New cytotoxic diterpenylnaphthohydroquinone derivatives obtained from a natural diterpenoid

Article abstract of DOI:10.1016/j.bmc.2007.06.005

Diterpenylquinone/hydroquinone derivatives were prepared through Diels-Alder cycloaddition between natural myrcecommunic acid or its methyl ester and p-benzoquinone (p-BQ), using BF₃·Et₂O as catalyst or under microwave (Mw) irradiati

Full text of DOI:10.1016/j.bmc.2007.06.005

Bioorganic & Medicinal Chemistry 15 (2007) 5760–5774
New cytotoxic diterpenylnaphthohydroquinone derivatives
obtained from a natural diterpenoid
a,
a
a
*
´
´
Jose M. Miguel del Corral, M. Angeles Castro, M. Lucena Rodrıguez,
Pablo Chamorro,a Carmen Cuevasb and Arturo San Felicianoa
aDepartamento de Qu´ımica Farmace´utica, Facultad de Farmacia, Universidad de Salamanca, E-37007 Salamanca, Spain
bPharmaMar S.A., Avda. de los Reyes, 1 P.I. La Mina-Norte, 28770 Colmenar Viejo, Madrid, Spain
Received 5 March 2007; revised 31 May 2007; accepted 5 June 2007
Available online 8 June 2007
Abstract—Diterpenylquinone/hydroquinone derivatives were prepared through Diels–Alder cycloaddition between natural myrce-
communic acid or its methyl ester and p-benzoquinone (p-BQ), using BF3ÆEt2O as catalyst or under microwave (Mw) irradiation.
Acetyl, methyl and benzyl derivatives of several diterpenylnaphthohydroquinone were prepared from cycloadducts following two
basic synthetic strategies, either protection before aromatisation or viceversa. Some of them were further functionalised at the
B-ring of the decaline core. Most of the new compounds were evaluated and some of them resulted cytotoxic against several tumour
cell lines with IC50 values under the lM level.
ꢀ 2007 Elsevier Ltd. All rights reserved.
OH
1. Introduction
O
HO
OH
HO
Terpenylquinones constitute an interesting group of
marine natural products,1 for which a wide variety of
biological activities have been described, including
anti-inflammatory, antifungal, anti-HIV and most often
antineoplastic activities. Their cytotoxicity has been jus-
tified on their ability for undergoing redox cycling and
on the generation of reactive oxygen species, which
would damage tumour cells.2 Most of these meroterpe-
noids are characterised by possessing a benzoquinone
(BQ) fragment attached to a terpenoid, which usually in-
cludes a decaline core, mostly without other functions,
as in the case of avarol and avarone (Fig. 1), which pres-
ent cytotoxic and antiviral activities.3 A few reported
examples present oxygenated functions at the decaline
moiety, as it is the case of the cytotoxic drimanoid
yahazunol4 (Fig. 1). The 1,4-naphthoquinone system
(1,4-NQ) is ubiquitously distributed in nature5 and it
is also present in numerous biologically active com-
pounds, such as menadione (vitamin K3) and drugs like
the antiparasitic atovaquone (Fig. 1).6 Both facts
O
H
OH
H
H
Avarol
Avarone
Yahazunol
O
O
OH
O
O
Cl
Menadione
Atovaquone
Figure 1. Structures of biologically active quinones.
prompted us, a few years ago, to study the influence of
the terpenyl and quinone sizes and functionalities on
the bioactivity of this type of meroterpenoids. Thus, sev-
eral monoterpenylbenzo-,7 naphtho-8 and anthraqui-
nones8d,9 (AQ) with different functions in the side
chain and in the quinone ring were prepared and their
antineoplastic cytotoxicity assayed. Additionally, sev-
eral diterpenyl-NQ and AQs were also synthesised.10
In those studies it became clear that all the compounds
Keywords: Terpenylquinones; Myrcecommunic acid; Allylic oxidation;
Cytotoxicity.
*
Corresponding author. Tel.: +34 923 294528; fax: +34 923 294515;
e-mail: jmmcs@usal.es
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.005

J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
5761
prepared were more cytotoxic than the 1,4-NQ itself,
being the NQ system more interesting than BQ and
AQ, with cytotoxic potencies in the lM range. The most
active compounds had the naphthalene ring either as
1,4-NQ or as diacetyl naphtho-1,4-hydroquinone (1,4-
NHQ). Also, few of those diterpenyl-NQ derivatives
had oxygenated the C-17 position of the terpenoid and
showed a very interesting cytotoxicity and selectivity.10
fore purification, because if the cycloaddition product
is subjected to chromatography on silica, it enolises to
the corresponding hydroquinones, which can undergo
further autoxidation to dihydronaphthoquinones.10 In
order to optimise the synthesis of cycloadducts 3 and 4
and looking for shortening the reaction time, two
Diels–Alder procedures were considered, one in ethereal
solution using BF3ÆEt2O as catalyst and other, under
Mw irradiation in absence of solvent. Some variations
were tested in both procedures, such as the p-BQ/diene
ratio, the type of solid support and the irradiation
power. The proportion of components in the crude reac-
These facts prompted us to continue our research and
to prepare several new series of NHQ/NQ prodrugs, in
order to extend the evaluation to in vivo assays. With
this aim, we planned to block the redox-cycling ability
of the hydroquinone hydroxyls through their protec-
tion with groups displaying different chemical and met-
abolic stability. The groups selected to protect the
hydroquinone moiety were the acetyl, benzyl and
methyl groups. Acetates are hydrolysable and remov-
able, either in vitro or in vivo and, consequently, they
could easily liberate the active hydroquinone. Indeed,
saponification would occur progressively at neutral
pH, along the 3 days needed for culminating the
in vitro assays and also, according to common meta-
bolic pathways,11–14 they would be removed in vivo un-
der the action of lipases and esterases. On the other
hand, methyl ethers of NHQ are chemically stable un-
der the in vitro evaluation conditions, and the methyl
group could be removed as a formaldehyde unit,
through in vivo metabolic cytochrome oxidation and
acetal hydrolysis.12 At an intermediate position, the
benzyl group can be more easily oxidised and degraded
than the methyl by chemical reagents, but the benzylic
protons would be less accessible to the in vivo oxida-
tive action of macromolecular enzymes than those of
the methyl group.13
1
tion products was established by H NMR analysis of
the mixtures and the results found are summarised in
Table 1.
Considering cycloadditions under Mw irradiation,16 in
all cases the reaction of 1 (or 2) with p-BQ (entries 1–8
or 9, Table 1) led to the corresponding cycloadduct 3
(or 4) as the main reaction product. However, when sil-
ica or alumina was used as solid supports, compound 3
was accompanied by its autoxidation products 5 and 6.
In some cases, when SiO2 was used (entries 2 and 5 of
Table 1), the quinone 5 was the major component. It
should also be mentioned that with Al2O3 as support,
the cycloaddition was not completed, remaining a part
of the starting diene unreacted, even with doubled
amount of p-BQ (entries 1, 3 and 4 of Table 1). On
the other hand, the cycloadduct 3 (or 4) was obtained
as the unique product (entries 6–9 of Table 1) when
using an excess of p-BQ (P1.5 equiv) and montmoril-
lonite K-10 as solid support. With this support, only
when the p-BQ/diene ratio was 1:1 (entry 7 of Table
1), some unreacted diene was detected in the cycloaddi-
tion crude. The good results obtained with K-10 should
be due to the acidity of this support, comparable to that
of mineral acids.16
Complementarily to those hydroxyls’ protections, sev-
eral transformations of the B-ring in the decaline moiety
were also planned in order to prepare yahazunol ana-
logues, for ascertaining the influence of oxygen func-
tions located around the position C-8 on the
cytotoxicity. In addition, the Diels–Alder reaction con-
ditions have been analysed, in order to optimise the
yields of diterpenyl-NHQs, as well as those of their sub-
sequent transformations.
Similarly, the reaction between 1 (or 2) and p-BQ in
ether, using BF3ÆEt2O as catalyst, led to the cycloadduct
3 (or 4) as the unique detected product in the reaction
crude in all of the cases (entries 10–13 of Table 1), mak-
ing this conditions even better than those of the Mw/K-
10 procedure. Although the Mw irradiation has the
advantage of shortening the reaction time, this proce-
dure needs an excess of the p-BQ that difficults the puri-
fication of the final products while the aromatised
products also appear with variable proportions in the
reaction crude.
2. Results and discussion
2.1. Chemistry
Acetylated and methylated derivatives of hydroquinones
8–10 were prepared from cycloadducts 3 and 4, follow-
ing two basic synthetic strategies (see Table 1). In the
first one, the acetylation or the methylation was accom-
plished prior to completion of the aromatisation (meth-
ods A and D). In the second, the oxidation to the NQ
was followed by the reduction to the NHQ and the final
acetylation or methylation (methods B and C). Method
A applied to cycloadducts 3 and 4, obtained through
Mw irradiation (Table 1, entries 8 and 9), afforded the
corresponding acetylated derivatives 8 and 9 in good
yield, though they were accompanied by the correspond-
ing autoxidation quinones NQ 6 and 7, respectively.
2.1.1. Diels–Alder cycloaddition. Diterpenylquinone/
hydroquinone derivatives have been prepared through
a Diels–Alder cycloaddition between myrcecommunic
acid 1 or methyl myrcecommunate 2 and p-benzoqui-
none (p-BQ). The natural labdane acid used as starting
material was isolated from berries of Juniperus oxyce-
drus.15 It was transformed into its methyl ester by treat-
ment with an ethereal solution of diazomethane. The
condensation of 1 and 2 with p-BQ gave the cycload-
ducts, 3 and 4, respectively. The presence and propor-
tion of the original diketone cycloadducts in the crude
1
reaction product had to be estimated by H NMR, be-

