On the active principles of the euphorbiaceae, IX. Ingenane type diterpene esters from five euphorbia species

Full text of DOI:10.1515/znb-1984-0525

On the Active Principles of the Euphorbiaceae, IXa
Ingenane Type Diterpene Esters from Five Euphorbia Species
H. Gottab, W. Adolf, H. J. Opferkuch, and E. Hecker*
Institut für Biochemie, Deutsches Krehsforschungszentrum, D-6900 Heidelberg, FRG
Z. Naturforsch. 39b, 683-694 (1984); received December 5. 1983
Diterpene Esters, Ingenol Derivatives, Skin Irritation, Euphorbiaceae
Investigations of E. antiquorum, E. helioscopia, E. palustris, E. peplus and E. quadrialata for
irritant and tumor promoting constituents afforded several new ingenane type diterpene esters
derived from the parent alcohols ingenol and 20-deoxyingenol and from the hitherto unknown 20-
deoxy-16-hydroxyingenol and 20-deoxy-13.16-dihydroxyingenol. The irritant activities of the
natural compounds are reported together with some aspects on structure activity relationships.
Introduction
We now report on the isolation of new ingenane
type diterpene esters from the three European
species E. helioscopia, E. palustris and E. peplus.
Some of their esters or parent alcohols are identical
with those isolated for comparison from the African
succulent E. quadrialata and from the Indian succu-
lent E. antiquorum. The active principle of a com-
mon Thai drug derived from E. antiquorum was de-
scribed elsewhere [14].
The plant family Euphorbiaceae consists of about
290 genera. Among them, the largest genus Euphor-
bia comprises about 1600 species [1], occurring as
succulent or non-succulent plants in most parts of the
world. Plant parts of E. species are well known to be
irritant. They are often used in folk medicine against
all kinds of diseases [e.g. 2—4]. Many E. species
growing wide-spread as weeds are held responsible
for poisoning of live-stock [5].
During the last 15 years, in investigations of the
chemical nature of the toxic principles of Euphor-
biaceae for obvious reasons preferentially the latices
of many of the large succulent and perennial Euphor-
Results
From E. peplus and E. quadrialata extracts of the
latices, from the other plants extracts of the aerial
parts were investigated. The constituents of all ex-
bia species indigenous to Africa were used. They tracts were isolated in separation procedures using
were shown to contain highly irritant and frequently
tumor promoting diterpene esters of the tigliane, in-
genane and daphnane type (for reviews see [6—8]).
Only few investigations were carried out with her-
baceous E. species indigenous to Europe occurring
mostly as weeds. From European species, as active
constituents so far esters of ingenol and of 16-hy-
droxyingenol have been isolated from E. lathyris [9]
and E. esula [10] and esters of 13-hydroxyingenol
and 13,19-dihydroxyingenol from E. cyparissias [11].
Moreover, esters of 20-deoxyingenol and 4-deoxy-
phorbol were obtained from E. biglandulosa [12] and
esters of 12-deoxyphorbol from E. helioscopia [13].
multiplicative distributions and chromatographic
methods monitored by the standard assay for irritant
activity on the mouse ear [15]; for further experimen-
tal details see also [16]. All factors (ID50<50 nmoles/
ear) and compounds (practically non-irritant,
ID50>50 nmoles/ear) are characterized with the first
or the first two letters of the plant species from which
they were isolated (letters for factors in bold-faced
type). Their structures and irritant activities are sum-
marized in the Table.
1. Derivatives o f ingenol (1, Fig. 1)
From the latex of E. antiquorum and from a Thai
purgative drug derived from it, Euphorbia factor A n t
was isolated and characterized as 3-O-angeloylin-
genol (2). It forms readily the 5.20-0-isopropylidene
derivative 3 upon treatment with acetone/p-toluene
sulfonic acid hydrate [14]. Preparation of the
acetonide from a crude irritant fraction obtained
from stems of E. antiquorum, containing i.a.
a VIIIth Communication, see: E. H. Seip, H. H. Ott, and
E. Hecker, Planta Med. 49, 199 (1983).
b Part of dissertation, see 1. c. [16].
Reprint requests to Prof. Dr. E. Hecker.
0340-5087/84/0500-0683/$ 01.00/0
Unauthenticated
Download Date | 10/29/18 8:13 AM

684
H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
Table. Structure and irritant
Parent alcohol Acid moiety
Factor/
compound
ID5oa
ID24 a
activities (ID 5qand ID^,) of fac-
tors or compounds isolated
from three European and two
(nmoles/ear) (nmoles/ear)
ingenol
ingenol
3-angelate
0.04
0.12
0.06
(= Anx) (2)
H,
Indian/African
Euphorbia
3-decatrienoate,
3-dodecatetraenoate
3-decadienoate
3-decadienoate
H3 (8, 9)
0.22
h 2,
species; standards: TPA (12-0-
tetradecanoylphorbol-13-ace-
tate), ID 5q: 0 .0 10 nmoles/ear,
ID^: 0.016 nmoles/ear [29] and
3-TI (3-O-tetradecanoylinge-
nol), ID2^: 0.011 nmoles/ear
ingenol
ingenol
He (10a)
(10b)
Pet (12)
0.03
0.08
n. d.
0.17
10
9.0
4.1
20
> 100
0.06
0.08
0.09
ingenol
20-acetate-3-angelate
Pe2
20-deoxyingenol 3-angelate
20-deoxyingenol 5-angelate
20-deoxyingenol 5-decatrienoate
20-deoxyingenol 5-benzoate
20-deoxyingenol 5-hexanoate
20-deoxyingenol 3.5-dibenzoate
(= H8) (17)
0.48
> 100
[18]-
(18)
(20)
(19)
(23)
(24)
(25)
h 4
h 7
P2
P7
P«
> 100
> 100
>100
> 100
> 100
20-deoxyingenol 5-benzoate-3-hexanoate > 100
P9
(30)
(33)
(32)
20-deoxy-16-
hydroxyingenol
20-deoxy-16-
hydroxyingenol
20-deoxy-16-
16-acetate-3-angelate
n. d.
2.5
> 100
12.0
5.0
> 100
Q.
P6
Pi
3,16-dibenzoate
5,16-dibenzoate
hydroxyingenol
(40)
(41)
(39)
20-deoxy-13,16- 3,16-dibenzoate-
dihydroxyingen. 13-phenylacetate
20-deoxy-13,16- 13-acetate-3,16-
dihydroxyingen. dibenzoate
20-deoxy-13,16- 3,13,16-tribenzoate
dihydroxyingen.
>
>
50
50
>
50
P3
P4
P5
>
50
18
n.d. = not determined.
a Standard deviation o: 1.3;
level of significance a —0.05.
n. d.
Fig. 1. Structure of ingenol (1)
and its derivatives 2— 12 .
chlorophyll, proved useful for isolation and charac-
terisation of A n( as 3, which could easily be sepa-
rated by TLC.
A more refined separation procedure with E. he-
lioscopia (for details see [16]) afforded several fac-
tors (Hj—H3, H5, H6), characterized as 3-acylates of
ingenol (1) with unsaturated acid moieties. Accord-
ing to spectral data and chromatographic properties
factor Hj is identical with Anlf Again, Hj furnished
the isopropylidene derivative 3, which upon transes-
terification with sodium methanolate afforded 5.20-
O-isopropylideneingenol 4 [17]. Transesterification
of the mixture H2, H3 not separable by TLC (silica
gel) with 0.1 M NaOCH3/MeOH yielded ingenol
(characterized as its 3.4:5.20-di-0-isopropylidene
derivative 5 [17]) and unsaturated methyl esters
showing UV absorbance at 346 (tetraenoate) and
307 nm (trienoate). After catalytic hydrogenation
they were identified by GLC analysis as methyl de-
canoate and dodecanoate. H2, H3 afforded the mix-
ture of acetonides 6, 7 with a sharp singlet at
5.60 ppm (3-H) in the NMR spectrum. Thus H2 rep-
resents 3-0-(2.4.6-decatrienoyl)ingenol (8) and H33-
0-(2.4.6.8-dodecatetraenovl)ingenol (9). Factors H5
1:
2:
3:
4:
5:
6 :
R '-R 4 =
H
R 1 = angeloyl, R;- R 4 = H (Anj = H,)
R 1 = angeloyl, R2 = H. R \ R4 = isopropylidene
R 1 = R2 = H. R3, R4 = isopropylidene
R 1, R2 = R3, R4 = isopropylidene
R 1 - C O (C H =C H )3-(C H 2)2-C H 3. R2 = H,
R3, R4 = isopropylidene
R 1 = C O (C H =C H )4-(C H 2)2-C H 3, R2 = H,
R3, R4 = isopropylidene
R 1 = CO(CH = CH )3-(C H ,),-C H 3, R2- R 4 =
(H2)
R 1 = C O (C H =C H )4-(C H ,> -C H 3, R2- R 4 =
7:
8:
9:
H
H
(H3)
Z/E
10a,b: R 1 = C O (C H = C H ),-(C H ,)4-C H 3,
R2- R 4 = H (H5 and H„)
Z/E
1 1a,b :R 1 = CO(CH = CH)2-(C H 2)4-C H 3. R2 - H,
R3, R4 = isopropylidene
12:
R 1 = angeloyl. R2 = R3 = H. R4 = COCH3 (Pe,)
Unauthenticated
Download Date | 10/29/18 8:13 AM

685
H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
and H6 seem to be geometric isomers of 3-0-(2.4-
decadienoyl)ingenol (10a,b). Differences in the
chemical shifts in the NMR spectra of the acetonides
11a and lib show up only in the region of
d >5.6 ppm. After transesterification of 11a and lib
and catalytic hydrogenation of the resulting methyl
esters in both cases decanoic acid methyl ester was
identified as the major compound by GLC.
