Further rearranged prenylxanthones and benzophenones from Garcinia bracteata

Tetrahedron 61 (2005) 8529–8535

Further rearranged prenylxanthones and benzophenones from Garcinia bracteata Odile Thoison,a Dao Dinh Cuong,b Anthony Gramain,a Ange`le Chiaroni,a Nguyen Van Hungb and Thierry Se´veneta,* a

Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette Cedex, France b Institute of Chemistry, NCST, Hoang Quoc Viet Road, Nghia Do, Cau Giay, Hanoi, Vietnam Received 10 February 2005; revised 25 May 2005; accepted 31 May 2005 Available online 18 July 2005

Abstract—New rearranged polyprenylxanthones, namely garcibracteatone, neoisobractatins A and B, and xerophenone C were isolated from the leaves and bark of a vietnamese Garcinia, Garcinia bracteata, together with 5-O-methylxanthone V1, bracteaxanthones I and II, and the known nemorosonol and simple xanthones. Neoisobractatins A and B exhibit a significant cytotoxic activity on KB cells. A biogenetic hypothesis is proposed, which explains the possible origin of these so-called cage-xanthanoids. q 2005 Elsevier Ltd. All rights reserved.

1. Introduction In continuation of our preliminary investigations on the leaves of Garcinia bracteata Wu ex Li (Clusiaceae)1 we isolated from the bark of the plant new prenylxanthones, garcibracteatone 1, xerophenone C 2, 5-O-methylxanthone V1 3, together with the known nemorosonol 4,2,3 and 10-Omethylmacluraxanthone.4 From the leaves of the plant, neoisobractatins 5 and 6, bracteaxanthones I 7, and II 8, and the known macluraxanthone,5 cudraxanthone R6 and gerontoxanthone I,7 were isolated. In this paper, we report the isolation, structure elucidation and biological evaluation on KB cells culture of the new compounds. A hypothetic mechanism is formulated to explain the biogenesis of the tetraprenylbenzophenones 1, 2, and 4 in the plant, and of the triprenylxanthones 5 and 6, which are new examples of the so-called cage-xanthanoids.8–10 2. Results and discussion 2.1. Isolation Dried and powdered leaves were extracted with EtOAc then EtOH as previously described.1 Bark of the plant was extracted by the same method. Both extracts were Keywords: Clusiaceae; Garcinia bracteata; Garcibracteatone; Xerophenone C; Neoisobractatins; Nemorosonol; Bracteaxanthones; Xanthones; Benzophenones. * Corresponding author. Tel.: C33 1 69823103; fax: C33 1 69077247; e-mail: [email protected] 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.05.091

chromatographed on silica gel, and compounds 1–4 and 10-O-methylmacluraxanthone were isolated from the EtOAc extract of the bark. Compounds 5–8, macluraxanthone, cudraxanthone R, and gerontoxanthone I were isolated from the EtOAc extract of the leaves. The structures of 1–8 have been determined by a combination of 1H, 13C, and 2D NMR techniques. X-ray analysis of the diastereoisomer 5 gives its relative configuration. 2.2. Structure elucidation Compound 4 was first isolated and compared to nemorosonol, which was previously isolated from Clusia nemorosa, and re-isolated in the present work from G. bracteata. The NMR data and HMBC correlations were identical.2,3 So compound 4 is nemorosonol. Garcibracteatone (1), gave a molecular peak at m/z 500 (EIMS) corresponding to the molecular formula C33H40O4. The IR spectrum indicated the presence of three ketone functions at 1739, 1708, and 1675 cmK1. The 13C NMR spectrum allows to number 33 carbons of which seven signals correspond to methyl substituents, eight methine of which four are aromatic and two olefinic, 13 quaternary carbons of which three are keto groups, and five methylene carbons (Table 1). In the HMBC NMR spectrum, the presence of two isoprenyl chains was deduced from the observation of a broad triplet of 2H at 5.02 ppm (H-12, H-22) correlating with two methylene signals at 2.28 (d, CH2-11) and at 2.25 (HA-21) and 2.10 (HB-21) (Table 1). Four aromatic protons coupling together correspond to a

