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Four cytotoxic annonaceous acetogenins from the seeds of Annona squamosa

Four new annonaceous acetogenins (ACGs), squamocin-I (1), II (2) and III (3) and squamoxinone-D (4), together with seven known ACGs (5–11), were isolated from the seeds of Annona squamosa. The structures of all isolates were elucidated and characterised by spectral and chemical methods. Compounds 1–4 were evaluated for their cytotoxicities against Hep G2, SMMC 7721, BEL 7402, BGC 803 and H460 human cancer cell lines. Compound 1 exhibited better potent activity than the positive compound and compound 3 shows selectively cytotoxical activity against H460 with IC50 values of 0.0492 μg/ml.


Introduction
The annonaceous acetogenins (ACGs) are now some of the most rapidly growing classes of new natural products and offer exciting cytotoxic in anti-tumour, anti-malarial, antimicrobial, antiprotozoal and pesticidal activities. They may have a special promise of becoming new chemotypes for anti-tumour and pesticidal agents (Bermejo et al. 2005;McLaughlin 2008;Kojima & Tanaka 2009). In our previous study, structure -activity relationships of ACGs from the seeds of Annona squamosa have been studied (Yang et al. 2009;Miao et al. 2014). Now four new tetrahydrofuran (THF) ACGs, squamocin-I (1), II (2) and III (3) and squamoxinone-D (4), together with seven known ACGs (5-11), were isolated from an ethyl acetate extract of the seeds of this species. We report the isolation and structural elucidation of these compounds, as well as the cytotoxic activities of compounds 1 -4 against five tumour cells. All of the isolated compounds 1-4 showed potent cytotoxicities, while some showed selective cytotoxicities against the human tumour cell lines Hep G2, SMMC 7721, BEL 7402, BGC 803 and H460.