Factor Pej from E. peplus (M* at m/e 472) shows
similar UV data as A n^ In the NMR spectrum an
additional singlet of an acetyl group appears at
2.09 ppm and the signal for 20-H2 at 4.65 ppm. It
was shown by selective acetylation [18] of An! that
factor Pej represents 20-0-acetyl-3-0-angeloylin-
genol (12)*. A compound with the same structure,
tentatively assigned, was isolated from Euphorbia
kamerunica [21].
2. Derivatives o f 20-deoxyingenol (13, Fig. 2)
Three further ingenane type angelates with a
molecular weight of 414 are Euphorbia factors H4,
H8 and Pe2. Pe2 and H8show the same spectroscopic
and chromatographic properties and are therefore
identical. Their NMR spectra differ from that of Anx
mainly by the absence of the signal of 20-H2, whereas
an additional broad singlet of a methyl group appears
at 1.80 ppm superimposed with that of 19-H3. De-
coupling experiments establish this signal to indicate
a 2O-H3 group. This assignment also explains the
diamagnetic shift of protons 5-H and 7-H of about
0.3 ppm. Transesterification of Pe2 afforded the
crystalline parent compound 20-deoxyingenol (13)
[22], which was characterized as the 3,5-diacetate
and the 3.4-O-isopropylidene derivative 15.
The difference between H8 and H4 is revealed by
the NMR spectra. The sharp singlet at 5.45 ppm is
consistent with the presence of a 3-acylate for H8. In
the spectrum of H4 this signal is replaced by a broad
singlet at 5.25 ppm similar to 5-acylates in the in-
genol series [18, 20]. An additional support for these
assignments was given by treating both factors with
acetone/p-toluenesulfonic acid hydrate. Only H4 af-
forded an acetonide 16. Therefore H8 and Pe2 repre-
sent the 3-angelate 17 and H4 the 5-angelate 18 of 20-
deoxyingenol (13).
Fig. 2. Structures of 20-deoxyingenol (13) and its deriva-
tives 14—25.
Two further Euphorbia factors were identified as
5-acylates of 13. P2was characterized as the benzoate
19, H7 as the 2.4.6-decatrienoate 20. In both cases
position of the acid moieties were ensured by prepar-
ing the acetonides 21 and 22, respectively. Transes-
terification of H7 yielded 20-deoxyingenol (13) and a
corresponding unsaturated methyl ester showing UV
absorbance at 302 nm. After catalytic hydrogenation
it exhibited an identical retention time as methyl de-
canoate. Euphorbia factor P7 must be a 5-hexanoate
23 according to the mass spectrum (M~ at m/e = 430)
and the NMR spectrum (5-H at 5.20 ppm). As indi-
cated by NMR the acid residue is probably branched.
13: R 1—R3 -
14: R 1 — R3 = COCH3, R2 = H
H
15: R 1, R2 = isopropylidene, R3 = H
16: R 1, R2 = isopropylidene, R3 = angeloyl
17: R 1 = angeloyl. R2 = R3 = H (H„ = Pe2)
18: R 1 = R2 = H, R3 = angeloyl (H,)
19: R 1 - R2 = H, R3 = benzoyl (P2)
20: R 1= R2 = H, R3 = C O (C H =C H )3-(C H 2)2-C H 3(H7)
21: R 1, R2 = isopropylidene, R3 = benzoyl
22: R 1, R2 — isopropylidene,
R3 = C O (C H =C H )3-(C H ,)2-C H 3
23: R 1, R2 = H, R3 = COC5H„ (P7)
24: R 1 = R3 = benzoyl, R2 = H (P8)
25: R 1 = COC5H „, R2 = H, R3 = benzoyl (P9)
* A recent report [19] describes the isolation of a com -
pound from Euphorbia canariensis which was assigned
the structure of the isomeric 3-0-acetyl-20-0-angeloyl-
ingenol. The spectral data are identical with those of Pex.
Positioning of the acid moieties [19] was deduced from a
selective hydrolysis reaction affording 20-0-angeloylin-
genol. Under such reaction conditions (0.1 M KOH),
however, migration of the acyl residues in ingenol esters
from 0 -3 to 0-20 are well known [18, 20]. Hence, the
20-angelate could have been generated from a factor
identical with Pexby base catalyzed rearrangement of the
primary product of hydrolysis, i. e. 3-O-angeloylingenol.
Also two 3.5-diesters of 13, compounds P8 and P9,
were isolated from E. palustris. P8 shows spectral
data identical with those of the 3.5-dibenzoate 24
prepared from 13 by treatment with benzoyl chloride/
pyridine. P9 is a hexanoate-benzoate with a probably
branched aliphatic chain like in P7. In the NMR spec-
trum of P9 a diamagnetic shift for the signals of 1-H
(0.16 ppm) and 3-H (0.32 ppm) compared to those
in the spectrum of P8 is observed. This may indicate
Unauthenticated
Download Date | 10/29/18 8:13 AM

H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
686
that 3-OH in P9 is esterified with the aliphatic acid,
i. e. compound P9 represents structure 25.
pene parent alcohol of esters occuring in E. margina-
ta [24],
The positions of the different acid residues in Qi
were investigated by mild base catalyzed transesterifi-
cation. Treatment with 5 x 10-'1M NaOCH3/MeOH
afforded the 3-angelate 27. In a smaller amount, the
5-angelate 28 was obtained probably formed by acyl
migration as known for ingenol-3-acylates [18]. In
about the same amount also the parent alcohol 26
was isolated and characterized as the 3.5.16-triace-
tate 29. Hence, spectral data and chemical reactions
prove structure 30 for Euphorbia factor Qj.
Fig. 3. Structure of 20-deoxy-16-hydroxyingenol (26) and
its derivatives 27—36 and of 20-deoxy-13.16-dihydroxy-
ingenol (37) and its derivatives 38—41.
R5 17
26 was also shown to be the parent alcohol of two
dibenzoates, factor P6 and compound Pj. An AB-
system at 4.46 ppm in the NMR spectrum of P6and a
broad singlet at 4.55 ppm in that of Pj indicate ben-
zoylation of the primary hydroxyl group in both
cases. The other ester positions are distinguishable
like in the series of 20-deoxyingenol. Whereas P6
shows a sharp singlet at 5.73 ppm, indicating 3-H and
superimposed with the multiplet of 7-H. P] has a
comparable but broader singlet at 5.46 ppm, which
must be due to the proton 5-H. Formation of the
acetonide 31 from P] is an additional evidende that Pt
represents the 5,16-dibenzoate 32 since only this sub-
stitution type (out of six possible diesters) should
afford an acetonide according to the Dreiding-Model.
Factor P6 therefore is the isomeric 3,16-dibenzoate
33.
26: R ‘- R 5 -
27: R 1 = angeloyl, R2—Rs = H
H
28: R 1 - R2 = R4 = R5 = H. R3 = angeloyl
29: R 1 = R3 = R4 = COCH3, R2 = R5 = H
30: R 1 = angeloyl. R2 = R3 = R5 = H. R4 = COCh3 (Q,)
31: R 1. R2 = isopropylidene. R3 = R4 = benzoyl. R5 = H
32: R 1 = R2 = R5 = H, R3 = R4 = benzoyl. (Pjj
33: R 1 = R4 = benzoyl. R2 = R3 = R5 = H (P6)
34: R ’- R 3 = R5 = H, R4 = benzoyl
35: R 1. R2 = isopropylidene. R3 = R5 = H. R4 = benzoyl
36: R 1, R2 = isopropylidene. R3—R3 =
37: R '-R 4 = H. R5 = OH
38: R 1 = R4 = benzoyl. R2 = R3 = H,
R5 = benzoyloxy (Ps)
39: R 1 = R4 = benzoyl. R2 = R3 = H.
R5 = OCOCH2C6H, (P3)
40: R 1= R4 = benzoyl. R2 = R3 = H, R5 = OCOCH, (P4)
41: R 1 = R2 = H. R3 = R4 = benzoyl. R5 = OCOCH3
H
The dibenzoates P] (32) and P6 (33) gave the
16-benzoate 34 after short treatment (1 h) with
0.1 M NaOCH3/MeOH. 34 afforded the isopropyl-
idene derivative 35, which upon transesterification
furnished 36. Complete transesterification of Pj or P6
occurred within 24 h to yield the parent alcohol 26
3. Derivatives o f 20-deoxy-16-hvdroxyingenol
(26, Fig. 3)
Euphorbia factor Qx, an angelate-acetate was iso-
lated from E. quadrialata. The chemical shifts of the
signals in the NMR spectrum, however, are similar which was characterized as its 3.5.16-triacetate 29.
to those in the spectrum of H8. Decoupling experi-
ments give proof for a 20-H3 group; furthermore an
AB-system appears at 4.16 ppm, which is due to a
hydroxymethyl group in the ingenane skeleton. The
4. Derivatives o f 20-deoxy-l3.16-dihydroxyingenol
(37, Fig. 3)
appearance of only one singlet of a cyclopropylic
methyl group and the provable doublet of 18-H3 at
0.95 ppm suggest this position to be at C-16 or C-17.
