Diterpenoids and triterpenoids from Euphorbia retusa

Article abstract of DOI:10.1021/np900127j

Six new ent-abietane lactones (1-6), three new esterified tetracyclic triterpenes (7-9), and seven known diterpenoids and triterpenoids were isolated from the roots of Euphorbia retusa. Their structures were elucidated by means of spectroscopic studies in

Full text of DOI:10.1021/np900127j

1258
J. Nat. Prod. 2009, 72, 1258–1264
Diterpenoids and Triterpenoids from Euphorbia retusa
Hamada Haba,† Catherine Lavaud,‡ Abdulmagid Alabdul Magid,‡ and Mohammed Benkhaled*,†
Laboratoire de Chimie et Chimie de l’EnVironnement (L.C.C.E), De´partement de Chimie, Faculte´ des Sciences, UniVersite´ de Batna,
Batna 05000, Algeria, and Laboratoire de Pharmacognosie, Institut de Chimie Mole´culaire de Reims, CNRS UMR 6229, BP 1039,
51097 Reims Cedex 2, France
ReceiVed March 1, 2009
Six new ent-abietane lactones (1-6), three new esterified tetracyclic triterpenes (7-9), and seven known diterpenoids
and triterpenoids were isolated from the roots of Euphorbia retusa. Their structures were elucidated by means of
spectroscopic studies including 1D and 2D NMR, mass spectrometry, chemical transformation, and comparison with
literature data.
Euphorbia retusa Forsk. (Euphorbiaceae) is distributed through-
out the Mediterranean region.1,2 Euphorbia is the largest Euphor-
biaceae genus with over 1000 species,2 and many of them have
been investigated chemically and pharmacologically due to carci-
nogenic and irritant properties of their latex.3-5 Diterpenes from
Euphorbia plants have been found to possess a number of interesting
biological activities.6-8 E. retusa is a perennial blue-green desert
herb that grows to about 40 cm in height, with long alternate leaves,1
and it contains a toxic and skin-irritant milky latex. This herb has
been used in folk medicine for treatment of warts, trichiasis, and
venomous bites.9 Previously, it was reported that the aerial parts
of E. retusa contained a number of common flavonol glycosides,10
triterpenoids,11,12 and fatty acids.12
Our present work describes the isolation and structure elucidation
of six new compounds with abietane lactone skeletons, 3,4,18ꢀ-
cyclopropa-8ꢀ-hydroxy-14-oxo-ent-abiet-13,15-en-16,12-olide (1),
3,4,18ꢀ-cyclopropa-14-oxo-ent-abieta-8,9,13,15-dien-16,12-olide
(2), 3,4,18ꢀ-cyclopropa-14-oxo-ent-abieta-7,13,15-dien-16,12-olide
(3), 3,4,18ꢀ-cyclopropa-7ꢀ-hydroxy-14-oxo-ent-abieta-8,9,13,15-
dien-16,12-olide (4), 3,4,18ꢀ-cyclopropa-14-oxo-ent-abiet-7-en-
16,12-olide (5), and 3,4,18ꢀ-cyclopropa-12ꢀ-hydroxy-ent-abiet-7-
en-16,14-olide (6), and three new esterified tetracyclic triterpenes,
24-methylenecycloartanyl formate (7), 24-methylenecycloartanyl
2′E,4′E-decadienoate (8), and tirucalla-7,24-dien-3ꢀ-yl 2′E,4′E-
decadienoate (9) from a dichloromethane extract of the roots of E.
retusa.
seven quaternary carbons (Table 1). Among these were lactone and
ketone carbonyls (δC 172.9 and 196.0), a vinylic methyl (δC
9.4),20,21 two methyl groups (δC 23.9 and 16.9), and one tertiary
1
OH-bearing carbon (δC 76.1). The H NMR spectrum in CDCl3
(Table 1) exhibited three signals at high field [δH 0.12 (1H, dd, J
) 5.7, 4.5 Hz, H-18 endo), 0.55 (1H, dd, J ) 9.3, 4.5 Hz, H-18
exo), and 0.71 (1H, dt, J ) 9.3, 5.7 Hz, H-3)] typical of a
cyclopropane ring as in spectra of abietane-type diterpenes,
suregadolides, isolated from Suregada multiflora.20,21 The 1H-1H
COSY spectrum of 1 revealed three proton-correlated fragments,
H-3/H-18 endo and exo, H-3/one of H-2 (R-oriented), H2-2/H2-1
for ring A, H-5/H2-6 and H2-6/H2-7 for ring B, and H-9/one of
H-11 (ꢀ-oriented), H2-11/H-12, H-12/H3-17 (homoallylic coupling)
1
for rings C and D. Detailed analysis of H NMR, 13C NMR, and
HSQC spectra of 1 allowed assignment of carbon and proton signals
of these fragments. HMBC correlations observed from the quater-
nary carbons C-4, C-10, and C-8 to H-3/H-5, H2-1/H-9, and H2-
7/H-9, respectively, allowed connection of these fragments and
established the partial structure of 1. The HMBC spectrum displayed
correlations characteristic of an abietane lactone between H3-17/
C-12, H3-17/C-13, and H3-17/C-16 (δC 172.9). The second carbonyl
(δC 196.0) was located at C-14, as indicated by its correlations with
H2-7, H-9, and H3-17. Correlations observed from C-8 (δC 76.1)
to H-9, H2-6, and H-11R placed the tertiary OH group at C-8.
