Synthesis and biological evaluation of dehydroabietic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.10.010

A series of C18-oxygenated derivatives of dehydroabietic acid were synthesized from commercial abietic acid and evaluated for their cytotoxic, antimycotic, and antiviral activities.

Full text of DOI:10.1016/j.ejmech.2009.10.010

European Journal of Medicinal Chemistry 45 (2010) 811–816
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of dehydroabietic acid derivatives
a
,
a
b
b
*
´
´
Miguel A. Gonzalez , David Perez-Guaita , Julieth Correa-Royero , Bibiana Zapata ,
**
Lee Agudelo b, Ana Mesa-Arango b, Liliana Betancur-Galvis b
,
a
´
Departamento de Qu´ımica Organica, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain
b Grupo de Bioactividad, Facultad de Medicina, Universidad de Antioquia, A.A1226, Medell´ın, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of C18-oxygenated derivatives of dehydroabietic acid were synthesized from commercial abietic
acid and evaluated for their cytotoxic, antimycotic, and antiviral activities.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 1 September 2009
Received in revised form
1 October 2009
Accepted 7 October 2009
Available online 13 October 2009
Keywords:
Abietic acid
Dehydroabietic acid
Cytotoxicity
Antimycotic activity
Antiviral activity
1. Introduction
Continuing our research program on the synthesis of bioactive
terpenoids, we were interested in confirming the results reported
Diterpene resin acids are important defense compounds of
conifers against potential herbivores and pathogens [1]. The bio-
logical activity of natural abietane acids has been reviewed [2].
Antimicrobial, antiulcer and cardiovascular activities are the most
representative for this class of diterpenoids. Dehydroabietic acid
(DHA)(Fig. 1, R]COOH), a natural occurring diterpene resin acid,
and its derivatives exhibit a broad spectrum of biological action. For
example, they have shown antiulcer [3], antimicrobial [4], anxio-
lytic [5], antiviral [6], antitumor [7], and cytotoxic activities [8].
Recent studies have demonstrated that DHA and some derivatives
are chemical modulators, particularly openers, of large-conduc-
tance calcium-activated Kþ channels (BK channels) [9]. This feature
makes DHA a new scaffold in the treatment of acute stroke,
epilepsy, asthma, hypertension, gastric hypermotility and
psychoses. Also, DHA was reported to have properties of enhancing
the inhibitory activity of anticancer drugs in cervical carcinoma
cells, hepatocellular carcinoma cells, or breast cancer cells [10].
However, the cytotoxic activity of easily available derivatives of
DHA have not yet been reported so far.
in a recent patent on the use of abietic acid 1 and derivatives as
antitumor agents [11] (Scheme 1). As a result, we studied a series of
abietic acid derivatives and found that methyl abietate 2 displayed
the highest cytotoxicity against HeLa cancer cells (CC50 11 mM), and
showed good selectivity towards non-cancerous cells (selectivity
index 13.7) [12]. Encouraged by these research results, DHA was
chosen as the starting material in screening a series of derivatives
for new potential bioactive compounds.
In this communication, we describe the syntheses of a number
of derivatives of DHA from commercially available (ꢀ)-abietic acid
(1) (Scheme 1) [13], and the results of preliminary evaluation of
their cytotoxic, antimycotic and antiviral activities. In this study, an
oxygenated moiety (such as methyl ester, alcohol, or aldehyde) was
introduced into the lipophilic dehydroabietane skeleton. In this
context, simple and sequential modifications were performed in
the molecule of DHA (4). Compound 4 and nine derivatives with
different functional groups at C7 and C18 were tested. All the
compounds were easily obtained in good yield, by standard or
reported chemical procedures. Some of the synthesized
compounds have been isolated as natural products, in particular,
dehydroabietinol (pomiferin A) (5) [14], dehydroabietinol acetate
* Corresponding author. Tel.: þ34 963543880; fax: þ34 963544328.
** Corresponding author. Tel.: þ57 45106059; fax: þ57 45106062.
E-mail addresses: miguel.a.gonzalez@uv.es (M.A. Gonza´lez), labeta@catios.udea.
edu.co (L. Betancur-Galvis).
