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Schweinfurthin D, A Cytotoxic Stilbenefrom Macaranga schweinfurthiiJohn A. Beutler a , Johnson Jato b , Gordon M. Cragg c & MichaelR. Boyd da SAIC-Frederick, Frederick Cancer Research and DevelopmentCenter , Frederick, Maryland, 21702-1201b Faculty of Medicine and Biomedical Sciences, B.P. 92 ,University of Yaounde , Yaounde, Cameroonc Natural Products Branch, DTP, DCTD, NCId Laboratory of Drug Discovery Research & Development,Developmental Therapeutics Program, Division of CancerTreatment and Diagnosis , National Cancer Institute, FrederickCancer Research and Development Center , Building 1052, Room121, Frederick, Maryland, 21702-1201Published online: 04 Oct 2006.

To cite this article: John A. Beutler , Johnson Jato , Gordon M. Cragg & Michael R. Boyd (2000)Schweinfurthin D, A Cytotoxic Stilbene from Macaranga schweinfurthii , Natural Product Letters,14:5, 399-404, DOI: 10.1080/10575630008043774

To link to this article: http://dx.doi.org/10.1080/10575630008043774

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Nutural Product Letters Volume 14(5), pp. 399-404 Reprints available directly from the Publisher Photocopying permitted by license only

0 2000 OPA (Overseas Publishers Association) N.V. Published by license under

the Hanvood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.

Printed in Malaysia.

SCHWEINFURTHIN D, A CYTOTOXIC STILBENE FROM MACARANGA SCHKEINFURTHII

JOHN A. BEUTLER,' JOHNSON JATO,+ GORDON M. CRAGG,t MICHAEL R. BOYD*

Laboratory of Drug Discovery Research d Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick Cancer Research and Development Center, Building 1052, Room 121, Frederick, Maryland 21 702-1201

'SAIC-Frederick, Frederick Cancer Research and Development Center, Frederick, Maryland 21 702-1201

tFaculty of Medicine and Biomedical Sciences, B. P. 92, University of Yaounde, Yaounde, Cameroon

fNatural Products Branch, DTP, DCTD, NCI

(Received 3rd February 2000)

Abstract: A novel cytotoxic stilbene, schweinhrthin D, was isolated from

Macaranga schweinfirthii (Euphorbiaceae) and its structure was determined by

spectroscopic methods. Schweinhrthin D showed comparable potency to

schweinhrthin B.

Key Words: Stilbene, Cytotoxicity, Macaranga, Euphorbiaceae.

From a recollection ofMacaranga schweinfirthii leaves in Cameroon, undertaken to

provide schweinhrthin A for hrther biological evaluation, we isolated a previously unrecognized stilbene along with the known schweinhrthins A-C. Schweinhrthin D

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400 J.A. BEUTLER et al.

(1) was a yellowish, optically active glass, [aID +23 O , yielding a high-resolution mass

spectrum corresponding to C,,H,,O,, equivalent to schweinhrthin B (2) plus an

0-Me 0-Me

1

additional H,O. The 'H- and I3C-NMR spectra were nearly identical to those of

schweinfurthin B (Table l), particularly for the tricyclic portion of the molecule,

suggesting hydroxylation on the geranyl moiety. The presence of an extra oxygenated

carbon shift at 6 71.5 (s), and the absence of one of the characteristic doublets of

triplets at 6 5.25 associated with H-2" and H-7" in schweinfurthin B prompted us to

analyze the 2-D NMR spectra more closely.

The signal at 6 5.25 (td, lH, J=7,1 Hz) was correlated in the GHMBC spectrum with

the methylene carbon at 6 41.3 (C-5'7 as well as the 4"-Me at 6 16.1, indicating that

these signals were associated with the inner prenyl unit. The H-5" signal (6 1.95) was

correlated to C-4", C-6" and C-7" (6 44.3). The methyl signals at 6 1.16 (6H) showed

strong GHMBC correlations to the tertiary alcohol at 6 71.5, as well as to the

methylene carbon at 6 44.3 (C-7"). Thus the final methylene C",

carbon at 6 24.0 could be placed between the upfield

methylenes to complete the second prenyl chain. 'H-NMR

shifts for this portion of 1 were also compared to those

reported for the simple orcinol derivative 3,' with excellent YH correspondence. The absolute stereochemistry of the

schweinfurthin series remains to be determined

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SCHWEINFURTHIN D. A CYTOTOXIC STILBENE

Table 1. 'H- and "C-NMR Spectra for 1,6 in pprn, CD,OD, 125 MHz

10 I

Carbon No 13 C shfl ' for 1 'H h ' f t f 1 (GHSOC) I3C shift for 2 1

I 2 3 4 4a 5 6 7 8 8a 9 9a 1 Oa 11 12 13 I ' 2' 3'

4', 8' S', 7'

6' I " 2" 3" 4" 5" 6" 7" 8" 9" lo"

39.2 78.8 71.8 44.8 78.1 150.2 108.4 130.8 121.7 124.4 23.7 48.7 137.6 16.5 29.4 22.0 128.6 127.7 143.4