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disubstituted aromatic ring. These data and the observation of other correlations lead to a nemorosonol-type structure with only two isoprenyl chains, and a possible rearrangement of the third one. In COSY and HMBC spectra, correlations between H-30 and C-18, which couples itself with CH3-19 and CH3-20, between H-17, C-8, and C-18, lead us to propose for compound 1 the structure of garcibracteatone as indicated. The relative configuration of (1) is possibly similar to the nemorosonol one (4) of which the relative configuration is known, all the stereocenters being determined except C-17 (see after), NOESY experiments show correlations between CH3-20 and OH-7, CH3-19, and H-17. Biogenetically, it can be hypothetized that garcibracteatone comes from nemorosonol 4, also isolated from the plant, by a cycloaddition involving carbons C-17 and C-18 on one hand, C-28 and C-29/C-8 and C-27 on the other hand, followed by oxidation of the enol (Scheme 1). Xerophenone C (2) gave a peak [M]C% at m/z 518 (EIMS), which matched the molecular formula C33H42O5. The

presence of the unsubstituted phenylketone/enol was shown by a peak at m/z 105, C6H5COC, and by five aromatic protons in the NMR spectrum. The 1H NMR spectrum in CDCl3 shows unresolved signals. In pyridined5, all the signals are well resolved, which lead us to suppose the presence of tautomeric forms (Table 1). The presence of three isoprenyl chains was deduced from the observation of three vinyl protons at 5.94 ppm (H-12), 5.85 (H-17), and 5.07 (H-22), correlating in COSY with three methylenes and six methyls, respectively. The 13C NMR spectrum allows to number 33 carbons of which five CH2 (three for the three isoprenyl chains, one isolated CH2 and the last one connected to a CH), seven methyls, nine CH, and 12 quaternary carbons (Table 1). HMBC correlations allow to connect the quaternary carbons with the isoprenyl chains. This product is very close to xerophenones A and B isolated from Clusia portlandiana,11,12 the only difference being a double bond between C-22 and C-23, instead of C-23 and C-24. NOESY experiments show correlations between H-5 and CH2-16, meaning that H-5 has the same orientation that the isoprenyl chain in C-3, between H-29/

O. Thoison et al. / Tetrahedron 61 (2005) 8529–8535

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Table 1. 1H and 13C NMR data of benzophenones 1 (CDCl3) and 2 (pyridine-d5) 1a

Position 1

H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 OH-7 a b

1.60 m 2.00 m 1.85 m

1.60 m 1.80 m 2.28 2H d (7.5) 5.02 br t 1.64 3H s 1.56 3H s 2.00 m 2.20 m 2.63 dd (8, 10) 1.31 3H s 0.96 3H s 2.10 m 2.25 m 5.02 br t 1.68 3H, s 1.62a 3H s 1.49 3H s

7.32 d (9) 7.54 dd (9, 9) 7.32 dd (9, 9) 7.67 d (9) 3.10

13

C

63.3 213.4 70.3 32.6 57.0 47.6 91.9 69.3 203.5 47.6 25.2 119.0 134.3 25.9 17.9 29.1 56.8 26.2 29.8 33.1 123.1 132.4 25.9 18.1 18.6 200.3 136.4 150.3 123.5 133.7 126.9 126.6

2b 1

HMBC

2, 3, 5, 6 2, 3, 5, 7

H

1.45 m 2.32 m 1.70 m

13

C

59.8 105.7 54.3 35.4 41.9 81.5 203.4 111.4 198.6 41.9

HMBC

3, 7, 21 2, 3, 5, 6, 16

1, 5, 6, 7, 9, 26 1, 5, 6, 9, 26 1, 9, 10, 12, 13 14

1.96 d (13) 2.54 d (13) 3.02 2H d (6.5) 5.94 t (7)

12, 13, 15 12, 13, 14 2, 3, 17, 18 2, 3, 17, 18 7, 8, 9, 16, 18, 19, 20 37.3 17, 18, 20, 29 17, 18, 19, 29 22, 23 22, 23

1.74 3H s 1.61 3H s 3.19 2H d (6.5)

35.9 122.2 132.5 26.1 17.8 26.4

5.85 t (6.5)

123.7

19, 20

133.1 26.1 17.8 28.9

17, 18, 20 17, 18, 19

122.7

21, 24, 25

25.7 17.6 25.2 193.4 138.3 128.9 128.0 131.5 128.0 128.9

22, 23, 25 22, 23, 24 5, 6, 10

22, 23, 25 22, 23, 24 5, 6, 7, 10

18, 28 29, 33 28, 30 29, 31 3, 7

1.74 3H s 1.71 3H s 1.53 m 2.11 m 5.07 t (7) 2.5 1.69 3H s 1.51 3H s 1.47 3H s 7.83 d (7) 7.41 m 7.41 m 7.41 m 7.83 d (7)

1, 2, 5, 6, 9, 11, 26 1, 2, 5, 6, 9, 11 1, 2, 9, 10, 12, 13 11, 14, 15 12, 13, 15 12, 13, 14 2, 3, 4, 7, 17, 18

27, 31, 33 28, 32 29, 33 28, 30, 33 27, 29, 31

CDCl3. Pyridine-d5.

