Phytochemistry Letters 3 (2010) 75–78
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Turrianes from Kermadecia rotundifolia as new acetylcholinesterase inhibitors Mehdi A. Beniddir, Anne-Laure Simonin, Marie-The´re`se Martin, Vincent Dumontet, Cyril Poullain, Franc¸oise Gue´ritte, Marc Litaudon * Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, 1, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2009 Received in revised form 11 December 2009 Accepted 17 December 2009 Available online 29 December 2009
Four new kermadecins, together with the known kermadecins A, B and D have been isolated from the Kermadecia rotundifolia ethyl acetate bark extract. These compounds are derivatives of the (20membered-o,-p)cyclophane skeleton and belong to the turriane family. The structures were elucidated by mass spectrometry, extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. Isokermadecin D (2) and kermadecins D and J (7 and 4) possess significant inhibitory effect on acetylcholinesterase. ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Kermadecia rotundifolia Proteaceae Kermadecin Turriane Cyclophane Acetylcholinesterase
1. Introduction In the course of automated screening for small-molecules for acetylcholinesterase inhibitory activity, a significant activity was observed for kermadecin D (7), a cyclophane-type compound isolated from the bark of Kermadecia elliptica (Jolly et al., 2008). With the aim to discover analogues of kermadecin D, we performed a chemical investigation of Kermadecia rotundifolia Brongniart & Gris, an endemic species to New Caledonia with very similar morphological characteristics to K. elliptica. The genus Kermadecia (Proteaceae), contains twelve species, of which four are endemic to New Caledonia (Virot, 1968). No report is mentioned regarding their utilisation by traditional healers. K. rotundifolia, may reach 20 m in height, possesses large orbiculate to ovalate leaves, and white to yellowish small flowers are organized in branched racemes 15–40 cm long. This rare species is mainly distributed in the Northern part of the main highland. Until now K. elliptica is the only species that has been investigated chemically and biologically (Jolly et al., 2008). The isolated compounds are new bisresorcinol derivatives having a 20-membered-o,-p-cyclophane skeleton or a biaryl-ether containing macrocycle in their structure. We wish to report in this paper the isolation and characterization of four new kermadecins
* Corresponding author. Tel.: +33 1 69 82 30 85; fax: +33 1 69 07 72 47. E-mail address:
[email protected] (M. Litaudon).
(1–4) together with their acetylcholinesterase inhibitory activities and those of three other known kermadecins (5–7). 2. Results and discussion This study was accomplished with the aid of HPLC, LC–APCI-MS and NMR analysis, and led to the isolation and identification of four new kermadecins (1–4) together with the known kermadecins A, B and D (5–7). In order to determine the presence of common fragments in this chemical series, we applied the LC/MS–MS method developed in our previous study on K. elliptica (Jolly et al., 2008). In APCI positive-ion mode, LC/MS–MS analysis of compounds 1–4 indicated the presence of ions resulting from the systematic loss of fragments 182 (C13H26) (1, 2 and 4) or 180 (C13H24 for compound 3), mass units in favour of a 14 carbons aliphatic chain, with an additional double bond in the case of 3. In negative-ion mode, LC/MS–MS analyses of the quasimolecular peak [MH] of 2 and 3 showed the presence of an ion at m/z = 369 corresponding to the loss of a fragment of 108 mass units, suggesting the loss of a dimethylpyran ring. Kermadecins A, B and D (5–7), previously isolated from K. elliptica (Jolly et al., 2008) were identified by comparison of their spectroscopic data (HR-MS, 1H and 13C NMR). The HRESIMS of compound 1 indicated an ion peak m/z 491.2789 [MH] (calcd. for 491.2797) giving the molecular formula C31H40O5. The IR spectrum of 1 showed absorption bands at 3600 cm1 for hydroxyl groups, 1608 and 1424 cm1 for an