5762
J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
Table 1. Quinones and acetylated and methylated hydroquinones obtained from myrcecommunic acid
O
O
O
O
O
16
12
13
1'
6'
11
10
2'
3'
20
1
4
9
14
15
2
3
5'
4'
8
O
7
6
H
H
H
H
COOR
18
R
H
COOR
COOR
COOH
R
R
H
19
5
6
7
1
2
H
Me
3
4
Me
Me
OR'
OR'
O
O
MeO
17
H
COOR
OMe
R
H
R'
Ac
Ac
8
9
R
Me
R
Me
10 Me
12
11 Me
Me
Entry
Diene
p-BQ/1(2) Ratio
Solid support
Conditions
Time
Products (ratio)
Method
Products (yields)
1
2
3a
4a
5a
6
1
1
1
1
1
1
1
1
2
1
2
2
2
2:1
2:1
Al2O3
SiO2
Mwb
8 min
8 min
4 min
4 min
8 min
4 min
4 min
8 min
8 min
24 h
1/3/5 (1:2:1)
3/5 (1:2)
—
—
—
—
—
—
—
A
—
—
Mwc
2:1
2:1
Al2O3
Al2O3
SiO2
Mwc
1/3/5/6 (1:2:2:1)
1/3/5 (2:2:3)
3/5 (1:5)
—
—
Mwb
2:1
2:1
Mwc
—
—
K-10
K-10
K-10
K-10
Mwc
3
1/3 (1:2)
7
1:1
Mwc
—
8
1.5:1
1.5:1
1:1
Mwc
3
4
3
4
4
4
6 (9%), 8 (68%)
7 (10%), 9 (69%)
8 (60%)
9
Mwc
A
10
11
12
13
BF3/Et2O
BF3/Et2O
BF3/Et2O
BF3/Et2O
B
1:1
24 h
24 h
B
9 (55%)
10 (58%), 12 (7%)
10 (25%), 11 (8%)
0.9:1
1:1
C
24 h
D
Method A: (i) Ac2O, py, rt, 12 h; (ii) DDQ, benzene, rt, 0.5–1 h.
Method B: (i) MnO2, benzene, reflux, 16 h. (ii) Na2S2O4, dioxane, H2O, rt, 2 h; (iii) Ac2O, py, rt, 12 h.
Method C: (i) MnO2, benzene, reflux, 16 h; (ii) Na2S2O4, dioxane, H2O, rt, 2 h; (iii) MeI, K2CO3, DMF, 110 ꢁC, 1 h.
Method D: (i) Me2SO4, NaOH, EtOH, reflux, 1.5 h; (ii) DDQ, benzene, rt, 0.5–1 h.
a Suspended in Hex overnight before the extraction with EtOAc.
b Power, 750 W.
c Power, 500 W.
When method B, consisting of MnO2 oxidation, fol-
lowed by reduction with sodium ditionite and acetyla-
tion, was applied to the cycloadducts 3 and 4 (Table 1,
entries 10 and 11), only the desired acetylated naphtho-
hydroquinones 8 and 9 were, respectively, isolated,
although the yield was somewhat lower than with meth-
od A. When the crude cycloadduct 4 was submitted to
method C, MnO2 oxidation, reduction and methylation
(Table 1, entry 12), unexpectedly, the methylated deriv-
ative 10 was accompanied by a small amount of the
symmetrical diterpenylanthraquinone 12. The formation
of this AQ should be consequence of a second Diels–Al-
der reaction between 4 and the residual unreacted diene–
ester 2, due to the relative starting excess of the latter
(Table 1, entry 12, 0.9:1 molar ratio). As an alternative,
method D, consisting in subjecting the crude reaction
product to methylation before the oxidation, was ap-
plied to compound 4; however, the yield was lower
and the side-chain suffered dehydrogenation leading to
compound 11, due to an extended DDQ oxidation.
2.1.2. Optimisation of diterpenylnaphthohydroquinone
synthesis. Methods A–D, leading directly to the final ter-
penylhydroquinone derivatives 8–10, were conducted
without purification of the intermediates; so we decided
to isolate those intermediate diketones 3 and 4 and to
optimise the remaining steps for the preparation of the
acetylated, methylated and benzylated NHQ derivatives
(Scheme 1). Thus, we followed the two strategies men-
tioned above, either protection before aromatisation or
vice versa. The dihydronaphthalene diacetates 13 and
14 were obtained in good yields by direct acetylation
of cycloadducts 3 and 4, respectively (Scheme 1, entries
1 and 2). The direct methylation of cycloadduct 4
(Scheme 1, entry 4) afforded 15 in moderate yield only.
Due to that, compound 3 was previously enolised by
keeping it with silica gel and quickly treating with di-
methyl sulfate. However, under these conditions the par-
tial methylation of the carboxylic group at C-19 was
observed. This prompted us to bring methylation to
completion by treating the mixture with diazomethane,

J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
5763
OR'
OR'
OR'
H
16
15
1'
R
H
R'
Ac
COOR
13
17
6'
5'
11
20
5
O
O
13
1
9
3'
14 Me Ac
15 Me Me
OR'
R'
Ac
Ac
Me
Bn
Bn
7
3
H
O
R
H
Me
COOR
18
H
19
8
R
COOR
9
3
4
H
10 Me
16 Me
17 Bn
Me
O
H
COOR
R
6
H
7
Me
Final products
(Yields)
Reaction
conditions
Product
(Yield)
Further reaction
conditions
Entry
Substrate
1
2
i)
i)
d)
d)
3
4
13 (78 %)
14 (77 %)
8 (91 %)
9 (46 %)
3
a), b), c)
3
15 (54 %)
d)
10 (51 %)
4
5
6
4
3
4
b)
e)
e)
15 (39 %)
6 (84 %)
7 (86 %)
f), h)
f), g)
f), h)
16 (46 %), 17 (22 %)
10 (56 %)
16 (67 %)
Scheme 1. Preparation of acetylated, methylated and benzylated hydroquinones from cycloadducts 3 and 4. Reagents and conditions: (a) SiO2,
benzene, rt, 7 h; (b) Me2SO4, NaOH, EtOH, reflux, 1.5 h; (c) CH2N2, ether, 12 h; (d) DDQ, benzene, rt, 0.5–1 h; (e) MnO2, benzene, reflux, 16 h; (f)
Na2S2O4, dioxane, H2O, rt, 2 h; (g) MeI, K2CO3, DMF, 110 ꢁC, 1 h; (h) NaH, DMF, 0 ꢁC, 15 min then BnBr, rt, 1 h; (i) Ac2O, py, rt, 12 h.
thus improving the yield in the trimethyl derivative 15
up to 54% (Scheme 1, entry 3). Further treatment of
compounds 13–15 with DDQ afforded the NHQ deriva-
tives 8–10 in moderate to good yields.
They possess the exocyclic D8(17) double bond that facil-
itates the introduction of the desired functionalities.
The first transformation performed was the allylic oxi-
dation17 of the diterpenyl-NHQ derivatives 9, 10 and
16 with SeO2 under different reaction conditions
(Scheme 2). When the diacetate 9 was treated with
SeO2 excess (entry 1, Scheme 2), the 7a-hydroxyl deriv-
ative 18 was obtained in good yield. When the same con-
ditions were applied to the dimethyl ether 10 (entry 2,
conditions a) and to the dibenzyl ether 16 (entry 3),
the corresponding alcohols 19 and 20 were obtained,
respectively, but in these cases, the other possible allylic
oxidation products, the tertiary 9a-hydroxyl derivatives
22 and 23, were also obtained. Trying to improve the
yields on the latter cases, new conditions were applied
to compound 10 using SeO2 and t-BuOOH (entry 2,
conditions b), however, the yield of the oxidation was
somewhat lower and compound 19 was accompanied
by the allylic peroxide 21.
The alternative procedure started with the oxidation9 of
the cycloadducts 3 and 4 with MnO2 to give NQs 6 and
7, respectively, in good yields (Scheme 1, entries 5 and
6). Reduction of NQ 7 followed by methylation with
MeI or benzylation with BnBr gave, respectively, 10
and 16 in acceptable yields (Scheme 1, entry 6). At-
tempts to obtain the dibenzylated hydroquinone from
NQ 6, in presence of the free carboxylic acid at the ter-
penic core, were not too successful because a mixture of
methyl (16) and benzyl (17) esters was obtained (Scheme
1, entry 5). The formation of both esters was due to the
partial benzylation of the carboxylic group, similarly to
that occurring in the methylation of cycloadduct 3,
while methylation of the remaining free carboxylic acid
occurred during hydride excess destruction with metha-
nol, in the workup after benzylation.
The absolute configuration at C-7 in compounds 18–20
was deduced from the analysis of the pattern of the H-7
signal in their 1H NMR spectra, that derived from small
Jvec coupling constants of 2.6–3.1 Hz, characteristic for
equatorial protons.17c,18 This was in accordance with
the mechanism of the SeO2 oxidation,19 that also served
to deduce the C-9 configuration proposed for com-
pounds 22 and 23.
2.1.3. Preparation of NHQs oxidised in the terpene
fragment. As mentioned above, another objective of this
research was focused on the introduction of oxygen
functions at C-7 of the terpene core in order to compare
the activity of the resulting compounds with that of yah-
azunol. We chosed diterpenylhydroquinones 9, 10 and
16 as the starting materials for those transformations.