Finally, the Me-19 protons were found to be correlated with C-3,
C-4, and C-18, while H-3 showed correlations with C-1, C-5, and
C-18. The relative configuration of 1 was determined from the
NOESY spectrum and the values of the coupling constants. The
orientations of H-12 and H-9 were established as axial and
equatorial, respectively, in ring C from the coupling constants and
multiplicities of H-9ꢀ (δH 1.82, d, J ) 6.3 Hz), H-11R (δH 2.66,
dd, J ) 12.0, 7.7 Hz), H-11ꢀ (δH 2.14, td, J ) 12.0, 6.3 Hz), and
H-12R (δH 5.39, br, ddq, J ) 12.0, 7.7, 2.0 Hz), which were similar
to those of reported abietane lactones having H-12R.14,20-24 NOE
correlations (Figure 1) observed between H-12R/Me-20, Me-20/
Me-19, and Me-19/H-3 indicated R-orientation of all these protons
and consequently ꢀ-orientation of the cyclopropane ring. The NOE
of H-5 with H-18 endo and with H-9ꢀ indicated that H-5 was
ꢀ-oriented and the A/B ring junction was trans. Considering the
constituents isolated so far from Euphorbia species, compound 1
was presumed to be an ent-abietane diterpene.13,14,24 The assigned
orientations of H-5, H-9, and Me-20 confirmed this to be a
compound belonging to the ent series.20-26 The orientation of the
OH group attached to C-8 was determined from the NOESY
spectrum recorded in DMSO-d6. Thus, the hydroxylic proton
displayed NOE effects with H-9ꢀ and H-7ꢀ and was therefore axial
(ꢀ-oriented). Compound 1 was thus identified as 3,4,18ꢀ-cyclo-
propa-8ꢀ-hydroxy-14-oxo-ent-abiet-13,15-en-16,12-olide and was
named retusolide A.
Results and Discussion
Purification of a dichloromethane extract of roots of E. retusa
by repetitive chromatographic separation provided nine new
compounds (1-9), and the known compounds were identified as
jolkinolide E,13 helioscopinolide E,14 24-methylenecycloartanol,15,16
24-methylenecycloartanone,17 cycloart-25-ene-3ꢀ,24-diol,18 cyc-
loeucalenol,12 and obtusifoliol.15 Physical and spectroscopic data
of the known compounds were identical with those published in
the literature.
Compound 1 was obtained as a colorless oil. The HREIMS of 1
indicated a molecular ion peak at m/z 330.1819, which corresponded
to the molecular formula C20H26O4. The IR spectrum showed
absorption bands at 3451 cm-1 for OH, 1765 cm-1 indicating an
R,ꢀ-unsaturated γ-lactone,8 and 1685 cm-1 also indicating an R,ꢀ-
unsaturated ketone.19 The 13C NMR spectrum of compound 1 in
CDCl3 was consistent with an abietane skeleton, with signals
corresponding to three methyl, six methylene, four methine, and
* To whom correspondence should be addressed. Tel: +213 33 86 89
46. Fax: +213 33 86 89 46. E-mail: mbenkhaled@yahoo.fr.
† University of Batna.
‡ CNRS UMR 6229.
10.1021/np900127j CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/11/2009

Diterpenoids and Triterpenoids from Euphorbia retusa
Journal of Natural Products, 2009, Vol. 72, No. 7 1259
Table 1. 1H and 13C NMR Data for 1
1a
1b
atom
H-1R
H-1ꢀ
H-2R
H-2ꢀ
H-3R
4
H-5ꢀ
H-6R
H-6ꢀ
H-7R
H-7ꢀ
ꢀ8-OH
H-9ꢀ
10
H-11R
H-11ꢀ
H-12R
13
δH (J in Hz)
δC
δH (J in Hz)
δC
1.76, dd (12.7, 5.9)
0.83, td (13.4, 6.0)
1.97, m
33.6
1.72, m
0.70, m
1.88, m
33.2
18.8
19.03
1.85, m
1.70, td (13.9, 6.9)
0.60, m
0.71, dt (9.3, 5.7)
19.1
15.7
51.1
22.7
19.0
15.8
50.5
22.8
1.28, dd (11.0, 2.7)
1.41, m
1.22, dd (14.3, 5.3)
1.23, dm (14.3)
1.77, m
2.45, dd (13.3, 3.9)
1.27, m
1.95, m
2.68, dd (12.7, 4.4)
1.43, m
not observed
1.82, d (6.3)
33.5
33.1
76.1
52.6
36.6
27.0
5.89, br, s
1.65, d (6.3)
75.8
52.8
36.3
26.9
2.66, dd (12.0, 7.7)
2.14, td (12.0, 6.3)
5.39, br, ddq (12.0, 7.7, 2.0)
2.51, m
1.93, td (11.9, 6.3)
5.44, br, ddq (11.9, 7.3, 2.0)
79.5
153.3
196.0
131.8
172.9
9.4
79.9
155.2
197.2
129.6
172.7
9.4
14
15
16
H-17
H-18 endo
H-18 exo
H-19
H-20
2.05, d (2.0)
0.12, dd (5.7, 4.5)
0.55, dd (9.3, 4.5)
1.00, s
1.84, d (2.0)
0.07, dd (5.6, 4.1)
0.43, dd (9.2, 4.1)
0.91, s
21.5
21.4
23.9
16.9
24.1
16.3
0.85, s
0.71, s
a Spectra were recorded in CDCl3. b Spectra were recorded in DMSO-d6.
Compound 2 was isolated as a white, amorphous powder and
exhibited a molecular ion peak at m/z 312.1723 (C20H24O3) in its
HREIMS, H2O less than that of 1. The IR spectrum had no OH
band, but showed a band at 1618 cm-1 (double bond). The 1H and
13C NMR signals of 2 were similar to those of 1 (Tables 2 and 3).
The only differences were the lack of signal at δ 76.1 (for C-8 in
1) and the appearance in the J-modulated 13C spectrum of two
signals attributed to olefinic carbons of a tetrasubstituted double

1260 Journal of Natural Products, 2009, Vol. 72, No. 7
Haba et al.
