Oxygenated diterpenes and other constituents from Moroccan Juniperus phoenicea and Juniperus thurifera var. africana

Article abstract of DOI:10.1016/j.phytochem.2004.07.021

Six new diterpenic acids isolated as their methyl ester together the isovalerate derivatives of p-methoxycinnamyl alcohol and 8-hydroxy-linalool, were isolated from the leaves of Juniperus thurifera and Juniperus phoenicea, grown in Morocco. The cytotoxicity of the abietane diterpenoids was tested against five cell lines. Six new diterpenic acids isolated as their methyl ester derivatives, i.e., methyl 12-oxo-8α,15-dihydroxyabiet-13-en-19-oate, methyl 12-oxo-8α-hydroxyabiet-13-en-19-oate, methyl 15-hydroperoxy- 8α,12α-epidioxiabiet-13-en-19-oate, methyl 15-hydroxy-8α, 12α-epidioxiabiet-13-en-19-oate, methyl 15-hydroperoxy-8α,14α, 12α,13α-diepoxiabietan-13-en-19-oate, and methyl 7α,12α-dihydroxysandaracopimarate, together with two new isovalerate derivatives of p-methoxycinnamyl alcohol and linalool, were isolated from the leaves of Juniperus thurifera var. africana and Juniperus phoenicea, grown in Morocco. The structures of these compounds were established by using spectroscopic techniques, including 2D NMR spectra. The cytotoxicity of the abietane diterpenoids was tested against five cell lines.

Full text of DOI:10.1016/j.phytochem.2004.07.021

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2507–2515
www.elsevier.com/locate/phytochem
Oxygenated diterpenes and other constituents from Moroccan
Juniperus phoenicea and Juniperus thurifera var. africana
a,
Alejandro F. Barrero , Jos e´ F. Qu ´ı lez del Moral , M. Mar Herrador ,
a
a
*
b
Mohamed Akssira , Ahmed Bennamara , Said Akkad , Mohamed Aitigri
b
b
b
a
Departamento de Qu ´ı mica Org a´ nica, Instituto de Biotecnolog ´ı a, Facultad de Ciencias, Universidad de Granada,
Campus Fuentenueva s/n, 18071-Granada, Spain
Laboratoire de Chimie Biorganique et Analytique, F.S.T., Universit e´ Hassan II-Mohammedia, BP146 20800 Mohammedia, Morocco
b
Received 18 May 2004; received in revised form 15 July 2004
Abstract
Six new diterpenic acids isolated as their methyl ester derivatives, i.e., methyl 12-oxo-8a,15-dihydroxyabiet-13-en-19-oate, methyl
2-oxo-8a-hydroxyabiet-13-en-19-oate, methyl 15-hydroperoxy-8a,12a-epidioxiabiet-13-en-19-oate, methyl 15-hydroxy-8a,12a-
1
epidioxiabiet-13-en-19-oate, methyl 15-hydroperoxy-8a,14a,12a,13a-diepoxiabietan-13-en-19-oate, and methyl 7a,12b-dihydroxy-
sandaracopimarate, together with two new isovalerate derivatives of p-methoxycinnamyl alcohol and linalool, were isolated from
the leaves of Juniperus thurifera var. africana and Juniperus phoenicea, grown in Morocco. The structures of these compounds were
established by using spectroscopic techniques, including 2D NMR spectra. The cytotoxicity of the abietane diterpenoids was tested
against five cell lines.
ꢀ
2004 Elsevier Ltd. All rights reserved.
Keywords: Juniperus thurifera; Juniperus phoenicea; Cupressaceae; Diterpenoids; Cytotoxic activity
1
. Introduction
species of J. phoenicea have already been studied, this
re-evaluation is directed to a comparison of how the
geographical situation affects the production of sec-
ondary metabolites. The mixture of the leaves and
cones of J. phoenicea is used as an oral hypoglycemic,
whereas the leaves are used against bronchopulmonary
diseases and as a diuretic (Bellakhder, 1997). The
essential oils of the leaves and cones have been proven
to possess antimicrobial activity (Stassi et al., 1996).
The essential oil of the wood J. thurifera var. africana
has been used as an abortive and menstruation regula-
tor (Bellakhder, 1997), whereas the wood tar is used in
veterinary science. The hexane and chloroform ex-
tracts of the leaves of European J. thurifera have been
tested on neoplastic KB cells, and show considerable
cytostatic activity (San Feliciano et al., 1989). Both
plants have been shown to accumulate terpenoids,
Continuing our studies on the chemical composition
of Spanish and Northern Moroccan plants with the
aim of finding both new natural compounds with
interesting biological activities and also investigating
the occurrence of natural terpenoids which could be
used as intermediates for the synthesis of added-value
compounds, we present herein the study of two Juni-
perus species growing in Morocco. On one hand, Juni-
perus thurifera L. var. africana Maire (Cupressaceae),
a tree endemic to North Africa, and on the other
hand, Juniperus phoenicea L. (Cupressaceae). Since
*
Corresponding author. Tel./fax: +34 958 243318.
E-mail address: afbarre@ugr.es (A.F. Barrero).
0
031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.07.021

2508
A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
1
6
5
phenylpropanoids and lignans (Hussein et al., 2003;
Comte et al., 1997; San Feliciano et al., 1991,
O
2
R
R
1
13
1
20
11
17
O
1
993a–c 1986, 1988; Cairnes et al., 1980; Barrero
1
O
1
4
2
3
9
6
1
0
8
et al., 2000) we have not found any article describing
the composition of the African trees. We report herein
the isolation of five new abietane diterpenoids (1–5), a
new pimarane derivative (6), and two new isovalerate
derivatives (7–8) from the hexane extracts of the leaves
of J. phoenicea and J. thurifera var. africana. We also
report the in vitro cytotoxicity of the new abietane
derivatives.
