Biochemical Systematics and Ecology 34 (2006) 78e82 www.elsevier.com/locate/biochemsyseco
Cornifolin, a new bis-tetrahydrofuran Annonaceous acetogenin from Annona cornifolia Luciana Alves Rodrigues Dos Santos, Maria Amelia Diamantino Boaventura, Lucia Pinheiro Santos Pimenta* Departamento de Quı´mica do Instituto de Cieˆncias Exatas da Universidade Federal de Minas Gerais, Av. Antoˆnio Carlos, 6627, Pampulha, CEP, 31270-910, Belo Horizonte, MG, Brazil Received 26 October 2004; accepted 12 May 2005
Keywords: Annona cornifolia; Annonaceae; Acetogenin; Seeds
1. Subject and source Seeds of Annona cornifolia A. St.eHil. were collected in Curvelo region, Minas Gerais State, from June to August 1998. The species was identified by M. C. R. Mello-Silva and a voucher specimen (BHCB 68114) deposited at the Instituto de Cieˆncias Biolo´gicas Herbarium, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. We report here the isolation and structure elucidation of one novel C-37 adjacent bis-tetrahydrofuran acetogenin, named cornifolin (1) from the ethanolic extract. The structure and relative stereochemistry of the acetogenin were elucidated based on 1D
* Corresponding author. Tel.: C55 31 34995754; fax: C55 31 34995700. E-mail address:
[email protected] (L.P.S. Pimenta). 0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.05.004
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and 2D NMR, including 1H NMR, 13C NMR, COSY, HMQC and ESIMS of the parent compound and/or simple chemical derivatives.
2. Previous work Acetogenins are limited to the Annonaceae family, especially the genus Annona (Santos et al., 1996; McLaughlin et al., 1998; Liu et al., 1999; Chang et al., 2003). A. cornifolia A. St.eHil. has not been investigated previously.
3. Present study The cleaned seeds were dried at 40 C, powdered (850 g) and extracted exhaustively using ethanol (F01, 120 g). This extract was dissolved in ethanol:water (7:3) and successively extracted with hexane and chloroform. Solvent removal under reduced pressure provided hexane (F02, 79.5 g), chloroform (F03, 7.3 g) and alcoholic (F04, 21.1 g) fractions. F03 was fractionated by normal phase MPLC and reverse phase HPLC, resulting in the isolation of cornifolin (20.0 mg, 1). The isolation of 1 was directed by a test for lethality to brine shrimp (BST). Compound 1 was isolated as a white wax. Its molecular weight was suggested by the mass peak at m/z 621.5 [M ÿ H]ÿ in ESIMS, which correspond to the formula C37H65O7. The signals of the terminal a,b-unsaturated g-lactone, without an OH group at C-4, in 1 H and 13C NMR spectra, were seen at d 6.98 (H-35), 4.99 (H-36), 2.26 (H-3), 1.40 (H-37) and at d 173.84 (C-1), 148.82 (C-35), 134.28 (C-2), 77.35 (C-36), 25.60 (C-3) and 19.15 (C-37), respectively (see Table 1). The presence of an adjacent bis-THF rings system with two flanking OH groups was suggested by the four carbon signals at d 83.30, 82.78, 82.48 and 82.14, which were correlated to the proton signals at d 3.83e3.94 (5H) in the HMQC spectrum. The one-bond 1He13C correlations detected in the HMQC spectrum, along with those observed in COSY, permitted the assignment of the signals at d 74.11 and 71.35 to oxygenated methine carbons and at d 3.40 and d 3.83e3.94 to hydrogens adjacent to the THF rings. COSY spectrum showed correlation between these signals at d 3.83e3.94 (5H) and the multiplet at d 3.40 (1H). The presence of a third hydroxyl group in the hydrocarbon chain was suggested by the signals at d 3.49 (1H) and d 71.70 (in 1H NMR and 13C NMR spectra, respectively). The placement of THF rings and the additional hydroxyl group (d 3.49 and d 71.70) was determined by a close examination of the ESIMS fragmentation of 1 and its TMS derivative (Fig. 1). The hydroxyl group was located at C7 on basis of fragments at m/z 184.7 and m/z 255, in ESIMS of 1 and its TMS derivative, respectively. The relative stereochemistries around the bis-THF rings were determined using the methodologies of Hoye and co-workers (Hoye and Zhuang, 1988) and Born et al. (1990), which compare the NMR data of the unknown acetogenin and its acetate derivative with a series of model compounds of known relative stereochemistry (Jossang et al., 1990; Cortes et al., 1991; Zeng et al., 1996). However, these models cannot
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Table 1 1 H NMR (400 MHz) for 1 and 1b and Proton/carbon
13
1 2 3 4 5 6 7 8 9e15 16 17 18 19e, 20e 19a, 20a 21 22 23e, 24e 23a, 24a 25 26 27 28e31 32 33 34 35 36 37 7AcO 17AcO 26AcO
173.84 134.28 25.60 27.35 21.98 37.44 71.70 37.22 29.12e29.69 33.17 74.11 83.30 28.37e28.90 28.37e28.90 82.48 82.14 28.37e28.90 28.37e28.90 82.78 71.35 32.42 29.12e29.69 31.80 22.56 14.02 148.82 77.35 19.15 e e e
a
13
C NMR (100 MHz) for 1 spectral dataa in CDCl3
C NMR of 1
1
1
e e 2.26 tt (8.56, 1.74, 1.74) 1.49e1.56 m 1.39e1.42 1.39e1.42 3.49 m 1.39e1.42 1.26 br s 1.39e1.42 3.40 m 3.83e3.94 m 1.61e1.66 m 1.83e1.98 m 3.83e3.94 m 3.83e3.94 m 1.61e1.66 m 1.83e1.98 m 3.83e3.94 m 3.83e3.94 m 1.39e1.42 1.26 br s 1.26 br s 1.26 br s 0.88 t (6.80) 6.98 q (1.74, 1.74) 4.99 qq (6.80, 1.74, 1.74) 1.40 d (6.80) e e e
e e 2.26 tt e e 1.49e1.62 4.60e4.91 1.49e1.62 1.25 br s e 4.60e4.91 3.94e4.04 1.95 m 1.95 m 3.80e3.90 3.80e3.90 1.95 m 1.95 m 3.94e4.04 4.60e4.91 e 1.25 br s e e 0.88 t 6.99 q 4.99 qq 1.40 d 2.02 s 2.07 s 2.04 s
H NMR of 1 (J in Hz)
H NMR of 1b
m m m
m m
m m
m m
Chemical shifts (d) are in ppm relative to TMS.