1874-3900/$ – see front matter ß 2009 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2009.12.003
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Table 1 13 C NMR spectroscopic data (125 MHz) in CDCl3 for compounds 1–3 and CD3OD for compound 4. Carbon
1
2
3
4
1 2 3 4 5 6 7–11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
34.5 31.3 27–29 27–29 27–29 27–29 27–29 27–29 27.0 27.7 145.5 135.8 183.7 116.3 152.4 183.5 109.1 149.0 108.4 154.6 110.7 126.0 116.6 128.7 76.3 28.2 28.1
30.7 31.2 27–30 27–30 27–30 27–30 27–30 27–30 27–30 35.8 147.1 107.9 160.5 99.5 156.2 108.5 134.5 148.0 108.7 150.0 108.9 137.5 116.5 128.8 76.5 28.0 28.0
32.9 30.8 27.7 130.9 131.1 29.0 27–29 27–29 29.4 35.1 144.4 107.4 153.8 107.3 153.8 107.4 108.4 153.3 108.2 154.2 109.2 146.1 115.7 129.2 76.3 27.8 27.8
31.2 31.7 31.3 28–30 28–30 28–30 28–30 28–30 31.8 36.8 146.4 108.5 161.6 100.0 159.3 109.5 134.6 151.8 102.6 156.0 108.6 139.0
aromatic ring and a strong band at 1632 cm1 suggesting a paraquinone moiety, confirmed in the 13C NMR spectrum by the presence of signals at 187.3 and 183.5 ppm for two ketones at C-17 and C-20, respectively. The spectral data of 1 were very similar with those of kermadecin F (8) (Jolly et al., 2008). In the 1H NMR, two singlets at d 6.38 (H-25) and 6.64 (H-16) suggested the presence of one pentasubstituted aromatic and one trisubstituted paraquinone ring. In addition, the presence of a dimethylpyran ring
fused to a phenol ring was confirmed with 1H and 13C NMR data (Table 1). In the HMBC spectrum, correlations from H2-1 to C-21, C-25 and C-26, H-16 to C-14, C-17, C-18 and C-20, and from H2-14 to C-16 and C-20 implied that the trisubstituted paraquinone was located as shown in structure 1. Other HMBC correlations were similar to those observed for kermadecin A (5), indicating that the dimethylpyran ring was fused to the aromatic ring in a similar way to 5. This was confirmed by the correlation between H-27 (d 6.57, d, J = 10 Hz) and the OH-22 (d 4.80) observed in the NOESY spectrum, obtained in CDCl3 at room temperature. Compound 1 was named isokermadecin F. Isokermadecin F, which possesses a chiral biaryl axis arising from the presence of the asymmetric paraquinone moiety, showed optical activity [a]D +24 (c = 1, CHCl3), of opposite sign to kermadecin F (8). Compound 2 was assigned the formula C31H42O4 based on HRESIMS. Most NMR signals of compound 2 were very similar to those of kermadecin D (7) suggesting the presence of the same substituted aromatic rings as in 7: one dimethylpyran ring fused to one aromatic ring and a long carbon chain attached to both aromatic rings. The ether linkage was suggested by the shift in the down field region of C-21 at d 134.5 (Ahmed et al., 2000), which in turn give a correlation to H2-1 in the HMBC spectrum. In addition, the chemical shifts of the two methyl groups at d 1.25, similar to the methyl signals in kermadecin B (6), and HMBC correlations between H-27 and C-22, C-23 and C-24, indicated that the dimethylpyran ring was fused to the aromatic ring in a similar way to 6. Thus compound 2, named isokermadecin D, possesses the structure depicted in Fig. 1. The molecular formula of 3, named kermadecin I, was C31H40O4 as indicated by HRESIMS. The 1H and 13C NMR spectra were very similar to those of kermadecin B (6) revealing symmetrical tetrasubstituted and pentasubstituted aromatic rings linked by a monounsaturated C14 aliphatic chain, and a dimethylpyran ring fused to an aromatic ring. In addition the presence of multiplet signals at d 5.22 and 5.34 (H-4 and H-5) in the 1H NMR spectrum and signals at d 130.9 and 131.1 (C-4 and C-5) in the 13C NMR spectrum were compatible with an additional double bond in the
Fig. 1. Structures of compounds 1–8.