OH
4
7
5
COOMe
COOMe
18
1
9
R = OH
R = H
1
2
R = OOH
R = OH
3
4
OH
O
O
OOH
OH
MeOOC
COOMe
5
2
. Results and discussion
6
OMe
Workup of the acidic part of the hexane extract
OH
from the leaves of J. phoenicea led, after esterification
with diazomethane, to the isolation of three new abie-
tanes and one new pimarane diterpenoids as their
methyl esters (1–2, 4 and 6, respectively). Two new
abietane acid derivatives, also isolated after esterifica-
tion with diazomethane (3 and 5), together with the
two new lynaloyl and cinnamyl isovalerates (7–8) were
isolated from the hexane extract of the leaves of J.
thurifera var. africana. In addition to the new
compounds, a number a known compounds were also
identified from these species; abietic (9) (Smith, 1978),
O
O
O
O
8
7
R
R1
4
-epi-abietic acid (10) (Tabacik and Laporthe, 1971),
abietinal (15) (Erdtman and Westfelt, 1963), dehydroa-
bietinal (16) (Fetizon et al., 1968), abietinol (19) (San
Feliciano et al., 1993a), 15-hydroxy-9a,13a-epidioxya-
biet-8(14)-en-18-oic acid (20) (Sy and Brown, 1988),
cis-(11) (Barrero et al., 1987) and trans-communic
CHO
COOH
11 R=CH , R =CH=CH
2
R
R1
R=CH3, R1= COOH
9
16
3
1
10 R=COOH, R1= CH3
R=CH3, R1= CHO
19 R=CH , R = CH OH
12 R=CH=CH2, R1=CH3
1
5
3
1
2
OH
O
O
(
12) acids (Lee et al., 1987), sandaraco (13) (Comte
et al., 1995), and isopimaric (14) acids (Antkowiak
et al., 1962), pimara-8(14),15-dien-18-ol (17) (Wenkert
and Buckwalter, 1972), pimara-7,15-dien-18-ol (18)
R1
R1
R
R
COOH
1
3
R=COOH, R1=H
17 R=CH2OH, R1=H
R=COOH, R1=OH
14 R=COOH, R1=H
18 R=CH2OH, R1=H
22 R=COOH, R1=OH
20
2
1
(
Wenkert and Buckwalter, 1972), 3b-hydroxysandara-
23 R=CH OH, R =OH
2 1
24 R=CH OH, R =OH
2
1
copimaric acid (21) (Doi and Kawamura, 1972), 3b-
hydroxyisopimaric acid (22) (Bruno et al., 1986),
pimara-8(14),15-dien-3b,18-diol (23) (San Feliciano
et al., 1988), pimara-7,15-dien-3b,18-diol (24) (San
Feliciano et al., 1988), linaloyl acetate (25) (Kaiser
and Lamparsky, 1977), phytol (26) (Sims and Pettus,
R
O
OAc
O
OH
R1
26
R2
2
2
8
9
R=H, R1=H, R2= H
R=H, R1=OMe, R2=OMe
30 R=OMe, R1=OMe, R2= OMe
O
2
5
27
O
R
O
R
O
OH
O
O
1
976), (E,Z)-2,4-decadienyl isovalerate (27) (Rickards
0
O
8
O
0
O
and Weiler, 1978), cinnamyl isovalerate (28), 3 ,4 -
dimethoxycinnamyl isovalerate (29) (San Feliciano
OH
O
32 R=H
MeO
OMe
MeO
OMe
OMe
0
0
0
33 R=OAc
34 R=OH
OMe
et al., 1986), 3 ,4 ,5 -trimethoxycinnamyl isovalerate
0
OMe
OMe
3
5
36 R=OH, 8(R)
37 R=H, 8(R)
8 R=OH, 8(S)
0
(
30) (San Feliciano et al., 1986), 3 ,4 -dimethoxycinna-
31
3
mol (31) (San Feliciano et al., 1986), elemol (32), 8a-
acetoxyelemol (33) (De Pascual-Teresa et al., 1977),
8a,11-elemdiol (34) (De Pascual-Teresa et al., 1977),
yatein (35) (Harmatha et al., 1982), podophyllotoxin
(
(
36) (Andrews et al., 1988), deoxypodophyllotoxin
37) (San Feliciano et al., 1990), and picropodophyllo-
Compound 1 exhibited the molecular formula
C H O , as deduced from its HRFABMS
2
1
32
5
+
([M + Na] , m/z 387.2148). The IR spectrum showed
toxin (38) (Fonseca et al., 1980).

A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
2509
ꢀ
1
absorption bands at mmax 3399 cm indicating a hyd-
were performed (Fig. 1). The configuration of C-4 and
C-8 were assigned based upon the enhancements ob-
ꢀ1
roxyl group and at 1724 and 1659 cm , attributable
to an ester group and to an a,b-unsaturated carbonyl
group, respectively. These carbonyl assignments were
supported by C NMR signals at d 201.7 and 177.4.
In the H NMR spectrum (Table 1), four tertiary methyl
served at dH 6.59 (H-14), and at dH 3.60 (CH OCO)
3
when Me-20 (dH 0.47) was irradiated. Furthermore,
the very high field chemical shift observed for Me-20
1
3
1
1
in the H NMR spectrum (d 0.47) was explained on
signals at d 0.47 (3H), 1.21 (3H), and 1.40 (6H), the lat-
ter ones attached to an oxygenated carbon, an oxygen-
ated methyl signal (d 3.60), and an olefinic proton
the basis of the shielding effect exerted towards this
methyl by both, the double bond at C-13 and the meth-
oxycarbonyl group at C-19.
1
3
signal (d 6.59) were observed. The C NMR spectrum
Table 2) showed 21 resonances and confirmed the pres-
The HRFABMS of compound 2 showed a signal at
m/z 371.2195, indicating a molecular formula of
(
1
13
ence of a trisubstituted double bond (d 145.8 and 146.3).