H-33 and H-11 and H-12, meaning that there is an hydrogen bond between CO-9 and OH-27 as observed for xerophenone A.11,12 We give it the trivial name of xerophenone C (2). Isolated from the leaves, neoisobractatins A (5) and B (6), gave a major peak in FABMS at m/z 465 [MH]C, which

Scheme 1.

matched the pseudomolecular formula C28H33O6. In the 1H NMR spectrum, the presence of additional methyl signals led to the conclusion that the product was a mixture of diastereoisomers. Re-chromatography on a graphite column allowed the isolation of the two separate diastereoisomers 5 and 6, one only crystallizing in heptane. The 1H and 13C

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O. Thoison et al. / Tetrahedron 61 (2005) 8529–8535

Table 2. 1H and

13

C NMR data of neoisobractatins 5 and 6 (CDCl3)

Position

5 1

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 OH-1

H

5.98 s

3.76 dd (4, 7) 7.21 d (7)

4.47 q (6) 1.39 d (6) 1.36 3H s 1.45 3H s 1.88 dd (10, 14) 2.48 d (14) 2.19 dd (5, 10) 1.37 3H s 1.39 3H s 2.07 dd (8, 14) 2.48 m 5.04 t (7) 1.62 3H s 1.74 3H s 12.7 s

6 13

C

166.0 92.3 168.5 114.1 155.2 199.8 79.4 44.8 134.1 134.8 177.2 101.8 83.7 43.4 91.2 14.2 20.7 25.5 33.0 42.3 83.7 26.7 29.6 30.2 117.3 136.3 18.1 25.8

1

H

5.99 s

3.76 dd (4, 7) 7.21 d (7)

4.50 q (6) 1.38 d (6) 1.21 3H s 1.57 3H s 1.88 dd (10, 14) 2.49 d (14) 2.21 dd (4, 10) 1.36 3H s 1.39 3H s 2.09 dd (8, 14) 2.49 m 5.03 t (7) 1.61 3H s 1.73 3H s 12.7 s

13

C

166.0 92.3 168.5 114.1 155.2 199.8 79.2 44.8 134.1 134.8 177.2 101.8 83.7 43.4 91.2 14.3 21.0 25.5 33.0 42.5 83.7 26.7 29.6 30.2 117.3 136.3 18.1 25.8

NMR spectra of both 5 and 6 were superimposable (except two methyls) and very close to those of isobractatin and 1-O-methylneobractatin, with a similar dihydrofurane substituted ring and a chelated hydroxy group as in isobractatin on one part, and a sequence CH2–CH–CH–CH] as in 1-O-methylneobractatin on the other part, which led us to propose that compounds 5 and 6 are new and correspond to neoisobractatin. The differences are observed in the 13C NMR spectrum for carbons C-13, C-14, and in the 1H NMR for CH3-

Figure 1. ORTEP diagram of compound 5.