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chain. In the 1H-1H COSY spectrum, the observation of correlations between protons H2-2 and H2-3 at d 1.42 and 1.86, respectively (2H each, m) with H-4 and H-5 at d 5.22 and 5.34, respectively (1H each, dt, J = 7.0 and 10 Hz), and between protons H2-2 and H2-1 at d 2.22 (2H, m), indicated that the position of the double bond was between C-4 and C-5. The presence of allylic carbon resonances at d 27.7 and 29.0, for C-3 and C-6, and olefinic carbon resonances at d 130.9 and 131.1, C-4 and C-5 (Rossi et al., 1982), together with the H-4/H-5 coupling constant (J = 10 Hz) allowed to propose the Z configuration for the geometry of the double bond. Other COSY and HMBC correlations were identical with those of compound 6 and allowed to propose the structure 3 depicted in Fig. 1. Compound 3 is therefore 4,5-dehydrokermadecin B. The molecular formula of 4 was determined as C26H36O4 on the basis of HRESIMS. In the 1H NMR spectrum, five aromatic protons appearing as two doublets at d 6.27 (1H, d, J = 2.0, H-23) and 6.20 (1H, d, J = 2.0, H-25), and three triplets at d 6.34 (1H, t, J = 2.1 Hz, H16), 5.93 (1H, t, J = 2.1 Hz, H-18) and d 6.23 (1H, t, J = 2.1 Hz, H-20) suggested 1,2,3,5-tetrasubstituted and 1,3,5 asymmetrical trisubstituted aromatic rings, respectively. Long-range correlations from H-25 to C-1, H2-1 to C-21 and C-25 and H-16 and H-20 to C-14 in the HMBC spectrum confirmed the location of the carbon chain between the two aromatic rings. The ether linkage was suggested by the shift in the down field region of C-17 and C-21 at d 161.6 and 134.5, respectively. Finally, in a NOESY experiment obtained in DMSO at room temperature, cross peaks between H-23 and the hydroxy groups OH-22 and OH-24, and OH-19 with H-18 and H-20 confirmed that compound 4, named kermadecin J, possesses the structure depicted in Fig. 1. The acetylcholinesterase (AChE) inhibitory activity was assayed by the method of Ellman et al. (1961) using AChE from Electrophorus electricus and tacrine as reference compound (IC50 = 0.115 mM). Kermadecins D and J, and isokermadecin D (7, 4 and 2) exhibited significant interaction with AChE (IC50 3.6 0.6, 3.4 0.3 and 3.4 0.8 mM, respectively), other compounds were poorly active or inactive. The results obtained for this chemical series suggest that an ether linkage is essential for a good inhibitory effect on AChE. The similar values of compounds 2 and 7 allowed us to conclude that there was no impact of the position of the dimethylpyran ring on the inhibitory activity. The study of K. rotundifolia has led to the isolation of four new (1–4) and three known kermadecins (5–7), of which kermadecins D and J (7 and 4) and isokermadecin D (2) are moderate acetylcholinesterase inhibitors. Their isolation is a consequence of a random screening in an acetylcholinesterase assay of our chemical library, and the use of LC/MS method to detect and to direct purification of compounds of this chemical series.
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4.5 mA; vaporizer temperature, 450 8C; capillary voltage, 15 V. Full scan mass spectra (50–800 mass units) were recorded alternatively in positive and negative mode. For MS2, the data dependent program was used so that the most abundant ions in each scan were selected and subject to MS2 analyses. The collisioninduced dissociation (CID) energy was adjusted to 50%. The isolation width of the precursor ion was 2.0 mass units. Kromasil analytic, semi-preparative and preparative C-18 columns (250 mm 4.6 mm; 250 mm 10 mm and 250 mm 21.2 mm ID, 5 mm Thermo1) were used for analytical and preparative HPLC separations using a ‘‘Waters autopurification system’’ equipped with a sample manager (Waters 2767), a column fluidics organizer, a binary pump (Waters 2525), a UV–Vis diode array detector (190– 600 nm, Waters 2996) and a Pl-ELS 1000 ELSD detector Polymer laboratory. 