Un nouvel inhibiteur de la croissance cellulaire, dénommé Rolliniastatine 2 a été isolé des graines de #Rollinia mucosa$ et sa structure déterminée. (Résumé d'auteur

Dufresne, C.

Leet, J.E.

Moretti, Christian

Pettit, G.R.

Polonsky, J.

Riesen, R.

Schaufelberger, D.

Schmidt, J.M.

Smith, C.R.

Horizon / Pleins textes

HETEROCYCLES, Vol. 28, No. 7, 1989 
ISOLATION AND STRUCTURE OF ROLLINIASTATIN 2 : A ‘NEW CELL 
GROWTH pTNHIBITORY ACETOGENIN FROM ROLLZNZA MUCOSA1 
George R. Pettit,’ Roland Riesen, John E. Leet, Judith Polonsky, Cecil R. Smith, 
Jean M. Schmidt, Claude Dufresne, Daniel Schaufelberger, and Christian Moretti 
Cancer Research Institute and Department of Chemistry, Arizona State University, 
Tempe, Arizona 85287-1604, U.S.A. 
Abstract - A new cell growth inhibitory acetogenin, designated rolliniastatin 2, has 
been isolated from seeds of the South American Rollinia mucosa (Annonaceae) and 
its structure determined. A diastereomeric relationship for rolliniastatin 1, 
rolliniastatin 2, and asimicin was proposed. 
The Rollinia genus (Annonaceae) is composed of some 65 species occurring principally from Central 
America to Argentina. 
Rollinia, and Uvaria genera have been found to contain members of a new class of biologically active bis- 
tetrahydrofurans. Our crystal smcture determination of rolliniastatin 12 provided the first definitive smcture in 
this new series.3-10 By x-ray crystallography, the relative stereochemistry of rolliniastatin 1 was established as 
4S*, 15S*, 16S*, 19R*, 20R*, 23S*, 24R*, and 36R*.2 Asimicin, recently isolated from Asimina triloba,8 
apparently differs from rolliniastatin 1 in its configuration at one or more of the chiral centers associated with the 
bis-tetrahydrofuran system. 
Further detailed fractionation of a hexane extract of Rolliniu mucosa seeds’ has led us to uncover companions 
of rolliniastatin 1, including asimicin and a new diastereomer of rolliniastatin 1, named rolliniastatin 2. 
Isolation of rolliniastatin 2 flash 
chromatography on silica gel, high-speed countercurrent distribution (J!ISCCD)11 with a multilayer coil planet 
centrifuge,lz column chromatography (LiChrom Sia) ,  and finally hplc (semipreparative with Partisil 10 silica gel 
followed by analytical with Partisil 5 silica gel) with the solvent system hexane - ethyl ether - methanol. With a 
hplc flow rate of 1.5 &min, emergence times (min) were: rolliniastatin 1.6.17; asimicin, 6.57; rolliniastatin 2, 
6.70. 
High resolution sp-shs  analysis of rolliniastatin 2 provided molecular formula C37H6607 (& 623.4885; 
calcd for C37Ha07 + H, 623.4866). The uv, ir, and e h s  spectra of rolliniastatin 2 were similar to. those 
reported for rolliniastatin 1 ,* asimicin,8 and 14-hydroxy-25-desoxyrollinicin.9 By thin-layer 
chromatographic comparison in dichloromethane - methanol (19:1), rolliniastatin 2 was hardly distinguishable 
The few species so far examined (chemically) from each of the Annona, Asimina, . 
was effected by consecutive applications of solvent partitioning, 
* Author to whom correspondence should be addressed. ORSTOM Fonds Documentaire 
from asimicin. However, the 400 h4Hz 1H- and 13C- nmr spectra of rolliniastatin 2, especially shifts for carbons 
19,20,21, and 22, were readily distinguishable from those of rolliniastatin 1 and asimicin. Distinct differences in 
the 6 1.5-2.1 region associated with methylene protons attached at C-17, C-18, C-21, and C-22 were noted. A 
diastereomeric relationship for the three acetogenins was deduced by comparing their 13C-nmr spectra. Chemical 
shifts for carbons 1-6 and 35-37 were found to be virtually identical. Accordingly, the y - lactone unit, carbons 3- 
6 in the side chain, and attachment of a hydroxyl group at C-4 were assumed to be the same in all three 
compounds. Confimation was obtained by nmr studies that included IH,IH-COSY, IH,1H-relayed-COSY13, 
and IH,13C-COSY experiments. Thus the same pattem of proton-proton and proton-carbon connections (Table 