14 and CH3-15 (Table 2). The two products are stereoisomers on C-12, each stereoisomer being isolated as a racemic mixture and we call them neoisobractatins A (5) and B (6). The structure of 5 isolated from the mixture 5 and 6 is confirmed by single-crystal X-ray structure analysis. Compound 5 crystallizes in the centrosymmetric space group P-1 of the triclinic system, implying that in the solid state neoisobractatin A is racemic. The depicted enantiomer (C12, S) (Fig. 1) can be directly compared to isobractatin.1 The left moieties of the molecules are identical, with the same intramolecular hydrogen bond observed between the hydroxyl group O1–H and the oxygen atom O9 (distances ˚ , angle O–H–OZ ˚ , HO1/O9Z1.84 A O1/O9Z2.563 A 146.38). Although, the right moieties look nearly similar, they are very different with respect to CO-5 position. Many xanthones have been isolated from leaves and bark of the plant. All of them have a UV spectrum characteristic of a 1,3,5,6-tetraoxygenated xanthone chromophore. Macluraxanthone,5 10-O-methylmacluraxanthone,4 cudraxanthone R,6 and gerontoxanthone I7 have been identified by comparison with the literature NMR data. The molecular formula C24H24O6 of compound 3 is derived from its EIMS (m/z 408 [M]C%) and HREIMS. The UV spectrum is characteristic of a 1,3,5,6-tetraoxygenated xanthone. The IR spectrum indicated the presence of a conjugated ketone at 1649 cmK1 and OH absorptions at 3516 cmK1. NMR data show that 3 possesses a xanthone skeleton, with a chelated OH at C-1, a prenyl substituent in position 4, and a dimethylpyran ring. The data are quite similar to that of xanthone V1 isolated from Vismia guineensis,13 except the presence of a methoxyle. NOESY correlations between the prenyl chain in C-4 and methoxyl allows to attach this methoxy at C-5 (Tables 3 and 4). This product is a O-methyl derivative of xanthone V1 and is named 5-O-methylxanthone V1 3. Two other xanthones isolated from the leaves present spectral data characteristic of a 1,3,5,6-tetraoxygenated

O. Thoison et al. / Tetrahedron 61 (2005) 8529–8535 Table 3. 1H NMR data of xanthones 3, 7, and 8 a

Position

3

7 8 10

6.99 d (9) 7.92 d (9) 6.75 d (10)

11 13 14

5.62 d (10) 1.48 3H s 1.48 3H s

15 16 17

3.52, 2H, d (7) 5.23, t (7)

18 19 OH-1 OCH3-5

1.69 s 3H 1.86 s 3H 13.2 s 4.13 s 3H

a b

b

Table 5. Cytotoxicity on KB cells b

7

8

Compound

KB (IC50 mg/mL)

6.96 d (9) 7.60 d (9) 2.83 dd (8, 14) 3.16 dd (2, 14) 4.39 br d (8) 1.86 s 5.03 s 4.83 s

6.88 d (9) 7.56 d (9) 2.57 dd (8, 17) 2.90 dd (5, 17) 3.80 br t 1.34 s 1.42 s

6.57 dd (10, 18) 4.88 d (10) 5.03 d (18) 1.75 3H s 1.75 3H s 14.07 s

6.64 dd (10, 18) 5.25 d (10) 5.43 d (18) 1.80 3H s 1.80 3H s

AcOEt leaf extract AcOEt trunk bark extract Garcibracteatone 1 Xerophenone C 2 5-O-methylxanthone V1 3 Nemorosonol 4 Neoisobractatin A 5 Neoisobractatin B 6 Bracteaxanthone I 7 Bracteaxanthone II 8

4 15 4.2 0.8 0.9 0.9 0.14 0.16 Inactive Inactive

3. Experimental

xanthone with a 1,1-dimethylallyl group located at C-4 as in gerontoxanthone I isolated from the same plant. Compounds 7 and 8 exhibit in mass spectrometry the same molecular weight at m/z 412 corresponding to C23H24O7, which is confirmed by HREIMS. 1D- and 2D NMR experiments show for compound 7 a five carbon chain with a 2H exo methylene at 4.83 and 5.03 ppm (singlets), and a CHOH at 78.3 ppm coupling with a CH2 at 23.1 ppm (Tables 3 and 4). Compound 7 is thus a new molecule named bracteaxanthone I. For compound 8, NMR data show that the exo methylene disappear, and that the C-12 is cyclised on the –OH on C-1 (Tables 3 and 4). This compound is a new molecule named bracteaxanthone II 8. 2.3. Biological activity

13

C NMR data for compounds 3, 7, and 8

Position

3a

7b

8b

1 2 3 4 4a 5 5a 6 7 8 8a 9 9a 10 11 12 13 14 15 16 17 18 19 OCH3

158.2 104.8 156.0 114.8 154.5 133.9 149.8 154.0 112.5 121.8 107.6 180.5 102.9 115.8 127.4 78.2 28.4 28.4 21.7 122.3 131.9 18.1 25.9 61.8

161.1 111.0 165.2 114.7 156.0 134.6 148.9 153.2 114.7 118.0 114.7 182.5 104.1 23.1 78.3 148.9 19.4 111.4 43.0 154.0 108.0 29.9 29.9

155.4 106.6 160.1 113.4 nd 135.9 147.0 151.9 114.9 118.6 118.6 176.2 nd 28.5 70.4 79.6 21.5 27.1 43.4 153.3 112.8 29.4 29.4

b

CDCl3. Acetone-d6.

