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(20) Fischer, R. A., Maurer, R. (1978) Aust. J. agric. Res. 29, 897.
(21) Clarckson, N.M., Rusell, J. S. (1976) Aust. J. agric. Res. 27,227.
(22) Yaniv, Z., M. Weissenberg, Palevitch, D. (1983) Acta Hort. 132,
217.
Abstract: A method for the isolation and preparation of 12-deoxyphorbol (1) from Euphorbium resin (Euphorbia resinifera Berg) was established to provide substantial amounts of 1-esters for bioassays. Acylation of 1 yielded 12-deoxyphorbol-13,20-diesters (2, esters with acids
CH3(CH2)COOH, n = 0,4,8, 12, 16,20; and with benzoic, oleic, elaidic and linoleic acid). Upon mild transesterification the 13,20-diesters 2
yielded the 13-monoesters 3. Both ester types were tested for their irritant activities, the 13-monoesters were also assayed for their tumor-
Introduction
experiment the 12-deoxyphorbol-13-tetradecanoate (3) appeared to exhibit a promoting activity (3) about as strong as that of
the well known standard promoter 12-O-tetradecanoylphorbol-13-acetate [Croton oil factor A1, TPA (5)]. To study structure activity relationships of 12-deoxyphorbol esters (6) in a systematic manner, a method to provide substantial amounts of
the diterpene parent 12-deoxyphorbol (1) from the easily ac-
18.
-0R2
12
13
(1,2)]. As far as tested, these esters showed more or less pronounced irritant activity on the mouse ear (24). Moreover,
some of the 13-monoesters tested also showed weak tumorpromoting activity on the back skin of mice (3,4). In a singular
O 6/
1: R1=R2=I-I
2: R'=R2=acyl
3: R1=H,R2=acyl
CH2OR1
20
(5) Foldesi, D., Lang, T., Kovacs, T. (1969) Herba Hung. 8,49.
(6) Fryer, R. F. (1972) Proc. Agron. Soc. N. Z. 2,73.
(7) Bernath, J., Foldesi, D. (1972) Herba Hung. 11,55.
66
sured with a Dupont CEC-21-110 B and 60 MHZ 'H-NMR-spectra
were measured with a Jeol HL C-60 spectrometer. The NMR-spectra
were recorded with tetramethylsilane (6 = 0.00 ppm) as internal stan-
C). Yields, R1 values and mass spectral data of all 13,20-diesters 2 prepared are compiled in Table I. For UV, IR and 'H-NMR data compare
the 13 ,20-diacetate (7) as a representative.
(a) 12-Deoxyphorbol-13-acetate. To a solution of 12-deoxyphorbol13,20-diacetate (2) (470 mg) in methanol (300 ml), 70% HCIO4 (0.4
ml) was added. The mixture was allowed to stand under nitrogen for 24
h. Sodium acetate (600 mg) was added, methanol was removed in vacuum and the residue was extracted with ethyl acetate. After drying and
evaporation of the solvent the crude product was purified by TLC (1/15
CH3OHJCH2CI2); yield: 210mg 13-acetate 3. MS: m/e 390 (Mt). 'HNMR (d5-pyridine): 1-H: 7.74 (m); 7.H: 6.1 (d, J78 = 5 Hz); 20-H2:
4.32 (s); 10-H: 3.7 (m); 8-H: 3.6 (m); 5-H2: 3.05 (s); 19-H3: 1.7 (m);
acetyl: 2.04; OH (exchangeable with D2O): 7.82 and 4.88 ppm (2 s). IR
(KBr): 3440 (OH), 1700 (CO), 1620 cm' (C = C). UV (methanol):
?.,,, (a) = 234 (5240), 333 nm (70).
(b) Monoesters 3 other than 13-acetate. To a solution of the 13,20diester 2(1.5 mmole in 10 ml of methanol), sodium methoxide in methanol (0.75 mmole, used as 10_2 M solution) was added. The reaction
mixture was kept at 5 C and tested by TLC (1/10 CH3OWCH2CI2)
every 30 mm in order to determine the time of optimal partial transesterification. The reaction was then quenched with acetic acid and the solvent was removed under reduced pressure. The residue was extracted
with ethyl acetate, washed with KHCO3 solution (5%) and finally with
water, The 13-monoesters 3 obtained were purified by TLC (3/I ether/
petroleum ether). Yields, Rf values and mass spectral data of all 13-monoesters 3 prepared are compiled in Table!. For UV, IR and NMR data compare the 13-monoacetate (above) as representative.