) 8.4, 7.3 Hz and δC 52.9, CH-13) instead of a vinylic methyl
group as in compounds 1-4. Scalar 1H-1H connectivities obtained
by COSY experiment allowed us to identify an expanded spin
system for rings C and D from H-9 (δH 2.23) to H3-17 (δH 1.43)
through H2-11 (δH 1.41 and 2.37)/H-12 (δH 5.10)/H-13 (δH 2.98)/
H-15 (δH 2.82). Association of all protons with the corresponding
carbons by HSQC experiment and analysis of the long-range
correlations in the HMBC spectrum led to the abietane lactone
structure of 5. Correlations observed in the NOESY spectrum
between H3-20, H-12, and H-13 indicated R-orientation of H-12
and H-13. The NOE correlation observed from H-12 to H3-17
suggested that H-15 and H3-17 were ꢀ- and R-oriented, respectively.
From these data, compound 5 was elucidated as 3,4,18ꢀ-cyclopropa-
14-oxo-ent-abiet-7-en-16,12-olide and was named retusolide E.
Figure 1. NOESY correlations of 1.
The molecular formula of compound 6, colorless oil, was
determined as C20H28O3 (m/z 316.2015, HREIMS). Its IR spectrum
displayed absorption bands at 3450 cm-1 (OH), 1757 cm-1
bond at δ 134.6 and 160.5. In the HMBC spectrum of 2, H2-1,
H3-20, and H-6ꢀ exhibited 3J interactions with C-9 and C-8,
respectively, while H2-7 and H2-11 displayed 2J and 3J correlations
with the two olefinic carbons. These correlations led to the
placement of the double bond in the B/C ring junction (δC 134.6,
C-8 and 160.5, C-9). The relative configuration of 2 was deduced
from analysis of NOE correlations and was the same as 1. Thus,
compound 2, named retusolide B, was elucidated as 3,4,18ꢀ-
cyclopropa-14-oxo-ent-abieta-8,9,13,15-dien-16,12-olide.
Compound 3, a white, amorphous powder, showed a molecular
ion peak at m/z 312.1716 corresponding to the same molecular
formula (C20H24O3) as 2. The EIMS, IR, UV, and NMR spectra of
3 were similar to those of 2, suggesting that 2 and 3 were
regioisomers. The 13C NMR spectrum (Table 3) revealed the
presence of one olefinic methine (δ 140.4) correlated in the HSQC
spectrum with the proton at δ 6.97 (dt, J ) 5.3, 2.4 Hz, H-7). This
proton coupled in the COSY spectrum with two geminal protons
H2-6 (δ 2.55, ddd, J ) 11.2, 5.6, 2.4 Hz and δ 2.36, m), which
were correlated with one methine proton (H-5, δ 1.65, dd, J )
11.3, 5.6 Hz). In the HMBC spectrum, the olefinic proton (H-7)
showed correlations with the C-14 carbonyl (δ 187.5) and with
the C-9 (δ 41.6) and C-5 (δ 44.8) methines. Compound 3 showed
the same relative configuration as 1 and 2, with a characteristic
NOE between H-5 and H-9. Consequently, compound 3 was
assigned as 3,4,18ꢀ-cyclopropa-14-oxo-ent-abieta-7,13,15-dien-
16,12-olide and was named retusolide C.
1
(lactone), and 1650 cm-1 (double bond). The H and 13C NMR
data of 6 (Tables 2 and 3) presented similarities with those of 3
and 5 with identical A and B rings of the ent-abietane skeleton.
Two secondary hydroxymethines were observed at δC 63.8 and 83.3
and one carbonyl at δC 181.7. These chemical shifts suggested the
presence of a lactone ring and an OH group. In the HMBC
spectrum, the hydroxymethines showed correlations with H-9 that
allowed placement of them at positions 12 and 14. The cross-peak
of the ethylenic proton H-7 (δ 5.99, m, W1/2 ) 11.0 Hz) with the
hydroxymethine at δC 83.3 (C-14) indicated that the D lactone ring
was fused on C-13 and C-14 to form a rearranged abietane lactone.
Consequently, C-12 was attached to a free OH group. The relative
configuration of 6 was determined from the NOESY spectrum and
by comparison with related ent-diterpenes.20-27 Correlations
observed between H3-20/H3-19/H-3/H-18 exo and H-9/H-5/H-18
endo proved that the relative configuration at C-3, C-4, C-5, C-9,
and C-10 was the same as that of the other compounds (1-5). The
NOE correlations of H-9 with H-5 and one of H-11 protons at δ
1.94 indicated that these three protons were ꢀ-oriented. The values
of the coupling constant of the two H-11 signals with H-12 (3.9
and 1.4 Hz) indicated that H-12 was R-equatorial on ring C in a
chair conformation. NOE effects observed between H-11R/H-12/
H-13/H-14 indicated that these protons were R-oriented. The
correlations from H3-17 to H-11ꢀ and H-9ꢀ suggested that H3-17
were ꢀ-oriented. The small value of the coupling constant between
H-13 and H-14 (J ) 4.4 Hz) implied a cis C/D ring junction.28
The structure of compound 6 was thus determined to be 3,4,18ꢀ-
cyclopropa-12ꢀ-hydroxy-ent-abiet-7-en-16,14-olide, and it was
named retusolide F.
Compound 4, a colorless oil, had the molecular formula
C20H24O4, as determined by HREIMS ([M]+, m/z 328.1682), 16
mass units more than that of 2. IR absorptions revealed the presence
of OH (3438 cm-1), R,ꢀ-unsaturated γ-lactone, and ketone (1767
and 1675 cm-1) groups.8,19 The H and 13C NMR spectra of 4
1
(Tables 2 and 3) showed high similarity to those of 2 and indicated
that the difference was mainly the presence of signals for a
hydroxymethine group (δH 4.72, br, s and δC 62.5). This supple-
mentary proton showed correlation with C-14 (δ 187.0) in the
HMBC spectrum and was attributed to H-7. Analyses of COSY,
HSQC, and HMBC experiments allowed assignments of all protons
and carbons. The NOESY correlations indicated that compound 4
possessed the same relative configuration as the compounds
described previously. The shape of the H-7 signal (broad singlet)
and the small values of its coupling constant with H-6 indicated
that H-7 was equatorial (R-oriented). The absence of NOE
correlation between ꢀ-oriented H-5 and H-7 confirmed this relative
configuration. Thus, compound 4 was identified as 3,4,18ꢀ-
cyclopropa-7ꢀ-hydroxy-14-oxo-ent-abieta-8,9,13,15-dien-16,12-
olide and was named retusolide D.