The presence of two quaternary oxygenated carbons (d
C H O . The H and C NMR spectra of this product
21 32 4
were very similar to those of 1 (Tables 1 and 2), the only
difference being the lack of the oxygenated function at
1
C-15 in 2. The H NMR signals corresponding to the
6
8.6 and 71.9) was also indicated. The combined analy-
sis of the HETCOR, COSY, HMBC spectra, and NOE-
DIFF experiments, permitted the assignment of an
abietane skeleton for this compound (Mei et al., 2002;
Lee and Cheng, 2001; Ohtsu et al., 2001): methyl 12-
oxo-8a,15-dihydroxyabiet-13-en-19-oate. The location
of the a,b-unsaturated ketone in 1 was established
according to its HMBC spectrum, in which cross-peaks
isopropyl group appeared at dH 1.01 (3H, d, J = 6.9
Hz), 1.05 (3H, d, J = 6.9 Hz) and 2.87 (1H, hp, J = 6.9
Hz). A new methine was observed at d 26.3 in the
C
1
3
C NMR spectrum. Accordingly, the structure of 2
was determined to be methyl 12-oxo-8a-hydroxyabiet-
13-en-19-oate. An epimer of 2 at C-4 was isolated form
Pinus sylvestris needles (Buratti et al., 1990).
were observed between dH 2.17 (H-7) and d 146.3 (C-
C
1
4), and between dH 6.59 (H-14) and dC 71.9 (C-15).
The long-range correlations of dC 68.6 (C-8) with dH
.59 (H-14), d 2.50 (H-11), and d 2.00 (H-6) indicated
The molecular formula of compound 3 was deduced
+
as C H O from its HRMS ([M + Na] , m/z
21
32
6
1
403.2093). Its H NMR spectrum was again similar to
that of ester 1, revealing – as in 1 – the presence of a
6
H
H
that the tertiary alcohol was at the C-8 position. Finally,
the location of the methoxycarbonyl group at the C-4
position was confirmed by the cross-peaks observed be-
tween d 177.4 (C-19) and d 1.21 (Me-18), and d 1.01
1
3
D -abietane skeleton supporting a methyl ester on a
quaternary carbon (dH 3.63, s, 3H) and an oxygenated
function at C-15 (d 1.34, s, 3H; d 1.45, s, 3H). Besides,
C
H
H
H
H
(H-3). In order to establish the relative configuration of
the stereogenic centers, different NOEDIFF experiments
a new secondary oxygenated carbon was observed (d
H
13
4.92, m). After comparing the C NMR data of
Table 1
1
H NMR data for compounds 1–5 (d in ppm, J in Hz)
Proton
1
1
2
3
4
5
a: 0.96–1.08 (m)
b: 1.81–1.88 (m)
a: 1.43–1.52 (m)
b: 1.76 (btt, 3.4, 13.9)
a: 0.96–1.08 (m)
b: 2.16–2.24 (m)
1.81–1.88 (m)
a: 1.95–2.03 (m)
b: 1.51–1.66 (m)
a: 2.17 (b dd, 3.5, 13.2)
b: 1.51–1.66 (m)
1.81–1.88 (m)
a: 0.85–1.10 (m)
b: 1.80–1.87 (m)
a: 1.42–1.52 (m)
b: 1.75 (btt, 3.6, 14.0)
a: 0.85–1.10 (m)
b: 2.16–2.24 (m)
1.80–1.87 (m)
a: 1.95–2.02 (m)
b: 1.53–1.66 (m)
a: 2.10–2.18 (m)
b: 1.53–1.66 (m)
1.80–1.88 (m)
a: 0.93–1.08 (m)
b: 0.93–1.08 (m)
a: 1.34–1.47 (m)
b: 1.72 (tq, 3.5, 13.9)
a: 1.34–1.47 (m)
b: 2.18 (bd, 3.2)
1.18–1.27 (m)
a: 1.82–1.97 (m)
b: 2.08 (m)
a: 1.34–1.47 (m)
b: 1.82–1.97 (m)
1.82–1.97 (m)
a: 0.95–1.11 (m)
b: 0.95–1.11 (m)
a: 1.20–1.46 (m)
b: 1.75 (tq, 3.5, 13.9)
a: 1.20–1.46 (m)
b: 2.22 (bd, 13.1)
1.18–1.27 (m)
a: 1.20–1.46 (m)
b: 2.09 (m)
a: 1.20–1.46 (m)
b: 1.85–1.99 (m)
1.85–1.99 (m)
a: 1.68–1.97 (m)
b: 0.97–1.13 (m)
a: 1.42–1.60 (m)
b: 1.68–1.97 (m)
a: 0.97–1.13 (m)
b: 2.25 (bd, 13.2)
1.22–1.33 (m)
a: 1.68–1.97 (m)
b: 2.00–2.12 (m)
a: 1.42–1.60 (m)
b: 1.68–1.97 (m)
1.68–1.97 (m)
2
3
5
6
7
9
1
1
a: 2.98 (dd, 6.4, 18.4)
b: 2.50 (d, 18.4)
a: 2.92 (dd, 6.4, 18.3)
b: 2.52 (d, 18.3)
a: 2.25 (ddd, 4.3, 9.4, 13.5)
b: 1.25 (dd, 5.1, 13.5)
4.92 (m)
a: 2.30 (ddd, 4.3, 9.3, 13.6)
b: 1.17 (ddd, 1.5, 5.5, 13.6)
4.92 (m)
a: 1.68–1.97 (m)
b: 2.00–2.12 (m)
3.22 (bd, 7.2)
12
14
15
16
17
18
20
6.59 (bs)
6.38 (bs)
6.18 (d, 1.8)
6.14 (d, 1.8)
3.35 (s)
2.87 (hp, 6.9)
a
b
c
d
1.40 (s)
1.40 (s)
1.21 (s)
0.47 (s)
3.60 (s)
1.01 (d, 6.9)
1.34 (s)
1.42 (s)
1.34 (s)
a
b
c
d
1.05 (d, 6.9)
1.45 (s)
1.44 (s)
1.49 (s)
1.21 (s)
0.44 (s)
3.60 (s)
1.21 (s)
0.35 (s)
3.63 (s)
1.23 (s)
0.39 (s)
3.66 (s)
1.26 (s)
0.69 (s)
3.67 (s)
MeOCO
a–d
Assignments with the same superscript letter may be interchanged.