Biological assays:
Irritant activity on the mouse ear and tumor promoting activity on the
back skin of mice were determined according to the standard procedures (5, 8). Experimental details for the latter activity may be taken from
Table II.
h, the reaction was quenched with acetic acid and the solvent was
removed under reduced pressure. The residue was chromatographed
on a polyamide (100 g) column using successively 1/3 water/methanol
(elution of crude 12-deoxyphorbol, 1) and 1/1 water/methanol (elution
of residual 12-deoxyphorbol-13-angelate, that was recycled).
12-Deoxyphorbol-13,20-diacetate (2)
To a solution of crude 1(2 g) in pyridine (6 ml), acetic acid anhydnde (3
ml) was added and the mixture was left for 24 h. After work-up, the
product was purified by TLC (1/4 ethyl acetate/benzene); yield: 170
mg. MS: rn/c = 432 (M). UV, IR and NMR data are fully identical
with those published earlier (7).
(KBr): 3400 (OH); 1690 (CO); 1620 cm' (C = C). UV: (methanol)
Xlna (a) = 233 (4600), 332 nm (60).
Results
12-Deoxyphorbol (1) may be prepared in quantities by isolation from Euphorbia resinifera resin as its 13,20-diacetate (2).
The hydrophobic triterpenes present in the methanol extract of
the resin were largely removed by partitioning between watermethanol and petroleum ether. Removal of the strong hydrophilic substances were affected by filtration on silica gel using
ethyl acetate as mobile phase. Subsequent chromatography on
silica gel lead to the isolation of a 12-deoxyphorbol ester fraction which amounts to 1.3 % by weight of the resin. Alkaline
(0.1 M sodium methoxide/methanol) transesterification of this
fraction provided 12-deoxyphorbol (1, for structural formulas
see Fig. 1) and 12-deoxyphorbol-13-angelate which evidently
products were separated by column chromatography on polyamide. The angelate was recycled whereas the crude 1 was
converted to its 13,20-diacetate 2, that is more stable than land
hence more suitable for storage.
Acylation of 1 with acid chlorideslpyridine afforded 13,20diesters 2 (Table I). By partial transesterification of the diesters
2 the 13-monoesters 3 were obtained (Table I). The mass spectra of the 13,20-diesters 2 and of the 13-monoesters 3 show the
expected molecular ions (Table I) and a similar fragmentation
pattern. The common ions at m/e = 330, 312 and 294 are characteristic for the 12-deoxyphorbol (1) moiety. The NMR spectra of the esters exhibit signals typical of this class of compounds (7).
from Merck. For analytical and preparative TLC, Merck silica gel
HF4 and PF4 were used, respectively. For column filtration or chro-
67
Table I. Yields, R, values and molecular ions of the 13,20-diesters 2 and 13-monoesters 3 prepared from 12-deoxyphorbol (1)
13,20-diesters 2
Acid moieties in
2 or3
Yield
Acetic
Hexanoic
Decanoic
Tetradecanoic
Octadecanoic
Docosanoic
Oleic
Elaidic
Linoleic
Benzoic
(%)
80
0.12
32
50
45
30
28
37
35
35
45
0.41
0.53
13-monoesters 3
Molecular ion
(m/e)
Yield
(%)
432
544
656
768
880
992
876
876
872
556
60
45
42
40
42
27
47
45
38
30
0.03
0.14
0.20
0.25
0.30
0.34
0.54
0.64
0.60
0.63
0.28
0.64
0.62
0.37
0.28
0.25
R'
Molecular ion
(m/e)
0.08
0.17
0.32
0.45
0.21
0.51
0.23
0.24
0.28
0.25
0.25
0.24
0.13
0.52
0.53
0.60
0.57
0.57
0.56
0.38
390
446
502
558
614
670
612
612
610
452
1/1
Table II. Tumor-promoting activities of the 12-deoxyphorbol-13-esters (3) prepared in the standardized assay (5, 8) on the back skin of mice.