Compound 5 possessed a molecular ion peak at m/z 314.1889
(C20H26O3) in the HREIMS, two mass units more than that of 3.
The IR spectrum contained bands at 1777 cm-1 (lactone) and 1665
cm-1 (R,ꢀ-unsaturated ketone).19 The 1H and 13C NMR spectra of
5 (Tables 2 and 3) were similar to those of 3. The main difference
was that 5 possessed a methyl group (H3-17, δH 1.43, d, J ) 7.3
Hz and δC 16.2) attached to an sp3 carbon methine (δH 2.98, dd, J
Compound 7 was obtained as a white, amorphous solid and
displayed a molecular ion at m/z 468.3970 in the HREIMS,
consistent with the molecular formula C32H52O2. The H NMR
1
spectrum of 7 (Table 4) showed signals corresponding to seven
methyl groups, one oxygenated methine attributed to H-3, two broad
singlets assignable to an exocyclic methylene group, and an
aldehydic deshielded signal. The 13C NMR spectrum (Table 4)
exhibited 32 signals, for seven methyl, 11 methylene including one
ethylenic carbon (dCH2), seven methine (among them one oxyme-
thine), and six quaternary carbons (among them two sp2). These
structural features confirmed the triterpene nature of 7 and were
closely similar to those of tetracyclic cycloartane-type triterpenes
such as 24-methylenecycloartanol isolated in this study and
identified previously from several Euphorbia species.15,16 The H
1
NMR spectrum of 7 included a low-field singlet (δ 8.18, H-1′) that
correlated with a carbon that resonated at δ 161.2 (C-1′) in the
HSQC experiment and with an oxymethine carbon signal at δ 80.8
(C-3) in the HMBC spectrum. These data indicated a formate group
(HCOO) at C-3 in compound 7.29 The chemical shift of H-3 (δ
4.75, dd, J ) 11.5, 4.7 Hz) confirmed the linkage of the formate
group at C-3. COSY, HSQC, and HMBC experiments allowed

Diterpenoids and Triterpenoids from Euphorbia retusa
Journal of Natural Products, 2009, Vol. 72, No. 7 1261
Table 2. 1H NMR Data for 2-5a and 6c
δH (J in Hz)
atom
H-1R
2
3
4
5
6
1.71, dd (12.5, 6.2)
0.90, ddd (12.5, 9.0, 6.4)
2.15, dd (13.7, 9.0)
1.90, dd (13.7, 6.4)
0.74, dt (9.3, 6.0)
1.33, dd (12.7, 2.7)
1.59, dddd (19.1, 12.7, 11.8, 5.7)
2.12, m
1.64, ddd (13.3, 5.1, 1.6) 1.70, m
0.83, td (13.3, 5.2)
2.01, tt (13.3, 5.1)
1.59, m
1.51, ddm (13.4, 3.2)
H-1ꢀ
H-2R
H-2ꢀ
H-3R
H-5ꢀ
H-6R
H-6ꢀ
7
0.95, m
2.15, tt (13.8, 6.0)
0.88, ddd (16.6, 11.0, 3.5) 0.70, td (13.4, 5.1)
1.96, tt (11.0, 5.7)
1.80, m
0.77, dt (9.2, 5.7)
1.59, dd (11.8, 4.7)
2.28, m
2.55, dm (17.6)
7.19, m
1.87, tt (13.4, 5.4)
1.69, dd (13.4, 3.2)
0.66, m
1.64, dd (12.6, 5.1)
2.08, tm (12.6)
2.28, dm (12.6)
5.99, m (W1/2 ) 11.0)
1.83, ddd (13.3, 5.2, 1.6) 1.95, dd (13.8, 6.2)
0.79, dd (8.9, 5.1)
1.65, dd (11.3, 5.6)
2.36, m
0.76, dt (9.2, 6.0)
1.76, br, d (13.9)
1.79, td (13.8, 4.4)
2.55, ddd (11.2, 5.6, 2.4) 2.25, m
2.59, dddd (18.2, 5.7, 3.6, 1.6) H-7R 6.97, dt (5.3, 2.4)
4.72, br, s H-7R
2.30, m H-7ꢀ
H-9ꢀ
2.40, dd (7.5, 2.7)
2.58, ddd (10.7, 7.1, 2.7) 3.27, dd (11.4, 6.4)
1.67, m 2.29, m
2.23, br, s
2.28, dd (13.1, 3.9)
1.09, td (13.1, 1.4)
1.94, dt (13.1, 3.9)
H-11R
H-11ꢀ
H-12R
H-13R
H-14R
15
3.21, ddd (15.8, 6.5, 1.6)
2.21, m
5.11, ddq (10.4, 6.5, 2.3)
1.41, dd (14.6, 3.7)
2.37, dm (14.6)
4.98, ddq (11.3, 7.1, 2.2) 5.11, br, ddq (10.1, 6.4, 2.3) 5.10, ddd (8.4, 3.7, 2.3) 4.13 overlapped
2.98, dd (8.4, 7.3)
2.23, dt (6.6, 4.3)
4.65, d (4.3)
2.72, quint (6.6)
H-15R
1.35, d (6.6)
0.08, dd (5.4, 4.5)
0.38, dd (9.1, 4.5)
0.98, s
2.82, quint (7.3)
H-15ꢀ
1.43, d (7.3)
0.17, dd (5.7, 4.5)
0.50, dd (9.2, 4.5)
1.06, s
H-17
2.22, d (2.3)
2.24, d (2.2)
0.16, dd (5.1, 4.6)
0.51, dd (8.9, 4.6)
1.06, s
2.25, d (2.3)
0.19, dd (6.0, 4.9)
0.61, dd (9.2, 4.9)
1.11, s
H-18 endo 0.08, dd (6.0, 4.4)
H-18 exo 0.59, dd (9.3, 4.4)
H-19
H-20
1.10, s
1.20, s
0.91, s
1.20, s
0.75, s
0.67, s
a Spectra were recorded in CDCl3. c Spectrum was recorded in CDCl3 + CD3OD.