Three other constituents of E. palustris, com-
pounds P3 and P4 and factor P5, exhibit strikingly
similar NMR spectra like factor P6 concerning the
Like in derivatives of 16-hydroxyingenol [23] an evi- characteristic signals between 3 and 7 ppm. Com-
dent shifting of the signal of 14-H to lower field can
be shown by decoupling experiments. Whereas in H8
this signal appears at 0.85 ppm it is now provable at
1.08 ppm. So it may be deduced that the oxygenation
has occurred at C-16. The parent compound 20-
deoxy-16-hydroxyingenol (26) was shown to be diter-
bined with the mass spectra, however, they give evi-
dence for triesters of a parent compound, which dif-
fers from 26 by an additional oxygen atom. P5(M~ at
mte — 676) shows signals of 15 aromatic protons in
the NMR which indicate a tribenzoate. Because no
new signal in the spectrum can be observed the addi-
Unauthenticated
Download Date | 10/29/18 8:13 AM

H. Gotta et al. • On the Active Principles of the Euphorbiaceae
687
tional oxygen atom should be attached to a tertiary
ready 5 hours after application (see Table), whereas
C-atom in the ingenane skeleton. The position is re- 3-O-tetradecanoylingenol (3-TI), as
a standard,
vealed by decoupling experiments. By irradiation at
4.3 ppm (8-H) the doublet at 1.61 ppm (J = 12 Hz)
is changed to a sharp singlet. This low field shift for
shows its maximum 24 h after application [18].
Among all factors isolated, 3-esters of ingenol exhibit
the highest irritant activity (see Table), though all of
14-H and the signals of the apparently more de- them are clearly less active than 3-TI after 24 h. Un-
shielded protons 11-H and 12-H2 indicate that the
oxygenation must have occurred at C-13. From these
saturation of the acid moieties, either in long chain
carboxylic acids (C10in factors H2, H5 and and C12
data and comparison with factor P6, P5 can be re- in factor H3) or in short chain acids (angelic acid in
garded as the 3.13.16-tribenzoate 38 of the new 20-
deoxy-13.16-dihydroxyingenol (37). The spectral
data of P3 and P4 prove them to be a dibenzoate-
phenylacetate 39 and a dibenzoate-acetate 40, re-
spectively, of the same parent alcohol. Probably the
acid moieties differing from those in P5 are located at
C-13, because the chemical shifts of 3-H and 16-H2
have not changed.
Chromatographic investigations suggest an insta-
bility of these compounds. After storage of P4for six
months in ethyl acetate the corresponding 5.13.16-
triester 41 was isolated in a yield of about 30%. Ob-
viously similar acyl migrations occur as in the series
of ingenol-3-acylates [18].
factor H xor A nx) reduces the exposure time to reach
the maximum of the irritant activity (/. e. minimum of
ID50) at 5 h. When Hj is esterified in 20-position with
acetic acid (as in Pe}) the activity measured 24 h af-
ter application on the mouse ear is comparable (ID5q
value was not determined).
3-Esters of 20-deoxyingenol still exhibit consider-
able irritant activity. Compared to 3-O-angeloylin-
genol, 3-0-angeloyl-20-deoxyingenol (Pe2 or H8) is
about four times less active. With phorbol-12.13-
diesters it was found that presence of a 20-hydroxyl
group is a prerequisite for irritant activity [27]. 5-
Esters of 20-deoxyingenol (H4, H7 and P2, P7) are
only weak irritants 5 h after application (ID5o be-
tween 4 and 20 nmoles/ear) and practically inactive
24 hours after application on the mouse ear
(IÜ 5o> 100 nmoles/ear). The 3,5-diesters of the same
parent alcohol, P8 and P9, are inactive.
Discussion
Esters of ingenol have been isolated from many
species of Euphorbiaceae in recent years (for review
see [6—8]). From the three European Euphorbia
species investigated here further esters of ingenol (1),
20-deoxyingenol (13), 20-deoxy-16-hydroxyingenol
(26) and 20-deoxy-13.16-dihydroxyingenol (37) were
Whereas 3.16-diesters of 16-hydroxyingenol are
highly active as irritants [23], 3.16-diesters of the new
parent alcohol 20-deoxy-16-hydroxyingenol (P6 and
0 0 only exhibit weak irritant activity, and the 5.16-
diester Pi of the same parent is practically non-ir-
obtained (for overview see Table). The same or simi- ritant.
lar esters of ingenol and 20-deoxy-16-hydroxyingenol
The 3.13.16-tribenzoate of the new parent alcohol
were shown to be present in E. antiquorum indigen- 20-deoxy-13,16-dihydroxyingenol (P5) is a weak irri-
ous to India and in the African species E. quadriala-
ta, respectively.
tant, too, and the aromatic/aliphatic 3.13.16-tries-
ters, P3and P4, are non-irritant. On the other hand it
is known that 3.13.19-triesters of 13.19-dihydroxy-
ingenol are highly irritant [11].
3-Angelates of ingenol and of 20-deoxyingenol
were previously also isolated from latex of E. para-
lias [28]. They were tested for irritant activity, but
the ID50values obtained are not comparable with the
In our investigation of E. helioscopia the presence
of 12-deoxyphorbol esters as reported by Evans et al.
[13] was not confirmed. The reason for this may be
the difference in origin of the plant specimens inves-
tigated: it is known, that e.g. latex from E. tirucalli
collected in Africa compared with latex collected in
Madagascar shows quantitatively different pattern of present data, perhaps due to use of different strains
diterpene esters of the tigliane and ingenane type of mice. Similarly as with factors Hj and H8, they
[25, 26]). Perhaps the biosynthetic routes producing exhibited 2 h after application on the mouse ear
these irritant diterpene esters depend on soil and/or
climate where these plants are grown.
Nearly all ingenane type factors isolated exhibit
higher irritant activity than at 24 h. However, 3-0-
angeloyl-20-deoxyingenol showed a ca. 20 fold high-
er activity than 3-O-angeloylingenol, which is differ-
their highest irritant activity on the mouse ear al- ing considerably from our data.
Unauthenticated
Download Date | 10/29/18 8:13 AM

H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
Experimental
exhaustively with methanol to yield 16.7 g of a
methanol extract. Partitioning between water and
ethyl acetate afforded 2.75 g of an ethyl acetate ex-
tract (ID50: 1.5 jug/ear). 1 g of this extract was sepa-
rated by prep. TLC in ether/petroleum ether = 4/1
and 30 mg of a crude irritant fraction were obtained,
still not homogeneous according to TLC. This frac-
tion was treated with acetone and catalytic amounts
of p-toluene sulfonic acid hydrate for 30 min. After
purification of the reaction product by prep. TLC in
ether/petroleum ether = 1/1 3.0 mg of an isopropyl-
idene derivative were obtained, Rf (ether/petroleum
ether = 1/1): 0.4; all spectral data are identical with
those described for compound 3 (see below).
E. helioscopia. 12 kg of aerial parts of E. helio-
scopia yielded 923 g of a methanol extract. Water and
ethyl acetate were added and the aqueous layer was
extracted with ethyl acetate for several times. The
ethyl acetate extract obtained (157 g, ID 5029 ^g/ear)
was distributed successively according to O’Keeffe in
the solvent systems A and B. The remaining irritant
active fraction (27.4 g, ID 506.4 //g/ear) was subjected
to a Craig distribution in system A with n = 4000
transfers. Then irritant principles were indicated in
the fractions of G = 0.06—0.18. From these frac-
tions by means of prep. TLC the Euphorbia factors
Hi, H4—H8and the mixture of Euphorbia factors H2,
H3 were isolated.
E. palustris. In an equivalent procedure irritant
principles were concentrated from the methanol ex-
tract (883 g) from 11.3 kg of E. palustris. The ethyl
acetate extract weighed 154 g. Aftertwo O'Keeffe dis-
tributions the active fraction (16.0 g. ID505.7 ,wg/ear)
was subjected to a Craig distribution with n = 3000
transfers. Irritant principles were found within the
fractions showing G = 0.05—0.11. From these frac-
tions the Euphorbia factors P2 and P5—P7 were iso-
lated besides the compounds Pj, P3, P4. Further simi-
lar compounds, P8 and P9 were isolated from more
hydrophobic non-irritant fractions which had been
separated after the second O’Keeffe distribution.
IR spectra were measured with a Perkin-Elmer
spectrometer model 521, the UV spectra (in
methanol) with a Beckman spectralphotometer Acta
M VI, the CD spectra with a dichrograph of Jobun
Yvon, Paris. Mass spectra were obtained with a Var-
ian spectrometer MAT 711. NMR spectra were run
in CDCI3 (unless otherwise stated) with instruments
from Bruker Physik AG, models HX-90 and MH-90.
Tetramethylsilane was used as an internal standard
(d = 0.00 ppm). OH-protons were detected by ex-
change with D20 .
For analytical and preparative TLC silica gel MN
P/UV254from Macherey & Nagel, Düren, was used as
well as silica gel plates 60 F254from E. Merck, Darm-
stadt. Columns were prepared with silica gel
0.063 —0.2 mm from Woelm, Eschwege. For GLC-
analyses a Becker-Packard chromatograph model
420 was employed with a column of 10% GESE on
chromosorb W/HP 80/100. Sports on TLC were vis-
ualized with vanillin-sulfuric acid.
