586
J . Org. Chem., Vol. 43, No. 4 , 1978
Kupchan et al.
New Cytotoxic Neolignans from Aniba megaphylla Mez.1*2 S. Morris I K ~ p c h a n Kenneth ,~ L. Stevens,*4Eric A. Rohlfing, Barry R. Sickles, Albert T. Sneden,* Richard W. Miller, and Robert F. Bryan* Department of Chemistry, Uniuersity of Virginia, Charlottesuille, Virginia 22901 Received June 30,1977
The isolation and elucidation of the structure and stereochemistry of megaphone ( l ) ,a new cytotoxic neolignan from Aniba rnegaphylla Mez., are reported. Chemical and spectral evidence supported structure 1 for megaphone, and a direct x-ray crystallographic analysis confirmed the structure and established the stereochemistry. Two additional new cytotoxic neolignans, megaphone acetate (2) and megaphyllone acetate (3), were isolated and their structures deduced in light of the structure of 1. The lack of activity of these neolignans as inhibitors of mitosis in sea urchin eggs is discussed in terms of structural features. An alcoholic extract of Aniba megaphylla Mez. (Lauraceae)5 was found t o demonstrate inhibitory activity in vitro against cells derived from human carcinoma of t h e nasopharynx (KB)? Systematic fractionation of the active ethanol extract (Chart I) guided by K B activity led t o the isolation of three new cytotoxic neolignans, megaphone (l),megaphone acetate (2), and megaphyllone acetate (3).
Chart I. Fractionation of the Cytotoxic Extract from A n i b a megaphylla Mez. d r i e d ground r o o t s o f A . meqaphylla ( 1 kg)
I I
hot 95% e t h a n o l e x t r a c t i o n
e t h a n o l i c e x t r a c t A ( 8 1 . 3 4) p a r t i t i o n between w a t e r and chloroform
I water soluble fraction B (12.2 9)
c h l o r o f o r r r so 1 u bl e fraction C (50.5 g)
I
We 1,
2_.
P = '-
3
R= C G C k 3
1
p a r t i t i o n between H,OIMethanol and S k e l l y s o l v e B
(1:9)
I
Chromatography of fraction H over silica gel gave a fraction which crystallized from chloroform-ether t o afford megaphone (1).On the basis of high-resolution mass spectrometry and confirmation by elemental analysis, megaphone was assigned the molecular formula C22H3006. The NMR spectrum exhibited signals a t 6 3.88 (6 H ) , 3.83 ( 3 H ) , and 3.46 ( 3 H), representing three aromatic and one aliphatic methoxyl groups and suggesting that megaphone was either a lignan or neolignan. The NMR spectrum also showed a singlet a t 6 6.66 (2 H ) and a doublet a t 6 4.64 (1 H ) which, when considered with the hydroxyl stretching band a t 3600 cm-l in the infrared (IR) spectrum, indicated a benzyl alcohol moiety with the aromatic ring symmetrically substituted. In order to confirm this partial structure, megaphone was oxidized using Jones reagent t o give d.ione 4., C22H2806. The two-proton singlet was
yype Mb
Me@ \
OMe 4
shifted downfield in the NMR spectrum of 4, and, in t h e IR spectrum, the hydroxyl band disappeared and a new carbonyl band appeared a t 1590 cm-l, thus confirming the presence of an aryl ketone. The IR spectrum of 1 showed the presence of an a,P-unsaturated carbonyl moiety (1670 cm-'), and the NMR spectrum exhibited a doublet a t 6 7.00 (1H, J = 10.5 Hz) and a doublet of doublets centered a t 6 6.02 (1H , J = 10.5,2.2 Hz) which could be assigned t o the a and p protons, respectively, in a cis-a,P-unsaturated carbonyl system. Oxidation of 1 t o afford 4 gave little change in either the IR or the NMR signals assigned t o this moiety, indicating t h a t the a,P-unsaturated carbonyl portion of the molecule was unaffected. Further inspection o f the NMR spectrum of 1 revealed the presence of an allyl group [6 5.83 (1H , m ) , 5.30 (1H, br), 5.19