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A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
Table 2
13
C NMR data for compounds 1–5 (d in ppm)
Carbon
1
2
3
4
5
1
2
3
4
5
6
7
8
9
39.6
18.9
37.6
43.7
55.1
21.1
42.7
68.6
55.4
38.9
35.7
39.7
19.0
37.7
43.7
55.3
21.3
43.1
68.9
55.6
39.0
34.9
198.6
148.2
144.5
26.3
21.2
21.9
37.9
18.7
37.9
43.9
55.5
20.6
32.6
76.9
49.6
37.4
25.0
72.2
146.3
129.0
81.9
22.7
23.0
28.8
177.5
13.0
51.4
38.0
18.7
38.0
43.9
55.6
20.6
32.7
76.8
49.7
37.3
25.6
72.6
40.0
19.3
38.1
44.1
55.0
22.6
35.5
59.2
47.1
39.4
20.5
49.8
62.1
56.7
81.7
21.2
21.6
29.0
10
11
12
13
14
15
16
17
18
19
20
201.7
145.8
146.3
71.9
28.9
29.0
149.6
124.6
70.8
28.2
28.3
28.8
177.5
13.1
51.5
a
a
a
b
b
c
c
d
d
e
e
28.2
28.9
177.5
15.3
177.4
15.5
51.4
177.5
14.8
51.4
CH
3
OCO
51.3
a–e
Assignments with the same superscript letter may be interchanged.
compounds 1 and 3 (Table 2), the most significant data
were the confirmation of the presence of a tertiary oxy-
genated carbon (d 72.2) in the latter; the two quaternary
oxygenated carbon corresponding to C-8 and C-15 were
again observed (d 76.9 and 81.9, respectively), while no
ketone carbonyl group signal was detected in 3. Accord-
ing to the molecular formula one unit of unsaturation
remained to be defined. As endoperoxide derivatives of
abietane have been reported as natural products, the
presence of a peroxide function between C-8 and C-12
was taken into account. The correlations observed in
the HMBC experiment between H-14 (dH 6.18) and C-
be b. These assignments were reached after noticing the
enhancement experienced by H-14 (d 6.18) when Me-20
was irradiated and the anomalously high field chemical
shift observed for Me-20 (d 0.35). The rest of selected
NOEs shown in Fig. 2 confirmed these assignments.
Compound 4 was also isolated in the present study on
J. phoenicea, and hence must be considered as a new nat-
ural product. The C-4 epimer of 4 was isolated by us
from the wood of Abies marocana (Barrero et al., 1994).
The spectral data for compound 5 (Tables 1 and 2)
suggest that, as 3, this compound is an abietane methyl
7
(dC 32.6), C-9 (dC 49.6), C-12 (dC 72.2), C-8 (dC
7
6.9), and C-15 (d 81.9) confirmed this assignment. Fi-
C
nally, the anomalous deshielding experienced by C-15
compared with that of 1 and other 15-hydroxyabietane
derivatives could be explained if a hydroperoxide func-
tion is located at this position, which, in addition, would
also lead to assign the sixth oxygen atom present in this
molecule. Treatment of 3 with LAH gave compound, 4,
with H and C NMR spectra practically identical to
those of 3. The only remarkable differences being the
shielding experienced by C-15, now appearing at d
O
OH
O
H
O
H
H
H
H
OH
Fig. 1. Selected NOEs observed for 1.
1
13
OOH
7
0.8, and the deshielding experienced by C-16 and C-
H
17 (signals at d 28.2 and 28.3). Compound 4 was con-
firmed to be the result of the chemoselective reduction
H
H
H
MeO C
of the hydroperoxide function of 3 after observing a
+
peak at m/z 387.2145 ([M + Na] ) in its HRFABMS,
2
O
H
O
H
which indicated a molecular formula of C H O . With
2
H
1
32
5
H
respect to the relative stereochemistry, the spatial dispo-
sition of the endoperoxide moiety was determined to be
a, while the methoxycarbonyl group was determined to
H
Fig. 2. Selected NOEs observed for 3.

A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
2511
ꢀ
1
ester derivative (mmax 1722 cm ; d 177.5 and 51.4; dH
dH 5.57 (H-14) and d 48.6 (C-9), 73.0 (C-12), and 84.9
C
C
3
1
.67, 3H, s) possessing a hydroperoxide function at C-
5 (d 81.7; d 1.34 and 1.49, both 3H, s). The rest of
(C-7). The latter two cross-peaks suggested the presence
of two hydroxyl groups at C-7 and C-12. The following
correlations confirmed this assignment: cross-peaks
C
H
the functionalities present in this molecule could be
determined to be two trisubstitued oxirane rings, as in-
ferred from the signals in their H [1H, d 3.22, bd
were observed between d 73.0 (C-12) and dH 1.49 (H-
C
1
11) and 1.07 (Me-17); while the signal corresponding
to C-7 (dC 84.9) showed correlations with the signals
of H-6 (dH 1.89), and H-14 (dH 5.57). The relative con-
figuration was assigned on the basis of the enhance-
ments observed in NOEDIFF experiments (Fig. 4).