1) was established for rolliniastatin 2 as for rolliniastatin 1.2 Although significant differences in shifts were 
observed for the bis-tetrahydrofuran systems, the three isomers apparently share the same regiochemical structure. 
OH 
ÔH 
Rollhiastatin 1,2, and asimicin. 
Recently, Annona 
reticulata. When we compared 
the 13C chemical shifts14 of this compound with those of rolliniastatin 2, it became apparent that they may be 
identical. The average difference between pairs of observed shifts was only 0.05 ppm, and in only one case was 
the difference as much as 0.12 ppm. As noted previously with the structural determination of rolliniastatin 1? 
eims fragmentation interpretations did not detect a hydroxyl group at C-4. While no specimen of 14-hydroxy-25- 
desoxyrollinicin was available for direct comparison, it now appears that the structure of this substance needs to 
be reassigned as a diastereomer of rolliniastatin 1. 
Rolliniastatin 2 was evaluated against the P-388 lymphocytic leukemia cell line and showed an impressive 
EDSO of 2.3 x 10-4 pg/ml.lS Further biological evaluation of rollhiastatins 1 and 2 as well as isolation and 
characterization of related cell growth inhibitory substances are in progress. 
Etse and Waterman9 reported the isolation of 14-hydroxy-25-desoxyrollinicin from 
The structure was assigned primarily on the basis of eims fragmentation. 
- 214 - 
HETEROCYCLES, Vol. 28, No. 7, 7989 
Table 1. Rolliniastatin 2 connectivities by high field (400 MHz) nmr in deuteriochloroform 
solution (6 in ppm downfield from TMS). 
Chemical shifts connected Carbon or proton connection 
A. Selected proton-carbon connections by H,C-COSY: 
6 7.17 + 151.8 H-35 + (2-35 
6 5.05 + 77.9 H-36 + C-36 
6 3.86 + 69.9 H-4 + C-4 
6 2.52, 2.39 + 33.2 H-3a,H-3b + C-3 
6 1.41 + 19.1 H-37 + (2-37 
B. Selected proton-proton connections by H,H- and H,H-relayed COSY 
67.17 + 5.05 H-35 + H-36 
6 7.17 3 2.52, 2.39 H-35 + H-3a, H-3b 
6 7.17 -+ 1.41 H-35 + H-37 
6 5.05 + 1.41 H-36 + H-37 
6 3.86 + 2.52, 2.39 H-4 -+ H-3a, H-3b 
6 2.52 + 2.39 H-3a + H-3b 
EXPERIMENTAL 
General Experimental Procedures. .All solvents employed for chromatography were redistilled. Thin layer 
chromatography (tlc) was performed on silica gel GHLF Uniplates (0.25 mm layer thickness) supplied by 
Analtech Inc., using dichloromethane - methanol (19:l) as mobile phase. Tlc results were interpreted under uv 
light and/or developed by anisaldehyde - acetic acid - sulfuric acid spray (heating at approximately 150 'C for 5 - 
10 min). A Gilson Model FC-SO fraction collector was used with column chromatography. Melting points 
(uncorrected) were determined with a Kofler-type hot stage apparatus. Uv spectra were recorded using a Hewlett- 
Packard S450A uv/vis spectrophotometer. The *H- and 13C- nmr spectra (tetramethylsilane as reference standard 
in deuteriochloroform solution) were obtained with a Briiker AM-400 spectrophotometer. The mass spectral 
measurements were performed with an MS-50 instrument at the NSF Regional Facility, University of Nebraska, 
Lincoln, NB. 
Rollinia milcosa . 
('herbier' no. CM927) is deposited with the Centre Orstom de Cayenne, Cayenne, French Guiana. 
Seeds of the Rollinia mucosa tree were collected in French Guiana. A voucher specimen 
- 215 - 
Extraction and Partitioning. Whole seeds (766 g) of Rollinia mucosa were ground to a coarse powder and 
extracted (vigorous stirring at ambient temperature for 18 hr) with hexane in portions using the ratio 150 g of 
seeds to 600-800 ml of hexane. The extraction was repeated twice. Solvent was removed in vacuo to yield a 
clear, light yellow oil (187 g). A portion of this oily hexane extract (1 1.6 g) was partitioned between hexane and 
methanol-water (9: 1) to provide 9.85 g and 2.05 g respectively of hexane and aqueous methanol fractions. 
Isolation of rolliniastatin 2. A 3.89 g aliquot of the methanol-water fraction was chromatographed 