Table 3. 13C NMR Data (δ) for 2-5a and 6b
indicated by HMBC correlations of H-2′ and H-3′ with carbonyl
C-1′. NOE interactions H-2′/H-4′ and H-3′/H-5′ indicated a trans
configuration of the double bonds. COSY and HSQC experiments
allowed assignments of protons and carbons to a 2′E,4′E-decadi-
enoyl ester. In the HMBC spectrum, H-3 (δH 4.70, dd, J ) 10.9,
4.7 Hz) correlated with the carbonyl group C-1′ (δ 167.2), implying
that the ester moiety was connected to C-3 of 24-methylenecy-
cloartanol. Therefore, the structure of 8 was determined to be 24-
methylenecycloartanyl 2′E,4′E-decadienoate.
atom
2
3
4
5
6
1
2
3
4
5
6
7
8
30.4
19.3
18.5
16.4
47.6
20.6
24.4
134.6
160.5
38.9
34.2
78.8
150.6
185.7
131.1
172.8
9.8
31.0
19.1
19.9
14.7
44.8
27.5
140.4
136.8
41.6
34.3
27.2
77.9
151.1
187.5
132.5
173.5
10.0
20.5
24.5
11.5
29.8
19.2
18.4
16.6
41.6
28.9
62.5
135.7
164.9
39.8
33.9
78.4
149.9
187.0
132.8
173.3
9.9
31.4
19.2
19.9
14.8
44.1
27.6
139.9
134.8
40.6
35.2
27.7
76.7
52.9
196.2
40.0
178.2
16.2
20.4
24.7
12.4
30.9
18.9
19.9
14.7
44.4
26.7
133.0
131.3
37.4
32.2
30.6
63.8
44.4
83.3
39.7
181.7
8.8
Compound 9 had a quasi-molecular ion [M + Na]+ at m/z
599.4792 (HRESIMS), which corresponded to the molecular
formula C40H64O2. Comparison of 1H and 13C NMR spectra (Table
9
10
11
12
13
14
15
16
17
18
19
20
1
4) suggested that 9 had the same ester moiety as 8. The H NMR
spectrum displayed signals due to two olefinic protons, a terminal
isopropylidene group, a secondary methyl, five tertiary methyl
groups, and an oxymethine. The 13C NMR exhibited resonances
typical of a tetracyclic skeleton possessing double bonds ∆7(8),24(25)
at δ 117.6 (C-7), 145.6 (C-8), 125.2 (C-24), and 130.9 (C-25).15
The COSY, HSQC, and HMBC experiments indicated that 9 was
a euphane- or tirucallane-type triterpenoid differing in configuration
at C-20 (20R/euphane30 and 20S/tirucallane31). Characteristic
NOESY interactions were detected between H3-30 (14ꢀ-Me) and
H-17ꢀ and between H3-21 (20-Me) and H-12R. These correlations
were consistent with those of tirucallane-type triterpenes.19,32
Absence of an NOE effect between H3-21/H-16 typical of euphane
compounds33 and the chemical shift of protons H3-21 at δ 0.94
confirmed that 9 belonged to the tirucallane rather than the euphane
series.19,32 Alkaline hydrolysis of 9 afforded tirucalla-7,24-dien-
3ꢀ-ol.34 These data led to characterization of 9 as tirucalla-7,24-
dien-3ꢀ-yl 2′E,4′E-decadienoate.
22.3
23.2
16.8
22.2
23.2
15.7
20.1
24.3
12.8
a Spectra were recorded in CDCl3. b Spectrum was recorded in CDCl3
+ CD3OD.
complete assignment of all protons and carbons. The relative
configuration of 7 was deduced from the NOESY spectrum and
conformed to that reported for 24-methylenecycloartanol. Alkaline
hydrolysis of 7 yielded 24-methylenecycloartanol, which was
determined by the 1H NMR spectrum and the value of [R]D.15 Thus,
the structure of 7 was established as 24-methylenecycloartanyl
formate.
Compound 8, a colorless gum, exhibited a quasi-molecular ion
[M + Na]+ at m/z 613 4971 in the HRESIMS, consistent with the
molecular formula C41H66O2. The 1H and 13C NMR shifts of 8 were
close to those of 7 (Table 4), suggesting that 8 was also a derivative
of 24-methylenecycloartanol. Alkaline hydrolysis of 8 gave 24-
methylenecycloartanol.15 The residue was a C10H15O unit. The 1H
and 13C NMR spectra of 8 displayed signals of an acyl ester moiety
including four olefinic methines at δH 5.86 (d, J ) 15.3 Hz, H-2′)
and δC 119.8 (C-2′), δH 7.29 (dd, J ) 15.3, 10.1 Hz, H-3′) and δC
144.7 (C-3′), δH 6.21 (dd, J ) 15.3, 10.1 Hz, H-4′) and δC 128.3
(C-4′), and δH 6.19 (dd, J ) 15.3, 7.0 Hz, H-5′) and δC 144.5 (C-
5′). These signals formed a conjugated 1,3-diene system, as
The phytochemical study of E. retusa resulted in the isolation
and characterization of ent-abietane-type diterpenes and tetracyclic
triterpenes with cycloartane, lanostane, and tirucallane skeletons.