(6) [15], 7a-hydroxydehydroabietinol (11) [16] and 7-oxodehy-
droabietinol (12) [17], however, few reports have appeared on their
biological activities.
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.010

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M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 45 (2010) 811–816
812
necessary to obtain six more derivatives. Thus, ester 3 was sapon-
ified with KOH in aqueous methanol to give dehydroabietic acid (4).
Reduction of 3 with LiAlH4 in dry tetrahydrofuran at reflux gave
dehydroabietinol 5 in 90% yield. Esterification of 5 with acetyl
chloride, benzoyl chloride and nicotinic chloride gave the corre-
sponding esters 6, 7, and 8, respectively. Finally, oxidation with
Dess–Martin periodinane [19] of 5 afforded aldehyde 9 in 95% yield.
On the other hand, reduction of ester 2 with LiAlH4 in dry
tetrahydrofuran at reflux gave alcohol (abietinol) 10 in quantitative
yield. Then, we reacted alcohol 10 with SeO2 to produce the
aromatization and simultaneous functionalization at C7
(Compound 11) [20]. Finally, alcohol 11 was oxidized with MnO2
and Dess–Martin periodinane to give the keto–alcohol 12 and the
aldehyde 13, respectively.
2.2. Biological evaluation
All compounds 1–13 (Scheme 1) were tested for antimycotic,
cytotoxic and antiviral activity with the exception of compound 8
which was not soluble under the experimental conditions. Firstly,
the compounds did not show antimycotic activity against Candida
parapsilosis, Candida krusei, Candida tropicalis and Candida albicans
Fig. 1. Chemical structure of some tested dehydroabietanes.
2. Results and discussion
2.1. Chemistry
in concentrations below 100 mg/mL (data not shown). MIC values
for the two reference antifungal drugs, amphotericine B and itra-
conazole (Sigma, New Jersey, USA), used as positive controls, were
within the established values for the AFST-EUCAST protocol. The
reaction of aromatization of abietanes did not improve anti-
Candida activity when we compare with the results of our previous
report [12]. In contrast, the dehydroabietanes 4 and 11 showed
anti-Aspergillus activity. The dehydroabietane 4 showed activity
The synthesis of the C18-functionalized dehydroabietanes used
in this work begins with the preparation of the required methyl
ester
2
(methyl abietate) from commercial (ꢀ)-abietic acid,
following a reported procedure (Scheme 1) [12]. Thus, abietic acid 1
was esterified by treatment with lithium hydroxide and methyl
sulphate to give ester 2 in quantitative yield. With the ester 2 in
hand, we carried out the reaction of aromatization to obtain the
dehydroabietane skeleton (Scheme 1). The ester 2 was treated in
against Aspergillus terreus with MIC value of 39.7
compound 11 showed activity against Aspergillus fumigates and
Aspergillus niger with MIC values of 50 and 63 g/mL, respectively.
mg/mL whereas
a
similar manner as pine rosin is treated to obtain dis-
m
proportionated rosin [18]. Thus, neat ester 2 was heated at 250 ꢁC in
the presence of 5% Pd/C to give methyl dehydroabietate 3 in 85%
yield. Then, we carried out the funtional group interconversions
Thus, the reaction of aromatization of abietanes improves anti-
Aspergillus activity when we compare with the results of our
previous report in which abietic acid 1 was not active.
Scheme 1. Reagents and conditions: a) LiOH, Me2SO4,DMF, 100%; b) 5% Pd/C, 250 ꢁC, 85%; c) KOH, MeOH, H2O, 75%; d) LiAlH4, THF, 90%; e) AcCl, BzCl, nicotinic chloride, Et3N, DCM,
75%, 70%, 80%, respectively; f) Dess–Martin periodinance, 90% for 9,85% for 13;g) SeO2, TBHP, DCM, 65%; h) MnO2, DCM, 60%.