Solvent systems used for multiplicative distribu-
tions: system A = petroleum ether/methanol/water
15/10/0.5; system B = tetrachloromethane/methanol/
water 4/2/0.16. Machinery and methods are described
elsewhere [15],
The separation procedures were accompanied by a
standardized test for irritant activity on the mouse
ear [15].
Plant materials. 1 kg of plant stems of E. anti-
quorum were purchased from F. G. Celo, Medicinal
Plants Inc., Zweibrücken. Latex of E. peplus was
collected in July and August in vineyards near
Heidelberg. The stalks were cut below the umbel and
the latex collected with glass capillary tubes. These
were crushed under acetone and the material was
extracted several times with acetone. Aerial parts of
E. helioscopia and E. palustris were purchased from
Heinrich Bornträger, Drugs and Spices, Offstein
(Germany). After collection all plant materials were
cut and preserved and stored under methanol. The
methanolic liquid of plant preparations was com-
bined with the methanol extracts (see below) of cor-
II. L a tic e s
responding plant parts.
A latex preparation of E. antiquorum (see [14])
E. quadrialata in methanol was provided by the late
Dr. P. R. O. Bally, Nairobi, Kenya.
E. peplus. 380 mg of an acetone extract of latex
(ID50 0.85 ^g/ear) was distributed in system A with
n = 35 transfers according to Craig. From the hy-
drophilic fraction (199 mg, ID50 0.38 ^g/ear) the
Euphorbia factors Pe! and Pe2 could be isolated by
means of prep. TLC.
E. quadrialata. Extraction of the latex preparation
(100 g) with acetone yielded 20 g of acetone soluble
material (IU: 6.6 wg/ear). By a Craig distribution us-
ing system A the acetone extract was separated into
Separation procedures
I. A e ria l P la n t p a rts
E. antiquorum. 1 kg of green stems of E. anti-
quorum, preserved under methanol were ho-
mogenized with an „Ultra Turrax“ and extracted
Unauthenticated
Download Date | 10/29/18 8:13 AM

689
H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
the non-irritant hydrophobic fraction (IU: >2000 jugl 71.81 (s); 66.87 (t); 43.48 (d); 38.41 (d); 31.00 (t);
ear) and the irritant hydrophilic fraction (2.5 g; ID50: 28.53 (q); 24.11 (s); 23.27 (d); 22.94 (d); 20.80 (q);
17.09 (q); 15.92 (q); 15.60 ppm (2 q).
2.2 jug/ear). From this fraction factor Ch (23 mg) was
isolated using prep. TLC.
a) P re p a ra tio n of th e a c e to n id e
3
of
fa c to r Hx: /^(petroleum ether/ether 1/1): 0.4. MS:
m/e = 470 (M+). UV: A(c) = 194 nm (19000); Amax
(c) = 209 (16800), 298 nm (400). XH NMR: 1-H:
6.05 (m); 7-H: 5.75 (m); 3-H: 5.63 (s); 20-H,: 4.18
(s); 8-H: 4.2—4.0 (superimposed); 5-H: 4.0 (s, br.);
19-H3: 1.8 (d, J = 1.5 Hz); 16-H3, 17-H3: 1.09 (s)
and 1.04 (s); 18-H3: 0.97 (d, 7 = 8 Hz); OH:
3.18 ppm; angelate: 1 olefinic H: 6.1—6.0 (m); 2
olefinic CH3: 2.1 —1.9 ppm; isopropylidene: 1.46 (s)
and 1.42 ppm (s).
Chemical reactions
Transesterification. About 10 mg of a sample were
treated with 1 ml of 0.1 M NaOCH3/MeOH for
1—5 h at room temperature. After addition of phos-
phate buffer, pH 6.7, the solution was extracted with
ethyl acetate. The organic layers were dried with
M gS04, and after evaporation of the solvent the pro-
ducts were isolated by prep. TLC in suitable solvent
systems.
Preparation of isopropylidene derivatives. The
sample was dissolved in a small volume of acetone
and then treated with a catalytic amount of p-toluene
sulfonic acid hydrate for 1—24 h at room tempera-
ture. The reaction was stopped by adding phosphate
buffer pH 6.7, the products were extracted with
ethyl acetate and purified by prep. TLC.
Acetylations. About 10 mg of a sample was dis-
solved in 5 ml of pyridine/acetic anhydride 3/1 and
kept at room temperature for 12—24 h. After hydro-
lization with phosphate buffer pH 6.7, the products
were extracted with ethyl acetate. The organic layer
was washed with 1 M HC1 then with buffer and final-
ly with water. After drying (M gS04) and evaporation
of the solvent, purification was carried out by prep.
TLC.
Hydrogenations o f unsaturated fatty acid methyl es-
ters. The transesterification products (usually 2 mg)
were dissolved in about 5 ml of ethyl acetate. After
adding a catalytic amount of palladium-charcoal the
reaction mixture was stirred for 2—3 h under hydro-
gen. At the end of the reaction the catalyst was re-
moved by filtration.
b) T ra n s e s te rific a tio n of 3 affo rd in g in-
g en o l-5 ,2 0 -a c e to n id e (4): All spectral data of 4
are identical with those described in [17].
M ix tu re of E u p h o rb ia fa c to rs H2, H3 (8, 9)
Rf (dichloromethane/acetone 4/1): 0.3—0.4. MS:
m/e = 522 (M +, H3), 496 (MT, H2) 330, 312.
*H NMR: 9 H (including 1-H, 7-H, 3-H and olefinic
H): 8.0-5.5; 5-H, 8-H, 20-H2: 4.2-4.0 (super-
imposed); 19-H3: 1.80 (s, br.); ca. 10 H: 1.7—1.2;
16-H3, 17-H3: 1.09 (s) and 1.07 (s); OH: 3.73 (s); 3.55
(s); 2.6—2.3 ppm (superimposed).
a) T ra n s e s te rific a tio n of fa c to rs H2, H3 af-
fording
1 and u n s a tu ra te d m eth y l e ste rs:
Characterisation of 1 by preparation of the ingenol-
3,4:5,20-diacetonide (5), spectral data identical with
those described in [17]. Catalytic hydrogenation of
the unsaturated methyl esters yielded methyl
dodecanoate (rel. int. 30%), methyl decanoate
(100%), others (< 3% ) as identified by GLC analysis
with authentic references.
b) P re p a ra tio n of th e a c e to n id e s 6, 7 of
fa c to rs H2, H3: MS: m/e = 562 (M+), 536 (M+). IR:
3520 (OH); 1712 (CO); 1608, 1590 cm-1 (C=C).
UV: X = 194 nm; Amax = 312, 354 nm. *H NMR:
10 H (incl. 1-H, 7-H): 8.0-5.5; 3-H: 5.60 (s); 5-H,
8-H, 20-H2: 4.25-4.0; 19-H3: 1.79 (d, 7 = 1.5 Hz);
16-H3, 17-H3: 1.06 (s) and 1.04 (s); OH: 3.20 (s);
isopropylidene: 1.47 (s) and 1.42 ppm (s).
1. Derivatives o f ingenol (1)
E u p h o rb ia fa c to r Aiix (2, d a ta see 1. c. [14])
E u p h o rb ia fa c to r Hi (2)
Rf (petroleum ether/ethyl acetate 1/1): 0.28. MS:
m/e = 430 (M+), 330, 312. IR (CH2C12): 3500 (OH),
1718 (CO), 1642 cm“1 (C=C). UV: X (e) = 194 nm
(18500); Amax (e) = 211 (16700), 296 nm (370). JH
NMR: 1-H, 7-H: 6.05 (m); 3-H: 5.53 (s), 20-H2: 4.14
(s); 5-H: 4.05 (s, br); 8-H: 4.2—4.0 (superimposed);
19-H3: 1.80 (d, 7 = 1.5 Hz); 16-H3, 17-H3: 1.06 (s)
and 1.09 (s); 18-H3: 0.96 (d, J = 7 Hz); OH:
3.52 ppm; angelate: 1 olefinic H: 6.15 (m); 2 olefinic
E u p h o rb ia fa c to r H5 (10a)
(dichloromethane/acetone 4/1): 0.36. MS: m/e =
498 (M+), 330, 312.
a)
P re p a ra tio n of th e a c e to n id e 11a of fac-
to r H5: ^(ether/petroleum ether 1/1): 0.5. MS: m/e =
538 (M+). UV: X (e) = 194 nm (15040); Amax (e) =
266 (14130), 314 nm (sh, 1660). JH NMR: 1 olefinic
CH3: 2.1-1.9 ppm (m). 13C NMR: 207.45 (s); 168.65 H: 7.62 (dd, J = 15 and 10 Hz); 5 H (including 1-H,
(s); 139.86 (s); 139.27 (d); 136.15 (s); 132.12 (d);
127.84 (d); 127.64 (s); 85.07 (s); 82.47 (d); 76.04 (d);
7-H): 6.25-5.65; 3-H: 5.60 (s); 5-H, 8-H, 20-H2:
4.2—4.0; 19-H3: 1.78 (d, 7 = 1.5 Hz); ca. 7 H: 1.3
Unauthenticated
Download Date | 10/29/18 8:13 AM

690
H. Gotta et al. • On the Active Principles of the Euphorbiaceae
(m); I6-H3, 17-H3: 1.08 (s) and 1.04 (s); ca. 5 H:
1.00—0.65; OH: 3.18 (s); isopropylidene: 1.47 (s)
and 1.42 ppm (s).