Related ent-abietane lactones have been reported previously in this
genus.8,13 However, the six new diterpenoids (1-6), named
retusolide A-F, belong to the rare class of ent-abietane-type
diterpenes containing a cyclopropane ring bridging C-3 and C-4
of the basic abietane skeleton20,21 and illustrate the interesting
chemodiversity of this species. In this plant, compounds having
ketone functions at C-14 were found (1-5). Compound 6 is the
first example of a rearranged ent-abietane lactone isolated from the
plant kingdom. The three esterified tetracyclic triterpenes (7-9)

1262 Journal of Natural Products, 2009, Vol. 72, No. 7
Haba et al.
Table 4. 1H and 13C NMR Data for 7, 8, and 9 (in CDCl3)
7
8
9
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3R
4
5R
6
7
8ꢀ
9R
10
11
1.33-1.70, m
31.5
26.9
80.8
39.4
47.1
20.9
25.78
47.8
20.2
25.79
26.4
1.31-1.70, m
1.71-1.86, m
31.6
26.9
80.3
39.6
47.1
20.9
25.8
47.8
20.1
25.9
26.5
1.30-1.74, m
1.72-1.78, m
36.8
24.2
80.7
38.0
50.7
23.7
117.6
145.6
48.8
34.8
18.1
1.75-1.85, m
4.75, dd (11.5, 4.7)
4.70, dd (10.9, 4.7)
4.65, dd (11.1, 4.1)
1.46, dd (12.2, 4.3)
0.87-1.64, m
1.13-1.39, m
1.57, dd (12.2, 4.8)
1.48, dd (12.5, 5.0)
0.86-1.64, m
1.15-1.38, m
1.57, dd (12.5, 4.3)
1.49, dd (13.0, 6.5)
2.01-2.19, m
5.30, br, q (2.7)
2.29, m W1/2 ) 25
1.56, m
1.17, m H-11ꢀ
2.05, m H-11R
1.69, m
1.18, m H-11ꢀ
2.05, dt (16.0, 9.0) H-11R
1.69, br, t (9.0)
12
13
14
15
16
17
18
19
32.8
45.2
48.8
35.5
28.1
52.2
17.9
29.7
32.5
45.2
48.8
35.5
28.1
52.2
17.9
29.8
1.67-1.83, m
33.7
43.5
51.1
33.9
28.2
52.9
21.8
13.1
1.34, m
1.37, m
1.52, m
1.33-1.97, m
1.66, m H-17R
1.02, s
0.64, br, d (4.1) H-19 endo
0.41, d (4.1) H-19 exo
1.45, m
1.33-1.98, m
1.67, br, t (12.0) H-17R
1.03, s
0.64, d (3.8) H-19 endo
0.41, d (3.8) H-19 exo
1.46, m
0.96, d (6.7)
1.20-1.64, m
2.17, m
1.35-1.98, m
1.52, q (10.0) H-17ꢀ
0.86, s
0.83, s
20
21
22
23a
23b
24
25
26
27
28
29
30
31a
31b
1′
36.1
18.3
34.9
31.3
36.1
18.2
34.9
31.2
1.44, m
35.9
18.3
36.1
24.9
0.94, d (7.1)
0.94, d (6.5)
1.09-1.50, m
2.10, m
1.92, m
5.16, t (6.6)
1.22-1.62, m
2.18, ddd (15.0, 10.5, 4.5)
1.94, m
1.93, m
156.9
33.7
21.9
21.8
25.3
15.1
19.3
105.9
156.9
33.8
21.9
21.8
25.4
13.9
19.3
105.9
125.2
130.9
25.7
17.6
27.6
15.9
27.2
2.29, sept (6.8)
1.09, d (6.8)
1.08, d (6.8)
0.93, s
0.96, s
0.95, s
4.76, br, s
4.72, br, s
8.18, s
2.29, sept (6.7)
1.09, d (6.7)
1.08, d (6.7)
0.92, s
0.99, s
0.96, s
1.74, s
1.66, s
0.91, s
1.01, s
1.02, s
4.77, br, s
4.72, br, s
161.2
167.2
119.8
144.7
128.3
144.5
32.9
28.4
31.3
22.4
14.0
167.1
119.7
144.7
128.3
144.5
32.9
28.4
31.3
22.4
14.0
2′
5.86, d (15.3)
7.29, dd (15.3, 10.1)
6.21, dd (15.3, 10.1)
6.19, dd (15.3, 7.0)
2.22, q (7.0)
1.49, m
1.35, m
1.37, m
0.93, t (6.6)
5.86, d (15.2)
7.29, dd (15.2, 10.0)
6.21, dd (15.2, 10.0)
6.19, dd (15.2, 6.9)
2.21, q (6.9)
1.47, m
1.33, m
1.36, m
0.95, t (6.5)
3′
4′
5′
6′
7′
8′
9′
10′
Oudjehih, Agronomic Department of the University of Batna. A voucher
specimen has been deposited in the herbarium of the Agronomic
Department under reference LCCE/373.