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M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 45 (2010) 811–816
813
Table 2
Next, the compounds 1–13 (Scheme 1) were tested in vitro for
potential antitumor and cytotoxic activities determining the
concentration of the compound that induces 50% growth inhibition
(IC50) of the HeLa and Jurkat tumor cell lines, and the Vero no-
tumor cell line (Table 1). All these compounds produced a dose-
dependent inhibition on the growth of the HeLa and Jurkat tumor
cell lines and Vero cell line, with R2 (coefficient of linear
regression) > 0.8. The dehydroabietane that showed the highest
antitumor in vitro activity on the HeLa and Jurkat tumor lines was
Cytotoxicity and anti-HSV-1 activity of dehydroabietane diterpenes on Veroe Cells
determined by the end-point titration technique.
Compound
CC100
g/mL)a
Viral Reduction
Factorb
Antiviral Activity
(m
g/mL)c
(
m
1d
>100
50
>50
>50
50
>50
>50
50
NA
100.5
–
–
6.25
–
–
–
25
50
–
2d
3
4
5
6
–
–
101
100.5
–
the dehydroabietane 5 with IC50 values of 13.0 ꢂ 2.8
mg/mL and
7
9.7 ꢂ 0.7
m
g/mL, respectively. However, the dehydroabietane that
9
showed the lowest cytotoxicity on non-cancerous cells (Vero cell
10d
25
25
101
–
6.25
–
line) and highest antitumor in vitro activity on the Jurkat tumor line
11
12
Heparin
Acyclovir
25
–
–
was the dehydroabietane 6 with IC50 values of 95.0 ꢂ 13.0
mg/mL
>100 I.U./mL
>600
102
104
0.5 I.U./mL
6.0
and 22.0 ꢂ 3.6 g/mL, respectively. This means that the dehy-
m
droabietane with the highest selectivity index (SI) was dehy-
droabietinol acetate (6) (SI ¼ 4.3). Comparing the cytotoxic activity
on HeLa cells of our report on abietane derivatives [12] with these
dehydroabietane derivatives, the compound with the highest
selectivity was methyl abietate (2) (SI ¼ 13.7), now the compound
dehydroabietinol (5) displayed the highest cytotoxic activity to
both HeLa and Jurkat cells.
a
Minimal toxic dose that detached 100% of the cell monolayer.
Ratio of the virus titer in the absence over virus titer in the presence of the tested
b
compound.
c
Maximal nontoxic dose that showed the highest viral reduction factor. N.A: no
activity.
d
These abietane activities were reported in a previous study on HeLa cells [12].
The dehydroabietanes did not show activity on HeLa cells.
e
In Jurkat cells, the cytotoxicity of dehydroabietinol 5 was higher
than that of methyl abietate (2). In contrast, the abietinol 10 was
more cytotoxic on Vero cells than on Jurkat cells. Specifically,
according to SI value the dehydroabietane derivatives are more
selective to Jurkat cells than abietane derivatives. Selectivity refers
to the ability of a compound to recognize its target without inter-
acting with other target. In this study, it is likely that the dehy-
droabietane derivatives recognize targets in Jurkat cells which are
not present in HeLa cells. The introduction of an oxygenated
functional group in position C-7 in compound 5 (compounds 11 and
12) led to less active compounds.
activity of abietic acid derivatives were consistent with this order of
activity but in these series of dehydroabietane derivatives is not
possible to establish this relationship.
The antiviral activity of the dehydroabietanes against herpes
simplex virus type 1 (HSV-1) was determined using a modified
end-point titration technique (EPPT)(Table 2) [12]. Only the dehy-
droabietanes 6 and 7 reduced the HSV-1 replication with values
below 100
mg/mL. The dehydroabietane that showed the highest
antiviral activity was dehydroabietinol acetate (6). According to
Vlietinck et al. [22], only the compounds with reduction factor (Rf)
of the viral titer over 1 ꢄ103 (Rf: ratio of the virus titer in the
absence over virus titer in the presence of the tested compound)
show relevant antiviral activity. The ester dehydroabietinol acetate
(6) was found to be slightly active against HSV-1 over infected
confluent monolayers of Vero cells with Rf value of 1 ꢄ101 at
Gigante and co-workers [21] evaluated the antitumoral in vitro
activity of different catechols from abietic acid. The order of activity
in the catechol series with different functional groups at position
C18 was ester > acid ꢃ alcohol. Our previous study on the biological
a concentration of 25 mg/mL. Comparing the antiviral activity of the
abietinol (10) [12] with dehydroabietinol (5), the aromatization of
Table 1
ring C did not improve the activity.