3.2. Plant material The bark of K. rotundifolia Brongniart & Gris was collected in 2006 at ‘‘Foreˆt Frouin’’, North Province. The corresponding voucher specimen DUM-0688 is kept at the Herbarium of the Botanical and Tropical Ecology Department of the IRD Center, Noumea, New Caledonia. 3.3. Extraction and isolation Air-dried material (1.6 kg) was extracted with a static highpressure high temperature extractor (HPHT), Zippertex1, developed in the ICSN Pilot Unit, using EtOAc (3 3.0 L, 1 h each, 100 bar, 40 8C), and concentrated under vacuum at 40 8C. The EtOAc extract (12.7 g) was submitted to filtration on polyamide using a 1:1 mixture of MeOH–EtOAc, and the filtered extract (11.0 g) was then subjected to silica gel column chromatography using a gradient of n-heptane-CH2Cl2 (80:20 to 0:100) and CH2Cl2–MeOH (100:0 to 80:20) to give 12 fractions (fractions A to L) according to their TLC profile. Additional column chromatography using silica gel with the same gradient was performed on fractions F and G to give 9 and 8 subfractions, respectively. Subfraction F-8 (91 mg) was subjected to preparative HPLC using an isocratic mobile phase MeCN–H2O 85:15 at a flow rate of 21.0 mL min1 to afford isokermadecin F (1) (tR 22.3 min, 22.0 mg), isokermadecin D (2) (tR 25.5 min, 2.4 mg) and kermadecin I (3) (tR 9.8 min, 1.8 mg). Subfraction G-12 was subjected to preparative HPLC with a solvent system of A (MeCN) and B (H2O) with a gradient elution of 80–100 of A over 35 min to afford kermadecin J (4) (tR 13.9 min, 7 mg). 3.4. Isokermadecin F (1)
3. Experimental 3.1. General experimental procedures IR spectra were obtained on a Nicolet FTIR 205 spectrophotometer and UV spectra on a PerkinElmer Lamba 5 spectrophotometer. The NMR spectra were recorded on a Bruker spectrometer at 500 MHz for 1H and 125 MHz for 13C using CDCl3 (1–3, 5–7), CD3OD and DMSO-d6 (4) as solvents. Chemical shifts (relative to TMS) are in ppm, and coupling constants in Hz. ESIMS was obtained on a LCT mass spectrometer. HRESIMS were run on a ESITOF spectrometer (LCT; Waters). LC–MS analyses were performed using a Finnigan Surveyor HPLC system and ion trap MSn detector (Finnigan LCQdeca). The Finnigan detector was equipped with an atmospheric pressure chemical ionisation (APCI) source operating under the following conditions: sheath gas flow rate, 80 (arbitrary units); N2, capillary temperature, 250 8C; discharge current,
Colourless amorphous solid; [a]25D +24 (c 1.0, CHCl3) UV (MeOH) lmax (log e), 224 nm (4.23), 262 nm (4.1); IR ymax 3600, 1632, 1608 and 1424 cm1; 1H NMR (CDCl3, 500 MHz) 1.10–1.4 (18H, m, H-3-H-11), 1.24 (2H, m, H-12), 1.40 (2H, m, H-2), 1.41 (3H, s, Me-30), 1.43 (3H, s, Me-31), 1.61 (2H, m, H-13), 2.21 (2H, m, H-1), 2.26 (1H, m, H-14a), 2.84 (1H, dt, J = 7.0, 15 Hz, H-14b), 4.80 (1H, s, OH-22), 5.52 (1H, d, J = 10 Hz, H-28), 6.38 (1H, s, H-25), 6.64 (1H, s, H-16), 6.57 (1H, d, J = 10 Hz, H-27), 7.21 (1H, s, OH-19). 13C NMR (CDCl3, 125 MHz), see Table 1; HRESIMS m/z [MH] 491.2789 (calcd. for C31H39O4, 491.2797). 3.5. Isokermadecin D (2) Pale purple amorphous solid; UV (MeOH) lmax (log e), 280 nm (3.87); IR ymax 3227, 2945, 1651 and 1448 cm1; 1H NMR (CDCl3, 500 MHz), 1.21 (2H, m, H-3), 1.25 (6H, s, Me-30 et Me-31), 1.20–