Enhancement of the signals corresponding to Me-19
and Me-17, observed after irradiating Me-20, were
fundamental for the assignment of the stereochemistry
1
3
(
J = 7.2); 1H, d 3.35, s], and C (two tertiary carbons
at d 49.8 and 56.7, and two quaternary carbons at 59.2
and 62.1) NMR spectra. The location of the epoxide
rings was established upon HMBC by observing correla-
tions between C-13 (d 62.1) and Me-16 and Me-17 (dH
1
H-11. Accordingly, the structure of 5 was determined to
be methyl 15-hydroperoxy-8a,14a,12a,13a-diepoxiabie-
tan-13-en-19-oate. However, the molecular ion was not
observed in the HR mass spectrum, the fragment pre-
senting the highest m/z appeared at 387. This fragment
corresponds to a loss of an oxygen atom from the molec-
ular ion, which indicated a molecular formula of
C H O . Finally, the relative stereochemistry of the
C
.34 and 1.49); and between C-8 (d 59.2) and H-6 and
C
1
at C-4, C-10, and C-13. The H NMR coupling constant
values measured for H-7 (t, J = 2.5 Hz), and H-12 (dd,
J = 4.0, J = 13.2 Hz) confirmed the spatial disposition
attributed to these protons.
Compound 7 showed a signal of the molecular ion at
m/z 248.1413 in the HRMS, indicating a molecular for-
1
mula of C H O . The H NMR spectrum showed sig-
2
1
32
5
15 20
3
chiral centers was confirmed by NOEDIFF experiments
Fig. 3).
Compound 6 exhibited the molecular formula
C H O , as deduced from its HRFABMS
nals due to the presence of a para-substituted aromatic
ring at d 7.36 (2H, d, J = 8.7 Hz) and 6.88 (2H, d,
J = 8.7 Hz), two olefinic proton at d 6.63 (1H, bd,
J = 15.8 Hz) and 6.18 (1H, dt, J = 15.8, 6.6 Hz), an oxy-
genated allylic methylene at 4.73 (2H, bd, J = 6.6 Hz),
and a methoxy group at 3.83 (3H, s), all of them attrib-
utable to a 4 -methoxylcinnamyl moiety. The C NMR
signals are in agreement with this assignment. On the
other hand, the signals appearing at dH 1.00 (6H, d,
J = 6.5 Hz), 2.17 (1H, m), and 2.26 (2H, d, J = 7.5
(
2
0
30
3
+
(
[M + Na] , m/z 371.2199). The most significant absorp-
tions in the IR spectrum appeared at m 3416, 1723,
max
ꢀ
1
and 1635 cm , which can be attributed to a hydroxyl
0
13
1
group, an ester carbonyl and a double bond. The H
NMR spectrum shows signals due to three methyl
groups, which appear as singlets at d 0.87, 1.07, and
1
.25, two secondary oxygenated carbons (d 3.61, dd,
Hz), together with the signal at d 173.0 confirmed the
C
J = 4.0, J = 13.2 Hz, and d 4.42, t, J = 2.5 Hz), an
ABX system corresponding to a vinyl moiety attached
to a quaternary carbon (A: d 5.13, dd, J = 1.0, J = 17.4
Hz; B: d 5.17, dd, J = 1.0, J = 10.8 Hz; X: d 5.81, dd,
J = 10.8, J = 17.4 Hz), and an olefinic proton (d 5.57,
presence of an isovalerate residue. Thus, the structure
0
of 7 was assigned to be of 4 -methoxycinnamyl isovaler-
ate.
As in 7, the NMR spectral data for compound 8 con-
firmed, the presence of an isovalerate moiety. The rest of
the signals could be attributed to a linaloyl residue
which has been oxygenated on one of the geminal
methyl groups (Tschesche et al., 1977). The assignment
of the double bond geometry was achieved on the basis
1
13
d, J = 2.2 Hz). Complete analysis of its H and
NMR spectra suggested a pimarane skeleton for 6
Barrero et al., 2003; Chang et al., 2000). The location
C
(
of the ester group was assigned according to the correla-
tions found in the HMBC spectrum between C-18 (dC
1
3
of the shielded C chemical shift observed for the vinyl
methyl (d 13.9), which suggested a syn disposition of this
methyl and the hydrocarbon chain. Accordingly, the
structure of 8 was assigned as 8-isovaleroyloxylinalool.
1
80.3) and Me-19 (dH 1.25). The position of the olefinic
proton at C-14 was determined from correlations be-
tween d 1.07 (Me-17) and d 137.7 (C-14), and between
H
C
OOH
H
H
H
H
H
OH
H
O
MeO C
2
H
H
O
H
H
H
H
H
H
H
MeO C
2
H
H
HO
Fig. 3. Selected NOEs observed for 5.
Fig. 4. Selected NOEs observed for 6.

2512
A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
Table 3
Cytotoxic activity of compounds 1–5, IC50 (lg/ml)
A-549
H-116
PSN1
T98G
SKBR3
1
>5
>5
5
>5
>5
0.1
2.5
2.5
2.5
>5
>5
0.1
5
>5
5
>5
>5
0.01
5
>5
>5
>5
>5
>5
0.05
2
>5
>5
>5
>5
2.5
3
4
5
Cycloheximide
Abietane diterpenoids have been reported to possess
several biological properties such as antimicrobial,
antiulcer, and cardiovascular activities (Tan et al.,
4. Experimental
4.1. General
2
2
002), activity as antitumor promoters (Ohtsu et al.,
001), antileishmanial activity (Tan et al., 2002), anti-
Optical rotations were determined with a polarimeter
Perkin–Elmer model 141, using CHCl as the solvent.