Cytotoxic activity of dehydroabietane diterpenes determined by the MTT technique
expressed as IC50
(m
g/mL).a
Cell linesb
Vero
3. Conclusions
Compound
HeLa
IC50
Jurkat
SId
In conclusion, we have prepared and tested several dehy-
droabietanes for their antitumor, antifungal and antiviral activities
in vitro. In general, the aromatization in ring C of abietic acid led to
less active compounds. Also, the antitumor activity of the dehy-
droabietanes was more selective to Jurkat cells than HeLa cells.
Dehydroabietinol acetate (6) presented the highest SI on Jurkat
cells and it also showed the highest anti-herpes activity. These
results confirm that abietane and dehydroabietane-type diterpenes
do have interesting antitumor properties and encourage us to
research about what targets are recognized for these diterpenes in
HeLa and Jurkat cells. Furthermore, these results also encourage us
to synthesize additional abietane and dehydroabietane derivatives
with the aim of obtaining compounds more potent biologically.
c
IC50
IC50
SId
1e
53.0 ꢂ 5.6
49.0 ꢂ 3.0
36.0 ꢂ 5.8
91.0 ꢂ 7.5
38.0 ꢂ 6.5
95.0 ꢂ 13.0
15.0 ꢂ 0.6
3.6 ꢂ 1.0
3.5
13.7
1.3
0.9
2.9
3.4
ꢃ32.0
–
2e
24.0 ꢂ 1.5
21.0 ꢂ 3.4
28.0 ꢂ 2.7
9.7 ꢂ 0.7
22.0 ꢂ 3.6
2.0
1.7
3.2
3.9
4.3
3
4
5
6
7
8
28.0 ꢂ 5.0
101.0 ꢂ 10.3
13.0 ꢂ 2.8
28.0 ꢂ 4.7
ꢃ200
ꢃ200
–
57.0 ꢂ 11.0
3.5
f
f
f
f
f
9
32.0 ꢂ 5.0
13.0 ꢂ 2.4
30.0 ꢂ 5.4
27.0 ꢂ 5.0
36.0 ꢂ 5.5
1.1 ꢂ 0.2
24.0 ꢂ 4.4
5.2 ꢂ 0.5
1.3
2.5
1.5
0.9
1.6
2.2
17.0 ꢂ 0.6
15.0 ꢂ 3.6
12.0 ꢂ 1.0
17.0 ꢂ 0.3
12.0 ꢂ 1.5
–
1.9
0.9
2.5
1.6
3.0
10e
11
12
13
20.0 ꢂ 3.8
30.0 ꢂ 1.2
23.0 ꢂ 4.3
0.05 ꢂ 0.01
Vincristinea
a
Concentration of compounds that induces 50% growth inhibition in 48 h.
HeLa, human cervix epitheloid carcinoma ATCC CCL-2; Vero, Cercopithecus
b
4. Experimental
aethiops African green monkey kidney ATCC CCL-81. Jurkat, human acute T cell
leukemia ATCC TIB-152.
4.1. Chemistry
c
IC50 values are expressed as the mean ꢂ S.E.M. of at least four dilutions by
quadruplicate.
Optical rotations were determined using a 5-cm path-length
cell, using dichloromethane as solvent (concentration expressed in
d
SI, selectivity index is defined as VERO IC50 over HeLa IC50
These compounds were tested in a previous report [12].
This compound did not dissolved in DMSO.
.
e
f
g/100 mL). [
a
]D-values are given in 10ꢀ1 deg cm2 gꢀ1. NMR spectra

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M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 45 (2010) 811–816
814
were recorded on a 300 MHz spectrometer with tetramethylsilane
as an internal standard. All spectra were recorded in CDCl3 as
solvent unless otherwise described. Complete assignments of 13C
NMR multiplicities were made on the basis of DEPT experiments.