OH); 1712 (CO); 1639 cm' 1 (C=C). UV: A (e) =
195 nm (14690); Amax (e) = 212 (14690), 280 nm (sh,
400). ‘H NMR: 1-H: 6.06 (d, J = 1.5 Hz); 7-H: 5.75
(m); 3-H: 5.45 (s); 8-H: 4.02 (m); 5-H: 3.69 (d, J =
7 Hz); 19-H3, 20-H3: 1.80 (s, br.); OH: 3.44 (s); 3.17
(d, J = 7 Hz); angelate: 1 olefinic H: 6.16 (m); 2
olefinic CH3: 2.02-1.93 ppm (m); 13C NMR: 207.06
(s); 168.71 (s); 139.47 (d); 137.58 (s); 136.57 (s);
132.71 (d); 127.51 (s); 124.07 (d); 85.07 (s); 83.19
(d); 77.08 (d); 71.88 (s); 43.41 (d); 38.73 (d); 31.07
(t); 28.53 (q); 24.05 (s); 23.33 (d); 23.14 (d); 21.97
(q); 20.73 (q); 17.04 (q); 15.86 (q); 15.53 (2 q) ppm.
a) T ra n s e s te rific a tio n of fa c to r Pe2 a ffo rd -
ing 2 0 -d eo x y in g eo l (13): M. p. 205—208 °C
(from ethyl acetate). R f (ether/petroleum ether 3/1):
0.26, staining: orange-brown. MS: m/e = 332 (M+).
UV: A (e) = 193 nm (14510); Amax (e) = 292 nm
(237). lU NMR: 1-H: 5.97 (m); 7-H: 5.73 (m); 3-H:
4.40 (s); 8-H: 3.99 (m); 5-H: 3.41 (d, br., J = 9 Hz);
19-H3: 1.86 (d, J = 1.5 Hz); 20-H3: 1.78 (s, br.); 16-
H3, 17-H3: 1.12 (s) and 1.07 (s); 18-H3: 0.96 (d, J =
7 Hz); OH: 4.07 (d); 2.56 (d, J = 9 Hz); 2.3 ppm
(superimposed). *H NMR (C5D5N): Chemical shifts
and multiplicity are identical with those published by
Uemura et. al. [22],
b)
T ra n s e s te rific a tio n of 11a a ffo rd in g in-
g e n o l-5 ,2 0 -aceto n id e (4) and u n s a tu ra te d
m eth y l e ste rs: Spectral data of 4 were identical
with those described in [17]. Catalytic hydrogenation
of the unsaturated methyl esters yielded methyl de-
canoate (rel. int. 100%) and others (17%, 19%,
25%, < 5% ) as identified by GLC analysis with au-
thentic references.
E u p h o rb ia fa c to r H6 (10b)
(dichloromethane/acetone 4/1): 0.41. MS: m/e =
498 (M+).
a) P re p a ra tio n of th e a c e to n id e lib of
fa c to r H6: Rf (ether/petroleum ether 1/1): 0.5. MS:
m/e = 480 (M+-acetone). UV: A = 269, 320 nm (sh).
‘H NMR: 1 olefinic H: 7.2-6.8 (m), 5 H (including
1-H and 7-H): 6.1—5.6 ppm. All other data as in the
spectrum of 10b.
b) T ra n s e s te rific a tio n o f l l b a ffo rd in g in-
g e n o l-5 ,2 0 -a c eto n id e (4) and u n sa tu ra te d
m eth y l e ste rs: 4 die cochromatograph with an au-
thentic sample, staining: brown. Catalytic hydroge-
nation of the unsaturated methyl esters yielded
methyl decanoate (rel. int. 100%) and others (13%,
44%, 13%, 14%, <10% ) as identified by GLC
analysis with authentic references.
b) P re p a ra tio n of th e a c e to n id e 15 of 20-
d eo x y in g e n o l (13): Rf (petroleum ether/ether 3/2):
0.35. MS: m/e = 372 (M+). IR (KBr): 3465 (OH);
1717 cm-1 (C = 0 ). UV: A (e) = 193 nm (12750); Amax
(e) = 292 nm (240). !H NMR: 1-H, 7-H: 5.80 (m,
superimposed); 3-H: 4.67 (s); 8-H: 4.00 (m); 5-H:
3.78 (d, J = 8 Hz); 19-H3, 20-H3: 1.85 (m, superim-
posed); 16-H3, 17-H3: 1.16 (s) and 1.04 (s); OH: 2.45
(d, J = 8 Hz); isopropylidene: 1.53 (s) and 1.51 ppm
(s). 13C NMR: 207.45 (s); 137.65 (s); 136.93 (s);
130.83 (d); 126.08 (d); 113.86 (s); 96.38 (s); 89.69
(d); 75.78 (d); 74.87 (s) 44.19 (d); 38.67 (d); 31.65
(t); 28.73, 27.88, 27.04. 23.92 and 23.46 (2 d and
4 q); 23.01 (s); 18.00 (q); 15.40 (q); 15.27 ppm (q).
E u p h o rb ia fa c to r Pe! (12)
Rf (petroleum ether/ether 1/3): 0.31. MS: m/e =
472 (M+), 312, 294. IR (CH2C12): 3550, 3550-3300.
(OH, br.); 1716 (CO); 1640 cm" 1 (C=C). UV
(CH2C12): A (e) = 195 nm (14360); Amax (e = 212
(13800); 295 (310); 330 nm (190). ‘H NMR: 1-H,
7-H, 1 olefinic H: 6.2—6.0 (superimposed); 3-H: 5.60
(s); 20-H2: 4.65±0.13 (/AB = 13 Hz); 8-H: 4.15 (m);
5-H: 3.93 (s, br.); 19-H3: 1.83 (s, br.); 16-H,, 17-H3:
1.09 (s) and 1.07 (s); 18-H3: 0.98 (d, J = 7 Hz); OH:
3.8 (br., 1 H); 3.58 ppm (s); acetate: 2.09; angelate:
2 olefinic CH3: 2.09—1.97 ppm (m).
c) A c e ty la tio n of 2 0 -d eo x y in g en o l (13) af-
fo rd in g th e 3 .5 -d ia c e ta te (14): Rf (ether/petro-
leum ether 1/1): 0.55. MS: m/e = 416 (M+). IR
P a rtia l sy n th esis of 12 from fa c to r Aitj:
(KBr): 3490 (OH); 1738, 1713 cm" 1(CO); UV: A(f)
a)
111 mg of Anj (2) were dissolved in 10 ml of di-
chloromethane/pyridine 4/1 and treated with 400 mg
of acetic anhydride for 1 h at room temperature.
Yield: 92 mg of 12 (76%). MS- and 'H NMR data
are identical with those described for Pej.
=
193 nm (14160); Amax (c)
=
290 nm (210);
JH NMR: 1-H: 6.11 (d, J = 1.5 Hz); 7-H: 5.89 (m);
5-H: 5.23 (s, br.); 3-H: 4.95 (s); 8-H: 4.2 (m); 19-H3:
1.78 (s. br.); 20-H3: 1.58 (s. br.); 16-H,, 17-H,: 1.11
(s) and 1.07 (s); 18-H3: 1.00 (d. J = 7.5 Hz); 14-H:
0.88 (superimposed); OH: 3.23 (s); 2 acetates: 2.28
(s); 2.16 ppm (s). CD (MeOH): A (J e) = 302
(+0.475), 270 (-0.704), 242 (+0.290), 224 nm
(-2.43).
2. Derivatives o f 20-deoxyingenol (13)
E u p h o rb ia fa c to r Pe2 (H8) (17)
Rf (ether/petroleum ether 1/1): 0.30. MS: m/e =
414 (M+), 314, 296. IR (CH2C12): 3560, 3510 (br.,
d) B e n z o y la tio n of 2 0 -d eo x y in g en o 1 (13)
a ffo rd in g th e 3 ,5 -d ib e n z o a te (24): Benzoyla-
Unauthenticated
Download Date | 10/29/18 8:13 AM

H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
691
tion was carried out like the acetylation with benzoyl
chloride instead of acetic anhydride. The spectral
esters (UV: X = 302 nm) yielded methyl decanoate
(rel. int. 100%) and others (< 5%) as identified by
data of 24 are identical with those of compound P8 GLC analysis with authentic reference.
(see below).
E u p h o rb ia fa c to r P2 (19)
E u p h o rb ia fa c to r H4 (18)
Rf (ether/petroleum ether 1/1): 0.31. MS: m/e =
436 (M*), 314, 296. IR (CH2C12): 3590, 3490 (OH);
1715 (CO); 1598 cm"1 (C=C). UV: X (e) = 197 nm
(34930); Amax (e) = 229 (14000); 267 (sh, 1070), 273
(1110), 280 (970), 300 nm (sh, 280). !H NMR: 1-H:
6.02 (d, J = 1.5 Hz); 7-H: 5.93 (m); 5-H: 5.44 (s,
br.); 8-H: 4.3 (m); 3-H: 3.86 (s); 19-H3: 1.84 (s, br.);
20-H3: 1.62 (s, br.); 16-H3, 17-H3: 1.18 (s) and 1.08
(s); 18-H3: 0.98 (d, J = 7 Hz); OH: 4.07 (s, br.);
2.8—2.2 (superimposed); benzoate: 8.3—7.3 ppm
(m).