distinguish if the relative stereochemistry of threoetransethreoetranseerythro is from C-17 to C-26 or from C-26 to C-17 and we cannot predict the relative configuration of the hydroxyl group in the hydrocarbon chain. The acetate derivative 1b was prepared and its NMR data are reported in Table 1. 13C NMR/1H NMR signals at d 74.11/3.40 for C17/H17 and d 83.3/3.83e3.94 for C18/H18 suggested a 17/18 stereorelationships as threo. The 13C NMR/1H NMR signals at d 71.35/3.83e3.94 for C26/H26 and d 82.78/3.93e3.94 for C25/H25 suggested a 25/ 26 stereorelationships as erythro. The 1H NMR signal of 1b at d 3.94e4.04 for H18 and H25 and d 3.80e3.90 for H21 and H22 indicated trans configuration for both THF rings (Hoye and Zhuang, 1988). Cornifolin (1) was extremely cytotoxic to brine shrimp DL50 2,29 x 10ÿ1 mg/mL. This is the first report on the isolation of an annonaceous acetogenin from the species A. cornifolia A. St.eHil.
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A
m/z 154
m/z 309
m/z 213
37 35
CH3(CH2)7 34 26
(CH2)9 25 O 22 21 O 18 OH
36 O
7 1
17
O
OH
OH
m/z 184
m/z 339
-H2O
m/z 166
B 37 35 CH3(CH2)7 34 26
(CH2)9 25 O 22 21 O 18 OSi(CH3)3
36 O
7 1
17 OSi(CH3)3
O
OSi(CH3)3 m/z 255
m/z 283
Fig. 1. Diagnostic ESIMS fragment ions (m/z). (A) Cornifolin (1) and (B) TMSi derivative (1a). Cornifolin (1): White solid; mp 33.7e34.3 C (ethyl acetate): [a]D 25: C18.5 ÿ1 (CHCl3, c 0.20). IR (cm ): 3420, 2910, 2845, 1745, 1650, 1460, 1315, 1200, 1070, 720. ESIMS m/z (relative intensity, %): 621.4 [M ÿ H]ÿ (100), 603.4 (4), 309.0 (4), 293.0 (5), 184.7 (82), 166.6 (25), 154.7 (35), 140.7 (21), 126.7 (72), 112.6 (54). TMSi-ether derivative (1a): It was prepared by treating 2 mg of 1 with 20 mL of N,O-bis-(trimethylsilyl)acetamide and 2 mL of pyridine and heating at 70 C for 30 min to yield the respective tri-TMSi derivative. ESIMS m/z (relative intensity, %): 837 [M ÿ H]ÿ (!1), 283 (100), 281 (70%), 255 (45).
37
threo erythro CH3(CH2)7 34 26
threo
35 (CH2)9
25 O 22 21 O 18 OR trans
1 OR
trans
Compound 1 1a 1b
O
7
17 OR
36
R H TMSi Ac
O
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Triacetylcornifolin (1b): It was prepared from 1 with Ac2O/Pyr. 1H NMR (400 MHz, CDCl3): Table 1.
4. Chemotaxonomy significance Annonaceous acetogenins constitute a series of C-35/C-37 natural products derived from fatty acids. Their structure is usually characterized by a long aliphatic chain bearing terminal methyl-substituted a,b-unsaturated g-lactone ring (sometimes rearranged to a ketolactone), with one, two or three tetrahydrofuran and/or tetrahydropyran rings and some substituents along the chain, particularly hydroxyls, acetoxyls, ketones, epoxides and/or double bonds being present (Alali et al., 1999). The annonaceous acetogenins have been isolated, so far, from the genera Annona, Anomianthus, Asimina, Disepalum, Goniothalamus, Rollinia, Polyalthia, Porcelia, Uvaria and Xylopia. The genus Annona comprises approximately 120 species and, from 1982 until 2002, 17 species of Annona were investigated, yielding 261 different annonaceous acetogenins. The isolation of a new acetogenin in A. cornifolia, about which there are no phytochemical reports, confirms this genus as a promising source of these compounds. These metabolites have drawn much attention because of their potent and varied biological activities, such as cytotoxic, antitumoral, antimalarial, antifeedant, antimicrobial, pesticidal and immunosuppressive activities.
Acknowledgements The authors thank the Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (CNPq) for fellowship to L.A.R.S. and to Dr. Edson Rodrigues Filho (UFSCar) for the mass spectra.
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