possessing a cycloartane or tirucallane genin are used as chemot-
axonomic markers of the genus Euphorbia.35
Experimental Section
Extraction and Isolation. Powdered roots (600 g) of E. retusa were
extracted with CH2Cl2 (2 × 5 L) at room temperature during 3 days to
obtain a crude extract (10 g). A portion of the extract (3 g) was subjected
to silica gel vacuum liquid chromatography (VLC) (50 × 50 mm;
fractions of 100 mL) using a gradient of n-hexane/EtOAc (100:0 to
0:100). Fractions having similar TLC profiles were pooled to give nine
fractions. Fraction 2 was subjected to silica gel CC using n-hexane/
EtOAc (100:0 to 0:100) as eluent to afford 17 fractions. Fractions eluted
with n-hexane/EtOAc (98:2) gave 60 mg of 24-methylenecycloartanol
in pure form. Preparative TLC of fractions eluted with n-hexane/EtOAc
(99.5:0.5), developed with a mixture of cyclohexane/toluene/EtOAc
(18:1.5:0.5), allowed isolation of compounds 7 (6.3 mg), 8 (5.1 mg),
and 9 (6.8 mg). Fractions eluted with n-hexane/EtOAc (99:1) were
separated by silica gel CC using a gradient of n-hexane/CHCl3 (100:0
to 90:10). Fractions eluted with n-hexane/CHCl3 (97:3) provided 24-
methylenecycloartanone (7.5 mg). Preparative TLC of fractions eluted
with n-hexane/EtOAc (97:3), developed with cyclohexane/EtOAc (85:
15), afforded a mixture of two compounds, cycloeucalenol and
obtusifoliol (10.6 mg). The fraction F-3 was subjected to silica gel CC
eluting with cyclohexane/EtOAc (100:0 to 90:10) to afford 12 fractions.
Fractions eluted with cyclohexane/EtOAc (99:1) were purified using
silica gel CC and elution with n-hexane/EtOAc (98:2), which yielded
jolkinolide E (13.5 mg). Fractions eluted with cyclohexane/EtOAc (98:
General Experimental Procedures. The optical rotations were
measured on a Perkin-Elmer 241 polarimeter. UV spectra were obtained
using a Kontron UVS900 lite, Uvikon 941 spectrophotometer. IR
1
spectra were measured on an Avatar 320 FT-IR spectrometer. H and
13C NMR spectra were recorded on a Bruker Avance DRX 500 NMR
spectrometer in CDCl3, CD3OD, or DMSO-d6 (1H at 500 MHz and
13C at 125 MHz). 2D NMR experiments were performed using standard
Bruker microprograms (XWIN-NMR version 2.6 software). EIMS and
HREIMS were recorded using a GCT Micromass apparatus. ESIMS
were obtained using a MSQ Thermofinnigan instrument. HRESIMS
experiments were recorded using a Micromass Q-TOF instrument.
Column chromatography (CC) was carried out on Kieselgel 60 (70-230
mesh, Merck) or LiChroprep RP-18 (40-63 µm, Merck). HPLC was
performed on a Dionex apparatus equipped with an ASI-100 autosam-
pler, a P580 pump, a diode array detector UVD 340S, and Chromeleon
software. An Interchim column (UP5ODB.25M, 250 × 10 mm, 5 µm)
was used for semipreparative HPLC using isocratic elution (MeCN/
H2O, 4:1) at 25 °C and a flow rate of 5 mL/min; the chromatograms
were monitored at 205, 225, 254, and 280 nm. TLC was carried out in
silica gel plates (Kieselgel 60 F254 Merck).
Plant Material. Roots of E. retusa were collected during May 2005
in the vicinity of Biskra (Algeria). The plant was identified by Pr. Bachir

Diterpenoids and Triterpenoids from Euphorbia retusa
Journal of Natural Products, 2009, Vol. 72, No. 7 1263
2) were purified by semipreparative HPLC using isocratic elution
(MeCN/H2O, 4:1), yielding 4.5 and 4.3 mg of pure compounds 2 and
3, respectively. Fractions F-4 and F-5 were mixed and applied to silica
gel CC eluting with n-heptane/EtOAc (100:0 to 80:20) to give 19
fractions. Fractions eluted with n-heptane/EtOAc (95:5) were purified
using silica gel CC and elution with CH2Cl2/EtOH (99.3:0.7), to provide
6.8 mg of cycloart-25-ene-3ꢀ,24-diol. Fractions eluted with n-heptane/
EtOAc (93:7) were submitted to silica gel CC using a gradient of
cyclohexane/EtOAc (100:0 to 80:20) to afford seven fractions. Purifica-
tion of fractions eluted with cyclohexane/EtOAc (95:5) by semiprepara-
tive HPLC eluting with an isocratic system (MeCN/H2O, 4:1) yielded
7.6 mg of compound 6. Fractions eluted with n-heptane/EtOAc (90:
10) were subjected to reversed-phase (RP-18) CC, using a gradient of
MeOH/H2O (60:40 to 100:0) as eluent, to provide compounds 1 (5.4
mg) and 4 (3.3 mg). Original fraction 6 was submitted to silica gel CC
eluting with cyclohexane/EtOAc (100:0 to 50:50) to obtain 10 fractions.
Fractions eluted with cyclohexane/EtOAc (90:10) were further purified
on RP-18 CC, with MeOH/H2O (60:40 to 100:0), to give compound 5
(3.6 mg). Original fraction 7 was applied to RP-18 CC eluting with
MeOH/H2O (40:60 to 100:0) to afford eight fractions. Fractions eluted
with MeOH/H2O (70:30) were purified by silica gel CC eluting with a
gradient of cyclohexane/EtOAc (100:0 to 70:30). Fractions eluted with
cyclohexane/EtOAc (90:10) contained 4.2 mg of helioscopinolide E.
Alkaline Hydrolysis. Each esterified triterpene, 7 (6.3 mg), 8 (5.1
mg), and 9 (6.8 mg), dissolved in CHCl3 (15 mL) was hydrolyzed
separately with 5% alcoholic KOH for 5 h at room temperature. The
reaction mixtures were exhaustively extracted with ethyl acetate (3 ×
20 mL). The EtOAc solubles were dried with anhydrous Na2SO4,
filtered, and evaporated in vacuo to give three fractions. After CC of
each fraction on silica gel, eluting with n-hexane and EtOAc (9:1), the
corresponding free alcohols were obtained.
