz, CD3OD) 174.5 (C-2), 62.5 (C-3), 46.5 (C-6), 31.0 (C-4), 24.5 (C-5). HRFABMS m/z 116.0713 [M + H]+ (calcd for C5H10NO2, 116.0712, +0.1 mmu) Compound 2: Brownish oil; 1H NMR (300 MHz, CDCl3) 5.30-5.40 (2H, m, H-9, H-10), 2.82-2.90 (1H, m, H-17), 2.67 (2H, t, J = 7.0 Hz, H-1), 1.95 - 2.05 (4H, m, H-8, H-11), 1.11-1.40 (22H, br s, alkyl chain), 1.04 (3H, d, J = 7.0 Hz, H-18); 13C NMR (75 MHz, CDCl3) 130 (C-9, C-10), 46.5 (C-17), 41.5 (C-1), 28-30 (alkyl chain), 27.0 (C-8, C-11), 23.0 (C-18); FAB-CID MS/MS m/z 283 [M + H]+ (100), 268 [M-CH3]+ (3), 239 (0.1), 224 (0.1), 210 (0.1), 196 (0.1), 182 (0.2), 169 (0.2), 155 (0.1), 140 (0.1), 114 (0.2), 44 (1.2). References
1. Happ, G. M.; Eisner, T. Science 1961, 134, 329-331. 2. Angela, G. K.; Meinwald, J. Chem. Rev. 1996, 96, 1105-1122. 3. Lebrun, B.; Braekman, J.-C.; Daloze, D.; Kalushkov, P.; Pasteels, J. M. Tetrahedron Lett. 1999, 40, 8115-8116. 4. Daloze, D.; Braekman, J.-C.; Pasteels, J. M. Chemoecology 1995, 5/6, 173-183. 5. Hunter, A.; Woodward, H. E. Biochem. J. 1941, 35, 1298-1306. 6. Gibbs, G.; Hateley, M. J.; McLaren, L.; Welham, M.; Willis, C. L. Tetrahedron Lett. 1999, 40, 1069-1072. 7. Hua, D. H.; Zhang, F.; Chen, J.; Robinson, P. D. J. Org. Chem.

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1994, 59, 5084-5087. 8. Hjeds, H.; Honore, T. Acta Chem. Scand. 1978, B32, 187-192. 9. Elming, N. Acta Chem. Scand. 1957, 11, 914. 10. Braconnier, M. F.; Braekman, J.-C.; Daloze, D.; Pasteels, J. M. Experientia 1985, 41, 519-520.

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11. Braconnier, M. F.; Braekman, J.-C.; Daloze, D. Bull. Soc. Chim. Belg. 1985, 94, 605-613. 12. Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin, J. L. Planta Medica 1982, 45, 31-34.