3.1. General Optical rotations at 208 were measured on a Perkin-Elmer 241 polarimeter. IR spectra were recorded on a PerkinElmer Spectrum BX FT-IR spectrometer and UV on a Varian Cary 100 spectrometer; EIMS were recorded on a Automass ThermoFinnigan. The NMR spectra were recorded on a Bruker AC-250 and AC 300 or AM 400. CC was performed using silica gel Merck H60. Compounds 5 and 6 have been purified by HPLC on Hypercarb column (5m, 10!150 mm), (CH2Cl2/AcOEt 3:1, 3 mL/mn), using a Waters equipment with a 600 E pump Autoinjector and a 996 photodiode array detector. 3.2. Plant material

Compounds 1–8 were evaluated against KB cell lines (Table 5). Many compounds are active, the most interesting

a

being compounds neoisobractatins A 5 and B 6, as for 1-Omethylneobractatin previously isolated from the leaves of the same plant.1

CDCl3. Acetone-d6.

Table 4.

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Leaves and bark of G. bracteata were collected at Pa` Co, Mai Chau, Hoa Binh province, 150 km west of Hanoi, North Vietnam, in September 1999. Identification was provided by one of us (A.G.) and Nguyen Huu Hien (Institute of Ecology, NCST, Hanoi). Voucher specimens (VN 578) are deposited in the Herbarium of the Institute of Ecology and Biological Resources, NCST, Hanoi, Vietnam. 3.3. Extraction and isolation The dried ground leaves of G. bracteata (2.88 kg) were extracted in a Soxhlet at room temperature with EtOAc and the extract was evaporated under vacuum (62 g, yield 2. 15%). Repeated column chromatography on silica gel afforded, (from heptane/EtOAc to EtOAc/MeOH), the previously isolated isobractatin, bractatin, 1-O-methylisobractatin, 1-O-methylbractatin, 1-O-methylneobractatin, 1-O-methyl-8-methoxy-8,8a-dihydro-bractatin,1 macluraxanthone, gerontoxanthone I, cudraxanthone R, and four new compounds, bracteaxanthones I 3 (0.040 g, Et2O/ cyclohexane 6:4) and II 4 (0.015 g, CH2Cl2/MeOH 9.5:0.5), and neoisobractatins A 5 and B 6 (0.025 and 0.025 g, respectively, heptane/EtOAc 9:1, then HPLC on Hypercarb column). The dried ground trunk bark of G. bracteata (1.9 kg) was extracted in a Soxhlet at room temperature with EtOAc and