105.8 x2 157.3 x2

116.0 23.2 124.7 134.9 16.1 41.3 24.0 44.3 71.5 29.2 29.2

3 31, rn 4 14,m

2 34 dd, 3, 14 Hz, 1 93 m

6 91 d 1 5 Hz

686 ,d 1 5Hz

2 7 7 m 1 7 6 m

1 1 0 s 3 H 1 l l s 3 H 1 4 1 s 3 H

6 86 d AB,16 Hz 6 77 d AB, 16 Hz

6 46 s 2H

3 31 obsc 5 25 td, 7, 1 Hz

1 7 7 s 3 H 1 95 m 1 4 7 m 1 3 8 m

1 1 5 s 6 H

39.1 78.7 71.7 44.7 78.0 1.50 1 108.3 130.6 121.7 124.3 23.9 48.6 137.5 16.2 29.3 21.9 128.5 127.6 143.3 105.8 157.2 115.9 23.2 124.5 134.8 16.5 40.9 27.1 125.5 131.9 17.7 25.8

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402 J.A. BEUTLER et ul

When tested in a 2-cell line, 2-day cytotoxicity assay,3 schweinhrthin D was

essentially equipotent with schweinfbrthin B. In the sensitive SF29S line the IC,, for

1 was 2 ng/ml and for 2, 3 ng/ml, respectively, while in the resistant A549 cell line,

the IC,, for 1 was 2.8 and for 2 ,5 .5 pg/ml, respectively. That this modification ofthe

geranyl moiety does not markedly affect cytotoxicity indicates that the addition of a

covalent linker at this site may afford access to affinity reagents usehl in elucidation

of the molecular target by which the schweinfurthins act. This is in line with our

recent unpublished results for vedelianin, a congener having a shorter C, side chain4,

which was found to be equipotent with schweinhrthin A, while maintaining the

difference in sensitivity between the SF29S and AS49 cell lines.

EXPERIMENTAL

General. NMR experiments were performed on a Varian Innova-500 system with a

gradient inverse detection probe with samples dissolved in d,-MeOH.

Plant material. Leaves of Macaranga schweinfurthii Pax were collected March

1999 in Korup National Forest, Cameroon and authenticated by Anacletus Koufani

and David Kenfack, with reference to the original collection by Duncan Thomas.

Initial recollection efforts in the vicinity of Yaounde, Cameroon did not yield samples

with detectable schweinhrthins, despite their morphological similarity. By collecting

the plant in the same location and in the same season as the original collection, we

were successhl in obtaining material with a substantial content of schweinhrthins.

Isolation. An improved isolation method was designed to facilitate large-scale

isolation of schweinhrthins. Ground, dry leaves were extracted with EtOH by

percolation at RT, then evaporated to yield an ethanolic extract. In four batches, a

total of 5 1 3 g of ethanolic extract was coated on a total of 440 g diatomaceous earth

(Dicalite Speed-Plus) and eluted successively with a total of 6.5 L hexane, 3.5 L

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SCHWEINFURTHIN D, A CYTOTOXIC STILBENE 403

CH,CI,, and 1.9 L MeOH. The CH,CI, eluates were evaporated and permeated in

three batches on a column of Sephadex LH-20 in CH,Cl,-MeOH (1 : 1 v/v). Fractions

were monitored by tlc for the schweinhrthins, which gave a characteristic red-orange

color with vanillin-H,SO, spray reagent on heating. Combined fractions containing

schweinhrthin B were pooled to yield 323 mg of crude schweinhrthins, which were

separated by C-18 RP HPLC (Rainin Dynamax 41.4 x 250 mm column, linear

gradient from 60-100% MeOH over 30 min) to yield 1 (8.5 mg) and 2 (40 mg).

Schweinfurthins A and C were obtained in similar fashion by HPLC of parallel

fractions from the LH-20 column step.

Schweinfurthin D (l), NSC# 71 3342, yellowish glass; [ct]D+23" (c=0.425, MeOH),

UV(Me0H) A,, 331 nm (log E 4.31), IR (film) 3391 (br) 1715, 1589, 1494, 1430,

1362, 1232, 1150, 1088, 1028, 957,909, 851, 759 cm"; 'H NMR see Table 1; I3C-

NMR see Table 1 ; FABMS (glycerol, positive mode) 580, 439, 425, 283, 271, 185,

HRFABMS (glycerol, positive mode) M' 580.3391 (calcd for C,,H,,O, 580.3400).

Cytotoxicity Bioassay. Two cell lines from the NCI 60-cell screen (SF-295 and

A549) were chosen for a two-day cytotoxicity assay3 for comparison of

schweinhrthin B with schweinhrthin D.

ACKNOWLEDGEMENTS

We thank Lewis Pannell for mass spectra, Jennifer Michael and Tanya Johnson for

cytotoxicity assays, Anacletus Koufani and David Kenfack for botanical identification,

Melvin Ngwang for plant extraction, and Odile Thoison (CNRS) for the sample of

vedelianin.

REFERENCES

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404 J.A. BEUTLER et (11.

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Journal of Natural Products, 62, 633-635.

4. 0. Thoison, E. Hnawia, F. Gueritte-Voegelein, and T. Sevenet (1992)

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