J values are given in Hz. Mass spectra (MS) were run by electron
impact (EI) at 70 eV. Reactions were monitored by thin-layer
chromatography (TLC) using Merck silica gel 60 F-254 in 0.25 mm-
thick plates. Compounds on TLC plates were detected under UV
light at 254 nm and visualized by immersion in a 10% sulfuric acid
solution and heating on a hotplate. Purifications were performed by
flash chromatography on Merck silica gel (230–400 mesh). All non-
aqueous reactions were carried out in an argon atmosphere in
oven-dried glassware. Commercial reagent grade solvents and
chemicals were used as received unless otherwise noted. Combined
organic extracts were washed with brine, dried over anhydrous
sodium sulphate, filtered and concentrated under reduced pres-
sure. Methyl abietate (2) was prepared from commercial abietic
acid (Aldrich) as reported by us [12]. All compounds prepared in
this work exhibit spectroscopic data in agreement with the
proposed structures.
J ¼ 10.8), 3.21 (1H, d, J ¼ 10.8), 2.85 (1H, m), 1.21 (6H, d, J ¼ 6.9), 1.21
(3H, s), 0.87 (3H, s); 13C NMR (75 MHz) dC 147.2 (s), 145.4 (s), 134.7
(s), 126.7 (d), 124.1 (d), 123.7 (d), 72.1 (t), 43.8 (d), 38.3 (t), 37.8 (s),
37.2 (s), 35.0 (t), 33.4 (d), 30.0 (t), 25.2 (q), 24.0 (q), 24.0 (q), 18.8 (t),
18.6 (t), 17.3 (q); HRMS (EI) m/z 286.2265 [M]þ, calcd for C20H30O:
286.2297.
4.1.4. 8,11,13-Abietatrien-18-yl acetate
(dehydroabietinol acetate, 6)
A solution of the alcohol 5 (300 mg, 1 mmol) and dimethyla-
minopyridine (12 mg, 0.1 mmol) in triethylamine (8 mL) at 0 ꢁC was
treated with acetyl chloride (355 mL, 5 mmol) dropwise. The
resulting mixture was stirred for 16 h. Then, it was diluted with
diethyl ether and washed with water and brine. The aqueous phase
was extracted twice with diethyl ether and the combined organic
extracts were dried and concentrated. The residue was purified by
chromatography eluting with hexane-ethyl acetate (7:3) to give
20
258 mg (75%) of acetate 6 as a slightly yellow oil: [
a]
D þ 8.5
(c 0.5); 1H NMR (300 MHz)
d
7.18 (1H, d, J ¼ 8.1), 6.99 (1H, dd, J ¼ 8.1,
1.5), 6.90 (1H, br s), 3.98 (1H, d, J ¼ 10.8), 3.69 (1H, d, J ¼ 10.8), 2.85
(1H, m), 2.02 (3H, s), 1.22 (6H, d, J ¼ 6.6), 1.21 (3H, s), 0.94 (3H, s);
13C NMR (75 MHz) dC 171.3 (s),147.1 (s),145.6 (s),134.7 (s),126.8 (d),
124.2 (d), 123.9 (d), 72.4 (t), 44.0 (d), 38.2 (t), 37.4 (s), 36.7 (s), 35.5
(t), 33.4 (d), 30.2 (t), 25.3 (q), 24.0 (q), 24.0 (q), 21.0 (q), 18.9 (t), 18.5
(t), 17.5 (q); HRMS (EI) m/z 328.2422 [M]þ, calcd for C22H32O2:
328.2402.