Rf (ether/petroleum ether 1/1): 0.29. MS: m/e
=
414 (M+), 314, 296. IR (CH2C12): 3590, 3490 (OH);
1715 (CO); 1640 c m '1 (C=C). UV: X (e) = 197 nm
(15520); Amax (e) = 214 (13300); 294 (330), 328 nm
(sh, 200). 'H NMR: 1-H: 5.98 (m); 7-H: 5.89 (m);
5-H: 5.25 (s, br.); 8-H: 4.22 (m); 3-H: 3.79 (s);
19-H3: 1.84 (s, br.); 20-H3: 1.60 (s, br.); 16-H3,
17-H3: 1.15 (s) and 1.07 (s); 18-H3: 0.98 (d, J =
7 Hz); 3.66 (s, imp.); OH: 3.91 (s); ca. 2.5 (superim-
posed); angelate: 1 olefinic H: 6.16 (m); 2 olefinic
CH3: 2.04—1.97 ppm (m).
a) P re p a ra tio n of th e a c e to n id e 21 of fac-
to rP 2:^(petroleum ether/ether3/2): 0.52. MS: m/e =
476 (M+). IR (CH2C12): 1715 (CO); 1598, 1580 c m '1
(C=C). UV: X (5) = 198 nm (43140); Amax (e) = 230
(14760), 274 (1050), 281 (880), 302 nm (sh, 210).
!H NMR: 1-H: 5.91 (d, J = 1.5 Hz); 7-H: 5.88 (m);
5-H: 5.50 (s); 3-H: 4.38 (s); 8-H: 4.04 (m); 19-H3:
1.86 (d, J = 1.5 Hz); 20-H3: 1.74 (s, br.); 16-H3,
17-H3: 1.16 (s) and 1.04 (s); 18-H3: 1.08 (d, J =
7 Hz); benzoate: 8.2—7.3 (m); isopropylidene: 1.50
(s) and 1.38 ppm (s).
a) P re p a ra tio n of the a c e to n id e 16 of fac-
to r H4: Rf (petroleum ether/ether 3/2): 0.55. MS:
m/e = 454 (M+). IR (CH2C12): 1710 (CO) cm-1. UV:
X (e) = 194 nm (15820); Amax (c) = 215 (sh, 12120);
297 nm (410). ‘H NMR: 7-H: 5.85 (m); 1-H: 5.78
(m); 5-H: 5.34 (s); 3-H: 4.33 (s); 8-H: 4.1 (m); 19-H3:
1.84 (d, J = 1.5 Hz); 20-H3: 1.78 (s, br.); 16-H3,
17-H3: 1.16 (s) and 1.03 (s); angelate: 1 olefinic H:
6.03 (m); 2 olefinic CH3: 2.04—1.97 (m); isopropyl-
idene: 1.46 (s) and 1.40 ppm (s).
E u p h o rb ia fa c to r H7 (20)
E u p h o rb ia fa c to r P7 (23)
Rf (ether/petroleum ether 1/1): 0.33. MS: m/e = 480
(M+), 314, 296. IR (CH2C12): 3590, 3490 (OH); 1715
(C = 0 ); 1610 cm-1 (C=C). UV (MeOH): X (e) =
194 nm (14360); Amax (c) = 258 (sh, 5300); 270 (sh,
7680); 282 (sh, 11080); 309 nm (18170). lH NMR:
ca. 8 olefinic H: 8.05—5.3 (m, including 1-H, 7-H);
5-H: 5.21 (s, br.); 8-H: 4.22 (m); 3-H: 3.79 (d, J =
4 Hz); 3.67 (s, imp.); 19-H3: 1.85 (s, br.); 20-H3:
1.62 (s, br.); 16-H3, 17-H3: 1.16 (s) and 1.07 (s,
Rf (ether/petroleum ether 1/1): 0.26. MS: m/e =
430 (M+), 314, 296. IR (CH2C12): 3590, 3490 (OH);
1715 c m '1 (CO). UV: X (e) = 193 nm (15660); Amax
(£) = 291 nm (280). !H NMR: 1-H 5.92 (d, J =
1.5 Hz); 7-H: 5.88 (m, superimposed with 1-H); 5-H:
5.20 (s, br.); 8-H: 4.22 (m); 3-H: 3.78 (d, J = 6 Hz,
br.); 4 H: 2.6-2.1 (m); 19-H3: 1.84 (d, J = 1.5 Hz);
20-H3: 1.63 (s, br.); ca. 17 H: 1.2-0.9; OH: 3.68 (s);
2.23 ppm (d, J = 6 Hz).
superimposed with m); OH: 3.91 ppm (d, J
=
7 Hz).
C o m p o u n d P8 (24)
a) P re p a ra tio n of the a c e to n id e 22 of fa c to r
H7: /^(petroleum ether/ether 3/2): 0.53. MS: m/e
=
Rf (ether/petroleum ether 1/1): 0.54. MS: parent
ion m/e = 540 (MT). IR (CH2C12): 3560 (OH); 1718
(CO); 1598, 1581 cm "1(C=C). UV: X (e) = 196 nm
(81650); Amax (e) = 230 (28720); 266 (sh, 1870); 274
(2040); 281 (1690); 305 nm (sh, 210). lH NMR: 1-H:
6.27 (m); 7-H: 5.93 (m); 5-H: 5.63 (s); 3-H: 5.36 (s);
8-H: 4.33 (m); 19-H3: 1.86 (d, J = 1.5 Hz); 20-H3:
1.60 (s, br.); 16-H3, 17-H3: 1.11 (s) and 1.07 (s); OH:
3.66 (s); 2 benzoates: 8.4—7.3 ppm (m). 13C NMR:
206.54 (s); 167.80 (s); 166.70 (s); 135.83 (s); 134.79
(s); 133.55 (s and d); 133.29 (d); 132.97 (d); 130.37
(2 d); 129.85 (2 d); 129.40 (s); 128.49 (2 d); 126.34
(2 d); 86.24 (s); 83.51 (d); 78.51 (d); 77.34 (imp.);
520. IR (CH2C12): 1715 (C = 0 ); 1610 cm '1 (C=C).
UV:A(£) = 197 nm (12990); Amax(e) = 258 (sh, 4050);
272 (sh, 5330); 282 (sh, 7000); 309 nm (11300).
!H NMR: 6 olefinic H: 7.7—5.9 (m, superimposed);
1-H, 7-H: 5.8 (m, superimposed); 5-H: 5.33 (s,
br.); 3-H: 4.31 (s, br.); 8-H: 4.05 (m); 3.66 (s,
imp.); 19-H3: 1.83 (s, br.); 20-H3: 1.71 (s, br.); 16-H3,
17-H3: 1.19 (s) and 1.04 ppm (s).
b) T ra n s e s te rific a tio n of fa c to r H7 a ffo rd -
ing 2 0 -d e o x y in g e n o l (13) and u n sa tu ra te d
m eth y l e s te rs : Spectral data of 13 see above.
Catalytic hydrogenation of the unsaturated methyl
Unauthenticated
Download Date | 10/29/18 8:13 AM

692
H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
72.27 (s); 43.61 (d); 38.98 (d); 31.20 (t); 28.47 (q);
24.37 (s); 23.27 (d); 23.07 (d); 21.38 (q); 17.09 (q);
15.56 ppm (2 q).
16-H2: 3.82 (s); 3-H: 3.75 (s); 19-H3: 1.85 (d, J =
1.5 Hz); 20-H3: 1.56 (s, br.); 17-H3: 1.15 (s); 18-H3:
0.95 (d, J = 7 Hz); 13-H, 14-H: 0.85 (superim-
posed); OH: 3.96; 2.45; angelate: 6.15 (m);
2.01-1.97 ppm (m).
C o m p o u n d P9 (25)
Rf (ether/petroleum ether 1/1): 0.61. MS: m/e =
534 (M+). IR (CH2C12): 3550 (OH); 1710 (C = 0 );
E u p h o rb ia fa c to r P6 (33)
1590, 1570 cm' 1 (C=C). UV: A (e)
=
195 nm
(43540); Amax (c) = 230 (13890), 274 (1410); 282
(1250); 297 nm (sh, 500). !H NMR: 1-H: 6.11 (m);
7-H: 5.86 (m); 5-H: 5.49 (s, br.); 3-H: 5.04 (s); 8-
Rf (ether/petroleum ether 3/1): 0.38. MS: m/e =
556 (M+), 312, 294. IR (CH2C12): 3560 (OH, br.);
1715 (CO); 1600, 1580 cm“1 (C=C). UV: A (e) =
H: 4.2 (m); 19-H3: 1.77 (d, /
= 1.5 Hz); 20-H3: 193 nm (80120); Amax (e) = 228 (29310), 266 (sh,
1420), 273 (1770), 280 (1380), 302 (sh, 210), 324 nm
(sh, 80). JH NMR: 1-H: 6.16 (m); 7-H: 5.76 (m);
3-H: 5.73 (s); 16-H2: 4.46±0.11 (7AB = 12 Hz); 8-H:
ca. 4.22 (superimposed); 5-H: 3.77 (d, J = 7 Hz);
19-H3: 1.84 (s, br.); 20-H3: 1.76 (s, br.); 17-H3: 1.22
(s); 18-H3: 1.06 (d, J = 7 Hz); OH: 3.67 (s); 3.22 (d,
7 = 7 Hz); 2 benzoates: 8.2—7.3 ppm (m).
a) T ra n s e s te rific a tio n (re a c tio n tim e 1 h)
o f fa c to r P6 affo rd in g 2 0 -d eoxy-16-hydroxy-
in g e n o l-1 6 -b e n z o ate (34): Rf (dichloromethane/
1.56 (s); 16-H3, 17-H3: 1.13 (s) and 1.07 (s);
6 H: 1.1—0.8; OH: 3.44 (s, br.); benzoate:
8.2—7.3 ppm (m).