(rapidly) on a column of silica gel. Elution with &chloromethane-methanol (97:3) led to seven fractions. The 
fourth and largest (1.59 g) was combined with a 1.52 g fraction similar in composition but prepared in a slightly 
different manner. The combined product was rechromatographed in the same manner with a solvent ratio of 99:l 
to yield five fractions. The f i s t  two (0.964 g) were combined with fractions (1.095 g) of similar compositions 
processed by a more complex procedure. The pooled material (1.824 g) was further separated by HSCCD1*?12 in 
a series of experiments where 0.7 to 0.8 g of fraction at a time was separated using the solvent system heptane - 
ethyl acetate - ethanol - water (10:3:4:2). The coil (365 ml capacity) was operated at SOO rotations /min with a 
flow rate of 3 ml / min. Fractions found to contain primarily rolliniastatin 2 were combined (0.26 g) and further 
enriched by Lobar-type column chromatography (silica gel; size B). Elution with hexane - ethyl ether - methanol 
(6 :2  1) at a flow rate of 6.0 ml / min afforded pure rolliniastatin 2 in fraction fifteen. The adjoining fractions from 
the Lobar column were subjected to semipreparative hplc on Partisil 10 (500 x 10.0 mm column) using hexane - 
ethyl ether - methanol (6:2:1) at 3 ml / min. Analytical hplc was performed with a Partisil 5 column (250 x 4.6 
mm) and the solvent system hexane - ethyl ether - methanol (7:2:1) at a flow rate of 1.5 ml / min. By analytical 
hplc, relative emergence times (min) were found to be: rolliniastatin 1, 6.17; asimicin, 6.57; and rolliniastatin 2, 
6.70. The total yield of pure rolliniastatin 2 was 23 mg. Recrystallization from chloroform gave a waxy solid 
melting at 73 - 76 O C ;  Rf 0.22 (dichloromethane - methanol 19:l); [aID*7 + 5.3" (c 0.23, CHCL); uv I,,, 
(CH30H) (log E )  210 (3.85); ir v,, (CHC13) 3450, 2925, 2855, 1755, 1720 (sh), 1615, 1465, 1320 cm-1; 
hreims, d z :  (C37H6607 molecular ion not observed), 604.4661 (0.2; M+-H20; calcd for C37H6406 M-H20: 
604.4703), 451.3109 (0.4), 433.2956 (6), 415.2860 (I l ) ,  381.2633 (3), 363.2560 (43), 345.2463 (19), 
312.2296 (19), 311.2254 (loo), 293.2252 (19); 1H nmr (400 MHz): 6 0.87 (3H, t, J = 6.8 Hz, H-34). 1.25 (32 
H, m, H-6 - 13, and 26 - 33), 1.42 (3H, d, J = 6.8 Hz, H-37), 1.46 (2H, m, H-5), 1.50 (2H, m, H-14), 1.7-2.0 
(8H, m, H-17, 18, 21, and 22), 2.39 (lH, dddd, J = 15.1, 3.5, 1.6, and 1.5 Hz, H-3b), 2.52 (lH, dddd, 15.1, 
3.5, 1.6, and 1.5 Hz, H-3a), 3.38 (lH, m, H-15), 5.05 (lH, ddd, J = 6.8, 1.5, and 1.5 Hz, H-36), 7.16 (lH, 
ddd, J = 1.5, 1.5, and 1.2 Hz, H-35); and 13C nmr (100 MHz): 174.4 (C-1), 151.7 (C-35), 131.1 (C-2), 83.1 
and 82.7 (C-16 and C-23), 82.4 and 82.2 (C-19 and C-20), 77.9 (C-36), 74.0 and 71.2 (C-15 and C-24), 69.9 
(C-4), 37.3 (C-5), 33.4 (C-25), 33.2 (C-3 and C-14), 31.8 (C-32), 29.6 to 28.3 (C-7 to C-12, C-21, C-22,'and 
C-27 to C-31), 28.8 and 28.3 (C-17 and C-IS), 25.9 (C-6 and C-13), 25.5 (C-26), 22.6 (C-33), 19.0 (C-37), 
. 
and 14.0 (C-34). 
- 216 - 
HETEROCYCLES, Vol. 28, No. 7 ,  7 989 
ACKNOWLEDGMENTS 
For support of this research our thanks and appreciation to: Grant CA-30311-01A3 to 03 awarded by the National 
Cancer Institute, DHHS, the Arizona Disease Control Research Commission, Eleanor W. Libby, the Waddell 
Foundation (Donald Ware), the Fannie E. Rippel Foundation, Herbert K. and Dianne Cummings (The Nathan 
Cummings Foundation), Polly J. Trautman, Lotte Flugel, Mary Dell Pritzlaff, the O h  Foundation (Spencer T. 
and Ann W.), the Robert B. Dalton Endowment Fund, and the Swiss National Science Foundation (to D. S.). 
We also thank Messrs P. J. Daschner and L. Williams for technical assistance. 
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Received, 9th February, 1988 
- 217 - 


Isolation and structure of rolliniastatin 2 : a new cell growth inhibitory acetogenin from Rollinia mucosa

Isolation and structure of rolliniastatin 2 : a new cell growth inhibitory acetogenin from Rollinia mucosa

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