5764
J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
OR
OR
16
1'
6'
11
10
20
OH
13
17
1
4
3'
a) or b)
5'
OOC(CH3)3
8
15
OR
3
OR
7
OH
R
H
H
H
H
COOMe
COOMe
COOMe
R
Me
Bn
R
Me
18
COOMe
R
19
22
23
21
Ac 18
Me 19
Bn 20
Ac
9
Me 10
Bn 16
c)
OR
d)
R
OR
OR
OR
Ac 24 (64 %)
Me 25 (70 %)
Bn 26 (68 %)
O
H
COOMe
OAc
H
COOMe
e)
f)
R
OAc
OH
COOMe
Ac 27 (98 %)
Me 28 (97 %)
Bn 29 (97 %)
O
H
H
COOMe
30 (80 %)
31 (24 %)
Reaction
conditions
Products
(Yield)
Entry
Substrate
1
2
9
10
a)
a)
b)
a)
18 (80 %)
19 (67 %), 22 (25 %)
19 (62 %), 21 (18 %)
20 (60 %), 23 (24 %)
3
16
Scheme 2. Preparation of oxidised derivatives of diterpenylnaphthohydroquinones. Reagents and conditions: (a) SeO2 (3.5 equiv), EtOH, 60 ꢁC,
16 h; (b) t-BuOOH, SeO2 (0.7 equiv), CH2Cl2, rt, 5 h; (c) DMSO, (COCl)2, CH2Cl2, ꢀ55 ꢁC, 30 min; then Et3N, 0 ꢁC, 30 min; (d) C6H6, aq 57% HI,
80 ꢁC, 10 min; (e) Na2CrO4, AcONa, AcOH, Ac2O, C6H6, 70 ꢁC, 8 h; (f) NaBH4, CeCl3, MeOH/THF, 1:1, 0 ꢁC, 45 min; then at rt, 3 h.
The allylic C-7 alcohols 18–20 were further oxidised to
the corresponding ketones 24–26. Attempts to oxidise
the alcohol 19 with PCC and PDC were unsuccessful,
leading to complex mixtures. However, on treating the
allylic alcohols 18–20 with DMSO/oxalyl chloride at
ꢀ55 ꢁC in presence of Et3N (Swern procedure), the cor-
responding a,b-unsaturated ketones 24–26 were ob-
tained as the unique products, with yields between
64% and 70%.
24% yield. As it would be expected, and unlike com-
pound 18, 31 possessed an equatorial 7b-hydroxyl
group, as it was confirmed by the coupling pattern of
the corresponding axial H-7 signal (d 4.09 ppm, dd,
1
J1 = 9.8 Hz, J2 = 6.2 Hz) in its H NMR spectrum.
2.2. Cytotoxicity
Most of the synthesised diterpenyl-NQ/NHQ derivatives
were evaluated in vitro for determining their antineo-
plastic cytotoxicity22 against the human cell lines: A-549
lung carcinoma, HT-29 colon carcinoma and MEL-28
malignant melanoma. The cytotoxicity results are shown
in Table 2 as GI50 values expressed in lM.
In order to obtain other functionality configurations we
double
(17)
planned the previous isomerisation of the D8
bond, to the more stable endocyclic tetrasubstituted po-
sition, that also would allow the oxidation at positions
C-7 and C-17. So the treatment of 9, 10 and 16 with
aq 57% HI at 80 ꢁC led to the corresponding D8 deriva-
tives 27–29. Nevertheless, when the previous allylic oxi-
dation conditions were applied to the D8 olefins, no
oxidation products were detected and the starting mate-
rial was recovered unreacted. Consequently, the intro-
duction of the hydroxyl group at C-7 was indirectly
achieved through reduction of the corresponding car-
bonyl group, which was easily formed by direct oxida-
tion with sodium dichromate. So, compound 27
treated with Na2CrO4 in benzene20 gave the desired ke-
tone 30. Several attempts to reduce the ketone function
by treating 30 with NaBH4 in different solvents and tem-
peratures were unsuccessful, and only when 30 was trea-
From these results, it can be stated that the compounds
assayed were cytotoxic and that the protection of the
hydroquinone hydroxyls, while blocking their redox
properties, displays an important influence on the activ-
ity. As it was expected for in vitro assays, the hydrolysa-
ble acetyl derivatives were the most potent, having GI50
values under the lM level, whereas the methylated and
benzylated hydroquinones were much less or almost
non-cytotoxic at the maximum concentrations tested.
It could be noted as exceptions the lM cytotoxicity lev-
els displayed by the methyl and benzyl ethers 25 and 26,
comparable to those of the diacetate 24, but this fact
would be justified on the basis of the intrinsic cytotoxic-
ity of the electrophilic a,b-unsaturated carbonyl, present
in the terpene core of these molecules.
21
ted with NaBH4 in the presence of CeCl3 in MeOH/
THF at 0 ꢁC, the reaction led to the C-7 alcohol 31 in

J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
5765
Table 2. Antineoplastic cytotoxicity for terpenylnaphthohydroqui-
O
OR'
16
15
none derivatives
12
13
1'
6'
11
10
2'
3'
20
Compound
A-549a
HT-29a
MEL-28a
1
4
9
2
3
5'
17
4'
8
6
7
>7.4
0.20
0.24
0.12
>6.7
>22.3
>4.1
2.5
5.7
5.7
O
OR'
7
0.20
0.24
0.50
>6.7
>22.3
>4.1
10.1
1.38
>5.0
4.4
0.20
0.24
0.12
>6.7
n.t.
6
H
H
8
COOMe
18 COOR
7
19
9
10
11
12
13
14
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
8
R = H, R' = Ac
8a R, R' = H
R = Me, R' = Ac
OAc
9
>4.1
5.0
0.51
>5.0
6.5
0.68
>5.0
n.t.
OAc
R
27 R = H2
30 R = O
H
COOMe
1.6
1.0
n.t.
Figure 2. The most potent NQ and NHQ derivatives.
>6.4
>4.9
>18.6
>21.5
12.4
2.3
>6.4
>4.9
>18.6
>21.5
12.4
4.4
>6.4
>4.9
>18.6
n.t.
In summary, we have prepared cytotoxic terpenyl-
NHQs protected as acetyl, methyl or benzyl derivatives
from the cycloadducts 3 and 4, obtained by Diels–Alder
addition between natural diterpenoids and p-BQ. The fi-
nal protected compounds were attained by two alterna-
tive procedures, involving hydroquinone protection and
aromatisation or vice versa. In general, the best results
were obtained through the second option, that is, via
NQ reduction and final hydroquinone protection. Addi-
tionally, several oxidised derivatives at the decaline core
were obtained through allylic oxidation procedures. The
bioevaluation revealed the interesting antineoplastic
cytotoxicity of the diacetyl NHQ derivatives, which,
while being enough stable for most chemical synthetic
work and ready to be hydrolysed by aqueous in vitro
or enzymatic in vivo media, constitute a group of good
candidates for further developments, in the search for
more potent and selective antineoplastic agents. In addi-
tion to the SAR mentioned above, the results found and
the global comparisons made contribute to support the
redox-based mechanism of action proposed for qui-
none/hydroquinone antineoplastics.2
9.0
n.t.
3.7
3.2
8.1
n.t.
n.t.
14.8
0.24
>6.7
15.2
0.19
4.8
0.24
>6.7
>16.6
0.19
4.8
n.t.
>6.7
n.t.
n.t.
n.t.
n.t., not tested.
a GI50 values expressed in lM.
Most of the SAR observations will mainly refer to struc-
ture variations in the decaline fragment of the diacetyl
NHQ derivatives. It can be seen that transformation
of the free carboxylic acid into the methyl ester enhances
by more than one order the cytotoxicity of diterpenyl-
NQ derivatives (6 vs 7), while this transformation seems
to have not a significant effect in the case of the diacylat-
ed NHQs (8 vs 9). The introduction of the 7a-hydroxyl
group slightly decreased the potency (9 vs 18). The same
happened with the carbonyl group at that position (9 vs
24), but the decrease attained more than one order of
magnitude for the 7b-hydroxyl group (9 and 17 vs 31).
The double bond isomerisation from the exocyclic to
the endocyclic position did not modify significantly the
activity (9 vs 27), while a slight increase was induced
by the presence of the 7-oxo function (24 vs 30).
3. Experimental
3.1. Chemistry
Optical rotations were determined on a Perkin-Elmer
241 polarimeter in CHCl3 and UV spectra on a Hitachi
100-60 spectrophotometer in CHCl3 solution. IR spectra
were obtained on a Niconet Impact 410 spectrophotom-
eter in NaCl film. All NMR spectra were recorded on
Bruker WP 200 SY (200 MHz for 1H and 50.3 MHz
The NQ 7 and the NHQs, 8, 9, 27 and 30 (Fig. 2) were
the most cytotoxic compounds with small differences in
potency. All of them displayed GI50 values under the
lM level and the NHQ derivatives share in common
the diacetate functionality. The practical equipotency
of NQ 7 and NHQ diacetate 9 could be explained
assuming that along the 3 days of the in vitro assay in
the atmospheric ambient, the acetate groups of NHQ
9 should be hydrolytically removed and the resulting
unprotected and less stable NHQ 8a would be oxidised
by air leading to NQ 7. This could support the design
hypothesis of this research, related to the prodrug nat-
ure of the protected NHQs, although for the methyl
and benzyl ethers it remains to be proven through
in vivo assays; for which, NHQ diacetate 8 and the di-
methyl and dibenzyl ethers 16 and 29 have been selected.
for 13C) or Bruker Avance 400 DRX (400 MHz for H
1
and 100 MHz for 13C) spectrometers in deuterochloro-
form using TMS as internal reference. Chemical shift
values are expressed in ppm and coupling constants
are reported in Hz. GSMS spectra were measured on a
Hewlett–Packard 5890 series II gas chromatograph
(5971 series mass selective detector) and electron impact
(EIMS) were run in a VG-MICROMASS ZAB-2F.
High-resolution mass spectra (HRMS) and the corre-
sponding low-resolution spectra (EIMS and FABMS)
were obtained on a VG TS-250 spectrometer by EI
and fast atom bombardment (FAB) working at 70 eV
and using nitrobenzyl alcohol as the matrix with xenon