3. Derivatives o f 20-deoxy-16-hydroxyingenol (26)
E u p h o rb ia fa c to r Ch (30)
R f (ether/petroleum ether = 4/1): 0.53. MS: m/e =
472 (M+), 330, 312. IR (KBr): 3490 (OH); 1730,
1718 (CO); 1640 cm-1 (C=C). UV: A (e) = 196 nm
(17880); Amax (e)
= 211 (15930), 285 nm (280).
acetone 4/1): 0.41. MS: m/e
= 452 (M+). IR
JH NMR: 1-H: 6.02 (d, J = 1.5 Hz); 7-H: 5.70 (m);
3-H: 5.44 (s); 16-H2: 4.16±0.1 (7AB = 12 Hz); 8-H:
4.12 (superimposed); 5-H: 3.67 (d, J = 7 Hz); 19-H3,
20-H3: 1.78 (m); 17-H3: 1.1 (s); 14-H: 1.08 (superim-
posed); I8-H3: 0.95 (d, J = 7 Hz); 13-H: 0.85
(superimposed); OH: 3.5 (s); 3.28 (d, J = 7 Hz);
acetate: 2.06 (s); angelate: 1 olefinic H: 6.12 (m); 2
olefinic CH3: 1.96—1.92 ppm (m).
(CH2C12): 3460 (br., OH); 1710 (CO); 1596 cm“ 1
(C=C). UV: A (e) = 195 nm (42130); Amax (e) = 228
(12520), 267 (sh, 770), 273 (850), 280 (730), 297 nm
(sh, 180). *H NMR: 1-H: 5.92 (d, J = 1.5 Hz); 7-H:
5.69 (m); 16-H2: 4.51 (s); 3-H: 4.40 (s); 8-H: 4.2 (m);
5-H: 3.43 (s, br.); 19-H3: 1.84 (d, / = 1.5 Hz); 20-H3:
1.72 (s, br.); 17-H3: 1.26 (s); 18-H3: 0.97 (d, J =
7 Hz); OH: 4.2 (br.) and 3.2—2.5 ppm (superim-
T ra n s e s te rific a tio n (re a c tio n tim e: 5 h)
posed); benzoate: 8.1-7.25 ppm (m).
a)
of fa c to r Ch a ffo rd in g 26, 27 and 28: In deviation
from the general instructions 5 x l0 “3 M NaOCH? in
methanol was used for transesterification.
b) P re p a ra tio n of th e a c e to n id e 35 from
34: Rf (ether/petroleum ether 3/1): 0.57. MS: m/e =
492 (M+). IR (CH2C12): 3560 (OH); 1710 (CO);
1600 cm“1 (C=C). UV: A (e) = 194 nm (43700);
Amax (e) = 228 (14670), 267 (sh, 770), 273 (920), 281
(810), 298 nm (sh, 200). *H NMR: 1-H, 7-H: 5.84
(m. superimposed); 3-H: 4.69 (s); 16-H2: 4.56±0.04
(/AB = 12 Hz); 8-H: 4.22 (m); 5-H: 3.77 (d, br., J =
8 Hz); 19-H3: 1.87 (s); 20-H3: 1.79 (s, br.); 17-H3:
1.22 (s); 18-H3: 0.98 (d, J = 7 Hz); OH: 2.50 (d, J =
8 Hz); benzoate: 8.2-7.3 (m); isopropylidene: 1.53
(s) and 1.47 ppm (s).
c) T ra n s e s te rific a tio n of 35 a ffo rd in g 20-
d e o x y -16-h y d ro x y in g e n o l-3 ,4 -a c e to n id e
(36): Rf (dichloromethane/acetone 4/1): 0.36. MS:
m/e = 388 (MT). IR (CH2C12): 3610, 3560 (br., OH);
1717 cm“1 (CO). UV: A (e) = 194 nm (11510); Amax
(e) = 290 nm (220). lU NMR: 7-H: 5.88 (m); 1-H:
5.83 (m); 3-H: 4.69 (s); 8-H: 4.2 (m); 16-H2, 5-H:
3.88 (superimposed); 19-H3, 20-H3: 1.87 (m.
superimposed); 17-H3: 1.17 (s); 18-H3: 1.00 (d, J =
7 Hz); OH: 2.8—2.5 (br.); isopropylidene: 1.56 (s)
and 1.51 ppm (s).
2 0 -D e o x y -1 6 -h y d ro x y in g e n o l (26)
26
was characterized as its 3,5,16-triacetate 29,
spectral data see below.
2 0 -D e o x y -1 6 -h y d ro x y in g e n o l-3 -a n g e la te
(27)
MS: m/e = 430 (M+).
NMR: 1-H: 6.08 (d, J =
1.5 Hz); 7-H: 5.75 (m); 3-H: 5.45 (s); 8-H: 4.2 (m);
16-H2: 3.85 ±0.1 (/AB = 12 Hz); 5-H: 3.65 (s); 19-H3,
20-H3: 1.8 (m); 17-H3: 1.15 (s); 18-H3: 0.95 (d, J=
7 Hz); 13-H, 14-H: 0.8; OH: 3.68, 3.12 ppm; ange-
late: 1 olefinic H: 6.17 (m);
2.0—1.96 ppm (m).
2 olefinic CH3:
2 0 -D e o x y -1 6 -h y d ro x y in g e n o l-5 -a n g e la te
(28)
MS: m/e 430 (MT). *H NMR: 1-H: 6.0 (d, J =
1.5 Hz); 7-H: 5.87 (m); 5-H: 5.25 (s); 8-H: 4.4 (m);
Unauthenticated
Download Date | 10/29/18 8:13 AM

H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
693
d) T ra n s e s te rific a tio n
(re a c tio n
tim e
17-H3: 1.22 (s); 18-H3: 1.06 (d, / = 7 Hz); 2 ben-
zoates: 8.2—7.2 (m); isopropylidene: 1.47 (s) and
1.38 ppm (s).
24 h) of fa c to r P6 affo rd in g 2 0 -d e o x y -16-hy-
d ro x y in g e n o l (26): Rf (ethyl acetate): 0.20, stain-
ing: dark-brown. MS: m/e = 348 (M+). IR (KBr):
3600-3200 (br., OH); 1705 c m '1(CO). UV: A (e) =
194 nm (11510); Amax (e) = 2900 nm (220). ]H NMR:
1-H: 5.97 (m); 7-H: 5.80 (m); 3-H: 4.42 (s); 8-H: 4.22
(m); 16-H2: 3.80±0.05 (/AB = 12 Hz); 5-H: 3.50 (s,
br.); 19-H3: 1.87 (s); 20-H3: 1.80 (s); 18-H3: 0.96 (d,
7 = 7 Hz); 4 OH: 3.4-2.1 ppm (br.). 13C NMR
(CD3OD): 210.63 (s); 141.22 (s); 140.70 (s); 129.79
(d); 123.74 (s); 86.18 (s); 81.24 (d); 77.27 (d); 73.96
(s); 63.30 (t); 44.52 (d); 40.81 (d); 31.65 (t); 31.13
(s); 24.96, 24.76, 24.57 (2 d and 1 q); 22.49 (q);
17.15 (q); 15.60 ppm (q).
4. Derivatives o f 20-deoxy-13,16-dihydroxyingenol
(37)
E u p h o rb ia fa c to r P5 (38)
Rf (ether/petroleum ether 3/1): 0.37, staining:
brown-grey. MS: m/e = 676 (M~), 328, 310. IR
(CH2C12): 3555, 3500 (br., OH); 1720 (CO); 1599,
1581 cm"1(C=C). UV: A (e) = 193 nm (96260); Amax
(£) = 196 (90190); 231 (37290), 266 (sh, 2510), 274
(2830), 281 nm (sh, 2370). !H NMR: 1-H: 6.18 (d,
J = 1.5 Hz); 3-H: 5.77 (s); 7-H: 5.74 (m, superim-
posed); 16-H2: 4.56±0.11 (7AB = 12 Hz); 8-H: 4.3
(superimposed); 5-H: 3.79 (s); 19-H3: 1.82 (d, J =
1.5 Hz); 20-H3: 1.72 (s, br.); 14-H: 1.61 (d, J =
12 Hz, partly superimposed); 17-H3: 1.29 (s); 18-H3:
0.99 (d, J = 7 Hz); OH: 3.7, 3.1-2.0 (superim-
posed); 3 benzoates: 8.25—7.35 ppm (m).
e) A c e tv la tio n of 2 0 -d e o x y -16-hydroxy-
in g e n o l
(26) a ffo rd in g
20-deoxy-16-hy-
(29): Rf
d ro x y in g e n o l-3 , 5 , 1 6 -tria c e ta te
(ether/petroleum ether 1/1): 0.18. MS: m/e = 474
(M+). IR (CH2C12): 3550 (OH); 1730 cm“1 (CO).