oxidant (Wang et al., 2002), tuberculostatic, antiplate-
let agregation activity, antiviral, and several others
3
IR spectra were recorded with a Perkin–Elmer model
983 G instrument as NaCl plates (films). NMR studies
(
Cousins, 1994). However, it has been the remarkable
1
were performed with a Bruker ARX 400 ( H 400
antitumor activity of abietane quinone derivatives re-
cently described in a patent (Kotoda et al., 2002),
which has encouraged us to test the cytotoxicity of
our abietanes 1–5. Five cell lines, A-549 (human lung
carcinoma), H-116 (human colon carcinoma), PSN1
1
3
MHz/ C 100 MHz) spectrometer, and a Bruker AMX
1
13
300 ( H 300 MHz/ C 75 MHz) spectrometer. Mass
spectra (EIMS, 70 eV) were run with a Hewlett–Packard
5972A mass spectrometer coupled to a Hewlett–Packard
5890A gas chromatograph. The accurate mass determi-
nation was carried out with an AutoSpec-Q mass spec-
trometer arranged in an EBE geometry (Micromass
Instruments, Mancester, UK) and equipped with a
FAB (LSIMS) source. Silica gel SDS 60 (35–70 lm)
was used for column chromatography.
(
human pancreatic adenocarcinoma), T98G (human
caucasian gioblastoma), and SKBR3 (human breast
carcinoma) were tested following established methods.
The results are summarized in Table 3. Compounds 1–
3
showed an inhibitory activity against H-116 at 2.5
lg/ml.
4
.2. Plant material
3
. Conclusion
Juniperus thurifera L. var. africana Maire (Cupressa-
ceae) was collected in August 2002, in the region of
Tamahdit, Middle Atlas, Morocco. J. phoenicea L.
(Cupressaceae) was collected in May 1998, in the region
of Ouarzzazzat, High Atlas, Morocco. Voucher speci-
mens are available for inspection at the herbarium of
the Scientific Institute of the University of Mohamed
V, Rabat.
Although the study of J. thurifera var. africana and
J. phoenicea led to the isolation of eight new natural
products as minor components, it can be concluded
from a comparative analysis with previously reported
studies that the content of terpenoid compounds in the
European and African species does not generally present
significant quantitative variations (San Feliciano et al.,
1
993a; Tabacik and Laporthe, 1971). Sandaracopimaric
4.3. Extraction and isolation
acid and various 4-epi-abietic acid derivatives are the
main components of both, European and North Africa
grown, J. phoenicea. On the other hand, both, the com-
ponents of J. thurifera var. africana have been shown to
be diterpenic acids, phenylpropanoids and lignans, a
composition very similar to that found in the European
variety.
Since the photoinduced reaction of abietadienes with
singlet oxygen is a known process (Barrero et al., 1991),
it should not be ruled out that compounds 3 and 5 are
artefacts of the Soxhlet extraction and/or manipulation
of the extract.
The air-dried leaves of J. phoenicea (0.5 kg) were ex-
tracted in Soxhlet apparatus with hexane resulting in 23
g of crude extract. This crude material was extracted
with 1 N NaOH, the aqueous layer acidified with HCl
and then extracted with t-BuOMe to give 2.9 g of an
acid fraction after removing the solvent. This acid frac-
tion was esterified with diazomethane and subsequently
subjected to column chromatography over silica gel
using mixtures of hexane/t-BuOMe of increasing polar-
ity as eluents. The less polar fraction (P1, hexane/t-
BuOMe, 1:2) was composed of 1250 mg of a mixture
of methyl ester derivatives of diterpenic acids, methyl
sandaracopimarate being the major one. The presence
Finally, three of the abietane derivatives show cyto-
toxic activity at 2.5 lg/ml.

A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
2513
ꢀ
1
+
of the methyl esters of 4-epi-abietic acid (10) and cis- and
trans-communic (11)–(12) acids as minor compounds in
this mixture was determined by comparison with stand-
ards. The six following fractions were eluted with hex-
ane/t-BuOMe, 1:2. P2 (83 mg) was rechromatographed
to isolate 7 mg of 6. P3 was containing 44 mg of 2. P4
1096, 997 cm ; EIMS m/z 349 [M ꢀ CH ] (52), 328
3
(77), 268 (42), 253 (100), 197 (44), 149 (82), 121 (97),
107 (59), 91 (75), 79 (77), 67 (60), 59 (80), 55 (87); HRF-
ABMS m/z 387.2148 (calc. for C H O Na, 387.2147).
2
1
32
5
4.5. Methyl 12-oxo-8a-hydroxyabiet-13-en-19-oate (2)
Colorless oil. [a] +18.3ꢁ (ca. 0.91, CH Cl ); IR (film)
(
65 mg) was a mixture with 2 as the main component.
Repeated column chromatography of P5 (57 mg) over
silica gel led to the isolation of 10 mg of 4. P6 (95 mg)
was a mixture containing 1. P7 consisted of 99 mg of 1.
The air-dried leaves of J. thurifera var. africana (1 kg)
were extracted in a Soxhlet apparatus with hexane result-
ing in 81 g of crude extract. A 20 g portion was subjected
to column chromatography over silica gel using mixtures
of hexane/EtOAc of increasing polarity as eluents. Eight
main fractions were collected, T1–T8. A mixture of 5.2 g
of abietic acid (9), trans-communic acid (12), and isopim-
aric acid (14) in a 7:2:1 ratio was obtained by crystalliza-
tion from T1 using a mixture of hexane–CH Cl (20:1).
D
2
2
mmax 3430, 2957, 2931, 1724, 1667, 1464, 1236, 1156,
1015, 977 cm ; HRFABMS m/z 371.2195 (calcd for
ꢀ
1
C H O Na, 371.2198).
32
2
1
4
4.6. Methyl 15-hydroperoxy-8a,12a-epidioxyabiet-13-en-
19-oate (3)
White powder. [a] +9.7ꢁ (ca. 1.03, CHCl ); IR (film)
mmax 3401, 2948, 1724, 1465, 1237, 1151, 1035, 971,
D
3
ꢀ
1
cm ; EIMS m/z 348 (13), 332 (6), 254 (28), 181 (9),
133 (21), 121 (58), 109 (29), 91 (45), 81 (40), 67 (35),
59 (39), 55 (44), 43 (100); HRFABMS m/z 403.2093
(calcd for C H O Na, 403.2097).