Positive control: 12-O-tetradecanoylphorbol- 13-acetate (TPA)
ester(3)
Application
Single
dose p
(nmole)
Acetate
Hexanoate
Decanoate
Tetradecanoate
Octadecanoate
Docosanoate
Oleate
Elaidinoate
Linoleate
Benzoate
TPA
Duration
At 12 weeks
(weeks)
200
40
40
80
100
80
80
80
80
160
45
48
48
42
39
26
23
23
24
48
48
Tumor
ratea
Tumor
0/28
0/28
8/28
0/28
yieldt'
2 or3
Acetic
Hexanoic
Decanoic
Tetradecanoic
Octadecanoic
Docosanoic
Oleic
Elaidic
Linoleic
Benzoic
TPA
13,20-diesters2
(nmole/ear)
0/28
0/24
0/28
0/24
100
86
12/27
17/24
16/27
29/27
96
65/24
51/26
0/24
67/24
77/26
72/27
86
10/27
10/27
25/27
21/27
4/28
0/27
8/28
0/27
22/26
16/27
0/25
8/28
14/28
11/26
0/24
17/24
Table HI. Irritant doses 50 (ID50, according to the standard procedure (5, 8)) of the 13,20-diesters 2 and 13-monoesters 3 of 12-deoxyphorbol (1) prepared as compared to 12-O-tetradecanoylphorbol-13acetate (TPA) (5)
Acid moieties in
Tumor
8/28
0/27
ID 13-monoesters 3
Tumor
rate
0/28
9/29
28/27
15/28
0/27
11/27
Histological diagnosisc
Promoting activity
At 20 weeks
yield
rate(%) miceinvest.
0/25
96
86
86
93
96
89
25/26
93
3/3
37/17
85/22
79/22
1 PEC
2PEC, 1 ADBAM
1 PEC
70/16
59/16
81/19
1 PEC
0
0
(nmole/ear)
2.3
0.19
0.26
0.050
0.14
2.5
0.087
0.078
0.062
4.4
1.8
1.0
5.4
0.26
5.1
4.0
0.4
23
57
0.016
Discussion
Table III shows that the 13,20-diesters 2 generally have lower
irritant activities than the corresponding 13-monoesters 3, except in case of the acetates which are comparably active. The
present set of esters clearly demonstrates that for maximal irritant activity the primary hydroxyl 20 must not be acylated (at
least not with an acyl residue identical with that in position 13).
A very similar relationship was discovered previously when
comparing phorbol-12,13-diesters to the corresponding phorbol-12,13,20-triesters (5, 6, 810). Within the 13-monoesters 3
it can be seen that the length of the acyl chain strongly affects
the irritant activity as is the case with phorbol-12,13-diesters
[bearing identical acyl chains (11)] or with ingenol-3-esters
12-Deoxyphorbol-13-
68
Fig. 2 Tumor rates and tumor yields as obtained in the standardized assay for tumor
TUMOR H
YIELD
promoting activity on the back skin of NMRImice (5,8); initiation: once a single dose of i =
100 nmole DMBA; promotion: twice weekly
a single dose p of 12-deoxyphorbol-13-ester
(3), or 12-O-tetradecanoylphorbol-13-acetate
30
1
50
WEEKS
(TPA). 12-deoxyphorbol-13-decanoate: p =
40 nmole; 12-deoxyphorbol-13-tetradecanoate: p = 80 nmole; 12-deoxyphorbol-13-octadecanoate: p = 100 nmole; TPA: p = 5 nmole.
\i
* 13-o4W.
A 13-.SIdMt
Ia-probe.
(%J
40 WEENS
Studies on 0-Methylfiavinantine
Acknowledgements
References
(1) Hecker, E. (1977) Pure Appl. Chem. 49, 1423.
(2) Evans, F. J., Soper, C. J. (1978) J. Nat. Prod. 41, 193.
(3) Gschwendt, M., Hecker, E. (1974) Z. Krebsforsch. 81, 193.
(4) Hergenhahn, M., Kusumoto, S., Hecker, E. (1974) Expenentia
30, 1438.
(5) Hecker, E., Schmidt, R. (1974) Progr. Chem. Org. Natur. Prod.
31, 377.
(6) Hecker, E. (1978) in Mechanisms of Tumor Promotion and Cocarcinogenesis, (Slaga, T. J., Sivak, A., Boutwell, R. K., eds.),
Vol. 2, p. 11, Raven Press, New York.
(7) Gschwendt, M., Hecker, E. (1969) Tetrahedron Lett. 3509.
Studies on 0-Methylfiavinantine
1. Analgesic Effect of 0-Methylfiavinantine in Mice
R. Ansa-Asamoah"3, G. A. Starmer2
Introduction
0-methylfiavinantine (OMF) (Fig. 1) is the major alkaloid of
the root bark of Rhigiocarya racemifera (Menispermaceae)
(33). The alkaloid possesses the morphinandienone structure
and its synthesis has been reported (19, 31). Morphinandienone-containing plants have widespread medicinal and recreational uses. The juice from the leaves of Rhigiocarya racemifera is
H3CO
2
OCH3
OMETHVLFI.AVINANTINE
Fig. 1.
NCH3
HO
OH
MORPHINE
69