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1.30 (18H, m, H-4-H-12), 1.45 (2H, m, H-2), 1.55 (2H, m, H-13), 2.37 (2H, m, H-1), 2.48 (2H, t, J = 6.6 Hz, H-14), 4.50 (1H, s, OH-19), 4.59 (1H, s, OH-24), 5.51 (1H, d, J = 10.0 Hz, H-28), 6.00 (1H, t, J = 2.1 Hz, H-18), 6.17 (1H, s, H-25), 6.23 (1H, t, J = 2.1 Hz, H-20), 6.42 (1H, t, J = 2.1 Hz, H-16), 6.55 (1H, d, J = 10 Hz, H-27); 13C NMR (CDCl3, 125 MHz), see Table 1; HRESIMS m/z [M+Na]+ 501.3106 (calcd. for C31H42O4Na, 501.2981). 3.6. Kermadecin I (3) Colourless amorphous solid; UV (MeOH) lmax (log e), 226 nm (4.28), 277 (3.67); IR ymax 2923, 1587 cm1, 1H NMR (CDCl3, 500 MHz), 1.29 (6H, s, Me-30 and Me-31), 1.14–1.28 (12H, m, H7-H-12), 1.42 (2H, m, H-2), 1.61 (2H, m, H-13), 1.84 (2H, m, H-6), 1.86 (2H, m, H-3), 2.22 (2H, m, H-1), 2.59 (2H, t, J = 6.1 Hz, H-14), 4.55 (2H, s, OH-17 and OH-19), 4.81 (1H, s, OH-24), 5.22 (1H, dt, J = 7.0, 10 Hz, H-4), 5.34 (1H, dt, J = 7.0, 10 Hz, H-5), 5.56 (1H, d, J = 10 Hz, H-28), 6.36 (1H, s, H-25), 6.39 (2H, s, H-16 and H-20), 6.59 (1H, d, J = 10 Hz, H-27); 13C NMR (CDCl3, 125 MHz), see Table 1; HRESIMS m/z [M+Na]+ 499.2807 (calcd. for C31H42O4Na, 499.2824). 3.7. Kermadecin J (4) Brown amorphous solid, UV (MeOH) lmax (log e), 275 nm (2.7); IR ymax 2922, 1592 and 1454 cm1; 1H NMR (DMSO-d6, 500 MHz), 1.20–1.30 (20H, m, H-3-H-12), 1.40 (2H, m, H-2), 1.51 (2H, m, H13), 2.26 (2H, m, H-1), 2.42 (2H, t, J = 6.6 Hz, H-14), 5.80 (1H, t, J = 2.1 Hz, H-18), 6.10 (1H, d, J = 2,1 Hz, H-25), 6.14 (1H, t, J = 2.1 Hz, H-20), 6.20 (1H, t, J = 2.1 Hz, H-16), 6.24 (1H, d, J = 2.1 Hz, H-23), 9.02 (1H, s, OH-24), 9.08 (1H, s, OH-19), 9.10 (1H, s, OH-22); 13C NMR (CD3OD, 125 MHz), see Table 1; HRESIMS m/z [MH] 411.2555 (calcd. for C26H35O4, 411.2535).
3.8. Acetylcholinesterase inhibitory activity Acetylcholinesterase (AChE) from E. electricus (C 2888) was purchased from Sigma. Inhibition of AChE activity was determined by the spectroscopic method of Ellman [13], using acetylthiocholine iodide as substrate, in 96-well microtiter plates. Each inhibitor was evaluated at ten concentrations (from 1 mg/mL to 0.05 mg/mL by diluting by a factor 3). IC50 values displayedqrepresent the ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P 2 mean standard deviation for three assays ðSD ¼ ðx xÞ ¯ =nÞ. Tacrine was used as reference compound. Acknowledgments Thanks to O. Thoison, ICSN, for her help in the LC–DAD-MS analysis. The authors are very grateful to North Province of New Caledonia who facilitated our field investigation. We express our thanks to Dr. J. Munzinger of the Botany and Plant Ecology Department, IRD (Institut de Recherche pour le De´veloppement), Noume´a, for his assistance in the identification of the plant. References Ahmed, A.S., Nakamura, N., Meselhy, M.R., Makhboul, M.A., El-Emary, N., Hattori, M., 2000. Phenolic constituents from Grevillea robusta. Phytochemistry 53, 149–154. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M.A., 1961. New and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Jolly, C., Thoison, O., Martin, M.-T., Dumontet, V., Gilbert, A., Pfeiffer, B., Le´once, S., Sevenet, T., Gueritte, F., Litaudon, M., 2008. Cytotoxic turrianes of Kermadecia elliptica from the New Caledonian rain forest. Phytochemistry 69, 533–540. Rossi, R., Carpita, A., Quirici, M.G., Veracini, C.A., 1982. Insect pheromone components: Use of 13C NMR spectroscopy for assigning the configuration of C5 5C double bonds of monoenic or dienic pheromone components and for quantitative determination of Z/E mixtures. Tetrahedron 38, 639–644. Virot, R., 1968. Proteaceae. In: MNHN (Eds.), Flore de la Nouvelle-Cale´donie 2. Association de Botanique Tropicale, Paris, pp. 78–101.