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the extract was evaporated under vacuum (60 g, yield 3. 1%). Repeated column chromatography on silica gel afforded (from CH2Cl2 to MeOH) 25 fractions. Further purifications by CC on silica gel afforded the known 10-Omethylmacluraxanthone, nemorosonol, and three new compounds, garcibracteatone 1 (0.270 g), 5-O-methylxanthone V1 3 (0.083 g) and xerophenone C 2 (0.048 g). 3.3.1. Garcibracteatone 1. White crystals, mp 168–169 8C; [a]25 D K1 (c 1.00, CHCl3); UV (MeOH) lmax (log 3): 253 (4.05), 202 (4.53); IR (CHCl3) nmax 1739, 1708, 1675 cmK1; 1 H NMR (CDCl3, 250 MHz) see Table 1; 13C NMR (CDCl3, 75 MHz) see Table 1; EIMS m/z 500 (20) [M]C%, 199 (80), 95 (60), 69 (100), 55 (80); HREIMS m/z 500.2927 (calcd for C33H40O4, 500.2942). 3.3.2. Xerophenone C 2. Whitish crystals, mp 145–146 8C; [a]25 D C105.7 (c 1.00, CHCl3); UV (MeOH) lmax (log 3): 284 (4.16), 246 (4.05), 203 (4.53); IR (CHCl3) nmax 1670, 1587, 1570 cmK1; 1H NMR (pyridine-d5, 250 MHz) see Table 1; 13C NMR (pyridine-d5, 75 MHz) see Table 1; EIMS m/z 518 (10) [M]C%, 147 (12), 105 (65), 69 (100), 55 (15); HREIMS m/z 518.3032 (calcd for C 33H 42 O5 , 518.3035). 3.3.3. 5-O-Methylxanthone V1 3. Amorphous powder; UV (MeOH) lmax (log 3): 336 (4.18), 281 (4.60); IR (CHCl3) nmax 3516, 1650, 1609, 1587 cmK1; 1H NMR (CDCl3, 250 MHz) see Tables 3 and 4; 13C NMR (CDCl3, 75 MHz) see Tables 3 and 4; EIMS m/z 408 (100) [M]C%, 393 (100), 365 (15); HREIMS m/z 408.1573 (calcd for C24H24O6, 408.1580). 3.3.4. Neoisobractatins A 5 and B 6 (mixture). Yellow crystals, mp (mixture) 182–183 8C; UV (MeOH) lmax (log 3): 334 (4.21), 259 (3.80); IR (CHCl3) nmax 1751, 1639 cmK1; 1H NMR of 5 and 6 (CDCl3, 250 MHz) see differences in Table 2; 13C NMR (CDCl3, 75 MHz), see differences in Table 2; HRFABMS [MH]C 465.2269 (5), 465.2264 (6) (calcd for C28H33O6, 465.2277). X-ray structure analysis of neoisobractatin A 5. Crystal data. Small yellow crystal of 0.10!0.13!0.26 mm3 grown from a mixture of heptane. C28 H32 O6, MwZ464.54, mp 182–183 8C. Triclinic system, space group P-1, ZZ2, ˚ , bZ10.282(6) A ˚ , cZ15.393(8) A ˚ , aZ aZ7.948(6) A ˚ 3, 90.93(3)8, bZ101.96(4)8, gZ99.87(3)8, VZ1210.7 A K3 ˚, dcZ1.274 g cm , F(000)Z496, l(Cu Ka)Z1.5418 A K1 mZ0.721 mm , decay: 7%. Data were measured with a Nonius-CAD4 diffractometer, up to qZ688 (K9%h%9, K12%k%12, l:0–18). 4465 data were collected leading to 4265 unique reflections, of, which 2334 were considered as observed having IR2s(I). The structure was solved by direct methods using program SHELXS8614 and refined by full-matrix least-squares, based upon unique F2 with program SHELXL93.15 The hydrogen atoms, located in difference Fourier maps, were fitted at theoretical positions and assigned an isotropic displacement parameter equivalent to 1.2 that of the bonded atom. Thus, refinement converged to R1(F)Z0.0764 for the 2334 observed reflections and wR2(F2)Z0.2460 for all the 4285 data with a goodness-of-fit S factor of 1.028. The residual ˚ K3. electron density was found between K0.40 and 0.36 e A

In the crystal packing, only van der Waals contacts are observed. Crystallographic data (excluding structure factors) for this structure have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 235853. Copies can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: C44 1223 336033 or e-mail: [email protected]). 3.3.5. Bracteaxanthone I 7. Amorphous powder; UV (MeOH) lmax (log 3): 330 (4.14), 254 (4.50); IR (CHCl3) nmax 1626, 1659 cmK1; 1H NMR (acetone-d6, 250 MHz) see Tables 3 and 4; 13C NMR (acetone-d6, 75 MHz) see Tables 3 and 4; EIMS m/z 412 (18) [M]C%, 341 (100), 285 (90), 273 (50), 153 (25), 71 (50), 69 (55), 55 (70); HREIMS m/z 412.1523 (calcd for C23H24O7, 412.1522). 3.3.6. Bracteaxanthone II 8. Amorphous powder; UV (MeOH) lmax (log 3): 317 (3.98), 290 (3.92), 252 (4.40); IR (CHCl3) nmax 1629, 1602 cmK1; 1H NMR (acetone-d6, 250 MHz) see Tables 3 and 4; 13C NMR (acetone-d6, 75 MHz) see Tables 3 and 4; EIMS m/z 412 (100) [M]C%, 397 (90), 285 (90), 341 (33), 325 (33), 285 (35), 55 (90); HREIMS m/z 412.1531 (calcd for C23H24O7, 412.1522). 3.4. KB cytotoxicity assay Experiments were performed in 96-well microtiter plates (2!105 cells mLK1). Cell growth was estimated by colorimetric assay based on conversion of tetrazolium dye (MTT) to a blue formazan product using live mitochondria.16 Eight determinations were performed for each concentration. Control growth was estimated at 16 determinations. Optical density at 570 nm corresponding to solubilized formazan was read for each well on a Titertek Multiskan MKII.

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