Table 3. 1H NMR (CDCl3-TMS, d ppm, J Hz) data for compounds 3–31
H
3
4
8
9
10
11
5
1.33 m
—
—
—
1.25 m
1.37 m
9
1.56 m
—
—
—
1.69 m
2.50 m
11
1.51 m
—
—
—
2.02 m; 1.69 m
2.97 m; 2.60 m
7.34 dd (8.6, 1.5)
8.11 d (8.6)
7.95 d (1.5)
4.97 br s; 4.72 br s
1.16 s
6.45 dd (15.6, 9.7)
6.59 d (15.6)
7.59 dd (8.8, 1.8)
8.12 d (8.8)
8.07 d (1.8)
4.80 br s; 4.62 br s
1.23 s
12
—
—
2.95 m; 2.61 m
7.38 dd (8.8, 1.5)
7.78 d (8.8)
7.55 s
2.97 m; 2.63 m
7.39 dd (8.8, 1.6)
7.78 d (8.8)
7.56 d (1.6)
4.97 br s; 4.68 br s
1.16 s
14
5.36 m
2.14 ma
2.40 ma; 2.08 ma
5.35 m
—
15
16
—
17
4.83 br s; 4.48 br s 4.82 br s; 4.46 br s
4.97 br s; 4.68 br s
1.20 s
18
1.23 s
0.58 s
6.66 s
1.17 s
0.48 s
6.65 s
20
20
30
50, 60
OAc
OMe
COOMe
0.61 s
0.51 s
0.51 s
0.74 s
7.22 d (8.0)
7.17 (8.0)
—
7.21 d (8.4)
7.16 d (8.4)
—
6.69 d (8.4)
6.63 d (8.4)
—
6.69 d (8.2)
6.64 d (8.2)
—
3.24–3.16 m
3.24–3.12 m
—
—
—
—
2.45 s; 2.46 s
—
2.45 s; 2.46 s
—
—
—
—
3.96 s; 3.95 s
3.61 s
3.96 s; 3.95 s
3.65 s
3.60 s
—
3.60 s
12
13
14
15
16
17
5
—
—
—
—
—
—
1.28 m
—
9
—
—
1.72 m
—
11
12
14
15
16
17
18
20
20
—
—
2.02 m; 1.72 m
2.97 m; 2.64 m
7.53–7.20 m
8.22 d (8.6)
8.05 br s
—
2.96 m
7.56 d (8.0)
8.21 d (8.0)
8.07 d (1.1)
—
2.99 m; 2.62 m
5.54 m
5.51 m
5.56 m
7.55–7.35 m
3.25–3.01 m
3.25–3.01 m
3.24–2.95 m
3.24–2.95 m
4.86 br s; 4.52 br s
1.17 s
3.32–3.12 m
3.32–3.12 m
4.87 br s; 4.58 br s
1.18 s
8.24 d (8.8)
8.06 br s
4.96 br s; 4.66 br s 4.88 br s; 4.54 br s
4.96 br s; 4.72 br s
1.16 s
4.96 br s; 4.72 br s
1.16 s
0.51 s
—
1.23 s
0.62 s
6.93 s
1.20 s
0.51 s
0.51 s
0.52 s
0.50 s
6.92 s
6.63 s
6.74 d (8.3)
6.69 d (8.3)
3.61 s
6.75 d (8.4)
30
—
6.69 d (8.4)
COOMe 3.60 s
—
3.61 s
3.61 s
—
OAc
OMe
OBn
—
—
—
2.31 s; 2.32 s
2.30 s; 2.32 s
—
—
—
—
—
—
—
3.79 s; 3.77 s
—
—
—
7.53–7.20 m (10H),
7.55–7.35 m(15H), 5.21 s (2H), 5.20 s (2H),
5.21 s (2H), 5.19 s (2H) 5.16 d (12.8, 1H), 4.99 d (12.8, 1H)
18
19
20
21
22
23
24
7
4.42 t (2.9)
2.89 m
4.44 t (3.1)
2.92 m
4.38 t (2.6)
2.94 m
—
—
—
—
12
14
15
16
17
18
20
20
2.85 m
2.87 m
—
—
7.38 dd (8.4, 1.5)
7.79 d (8.4)
7.52 br s
7.33dd (8.3, 1.3)
8.12 d (8.3)
7.92 d (1.3)
(7.51–7.38) m
8.24 d (8.8)
8.04 d (1.1)
5.16 br s; 4.83 br s
1.16 s
7.38 dd (8.6, 1.6)
8.12 d (8.6)
7.99 br s
7.37 dd (8.6, 1.6)
8.12 d (8.6)
7.99 br s
(7.50–7.37) m
8.23 d (8.8)
8.10 br s
5.09 br s; 4.88 br s
1.22 s
7.37 dd (8.8, 1.7)
7.80 d (8.8)
7.53 br s
5.86 br s; 5.23 br s
1.15 s
5.16 br s; 4.78 br s 5.18 br s; 4.82 br s
4.64 d (10.0); 4.47 d (10.0) 5.09 br s; 4.89 br s
1.15 s
1.16 s
1.23 s
1.22 s
0.49 s
0.49 s
0.50 s
0.84 s
0.64 s
0.63 s
0.66 s
7.21 d (8.0)
7.16 d (8.0)
2.45 s; 2.47 s
—
6.68 d (8.3)
6.63 d (8.3)
—
6.74 d (8.4)
6.68 d (8.4)
—
6.68 d (8.4)
6.64 d (8.4)
—
6.69 d (8.2)
6.63 d (8.2)
—
6.74 d (8.4)
6.69 d (8.4)
—
7.22 d (8.3)
7.17 d (8.3)
2.47 s; 2.45 s
—
30
OAc
OMe
3.96 s; 3.95 s
—
3.96 s; 3.95 s
3.96 s; 3.95 s
—

J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
5767
as the fast atom. Column chromatography (CC) was
performed on silica gel (Merck No. 9385). TLC were
carried aut on silica gel 60 F254 (Merck, 0.25 mm thick)
and were detected by UV and by spraying with 10%
(v/v) sulfuric acid in ethanol and subsequent heating.
HPLC analyses were carried out on a Waters Delta
600 high-pressure liquid chromatograph with a Waters
2996 spectrophotometric detector (200–600 nm) and
equipped with a Xterraꢂ Prep MS C18 column,
150 · 10 mm, flow rate 5 mL/min. Elemental analyses
were obtained with a Perkin-Elmer 2400 CHN. Solvents
and reagents were purified by standard procedures as
necessary.
3.1.1. Starting materials. Myrcecommunic acid 1 was
isolated from berries of J. oxycedrus and transformed
into methyl myrcecommunate
procedure.10
2
by
a
reported
3.1.2. Cycloadducts 3 and 4: General procedures for the
Diels–Alder cycloadditions. In the presence of BF3ÆEt2O.
To a solution of p-BQ (178 g, 1.65 mmol) in dry ether,
1 (499 mg, 1.65 mmol) or 2 (522 mg, 1.65 mmol) and
BF3ÆEt2O cat. were added. The mixture was stirred at
room temperature under argon atmosphere for 24 h,
then, it was diluted with ether, washed with brine, dried
over anhydrous Na2SO4 and the solvent evaporated off
to yield cycloadduct 3 or 4.
Compound 3. Six hundred and thirty-seven milligrams
(94%). IR cmꢀ1 (film): 3500–2500, 1680, 1470, 1380,
1180, 1030, 910, 850, 730. 1H NMR (Table 3). 13C
NMR (Table 4). HRMS (FAB, M+1): calcd C26H34O4
411.2535; found 411.2495.
Compound 4. Six hundred and eighty-six milligrams
(98%). IR cmꢀ1 (film): 1722, 1689, 1656, 1448, 1153,
1
1030, 887, 730. H NMR (Table 3). 13C NMR (Table
4). HRMS (FAB, M+1): calcd C27H37O4 425.2692;
found 425.2703.
Under microwave irradiation. Diterpenoids 1 or 2 and
p-BQ were dissolved in dichloromethane and the corre-
sponding solid support (five times the reactant weights)
was added and well mixed. Then, the organic solvent
was evaporated and dried material was subjected to
microwave irradiation for a specified time (2 min inter-
vals, see Table 1). The solid mixture was treated according
to the description in Table 1, and the reaction product was
extracted from the support with EtOAc. After evapora-
tion of the organic solvent the samples were analysed by
NMR to determine the product ratios (Table 1).
Spectroscopic data of 5 and 6 were in agreement with
those previously described by us.10
3.1.3. Diterpenylnaphthohydroquinone diacetate (8).
Method A. The cycloaddition product under microwave
irradiation between 1 (293 mg, 0.97 mmol) and p-BQ
(157 mg, 1.45 mmol) was treated with acetic anhydride
and pyridine. The mixture was kept at room tempera-
ture and darkness for 12 h. After this period, the reac-
tion mixture was quenched with ice, diluted in water