2
2
The mother liquors were concentrated and chromato-
graphed repeatedly to afford 43 mg of (E,Z)-2,4-decadie-
nyl isovalerate (27), 173 mg of abietinal (15), 90 mg of
linaloyl acetate (25), 10 mg of dehydroabietinal (16), 10
mg of cinnamyl isovalerate (28), 30 mg of elemol (32),
and 190 mg of 7. T2 was re-subjected to silica gel column
chromatography to yield a mixture (41 mg) of pimara-
2
1
32
6
4.7. Methyl 15-hydroxy-8a,12a-epidioxyabiet-13-en-19-
oate (4)
Colorless oil. [a] +72.4ꢁ (ca. 0.46, CHCl ); IR (film)
D
3
ꢀ
mmax 3444, 2932, 1723, 1649, 1463, 1235, 1170, 971 cm ;
+
1
8
(14),15-dien-18-ol (17) and pimara-7,15-dien-18-ol
18), and 32 mg of abietinol (19). T3 was again
subjected to column chromatography to give 110 mg of
(
EIMS m/z 349 [M ꢀ CH ] (10), 332 (13), 271 (8), 253
3
(17), 195 (14), 149 (83), 121 (100), 107 (52), 91 (84), 79
(76), 67 (77); HRFABMS m/z 387.2145 (calc. for
C H O Na, 387.2147).
0
0
3
1
,4 -dimethoxycinnamyl isovalerate (29), 36 mg of 8,
5 mg of 15-hydroxy-9a,13a-epidioxyabiet-8(14)-en-18-
2
1
32
5
oic acid (20), and 92 mg of an equimolecular mixture
of 3b-hydroxysandaracopimaric acid (21) and 3b-hyd-
roxyisopimaric acid (22). T4 was rechromatographed
over silica gel to yield 14 mg of 4-epiabietic acid (10),
4.8. Reduction of 3 with LAH
To a solution of 3 (28 mg, 0.08 mmol) in 6 ml of dry
THF was added 8 mg (0.20 mmol) of LAH. The mixture
0
0
0
9
0 mg of phytol (26), 130 mg of 3 ,4 ,5 -trimethoxycinn-
amyl isovalerate (30), and 250 mg of 8a-acetoxyelemol
was stirred at room temperature overnight. Std. NH Cl
4
(
(
33). T5 was constituted of 476 mg of 8a,11-elemdiol
34). T6 was composed mainly of yatein (35) (225 mg).
soln was added and the mixture extracted with t-
BuOMe. The combined extracts were washed with brine,
dried over Na SO , and concentrated to give 24 mg of 4.
T7 was subjected to repeated column chromatography
and various chemical derivatizations including esterifica-
tion with TMSCHN , acetylation and saponification to
yield 95 mg of 3, 4.5 mg of 4. In addition, 75 mg of yate-
ine (35), 312 mg of deoxypodophyllotoxin (37), 75 mg of
a mixture of pimara-8(14),15-dien-3b,18-diol (23) and
pimara-7,15-dien-3b,18-diol (24), as well as 10 mg of
3
Rechromatography of T8 led to the isolation of podo-
phyllotoxin (36) (248 mg) and picropodophyllotoxin
(
2
4
4.9. Methyl 15-hydroperoxy-8a,14a,12a,13a-diepoxyabie-
tan-13-en-19-oate (5)
2
White powder. [a]D +0.25ꢁ (ca. 0.40, CHCl ); IR
3
(film) m
3387, 2934, 2854, 1722, 1452, 1241,
max
ꢀ1
0
0
,4 -dimethoxycinnamol (31) could be obtained.
1163 cm ; EIMS m/z 348 (6), 332 (5), 269 (9), 254
(27), 223 (16), 163 (25), 133 (28), 121 (99), 109 (50), 91
(53), 79 (65), 67 (78), 59 (100), 55 (88); HRFABMS m/
z 387.2143 (calcd for C H O Na, 387.2147).
224 mg) (38).
2
1
32
5
4
.4. Methyl 12-oxo-8a,15-dihydroxyabiet-13-en-19-oate
1)
4.10. Methyl 7a,12b-dihydroxysandaracopimarate (6)
(
Colorless oil. [a]D +14.3ꢁ (ca. 1.2, CHCl ); IR (film)
3
Colorless oil. [a] +20.1ꢁ (ca. 0.66, CH Cl ); IR (film)
mmax 3416, 2927, 2853, 1723, 1635, 1440, 1253, 1160,
D
2
2
ꢀ1 1
mmax 3399, 2953, 2875, 1724, 1659, 1462, 1237, 1159,
1068, 994, 843 cm ; H NMR (CDCl , 400 MHz) d
3

2514
A.F. Barrero et al. / Phytochemistry 65 (2004) 2507–2515
0
.87 (3H, s, H-20), 1.07 (3H, s, H-17), 1.17–1.25 (1H, m,
H-1b), 1.25 (3H, s, H-19), 1.42–1.52 (1H, m, H-6b), 1.49
1H, bq, J = 13.7 Hz, H-11b), 1.52–1.81 (6H, m, H-1a,
(48), 57 (92), 43 (100); HRFABMS m/z 277.1784 (calc.
for C H O Na, 277.1779).