262 (0.56), 203 (1.20) nm; IR (CHCl3) λmax 2954, 2930, 2864, 1718,
1641, 1615,1460, 1376, 1244, 1145, 984 cm-1 1H and 13C NMR
;
(CDCl3), see Table 4; ESIMS m/z 613 [M + Na]+; HRESIMS m/z
613.4971 (calcd for C41H66O2Na, 613.4961).
Tirucalla-7,24-dien-3ꢀ-yl 2′E,4′E-decadienoate (9): colorless gum;
[R]25D -9.4 (c 0.37, CHCl3); UV (MeOH) λmax (log ε) 281 (0.30), 251
(0.50), 207 (1.24) nm; IR (CHCl3) λmax 2952, 2929, 2862, 1712, 1642,
1
1617, 1458, 1375, 1247, 1140, 990 cm-1; H and 13C NMR (CDCl3),
see Table 4; ESIMS m/z 599 [M + Na]+, 615 [M + K]+; HRESIMS
m/z 599.4792 (calcd for C40H64O2Na, 599.4804).
Acknowledgment. We wish to thank the Ministry of Higher
Education and Scientific Research (Algeria) for financial support. The
authors thank Dr. D. Harakat, P. Sigaut, and D. Patigny, Service de
Spectrome´trie de Masse, UMR CNRS 6229, Institut de Chimie
Mole´culaire de Reims (France), for performing the MS spectra.
Supporting Information Available: 1H NMR and 13C NMR spectra
of new compounds 1-9 and their tables with a full listing of 1H NMR,
COSY, HMBC, and NOESY spectroscopic data are available free of
charge via the Internet at http://pubs.acs.org.
References and Notes
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Retusolide A (1): colorless oil; [R]25 +29.5 (c 0.35, CHCl3); UV
D
(MeOH) λmax (log ε) 241 (0.64), 204 (0.53) nm; IR (CHCl3) λmax 3451,
1
2925, 2863, 1765, 1685, 1654, 1615, 1221, 1092, 1020 cm-1; H and
13C NMR (CDCl3 and DMSO-d6), see Table 1; EIMS m/z 330 [M]+
(10), 312 (40), 244 (43), 177 (100); HREIMS m/z 330.1819 (calcd for
C20H26O4, 330.1831).
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(8) Lee, C.-L.; Chang, F.-R.; Hseih, P.-W.; Chiang, M.-Y.; Wu, C.-C.;
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λ
max 2928, 2860, 1760, 1682, 1650, 1620, 1381, 1230, 1106, 1050 cm-1
;
1H and 13C NMR (CDCl3), see Tables 2 and 3; EIMS m/z 312 [M]+
(30), 297 (15), 223 (60), 205 (50), 148 (100); HREIMS m/z 312.1723
(calcd for C20H24O3, 312.1725).
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CHCl3); UV (MeOH) λmax (log ε) 256 (1.22), 206 (1.04) nm; IR (KBr)
¨
(12) Harraz, F. M.; Gu¨rek, F.; Oksu¨z, S.; Ulubelen, A. Turk. J. Chem. 1994,
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max 2926, 2858, 1765, 1658, 1618, 1322, 1219, 1096, 1018 cm-1; 1H
18, 251–257.
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297 (20), 257 (30), 223 (25), 205 (20), 148 (100); HREIMS m/z
312.1716 (calcd for C20H24O3, 312.1725).
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(MeOH) λmax (log ε) 275 (0.38), 206 (1.05) nm; IR (CHCl3) λmax 3438,
2926, 2865, 1767, 1675, 1645, 1384, 1329, 1258, 1158, 1125, 1079,
1013 cm-1; 1H and 13C NMR (CDCl3), see Tables 2 and 3; EIMS m/z
328 [M]+ (6), 310 (80), 265 (100), 267 (65), 242 (70), 227 (50), 149
(85); HREIMS m/z 328.1682 (calcd for C20H24O4, 328.1675).
¨
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Retusolide E (5): white, amorphous solid; [R]25 +9.2 (c 0.08,
D
CHCl3); UV (MeOH) λmax (log ε) 248 (0.36), 204 (0.60) nm; IR (KBr)
λmax 2923, 2853, 1777, 1665, 1635, 1580, 1449, 1380, 1250, 1080,
1010 cm-1; 1H and 13C NMR (CDCl3), see Tables 2 and 3; EIMS m/z
314 [M]+ (20), 299 (10), 279 (50), 167 (25), 149 (100); HREIMS m/z
314.1889 (calcd for C20H26O3, 314.1882).
Retusolide F (6): colorless oil; [R]25 -44.8 (c 0.24, CHCl3); UV
D
(MeOH) λmax (log ε) 281 (0.14), 241 (0.33), 205 (0.78) nm; IR (CHCl3)
λ
max 3450, 2926, 2850, 1757, 1625, 1453, 1381, 1184, 1018 cm-1; 1H
and 13C NMR (CDCl3 + CD3OD), see Tables 2 and 3; EIMS m/z 316
[M]+ (5), 298 (30), 266 (20), 159 (65), 121 (60), 107 (100); HREIMS
m/z 316.2015, (calcd for C20H28O3, 316.2038).
24-Methylenecycloartanyl formate (7): white, amorphous solid;
[R]25 +33.6 (c 0.36, CHCl3); UV (MeOH) λmax (log ε) 203 (0.60)
D
nm; IR (KBr) λmax 2928, 2865, 1727, 1641, 1465, 1376, 1213, 1198,
1
1175, 1040 cm-1; H and 13C NMR (CDCl3), see Table 4; EIMS m/z
468 [M]+ (2), 396 (20), 298 (20), 284 (100), 175 (10), 174 (15);
HREIMS m/z 368.3970 (calcd for C32H52O2, 468.3967).
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24-Methylenecycloartanyl 2′E,4′E-decadienoate (8): colorless
gum; [R]25D +32.4 (c 0.32, CHCl3); UV (MeOH) λmax (log ε) 280 (0.34),

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