Skellysolve B soluble fraction E ( 5 . 7 g)
Methanol s o l u b l e fraction D
I
1
p a r t i t i o n between HiO/Methanol ( ? : E ) and c a r b o n t e t r a c h l o r i d e
I
I
Methanol s o l u b l e fraction F
carbon t e t r a c 9 l o r l d e soluble fraction G
A
p a r t i t i o n between h 2 G l Methanol ( 4 6 ) and c h l o r o f o r m
Flethanol s o l u b l e 'raction I
chloroform soluble +raction P
(1H, br)] and a secondary methyl group [6 0.77 (3 H, d, J = 7.1 Hz)]. In the NMR spectrum of 4, the doublet for the secondary methyl group shifted to 6 1.18, indicating t h a t it was in close proximity to the benzylic alcohol. Additionally, in the NMR spectrum of 4, the signal for the proton on the carbon a t o the secondary methyl group appeared as a quartet a t 6 4.04 (1H, J = 7.5 Hz), indicating that this carbon was attached t o two quaternary carbons. The data thus accounted for all but one site of unsaturation, suggesting t h a t there was an additional ring which included the a$-unsaturated ketone. T h e 13C NMR spectrum of megaphone confirmed the presence of all the above moieties, and the presence of six quaternary carbons was indicatedfour in the aromatic moiety, one in the carbonyl group, and one in the cyclohexenone ring. Consequently, the cyclohexenone ring must also contain one methylene carbon. In addition, signals were observed for two carbons with general structure A. One signal was ascribed t o the benzylic carbon
A
0022-326317811943-0586$01.00/0 0 1978 American Chemical Society
3.Org. Chern., VoE.43, No. 4 , 1978 587
New Cytotoxic Neolignans
Table I. Atomic Parameters for the Nonhydrogen Atomsa Atom
x la
7706 (8) 8802 (9) 10168 (8) 10105 (9) 8684 (10) 7338 (9) 11672 (10) 12248 (12) 7345 (11) 5897 (8) 5845 (8) 6608 (10) 4182 (10) 5298 (12) 5062 (17) 4154 (9) 3527 (10) 1879 (11) 867 (10) 1306 (9) 3025 (9) -179 (14) 11579 (6) 11450 (7) 8770 (7) 5772 (6) 4429 (7) 358 (7)
Figure 1. Stereoscopic view (ORTEP) of the molecular conformation. and the other to the carbon in the cyclohexenone ring attached t o the methoxyl group. Oxidized megaphone (4)showed a one-proton doublet of doublets a t 6 2.90 ( J -= 10.5, 12.5 Hz) which could be assigned to one of the methylene protons in t h e cyclohexenone ring. Irradiation of this signal partially collapsed the multiplet a t 6 4.30 (MeO-CH), and irradiation of t h e multiplet at 6 4.30 partially collapsed thie doublet of doublets, thus confirming t h a t the methylene group was in close proximity t o the MeO-CH moiety. Hydrogenation of megaphone (1)in EtOH gave an oil which analyzed for C22H3.40,: (by high-resolution mass spectrometry) and showed hydroxyl bands in the IR spectrum [3600 (s) and 3425 cm-1 (br)] but no carbonyl group. Attempts t o oxidize the product with Jones reagent were unsuccessful, suggesting t h a t the secondary alcohol was no longer present. Therefore, a hemiketal structure, 5, was proposed for the product of the hydrogenation reaction. The NMR data were also consistent with such a structure.