1
5
26
3
(
H-2, H-3, H-11a), 1.89 (1H, bdt, J = 2.3, 14.7 Hz, H-6
a); 2.28–2.34 (1H, m, H-9), 2.38 (1H, dd, J = 2.2, 13.0
Hz, H-5), 3.61 (1H, dd, J = 4.0, 13.2 Hz, H-12), 3.68
4
.13. Cell proliferation assay
The 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazo-
(
(
3H, s, COOCH ), 4.42 (1H, t, J = 2.5 Hz, H-7), 5.13
3
lium (MTT; Sigma Chemical Co., St. Louis, MO) dye
reduction assay in 96-well microplates was used, essen-
tially as described (Mosmann, 1983). The assay is
dependent on the reduction of MTT by mitochondrial
dehydrogenases of viable cell to a blue formazan prod-
uct which come be measured spectofhotometrically.
1H, dd, J = 1.0, 17.4 Hz, H-16 trans), 5.17 (1H, dd,
J = 1.0, 10.8 Hz, H-16 cis), 5.57 (1H, d, J = 2.2, H-14),
5
1
.81 (1H, dd, J = 10.8, 17.4 Hz, H-15); C NMR
3
(
CDCl , 100 MHz) d 180.3 (s, COOCH ), 145.0 (d, C-
3
3
1
8
(
5), 137.7 (d, C-14), 133.1 (s, C-8), 114.6 (t, C-16),
4.9 (d, C-7), 73.0 (d, C-12), 52.5 (q, COOCH ), 48.6
d, C-9), 46.1 (s, C-4), 43.4 (s, C-13), 40.4 (d, C-5),
3
3
BAE cells (8 · 10 cells in a total volume of 200 ll of
3
DMEM/20% FBS) and tumor cells (4 · 10 A-549 cells
3
2
1
8.2 (t, C-1), 37.5 (s, C-10), 37.2 (t, C-3), 27.4 (t, C-6),
a
5.8 (t, C-11), 18.0 (t, C-2), 17.3 (q, C-17 ), 17.5 (q, C-
3
3
3
or 6 · 10 H-116, 6 · 10 PSN1, 6 · 10 SKBR3 and
3
6
· 10 T98G cells in a total volume of 200 ll of com-
a
a
9 ), 15.0 (q, C-20) ( assignments with the same super-
plete medium) were incubated in each well with serial
dilutions (5, 2.5, 1, 0.5, 0.1, 0.05 and 0.01 lg/ml) of
the tested compounds. After 2 days of incubation (37
script letter may be interchanged); HRFABMS m/z
3
71.2199 (calcd for C H O Na, 371.2198).
21 32 4
ꢁ
C, 5% CO in a humid atmosphere) 50 ll of MTT (5
2
0
4
.11. 4 -Methoxycinnamyl isovalerate (7)
mg/ml in PBS) were added to each well and the plate
was incubated for a further 2 h (37 ꢁC). The resulting
formazan was dissolved in 100 ll DMSO and read at
Obtained as colorless oil; IR (film) mmax 2960, 2872,
735, 1608, 1513, 1464, 1379, 1293, 1251, 1174, 1118,
04 cm ; H NMR (CDCl , 300 MHz) d 0.99 (6H, d,
J = 6.5, H-4 and H-5 ), 2.18 (1H, m, H-3 ), 2.26 (2H,
d, J = 7.0, H-2 ), 3.84 (3H, s, OMe), 4.73 (2H, dd,
J = 1.2, 6.6, H-1), 6.18 (1H, dt, J = 6.6, 15.8, H-2);
1
9
4
90 nm. All determinations were carried out in tripli-
ꢀ
1 1
3
cate. IC50 value was calculated as the concentration of
drug yielding a 50% of cell survival.
0
00
00
00
0
6.63 (1H, bd, J = 15.8, H-3), 6.88 (2H, d, J = 8.7, H-3
13
and H-5 ), 7.36 (2H, d, J = 8.7, H-2 and H-6 );
NMR (CDCl , 75 MHz) d 173.0 (s, C-1 ), 159.7 (s, C-
4 ), 134.0 (d, C-3), 128.9 (s, C-1 ), 127.9 (d, C-2 and
0
C-6 ), 121.2 (d, C-2), 114.1 (d, C-3 and C-5 ), 65.1 (t,
0
0
0
C
Acknowledgements
0
0
3
0
0
0
This work was supported by a grant from CICYT
(SAF2001-2639), the Spanish Ministerio de Ciencia y
Tecnolog ´ı a (Project BQU 2002-03211), from the
ESRSFC Ministry of the Moroccan Government (PRO-
TARS P2T2/07), and from the Junta de Andaluc ´ı a
(Consejer ´ı a de Presidencia, Programa de Cooperaci o´ n
Andaluc ´ı a-Marruecos). We also thank Instituto Biomar,
S.A. for the antitumor tests.
0
0
00
00
C-1), 55.4 (q, OCH ), 43.6 (t, C-2 ), 25.8 (d, C-3 ),
3
00
00
2
1
5
2.5 (q, C-4 and C-5 ); EIMS m/z 248 (40), 164 (38),
47 (87), 131 (24), 103 (32), 91 (37), 85 (100), 77 (18),
7 (87), 49 (97); HRFABMS m/z 248.1413 (calcd for
C H O , 248.1412).
1
5
20
3
4
.12. Isovaleroyloxylinalool (8)
Colorless oil. [a]D ꢀ1.4ꢁ (ca. 0.3, CH Cl ); IR (film)
2
2
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mmax 3429, 2958, 2920, 2851, 1733, 1644, 1609, 1463,
ꢀ
372, 1294, 1251, 1184, 1119 cm ; H NMR (CDCl ,
3
1 1
1
4
(
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0
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3H, s, H-10), 1.50–1.65 (2H, m, H-4), 1.62 (3H, bs, H-
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bt, J = 7.2, H-6), 5.89 (1H, dd, J = 10.7, 17.3, H-2);
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0
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6
1
5
9.9 (t, C-8), 43.5 (t, C-2 ), 41.6 (t, C-5), 27.9 (q, C-
0
0
0), 25.8 (d, C-3 ), 22.5 (t, C-4), 22.4 (q, C-4 and C-
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(
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