Results and discussion
Compound 1 was isolated as a white powder and gave a molecular formula of C 37 H 66 O 7 as deduced from HR-ESI-MS (m/z 645.1338 [M þ Na] þ , calcd for C 37 H 66 O 7 Na 645.4706). The UV maximum at 208 nm and a positive reaction to Raymond's reagent implied the presence of an a,b-unsaturated g-lactone. The proton signals in the 1 H NMR spectrum (Table S14) -3b), and in the 13 C NMR spectrum (Table S14) at d C 174.6 (C-1), 131.1 (C-2), 151.8 (C-35), 77.5 (C-36) and 19.0 (C-37) confirmed the presence of the terminal fragment with a hydroxyl group at the C-4 position (Morré et al. 1995). The signals at d H 3.41 and 3.85-3.94 as well as those at d C 83.2 (C-15), 82.8 (C-22), 82.5 (C-18), 82.2 (C-19), 74.1 (C-14) and 71.4 (C-23) in the 1 H and 13 C NMR spectra are the characteristic of the presence of an adjacent bis-THF rings system with two flanking hydroxyl groups (Chang & Wu 2001). The disposition of the adjacent bis-THF unit with flanking hydroxyls was placed at C-14 to C-23 according to the ESIMS fragment ion peaks at m/z 249, 319,337,349,185,401,419 and 437 (Figure S13). By careful comparison of 1 H and 13 C NMR data with a series of bullatacin-type compounds (Sahai et al. 1994), the relative stereochemistry from C-14 to C-23 of 1 was found to be threo/trans/threo/trans/erythro (Chen et al. 2011 (Table S14) at d C 173.8 (C-1), 134.3 (C-2), 148.8 (C-35), 77.4 (C-36) and 19.2 (C-37) confirmed the presence of the terminal fragment without a hydroxyl group at the C-4 position (Morré et al. 1995). The signals at d H 3.40 and 3.83-3.96 as well as those at d C 83.3 (C-13), 82.8 (C-20), 82.5 (C-16), 82.1 (C-17), 74.1 (C-12) and 71.4 (C-21) in the 1 H and 13 C NMR spectra are the characteristic of the presence of an adjacent bis-THF rings system with two flanking hydroxyl groups (Chang et al. 1998). The disposition of the adjacent bis-THF unit with flanking hydroxyls was placed at C-12 to C-21 according to the ESIMS fragment ion peaks at m/z 199, 181,223,399,363,345,229,211,449 and 413 ( Figure S13). By careful comparison of 1 H and 13 C NMR data with a series of bullatacin-type compounds (Sahai et al. 1994), the relative stereochemistry from C-12 to C-21 of 2 was found to be threo/trans/threo/trans/erythro (Chen et al. 2011). The signals at d H 3.83 -3.96 and 3.59 as well as those at d C 71.4 and 71.7 in 2 for C-21/C-22 were then concluded that the configuration of C-21/C-22 in 2 is threo (Yu et al. 1998).
Compound 3 was isolated as a white powder and gave a molecular formula of C 37 H 66 O 7 as deduced from HR-ESI-MS (m/z 645.1586 [M þ Na] þ , calcd for C 37 H 66 O 7 Na 645.4706). The UV maximum at 209 nm and a positive reaction to Raymond's reagent implied the presence of an a,b-unsaturated g-lactone. The proton signals in the 1 H NMR spectrum (Table S15) (Table S15) at d C 174.0 (C-1), 134.3 (C-2), 148.9 (C-35), 77.5 (C-36) and 19.2 (C-37) confirmed the presence of the terminal fragment without a hydroxyl group at the C-4 position (Morré et al. 1995). The signals at d H 3.39 and 3.83-3.94 as well as those at d C 83.3 (C-15), 82.8 (C-22), 82.6 (C-18), 82.3 (C-19), 74.2 (C-14) and 71.3 (C-23) in the 1 H and 13 C NMR spectra are the characteristic of the presence of an adjacent bis-THF rings system with two flanking hydroxyl groups (Chang et al. 1998). The disposition of the adjacent bis-THF unit with flanking hydroxyls was placed at C-14 to C-23 according to the ESIMS fragment ion peaks at m/z 263, 305, 323, 341, 403, 421, 533 and 551 ( Figure S13). By careful comparison of 1 H and 13 C NMR data with a series of bullatacin-type compounds (Sahai et al. 1994), the relative stereochemistry from C-14 to C-23 of 3 was found to be threo/trans/threo/trans/erythro (Chen et al. 2011). The absolute configuration of the secondary alcohol chiral centre at C-29 was not determined due to the difficult assignment of the diagnostic protons by Mosher method (Zhou et al. 2000).
Compound 4 was isolated as a white waxy solid and gave a molecular formula of C 37 H 66 O 6 as deduced from HR-ESI-MS (m/z 629.1680 [M þ Na] þ , calcd for C 37 H 66 O 6 Na629.4757). The UV maximum at 211 nm and a positive reaction to Raymond's reagent implied the presence of an a,b-unsaturated g-lactone. The proton signals in the 1 H NMR spectrum (Table S15) H-3b), and in the 13 C NMR spectrum (Table S15) at d C 174.6 (C-1), 131.2 (C-2), 151.8 (C-35), 78.0 (C-36) and 19.1 (C-37) confirmed the presence of the terminal fragment with a hydroxyl group at the C-4 position (Morré et al. 1995). The signals at d H 3.39 and 3.84 as well as those at d C 83.2 (C-13), 81.8 (C-16), 74.1 (C-12, 17) in the 1 H and 13 C NMR spectra are the characteristic of the presence of an mono-THF rings system with two flanking hydroxyl groups (Chang et al. 1998). The disposition of the mono-THF unit with flanking hydroxyls was placed at C-12 to C-17 according to the ESIMS fragment ion peaks at m/z 151, 177,231,249,267,339,269,251,337 and 301 ( Figure S13). By careful comparison of 1 H and 13 C NMR data with a series of annonacin-type compounds (Tam Vu et al. 1995), the relative stereochemistry from C-12 to C-17 of 4 was found to be threo/trans/threo (Chen et al. 2011). A cis double bond (Fang et al. 1993) in the aliphatic chain of 4 was evident from two olefinic proton signals at d H 5.34 and 5.34 and two carbon resonances at d C 130.0 (C-7) and 129.8 (C-8) in the 1 H and 13 C NMR spectra.
All 4-OH acetogenins that have been found so far have the R configuration at C-4 and the S configuration at C-36 (Alali et al. 1999). The structures of compounds 1 -4 are shown in Figure 1.
Bioactivity data obtained with 1 -4 are summarised in Table 1. They all showed potent cytotoxicities against five human tumour cell lines in culture. The bioactivities of many are better than fluorouracil. Although not all compounds have significantly cytotoxic activities against all cancer cell lines, some of them indicated high selective inhibition activities against the human tumour cell lines. For instance, compound 3 shows significant cytotoxical activity against H460 selectively. It indicates that ACGs which have -OH at the tail of carbon chain have high selective inhibition activities against H460. The cytotoxic activities of compounds 5-11 have been determined in previous study (Chen 2013).

Plant material
The seeds of A. squamosa were collected from Guangdong Province in July 2012. The sample was authenticated and deposited in the Pharmaceutical College of Nanjing University of Chinese Medicine, Jiangsu (No. 132).

Supplementary material
Experimental details relating to this paper are available online at http://dx.doi.org/10.1080/ 14786419.2015.1055490, alongside Figures S1 -S13 and Tables S14 and S15.

Disclosure statement
No potential conflict of interest was reported by the authors.