M e 0 , f l o M
MeO’
\ O M 5
Careful reexamination of the NMR spectrum of megaphone (1) revealed “spurious” signals, e.g., 6 0.60 (d, J = 7.5 Hz), 3.37 (s), and 6.48 (9). These signals could be accounted for by t h e presence of an equilibrium mixture of megaphone and its hemiketal, 6, in solution. The relative intensity of the signals ascribed t o 1 and 6 were found t o vary with both solvent and on L
-
1
OMe
6 temperature. In CDC13 a t 23 “ C the ratio of 1 t o 6 was found t o be 100:12, whereas in pyridine-d5 a t 23 “C the ratio was
vlb 1283 (7) 819 (8) 1067 (7) 1798 (7) 2225 (7) 1993 (8) -229 (10) 2991 (9) 3269 (9) 1123 (7) 1956 (7) 1472 (9) 3272 (8) 4225 (9) 5225 (11) 2398 (7) 3028 (7) 3144 (9) 2563 (10) 1797 (8) 1425 (7) 389 (9) 664 (6) 2060 (6) 2930 (6) 0000 ( - ) b 3475 (6) 815 (6)
2I C
5130 (8) 4937 (8) 5887 (8) 6915 (8) 7130 (8) 6191 (8) 4940 (10) 7447 (11) 8473 (9) 4084 (8) 2904 (8) 1828 (9) 1200 (9) 1576 (10) 1693 (13) 2366 (8) 3425 (8) 3297 (10) 2373 (10) 1429 (9) 1830 (8) 54 (12) 5804 (6) 7873 (6) 8184 (6) 3566 (6) 4384 (5) 1359 (6)
B 3.4 3.8 3.5 3.7 3.9 3.7 5.6 6.6 5.2 3.4 3.5 4.4 4.6 6.0 9.9 3.8 3.6 5.7 5.4 4.5 3.8 6.8 5.0 5.0 4.8 4.2 4.7 5.3
a Positional parameters for the hydrogen atoms (see supplementary material) are given as fractions of the unit-cell edges ( x104)with esd’s, in parentheses, on the same scale. Equivalent anisotropic thermal parameters (see supplementary material) a r ~ given in A2. Held fixed to define the origin.
*
85:lOO. The temperature dependence was shown by the ratio in pyridine-d5 a t 46 and a t 83 “C which was 100:95 and 100:54, respectively. In both megaphone hemiketal (6) and tetrahydromegaphone hemiketal(5), the methyl group occurs a t high field in the NMR spectrum, Le., 6 0.60 ( J = 7.5 Hz) and 0.62 ( J = 7.6 Hz), respectively, thus indicating a cis relationship t o the aromatic system, as found in similar neolignans from other Aniba species such as porosin.7~~ The coupling constant of the benzylic proton (6 5.25, J = 9.7 Hz) in 5 indicated a gauche arrangement t o the adjacent proton. Examination of the double doublet a t 6 2.90 (one proton of the ring methylene group) showed coupling constants of 12.7 and 10.5 Hz, one of which was the geminal coupling constant. The other, because of its magnitude, must be the result of a 1,2-trans diaxial interaction; hence, the aliphatic methoxyl group was in a n equatorial position. From these data the structure of megaphone could be assigned as 1. T o confirm this structure, and to establish t h e molecular conformation and absolute stereochemistry, a direct x-ray analysis of 1 was carried out. A stereoscopic viewg of the molecular structure found is shown in Figure 1, and atomic coordinates are given in Table I. Bond distances and angles and selected torsion angles are given in Figure 2, and additional torsion angles of interest are listed in Table 11. The molecular structure proposed is confirmed as correct. In the crystal the molecule adopts an extended stepped conformation with the bond C(a)-C(P) lying in a plane near normal t o t h e plane of t h e phenyl ring, t h e torsion angle C(l)-C(a)-C(p)-C(l’) being -143”. The more favorable, fully staggered, 180” alignment is prevented by the limiting intramolecular contact O(a)-H(G’a) of 2.54 A which prevents further rotation about C(a)-C(p). T h e hydroxy group O ( a )
588
J . Org. Chem., Vol. 43, No. 4, 1978
Kupchan e t al.
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