UV: A (e)
= 194 nm (11760); Amax (c) = 283 nm
(180). lH NMR: 1-H: 6.06 (d, J = 1.5 Hz); 7-H: 5.81
(m); 5-H: 5.20 (s, br.); 3-H: 4.91 (s); 8-H: 4.28 (m);
16-H2: 4.20 ± 0.08 (7AB = 12 Hz); 19-H3: 1.77 (d, J =
1.5 Hz); 20-H3: 1.56 (s); 17-H3: 1.13 (s); 18-H3: 0.98
(d, J = 7 Hz); OH: 3.20 (s); 3 acetates: 2.28 (s), 2.13
(s) and 2.09 ppm (s).
C o m p o u n d P3 (39)
Rf (ether/petroleum ether 3/1): 0.36. MS: m/e =
690 (M+), 328, 310. UV: A (e) = 194.5 nm (70080);
Amax (e) = 228 (21130), 268 (sh, 1500); 274 (1640),
281 nm (1390). lH NMR: 1-H: 6.10 (d, J = 1.5 Hz);
7-H, 3-H: 5.65 (m, superimposed); 16-H2:
4.41 ±0.10 (/AB = 12 Hz, superimposed with 8-H
and imp.); 5-H: 3.73 (s, br.); 19-H3: 1.84 (d, J =
1.5 Hz); 20-H3: 1.73 (s, br.); 14-H: 1.50 (partly
superimposed); 17-H3: 1.14 (s); OH: ca. 1.8
(superimposed); phenylacetate: arom. H: 7.28 (s,
partly superimposed); methylene group: 3.57 (s); 2
benzoates: 8.2—7.2 ppm (m).
C o m p o u n d Pi (32)
Rf (ether/petroleum ether 3/1): 0.48. MS: m/e =
556 (M+), 312, 294. IR (KBr): 3440 (br., OH); 1715
(CO); 1595, 1580 c m '1(C=C), UV: A (e) = 195 nm
(83220); Amax (e) = 229 (30140), 266 (sh, 1950), 273
(2160), 281 nm (1810). lH NMR: 1-H: 6.00 (d, J =
1.5 Hz); 7-H: 5.90 (m); 5-H: 5.46 (s, br.); 16-H2:
4.55 (s, br.); 8-H: 4.5 (superimposed); 3-H: 3.87 (s);
19-H3: 1.84 (s); 20-H3: 1.55 (s); 17-H3: 1.24 (s);
18-H3: 0.99 (d, / = 7 Hz); OH: 4.20 (s); 3.3 (br.); 2
benzoates: 8.3—7.3 ppm (m). 13C NMR: 206.93 (s);
167.15 (s); 166.44 (s); 139.66 (s); 135.70 (s); 133.62
(d); 130.11 (d); 129.66 (d); 129.33 (s); 129.20 (d);
128.62 (d); 128.49 (d); 124.85 (d); 85.07 (s); 79.87
(d); 77.34 (d); 72.98 (s); 66.48 (t); 43.61 (d); 39.45
(d); 30.87 (t); 27.82 (s); 24.44 (q); 24.05 (d); 23.85
(d); 21.45 (q); 17.03 (q); 15.47 ppm (q).
C o m p o u n d P4 (40)
Rf (ether/petroleum ether 3/1): 0.36. MS: m/e =
614 (M+), 328, 310. IR (CH,C12): 3680-3300 (br.,
OH); 1712 (CO); 1597, 1580* cm-1 (C=C). UV: A
(e) = 195 nm (62900); Amax (e) = 229 (22570), 266
(sh, 1860), 273 (2150), 280 nm (1940). !H NMR: 1-H:
6.12 (d, J = 1.5 Hz); 3-H, 7-H: 5.65 (m, superim-
posed); 16-H2: 4.43±0.10 (/AB = 12 Hz); 8-H: 4.25
(m); 5-H: 3.74 (s, br.); 19-H3: 1.85 (d, J = 1.5 Hz);
20-H3: 1.73 (s, br.); 14-H: 1.45 (d, J = 13 Hz);
17-H3: 1.26 (s); OH: 3.6 and 3.0—2.0 (superim-
posed); acetate: 2.03 (s); 2 benzoates: 8.2—7.3 ppm
(m).
a) Is o m e ris a tio n of co m p o u n d P4 a ffo rd -
ing 41: After storage of compound P4 (40) for six
months in ethyl acetate by tic an additional com-
pound was detected and isolated by prep. TLC to
yield 30% of 20-deoxy-13,16-dihydroxyingenol-13-
a) P re p a ra tio n of th e a c e to n id e 3 1 of com -
p o u n d P^ ^(ether/petroleum ether 1/1): 0.4. MS:
m/e
= 596 (M+). IR (KBr): 1715 (CO); 1595,
1580 c m '1(C =C ). UV: A (e) = 196 nm (76970); Amax
(e) = 230 (28100), 267 (sh, 1750), 273 (1970), 281 nm
(sh, 1650). lH NMR: 1-H: 5.93 (d, J = 1.5 Hz); 7-H:
5.91 (m, superimposed); 5-H: 5.52 (s); 16-H2:
4.58 ±0.16 (7ab = 12 Hz); 3-H: 4.39 (s); 8-H: 4.23
(m); 19-H3: 1.87 (d, J = 1.5 Hz); 20-H3: 1.73 (s, br.);
Unauthenticated
Download Date | 10/29/18 8:13 AM

694
H. Gotta et al. ■On the Active Principles of the Euphorbiaceae
4.17; 3-H: 3.83 (s); 19-H3: 1.84 (d, J = 1.5 Hz);
20-H3: 1.55 (s, br.); 17-H3: 1.39 (s); imp.: 1.35 (s);
18-H3:1.02 (d, J = 7 Hz); OH: 3.9 and 1.6; acetate:
2.03 (s); 2 benzoates: 8.2—7.3 ppm (m).
acetate-5,16-dibenzoate (41). Rf (ether/petroleum
ether 3/1): 0.37. MS: m/e = 614 (M+). *H NMR: 1-H:
5.98 (d, J = 1.5 Hz); 7-H: 5.81 (m); 5-H: 5.40 (s,
br.); 16-H2: 4.55 (s); 8-H: 4.4 (m); imp.: 4.22 and
[16] H. Gotta. Dissertation Universität Heidelberg (1978).
[17] H. J. Opferkuch, W. Adolf, B. Sorg, S. Kusumoto,
and E. Hecker, Z. Naturforsch. 36b, 878 (1981).
[18] B. Sorg and E. Hecker. Z. Naturforsch. 37b, 748
(1982).
[19] L. J. Lin and A. D. Kinghorn. J. Agric. Food Chem.
31, 396 (1983).
[20] B. Sorg and E. Hecker, Z. Naturforsch. 37b, 1640
(1982).
[1] A. Engler, Syllabus der Pflanzenfamilien, Berlin-
Nikolassee 1964, Gebr. Bornträger.
[2] J. M. Watt and M. G. Breyer-Brandwijk. The Medici-
nal and Poisonous Plants of Southern and Eastern A f-
rica. Edinburgh 1962. E. and S. Livingstone.
[3] J. L. Hartwell. Lloydia 32, 157 (1969).
[4] J. F. Morton, Atlas of Medicinal Plants of Middle
America. Bahamas to Yucatan, Springfield, Illinois
1981, Thomas, C. C. (ed.).
[21] K. A. Abo and F. J. Evans, Phytochemistry 21, 725
(1982).
[22] D. Uemura, H. Ohwaki. and Y. Hirata, Tetrahedron
Lett. 1974, 2527.
[23] H. J. Opferkuch and E. Hecker, Tetrahedron Lett.
1974, 261.
[5] A. A. Forsyth, British Poisonous Plants, p. 73 —75,
London 1968. Her Majesty Stationary Office.
[6] E. Hecker. Pure Appl. Chem. 49, 1423 (1977).
[7] E. Hecker, in Slaga, T. S.. A. Sivak. and R. K. Bout-
well (eds): Carcinogenesis, Vol. 2, Mechanism of
Tumor Promotion and Cocarcinogenesis, p. 11, Ra-
ven Press. New York 1978.
[24] H. G. Wippert, Dissertation Universität Heidelberg
1977.
[8] F. J. Evans and C. J. Soper. Lloydia 41, 193 (1978).
[9] W. Adolf and E. Hecker. Z. Krebsforsch. 84, 325
(1975).
[25] G. Fürstenberger and E. Hecker, Experientia 33, 986
(1977).
[26] G. Fürstenberger and E. Hecker, Tetrahedron Lett.
1977, 925.
[27] G. Fürstenberger and E. Hecker. Planta Medica 22,
241 (1972).
[10] E. Seip and E. Hecker. Planta Medica 46, 215 (1982).
[11] H. H. Ott and E. Hecker. Experientia 37, 88 (1981).
[12] G. Falsone. A. E. G. Crea. and A. E. Noack, Arch.
Pharm. 315, 1026 (1982).
[28] M. D. Saved. A. Riszk. F. M. Hammouda. M. M. El-
Missiry. E. M. Williamson, and F. J. Evans. Experien-
cia 36,' 1206 (1980).
[29] M. Hergenhahn. G. Fürstenberger. H. J. Opferkuch.
W. Adolf. H. Mack, and E. Hecker, J. Cancer Res.
Clin. Oncol. 104, 31 (1982).
[13] R. J. Schmidt and F. J. Evans. Contact Dermatitis 6,
204 (1980).
[14] W. Adolf. S. Chanai. and E. Hecker. J. Sei. Soc.
(Thailand) 9, 81 (1983).
[15] E. Hecker and R. Schmidt. Progr. Chem. Org. Natur.
Prod. 31, 377 (1974).
Unauthenticated
Download Date | 10/29/18 8:13 AM