Table 4. 13C NMR (CDCl3–TMS, d ppm) data for compounds 3–31
C
3
4
8
9
10
39.0
11
40.9
12
39.1
13
39.1
14
39.2
15
39.1
16
39.0
17
39.0
18
38.7
1
39.0
19.8
37.9
44.1
56.2
26.0
38.6
39.1
19.8
38.1
44.2
56.1
26.1
38.6
38.8
19.8
37.7
44.1
55.9
26.0
38.6
39.2
20.0
38.2
44.4
56.4
26.4
38.6
2
19.9
38.1
19.7
38.4
19.8
38.1
44.2
56.2
26.2
38.7
147.6
55.3
40.2
25.4
34.8
150.3
133.5
127.4
126.7
106.7
28.7
177.5
12.5
183.9
—
20.0
37.9
20.1
38.3
19.9
38.2
19.9
38.1
19.9
38.1
19.9
37.9
3
4
44.3
56.2
44.3
55.8
44.3
56.3
44.4
56.3
44.3
56.3
44.3
56.2
44.4
56.3
43.9
48.6
5
6
26.3
38.8
25.1
37.3
26.2
38.8
26.4
38.6
26.3
38.8
26.3
38.7
26.3
38.7
32.4
73.9
7
8
147.8
55.1
40.4
147.8
55.0
40.1
147.8
54.8
40.3
148.2
55.3
40.4
148.0
55.3
40.2
149.5
60.9
39.8
148.1
55.3
40.5
148.2
55.3
40.3
148.1
55.6
40.2
148.0
55.2
40.2
147.9
55.1
40.3
149.1
48.4
40.2
9
10
11
12
13
14
15
16
17
18
19
20
10
21.6
36.2
21.5
36.0
25.2
34.6
25.3
34.7
25.9
35.0
128.7
133.0
135.1
123.6
122.0
119.4
108.2
28.7
21.5
35.7
21.5
35.7
21.8
36.2
25.7
34.8
25.7
34.8
24.7
34.4
135.7
118.0
24.6a
27.3a
106.4
28.9
136.4
117.9
24.5a
27.3a
106.3
28.7
141.9
128.4
121.6
119.8
106.6
28.8
141.9
128.5
121.7
120.0
106.5
28.8
140.8
127.2
121.8
120.2
106.6
28.8
134.3
116.7
27.7
134.4
116.7
27.7
134.8
117.4
27.5
140.8
127.9
122.0
120.4
106.5
28.7
140.9
128.1a
122.0
120.5
106.7
28.9
141.6
128.3
121.8
120.0
109.4
28.5
25.2
106.5
29.0
25.2
106.4
28.8
25.3
106.5
28.8
183.9
12.7
177.8
12.5
183.7
12.7
177.6
12.7
177.8
12.6
177.7
13.5
184.0
12.9
177.8
12.7
177.8
12.6
177.7
12.6
177.0
12.8
177.8
11.6
200.0a
139.2
139.2
200.2a
46.2
200.1a
139.2
139.2
200.4a
46.2
143.9
116.6
117.6
144.2
126.1
127.7
—
144.1
116.7
117.6
144.5
126.3
128.0
51.0
149.2
102.3
103.2
149.6
124.7
126.5
51.1
149.5a
103.1
103.6
149.7a
125.3
126.5
51.1
146.2
119.9
119.9
146.2
128.6
128.9
—
146.2
119.9
119.9
146.2
128.6
128.7
51.1
151.0
106.8
106.8
151.0
124.6
124.9
51.1
148.5
104.0
105.2
148.9
126.8
127.2
51.0
148.5
104.0
105.2
148.9
125.1
126.8
—
143.9
116.7
117.7
144.4
126.2
127.7
51.2
20
30
—
40
183.7
131.5
134.1
51.0
—
50
60
46.8
46.7
51.1
COOMe
OAc
—
—
—
169.4
20.9
169.1
20.9
—
—
169.3
20.8
169.2
20.8
—
—
—
169.5
169.4
21.0
OMe
OBn
—
—
—
—
—
—
—
—
55.7
—
55.7
—
—
—
—
—
—
—
55.6
—
—
70.4, 70.5,
137.5,
137.6, 127.9a,
128.5(5C)a,
127.4(4C)a
—
—
—
70.5,70.4,
65.9,137.6,
137.5,136.1,
128.1(5C)a,
128.0(2C)a,
127.9(2C)a,
127.8(2C)a,
127.3(4C)a

19
38.7
20
38.7
21
36.8
22
31.6
23
36.4
24
25
40.1
26
40.2
27
37.7
28
37.7
29
37.7
30
36.2
31
37.5
1
40.2
19.7
38.7
44.1
53.2
37.7
2
19.9
38.0
19.9
38.0
19.5a
37.6
19.7
37.8
20.0
39.1
19.7
38.7
19.7
38.7
19.6
37.0
19.6
37.0
19.6
37.0
19.1
37.3
19.3
37.3
3
4
43.9
48.8
43.9
48.6
43.9
53.1
44.4
48.3
44.3
48.3
44.2
53.8
44.2
53.8
43.9
53.5
43.9
53.5
43.9
53.5
43.6
50.8
43.4
50.5
5
6
32.3
74.0
32.3
74.0
30.7
30.1
25.8
32.3
26.4
38.2
37.8
37.8
20.8
37.3
20.8
37.3
20.8
36.7
31.4
72.9
7
203.9
148.1
52.6
203.7
148.1
52.7
203.9
148.1
52.7
37.3
199.6
130.9
164.8
41.4
8
149.1
48.4
40.2
149.2
48.4
40.3
126.9
145.6
40.1
20.5a
148.5
79.6
43.9
148.1
79.3
43.3
127.5
138.6
39.7
127.3
138.9
39.7
128.5a
138.9
39.6
141.6
130.5
40.5
9
10
11
12
13
14
15
16
17
18
19
20
10
38.7
25.8
38.7
26.5
38.7
26.4
25.2
34.5
25.1
34.4
33.3
30.7
38.8
34.9
30.4
34.3
30.8
34.4
30.8
34.3
31.8
35.2
30.4
36.3
38.4
34.4
34.9
34.8
140.4
127.0
121.9
120.2
109.7
28.5
140.4
128.5a
122.2
120.5
109.7
28.5
140.2
126.9
121.9
119.8
75.8
140.5
127.0
122.0
120.1
110.4
29.0
140.9
127.8a
122.1
120.5
106.7
28.9
140.8
128.1
121.9
120.2
117.1
27.9
139.6
126.9
122.0
120.4
117.4
20.8
139.8
127.8a
122.3
120.5
117.4
28.0
142.0
128.3
121.7
119.4
19.8
140.7
126.9
121.9
119.7
19.9
140.9
127.7a
122.2
120.0
19.9
140.4
127.8
122.1
119.7
11.6
142.3
128.2
121.8
119.5
17.7
28.4
178.0
17.9
28.4
178.0
17.8
28.4
178.1
17.7
28.4
178.1
17.7
27.7
177.0
15.8
28.2
177.7
14.7
177.8
11.6
177.8
11.7
178.1
14.6
177.8
14.7
176.9
12.4
176.8
12.4
176.9
12.4
149.1
102.3
103.3
149.6
124.7
126.3
51.1
148.4
104.0
105.2
148.9
125.1
126.7
51.2
149.1
102.4
103.2
149.5
124.8
126.4
51.1
149.1
102.5
103.3
149.6
124.8
126.4
51.1
148.5
104.0
105.2
148.9
125.1
126.9
51.2
144.0
116.9
117.8
144.2
126.2
1247.7
51.4
149.2
102.6
103.5
149.6
125.0
126.5
51.3
148.4
104.2
105.3
148.8
125.2
126.8
51.5
143.9
116.7
117.7
144.3
126.1
127.8
51.2
149.1
102.4
103.2
149.6
124.7
126.5
51.0
148.9
104.1
105.3
148.5
125.1
127.1a
51.0
143.9
117.1
117.9
144.3
126.3
127.7
51.4
143.9
117.8
116.8
144.3
126.2
127.8
51.2
20
30
40
50
60
COOMe
OAc
—
—
—
—
—
169.4
21.0
—
—
169.4
21.0
—
—
169.2
20.9
21.0
169.4
20.9
OMe
OBn
55.6
—
—
55.7;
55.6
—
55.6;
55.7
—
—
—
—
55.7
—
—
—
—
55.6
—
—
—
—
—
70.4; 70.5;
137.5(2C);
127.8a,
127.3a(8C)
70.4(2C);
137.7(2C);
128.6a;
127.4a(8C)
70.6; 70.4;
137.5(2C);
128a;
127.3a(8C)
70.5; 70.3;
137.6;
137.5;
127.2a,
127.1a(8C)
—
—
C(CH3)3
C(CH3)3
—
—
—
—
26.5
80.1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
a Interchangeable assignments.

5770
J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
and extracted with EtOAc. Then the organic layer was
successively washed with 2 N HCl, aq satd NaHCO3
and brine and evaporated off to afford a residue that
was dissolved in benzene and DDQ (330 mg, 1.45 mmol)
was added. The mixture was stirred at room tempera-
ture for 0.5–1 h. Then it was filtered off, the organic sol-
vent was evaporated and the reaction product was
purified by CC, using mixtures of Hex/CH2Cl2, 2:8 as
eluent, to yield 610 (37 mg, 9%) and 8 (325 mg, 68%).
3.1.5. Methylated diterpenylnaphthohydroquinone (10).
Method C. The cycloaddition product between 2
(511 mg, 1.61 mmol) and p-BQ (157 mg, 1.45 mmol) in
presence of BF3ÆEt2O was oxidised with MnO2 (665 mg,
7.65 mmol) and reduced with Na2S2O4 (5.6 g), as de-
scribed above in method B, to give a reduction product
that was redissolved in DMF (15 mL) and MeI (545 lL,
8.75 mmol) and K2CO3 (966 mg, 7.9 mmol) were added.
The mixture was stirred at 110 ꢁC for 1 h. Then the reac-
tion was allowed to cool down to room temperature and
quenched by addition of a 2-N solution of HCl while
being vigorously stirred. The resulting solution was ex-
tracted with EtOAc and the combined organic layers were
washed with brine and dried over anhydrous Na2SO4. Re-
moval of the solvent gave a reaction product, which was
purified by CC using mixtures of Hex/EtOAc, 95:5 as elu-
ent, affording 10 (425 mg, 58%) and 12 (81 mg, 7%).
22
Compound 8. ½aꢁ +37.4ꢁ (589), +38.2ꢁ (578), +40.2ꢁ
D
(546), (c 0.84, CHCl3). UV kmax (e): 226(11800),
257(2200). IR cmꢀ1 (film): 3500–2500, 1766, 1693,
1
1460, 1366, 1053, 893. H NMR (Table 3). 13C NMR
(Table 4). Anal. (C30H36O6) C, H. HRMS (FAB,
M+1): calcd C30H37O6, 493.2590; found, 493.2576.
Method B. The cycloaddition product between 1 (293 mg,
0.97 mmol) and p-BQ (105 mg, 0.97 mmol) in presence of
BF3ÆEt2O was treated with MnO2 (379 mg, 4.3 mmol) un-
der refluxed benzene (20 mL) for 16 h. Then the mixture
was filtered through Celite, the solid washed with CH2Cl2,
and the organic solvent evaporated off. The crude product
was dissolved in dioxane (13 mL) and a solution of
Na2S2O4 (3.5 g) in H2O (15 mL) was added. The mixture
was kept stirring at room temperature under argon atmo-
sphere for 2 h, then it was diluted with EtOAc, washed
with brine, dried over anhydrous Na2SO4 and the organic
solvent evaporated off to yield a reduction product that
was acetylated with acetic anhydride and pyridine and
purified by CC using mixtures Hex/EtOAc, 8:2 as eluent,
yielding 8 (287 mg, 60%).
22
Compound 10. ½aꢁ +30.5ꢁ (589), +30.1ꢁ (578), +35.4ꢁ
D
(546), (c 0.94, CHCl3). UV kmax (e): 250 (9000), 319
(1800), 333 (1700). IR cmꢀ1 (film): 2845, 1723, 1603,
1463, 1363, 1271, 1152, 1097, 888. H NMR (Table 3).
1
13C NMR (Table 4). EIMS m/z: 450 (M+). HRMS
(EI): calcd. C29H38O4 450.2770; found, 450.2824.
22
Compound 12. ½aꢁ +54.6ꢁ (589), (c 0.78, CHCl3). UV
D
kmax (e): 267 (53800). IR cmꢀ1 (film): 1724, 1673, 1446,
1227, 1153, 931, 887. 1H NMR (Table 3). 13C NMR (Ta-
ble 4). HRMS (FAB, M+1): calcd. C48H61O6 733.4468;
found 733.4387.
Method D. The cycloaddition product between 2 (1.33 g,
4.20 mmol) and p-BQ (454 mg, 4.20 mmol) in presence
of BF3ÆEt2O was dissolved in EtOH (45 mL) and a 10-
N solution of NaOH (38 mL) was added dropwise while
the mixture was heated under reflux. After 10 min,
Me2SO4 (11.5 mL, 12.1 mmol) was added dropwise
and the stirring and heating was continued over 0.5 h.
Then the reaction was allowed to reach rt and was
quenched by the addition of a 2-N solution of HCl while
being vigorously stirred. The resulting solution was ex-
tracted with EtOAc and the combined organic layers
were washed with brine and dried over anhydrous
Na2SO4. Removal of the solvent gave 1.35 g of reaction
product. This residue was dissolved in benzene (50 mL)
and treated with DDQ (1.43 g, 6.30 mmol) following the
general procedure described above for 8. CC of the reac-
tion product, using mixtures of Hex/EtOAc, 95:5 as elu-
ent, afforded 11 (156 mg, 8%) and 10 (468 mg, 25%).
From 13: General procedure for aromatisation with DDQ.
To a solution of 13 (194 mg, 0.39 mmol) in benzene
(10 mL) was added DDQ (134 mg, 0.59 mmol). The
mixture was kept at room temperature for 0.5–1 h. Then
it was filtered, the organic solvent was evaporated and
the reaction product was purified by CC, using mixtures
of Hex/EtOAc, 8:2 as eluent, to yield 8 (175 mg, 91%).
3.1.4. Diterpenylnaphthohydroquinone diacetate (9).
Method A. The above method A was applied to the cyclo-
addition product obtained from microwave irradiation
between 2 (330 mg, 1.04 mmol) and p-BQ (168 mg,
1.56 mmol). CC of the crude product using mixtures of
Hex/CH2Cl2, 1:9 as eluent afforded 710 (44 mg, 10%)
and 9 (363 mg, 69%).
22
D
Compound 9. ½aꢁ +42.3ꢁ (589), +43.0ꢁ (578), +44.1ꢁ
(546), +50.3ꢁ (436), (c 0,89, CHCl3). UV kmax (e): 224
(1200), 254 (1800). IR cmꢀ1 (film): 1760, 1720, 1460,
1370, 1210, 1050, 920, 890. 1H NMR (Table 3). 13C
NMR (Table 4). HRMS (FAB, M+1): calcd C31H38O6
507.2747; found, 507.2701. Anal. (C31H37O6) C, H.
Compound 11. UV kmax (e): 240 (12,000), 268 (19,200).
IR cmꢀ1 (film): 2849, 1724, 1600, 1463, 1465, 1272, 1154,
1
1096. H NMR (Table 3). 13C NMR (Table 4). HRMS
(FAB; M+1): calcd C29H37O4 449.2692, found, 449.2692.
From 15. Treatment of 15 (150 mg, 0.33 mmol) with
DDQ (56 mg, 0.50 mmol) following the general proce-
dure described above for 8 afforded a reaction product
that was purified by CC, using mixtures of Hex/EtOAc,
95:5 as eluent, to yield 10 (76 mg, 51%).
Method B. Using the method B, compound 9 was ob-
tained in 55% overall yield from the cycloadduct 4.
From 14. Treatment of 14 (472 mg, 0.93 mmol) with
DDQ (315 mg, 1.39 mmol) as described above following
the purification by CC using mixtures of CH2Cl2/Hex,
9:1 as eluent yielded 9 (218 mg, 46%).
From 7. Compound 7 (823 mg, 1.96 mmol) in dioxane
(31 mL) was reduced with Na2S2O4 (6.8 g) in H2O

J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
5771
(35 mL) and the reduction product was treated with MeI
(590 lL, 9.45 mmol) and K2CO3 (1.03 g, 7.48 mmol) in
DMF as described before in method C giving a residue,
which was purified by CC using mixtures of Hex/EtOAc,
95:5 as eluent, to afford 10 (491 mg, 56%).
NaOH (2.8 mL) and Me2SO4 (850 lL, 0.90 mmol) as de-
scribed before in the first stage of method D. After re-
moval of the solvent the residue obtained was
esterified with ethereal solution of CH2N2 and when
the reaction was completed, the solvent was evaporated
and the product was purified by CC, using mixtures of
Hex/EtOAc, 98:2 as eluent, to yield 15 (61 mg, 54%).
3.1.6. Diterpenylnaphthoquinone (6): General Procedure
for oxidation with MnO2. Compound 3 (850 mg,
2.07 mmol) was treated with MnO2 (868 mg, 10.0 mmol)
under refluxed benzene (40 mL) for 16 h. Then the mix-
ture was filtered through Celite, the solid washed with
CH2Cl2, and the organic solvent evaporated off. The res-
idue was purified by CC using mixtures of Hex/EtOAc,
9:1 as eluent, to give 610 (711 mg, 84%).
3.1.11. Benzylated diterpenylnaphthohydroquinones (16
and 17). From 7. Treatment of 7 (286 mg, 0.68 mmol)
with Na2S2O4 (2.4 g) in dioxane/H2O (10 mL/13 mL)
as described before gave 275 mg of reduction product.
To a suspension of NaH (78 mg, 3,24 mmol) in DMF
(9 mL) under argon was added the reduction product
dissolved in DMF (3 mL) at 0 ꢁC. After 15 min of vigor-
ous stirring, BnBr (0.47 mL, 3.90 mmol) was added and
the reaction mixture was stirred at room temperature for
1 h. The reaction was quenched by the addition of
MeOH and the solvent was partially evaporated. The
residue was redissolved in Et2O and the resulting solu-
tion was washed with brine and dried over anhydrous
Na2S2O4. Removal of the solvent gave a residue, which
3.1.7. Diterpenylnaphthoquinone (7). Oxidation of 4
(850 mg, 2.0 mmol) with MnO2 (868 mg, 10.0 mmol) as
the general procedure described above followed by the
purification by CC using mixtures of Hex/EtOAc, 95:5
as eluent, yielded 710 (757 mg, 86%).
3.1.8. Dihydronaphthohydroquinone diacetate (13). Com-
pound 3 (263 mg, 0.64 mmol) was acetylated with acetic
anhydride and pyridine following the above general pro-
cedure for acetylation and purification by CC using mix-
was purified by CC using mixtures of Hex/EtOAc, 95:5
22
D
as eluent, to yield 16 (264 mg, 67%). ½aꢁ +30.7ꢁ (589),
+30.5ꢁ (578), +34.8ꢁ (546), (c 0.91, CHCl3). UV kmax
(e): 248 (13,200). IR cmꢀ1 (film): 2931, 2870, 2849,
1722, 1601, 1497, 1433, 1361, 1272, 1245, 1153, 1092,
tures of Hex/EtOAc, 8:2 as eluent afforded 13 (254 mg,
22
D
78%). ½aꢁ +40.7ꢁ (589), +42.5ꢁ (578), +48.5ꢁ (546),
1
+79.1ꢁ (436), (c 1.01, CHCl3). UV kmax (e): 260 (7200).
1076, 734, 696. H NMR (Table 3). 13C NMR (Table
IR cmꢀ1 (film): 3500–2550, 1780, 1690, 1650, 1200,
4). FABMS m/z: 603 (M+1)+. HRMS (FAB, M+1):
calcd C41H47O4 603.3474; found 603.3528.
1
1180, 1050, 900. H NMR (Table 3). 13C NMR (Table
4). Anal. (C30H38O6) C, H. HRMS (FAB, M+1) calcd
495.2746; found 495.2673.
From 6. The reduction of 6 (197 mg, 0.48 mmol) with
Na2S2O4 as described before followed by benzylation
according to the procedure described above gave a resi-
due which was purified by HPLC using mixtures of
3.1.9. Dihydronaphthohydroquinone diacetate (14). A
solution of 4 (495 mg, 1.16 mmol) in pyridine was trea-
ted with acetic anhydride following the first stage of
method A described above for 8. The obtained residue
MeCN/H2O 60/40 to yield 16 (133 mg, 46%) and 17
22
D
(72 mg, 22%). ½aꢁ +22.9ꢁ (589), +23.4ꢁ (578), +25.8ꢁ
was purified by CC using mixtures of Hex/EtOAc, 9:1
(546), +23.2ꢁ (436), (c 0.76; CHCl3). UV kmax (e): 249
(17,900). IR cmꢀ1 (film): 2933, 2870, 2848, 1720, 1601,
1497, 1433, 1361, 1272, 1244, 1127, 1091, 1076, 733,
22
D
as eluent, yielding 14 (472 mg, 77%). ½aꢁ +34.9ꢁ (589),
+36.7ꢁ (578), +41.7ꢁ (546), +71.3ꢁ (436), (c 1.0, CHCl3).
UV kmax (e): 265 (6400). IR cmꢀ1 (film): 1775, 1725,
696. H NMR (Table 3). 13C NMR (Table 4). FABMS
1
1
1650, 1370, 1200, 1180, 1030, 900. H NMR (Table 3).
m/z: 679 (M+1)+. HRMS (FAB, M+1): calcd C47H51O4
679.3787; found 679.3755.
13C NMR (Table 4). GCMS (220–290 ꢁC, 5 ꢁC/min,
HP-1 column), >99%, tR = 23.19 min, (m/z) 508 (M+).
Anal. (C31H40O6) C, H. HMRS (FAB, M+1) calcd
509.2903; found 509.2887.
3.1.12. Allylic alcohols (18–20): General procedure for the
allylic oxidation with SeO2. Conditions a. To a stirred solu-
tion of 9 (299 mg, 0.59 mmol) in EtOH (6 mL) was added
SeO2 (2.08 mmol, 3.5 equiv). The mixture was heated at
60 ꢁC and monitored by TLC until starting material dis-
appeared over 16 h. After evaporating the solvent, the res-
idue was redissolved in diethyl ether and the organic layer
was washed with brine, dried over Na2SO4, filtered and
evaporated off. The reaction product was purified by
3.1.10. Methylated dihydronaphthohydroquinone (15).
From 4. Treatment of 4 (414 mg, 0.97 mmol) with
Me2SO4 (3.6 mL, 3.80 mmol), a 10-N solution of NaOH
(11.8 mL) in EtOH (15 mL) as described before in the
first stage of method D, gave a reaction product which
was purified by CC using mixtures of Hex/EtOAc, 98:2
as eluent, to yield 15 (175 mg, 39%). IR cmꢀ1 (film):
2846, 1724, 1644, 1604, 1464, 1440, 1152, 1129, 888.
1H NMR (Table 3). 13C NMR (Table 4).
CC (eluent: Hex/EtOAc, 7:3) to yield 18 (246 mg, 80%):
22
½aꢁ +19.1ꢁ (589), +20.6ꢁ (578), +22.1o (546), (c 1.0,
D
CHCl3). UV kmax (e): 241 (7600), 277 (2900). IR cmꢀ1
(film): 3515, 2930, 2870, 1770, 1725, 1610, 1450, 1365,
1200, 1095, 1050, 900, 825, 735. RMN 1H: Table 3.
RMN 13C: Table 4. HMRS(ESI, M+23): calcd
C31H38O7Na 545.2509; found 545.2524.
From 3.To a stirred solution of 3 (105 mg, 0.25 mmol) in
benzene (20 mL) was added silica gel (1.1 g). The mix-
ture was stirred at room temperature under argon for
7 h. The resulting mixture was filtered and washed with
EtOAc. Removal of the solvent gave a residue that was
dissolved in EtOH (10 mL) and treated with 10 N
Following the conditions (a), CC of the product ob-
tained from 10 (258 mg, 0.59 mmol) yielded: (a) com-

5772
J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
pound 22 (70 mg, 25%) using mixtures of Hex/EtOAc,
95:5 as eluent: IR cmꢀ1 (film): 3529, 2944, 2873, 1723,
1632, 1603, 1463, 1455, 1428, 1368, 1271, 1243, 1195,
The organic layer was washed successively with 2 N
HCl, aq satd NaHCO3 and brine, dried over Na2SO4,
filtered and evaporated off affording a crude product,
1
1154, 1097, 975, 901, 831, 800, 719. RMN H: Table
which was purified by CC (eluent: Hex/EtOAc, 8:2) to
22
D
3. RMN 13C: Table 4. MS m/z (%): 467 (M+1)+ (20),
281 (13), 201 (86), 136 (66). HRMS (FAB, M+1): calcd
C29H39O5 467.2797; found, 467.2778; (b) compound 19
yield 24 (54 mg, 64%): ½aꢁ +28.3ꢁ (589), +28.9ꢁ (578),
(c 0.79, CHCl3). UV kmax (e): 240 (12300), 287 (4500).
IR cmꢀ1 (film): 2938, 2873, 2852, 1765, 1722, 1694,
1608, 1463, 1432, 1366, 1268, 1202, 1155, 1053, 896,
(184 mg, 67%) using mixtures of Hex/EtOAc, 8:2 as elu-
22
D
1
ent: ½aꢁ +6.9ꢁ (589), +7.3ꢁ (578), +9.3ꢁ (546), (c 0.71,
825. RMN H: Table 3. RMN 13C: Table 4. HRMS
CHCl3). UV kmax (e): 250 (14100), 319 (3100). IR
cmꢀ1 (film): 3427, 2934, 2872, 2852, 1723, 1603, 1464,
1450, 1368, 1331, 1271, 1243, 1195, 1152, 1097, 1040,
(FAB, M+1): calcd C31H37O7 521.2539, found,
521.2532.
1
973, 898, 800, 737, 720. RMN H: Table 3. RMN 13C:
The Swern oxidation was applied to obtain the follow-
ing derivatives:
Table 4. MS m/z (%): 466 (M)+ (21), 201 (28), 136
(40). HRMS (EI, M+): calcd C29H38O5 466.2719, found,
466.2754.
Compound 25 (46 mg, 70%, eluent: Hex/EtOAc 95/5)
22
from 19 (0.65 mg, 0.14 mmol): ½aꢁ
+31.6ꢁ (589),
Starting from 16 (356 mg, 0.59 mmol) and following
conditions a), CC (eluent: Hex/EtOAc, 95:5) of the reac-
tion product yielded: (a) compound 23 (87 mg, 24%):
UV kmax (e): 248 (17,400). IR cmꢀ1 (film): 3520, 3064,
3031, 2942, 2871, 1721, 1601, 1497, 1453, 1433, 1361,
1273, 1242, 1201, 1154, 1093, 1075, 905, 799, 733, 696.
+32.6ꢁ (578), +36.5ꢁ (546), +34.7ꢁD (436), (c 0.98,
CHCl3). UV kmax (e): 249 (21100), 319 (4400). IR
cmꢀ1 (film): 2936, 2873, 1723, 1695, 1603, 1463, 1426,
1368, 1272, 1243, 1231, 1194, 1151, 1097, 965, 803.
RMN H: Table 3. RMN 13C: Table 4. MS m/z (%):
1
464 (M)+ (14), 201 (100), 136 (56). HRMS (EI, M+):
calcd C29H36O5 464.2563, found, 464.2621.
1
RMN H: Table 3. RMN 13C: Table 4. MS m/z (%):
619 (M+1)+ (1), 307 (13), 154 (100), 91 (71). HRMS
(FAB, M+1): calcd C41H47O5 619.3424, found,
Compound 26 (58 mg, 68%, eluent: Hex/EtOAc, 9:1)
22
D
22
D
619.3452; (b) compound 20 (216 mg, 60%): ½aꢁ +3.8ꢁ
from 20 (84 mg, 0.14 mmol): ½aꢁ +24.2ꢁ (589), +24.3ꢁ
(589), +3.8ꢁ (578), +4.2ꢁ (546), (c 0.73, CHCl3). UV kmax
(e): 250 (15600), 319 (1100). IR cmꢀ1 (film): 3419, 3064,
3031, 2937, 2850, 1722, 1601, 1453, 1432, 1361, 1272,
1243, 1200, 1151, 1092, 1075, 910, 802, 734, 697.
(578), +28.5ꢁ (546), (c 0.80, CHCl3). UV kmax (e): 249
(20100), 319 (4300). IR cmꢀ1 (film): 2927, 2852, 1723,
1695, 1601, 1497, 1454, 1362, 1273, 1243, 1151, 1092,
982, 800, 734, 696. RMN 1H: Table 3. RMN 13C: Table
4. HRMS (ESI, M+Na): calcd C41H44O5Na 639.3081,
found, 639.3066.
1
RMN H: Table 3. RMN 13C: Table 4. MS m/z (%):
619 (M+1)+ (2), 307 (12), 154 (100), 91 (94). HRMS
(FAB, M+1): calcd C41H47O5 619.3424, found,
619.3473.
3.1.14. Diterpenylnaphthohydroquinones (27–29): Gen-
eral procedure for isomerisation. To a 10ꢀ1 M solution of
9 (105 mg, 0.23 mmol) in C6H6 (23 mL) was added HI
57% (0.53 mL, 4.01 mmol). The mixture was stirred at
80 ꢁC for 10 min. Then, ethyl acetate was added and
the organic layer was washed with aq satd NaHCO3,
brine, dried over Na2SO4, filtered and evaporated off
Conditions b. To a stirred solution of 10 (160 mg,
0.35 mmol) in CH2Cl2 (10 mL) SeO2 (27 mg, 0.24 mmol)
was added. Then a 6 M solution of t-BuOOH in decane
(180 lL, 1.08 mmol) dissolved in CH2Cl2 (5 mL) was
added dropwise under argon atmosphere and the stir-
ring was continued at rt for 5 h. Then it was diluted with
CH2Cl2 and the organic layer was washed with brine,
dried over Na2SO4, filtered and evaporated off. The
reaction product was purified by CC to yield: (a) com-
pound 21 (31 mg, 18%) using Hex/EtOAc, 95:5 as elu-
yielding the isomerised product 27 (0.23 mmol, 98%):
22
D
½aꢁ +42.1ꢁ (589), +43.1ꢁ (578), +48.5ꢁ (546), (c 0.62,
CHCl3). IR cmꢀ1 (film): 2950, 1765, 1725, 1600, 1500,
1460, 1365, 1200, 1050, 970, 910, 890, 795. RMN H:
1
Table 3. RMN 13C: Table 4. HRMS (FAB, M+1): calcd
C31H39O6 507.2747, found, 507.2702.
1
ent: RMN H: Table 3. RMN 13C: Table 4. MS m/z
(%): 538 (M)+ (35), 351 (7), 307 (11), 267 (11), 201
(100), 154 (68), 91 (67). HRMS (EI, M+): calcd
C33H46O6 538.3532, found, 538.3562; (b) compound 19
(102 mg, 62%) using Hex/EtOAc 8/2 as eluent.
Starting from 10 (113 mg, 0.23 mmol) and following the
above procedure for isomerisation the reaction product
yielded 28 (0.22 mmol, 97%): UV kmax (e): 252 (12600),
319 (2900). IR cmꢀ1 (film): 2934, 2872, 2851, 2832,
1724, 1602, 1463, 1450, 1271, 1242, 1192, 1097, 799,
3.1.13. a,b-Unsaturated ketones (24–26): General proce-
dure for Swern oxidation. To a precooled at ꢀ55 ꢁC and
stirred solution of 2 M oxalyl chloride (0.21 mL,
0.42 mmol) in dry CH2Cl2 (3 mL) a solution of DMSO
(60 lL, 0.83 mmol) in dry CH2Cl2 (1 mL)was added
dropwise. After 5 min at ꢀ55 ꢁC, a solution of 18
(73 mg, 0.14 mmol) in dry CH2Cl2 (2 mL) was slowly
added. The mixture was stirred at the same temperature
for 30 min, then triethylamine (0.20 mL, 1.44 mmol) was
added dropwise. The mixture was warmed to 0 ꢁC over
1 h, quenched with water and extracted with CH2Cl2.
1
719. RMN H: Table 3. RMN 13C: Table 4. MS m/z
(%): 451 (M+1)+ (40), 201 (85), 136 (43), 73 (100).
HRMS (FAB, M+1): calcd C29H39O4 451,2848, found,
451,2886.
Starting from 16 (141 mg, 0.23 mmol) and following the
procedure for isomerisation the reaction product yielded
29 (0.22 mmol, 97%): UV kmax (e): 249 (13,700). IR cmꢀ1
(film): 3063, 3030, 2926, 2869, 2851, 1723, 1600, 1497,
1452, 1432, 1361, 1272, 1192, 1161, 1092, 1075, 1076,

J. M. Miguel del Corral et al. / Bioorg. Med. Chem. 15 (2007) 5760–5774
5773
1
1028, 984, 802, 733, 696. RMN H: Table 3. RMN 13C:
Acknowledgment
Financial support for this work came from Junta de
´
Castilla y Leon (Spain) (SA 068/04 and SA114A06).
Table 4. HRMS (FAB, M+1): calcd C41H47O4 603.3474;
found, 603.3498.
3.1.15. a,b-Unsaturated ketone (30). To a stirred solution
of 27 (344 mg, 0.68 mmol) in C6H6 (15 mL), AcOH
(1.25 mL), Ac2O (1.90 mL), NaOAc (470 mg) and Na2-
CrO4 (625 mg, 3.86 mmol) were added. The mixture
was heated at 70 ꢁC for 8 h. After evaporating the sol-
vent, the residue was redissolved in EtOAc and the or-
ganic layer was washed with Na2S2O3 and brine, dried
over Na2SO4, filtered and evaporated off. The reaction
product was purified by CC (eluent: Hex/EtOAc, 8:2)
to yield 30 (283 mg, 80%): IR cmꢀ1 (film): 2940, 1765,
1725, 1660, 1610, 1450, 1365, 1200, 1050, 1010, 895,
825, 735. RMN 1H: Table 3. RMN 13C: Table 4. HRMS
(FAB, M+1): calcd C31H36O7 521.2539; found 521.2598.
References and notes
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rated off. The reaction product was purified by CC (elu-
22
ent: CHCl3/EtOAc, 8:2) to yield 31 (100 mg, 24%): ½aꢁ
D
+22.2ꢁ (578), +24.3ꢁ (546), (c 0.63, CHCl3). IR cmꢀ1
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1
1200, 1050, 915, 825, 735. RMN H: Table 3. RMN
7. (a) Molinari, A.; Oliva, A.; Reinoso, P.; Miguel del Corral,
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´
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3.2. Bioactivity: Cell growth inhibition assay
´
2001, 46, 33–39.
A colorimetric assay using sulforhodamine B (SRB) has
been adapted for a quantitative measurement of cell
growth and viability, following a previously described
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Using control OD values (C), test OD values (T) and
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