4.1.1. Methyl 8,11,13-abietatrien-18-oate
(methyl dehydroabietate, 3)
To neat methyl abietate (2) (4.0 g, 12 mmol) 5% Pd/C (200 mg)
was added and then heated to 240–250 ꢁC for 2 h. Then, the
mixture was cooled to rt and diluted with hexane-ethyl acetate 8:2
and purified by flash chromatography, using hexane-ethyl acetate
(8:2) as eluent, to give methyl dehydroabietate 3 (3.3 g, 85%) as an
4.1.5. 8,11,13-Abietatrien-18-yl benzoate (dehydroabietinol
benzoate, 7)
A solution of the alcohol 5 (300 mg, 1 mmol) and dimethyla-
minopyridine (12 mg, 0.1 mmol) in triethylamine (8 mL) at 0 ꢁC was
20
orange oil: [
a
]
D þ 52.0 (c 1.0); 1H NMR (300 MHz)
d 7.16 (1H, d,
J ¼ 8.1), 6.99 (1H, dd, J ¼ 8.1, 1.5), 6.88 (1H, br s), 3.65 (3H, s), 1.27
(3H, s),1.23 (3H, s),1.21 (6H, s); 13C NMR (75 MHz) dC 179.1 (s),146.9
(s), 145.7 (s), 134.7 (s), 126.8 (d), 124.1 (d), 123.9 (d), 51.9 (d), 47.6 (s),
44.8 (d), 37.9 (t), 36.9 (s), 36.6 (t), 33.4 (d), 29.9 (t), 25.1 (q), 24.0 (q),
24.0 (q), 21.7 (t), 18.6 (t), 16.5 (q); HRMS (EI) m/z 314.2231 [M]þ,
calcd for C21H30O2: 314.2246.
treated with benzoyl chloride (580 mL, 5 mmol) dropwise. The
resulting mixture was stirred for 16 h. Then, it was diluted with
diethyl ether and washed with water and brine. The aqueous phase
was extracted twice with diethyl ether and the combined organic
extracts were dried and concentrated. The residue was purified by
chromatography eluting with hexane-ethyl acetate (7:3) to give
286 mg (70%) of benzoate 7 as a slightly yellow oil: 1H NMR
4.1.2. 8,11,13-Abietatrien-18-oic acid (dehydroabietic acid, 4)
A mixture of ester 3 (200 mg, 0.63 mmol), KOH (85%, 1.0 g,
15 mmol), H2O (2 mL) and methanol (12 mL) was refluxed for 16 h.
Then, additional KOH (85%, 400 mg, 7 mmol) was added and reflux
continued for 5 h more. After this time, the reaction mixture was
then cooled, poured into aqueous HCl (1.5 M, 20 mL) and extracted
three times with ethyl acetate. The organic extract was dried over
MgSO4 and concentrated under reduced pressure to give the crude
acid which was purified by chromatography eluting with hexane-
(300 MHz)
d 8.01 (2H, m), 7.53 (1H, m), 7.41 (2H, m), 7.20 (1H, d,
J ¼ 8.1), 7.00 (1H, dd, J ¼ 8.1,1.5), 6.88 (1H, br s), 4.21 (1H, d, J ¼ 10.8),
3.98 (1H, d, J ¼ 10.8), 2.85 (1H, m), 1.25 (3H, s), 1.22 (6H, d, J ¼ 6.9),
1.04 (3H, s); 13C NMR (75 MHz) dC 166.6 (s),146.9 (s), 145.5 (s), 134.7
(s), 132.8 (d), 130.3 (s), 129.5 ꢄ 2 (d), 128.4 ꢄ 2 (d), 126.9 (d), 124.3
(d), 123.9 (d), 73.0 (t), 44.9 (d), 38.3 (t), 37.6 (s), 37.1 (s), 35.8 (t), 33.4
(d), 30.1 (t), 25.5 (q), 23.9 (q), 23.9 (q), 19.1 (t), 18.6 (t), 17.6 (q);
HRMS (EI) m/z 390.2537 [M]þ, calcd for C27H34O2: 390.2559.
ethyl acetate (3:7) to give acid 5 (142 mg, 75%) as a foam:
20
[
a]
D þ 59.7 (c 0.5); 1H NMR (300 MHz)
d
7.16 (1H, d, J ¼ 8.4), 6.99
(1H, dd, J ¼ 8.1, 1.5), 6.88 (1H, br s), 2.85 (1H, m), 1.28 (3H, s), 1.21
(6H, d, J ¼ 6.9); 13C NMR (75 MHz) dC 184.5 (s), 146.7 (s), 145.7 (s),
134.7 (s), 126.9 (d), 124.1 (d), 123.8 (d), 44.6 (d), 37.9 (t), 36.8 (s),
36.7 (t), 33.4 (d), 30.0 (t), 25.1 (q), 24.0 (q), 24.0 (q), 21.7 (t), 18.5 (t),
16.2 (q); HRMS (EI) m/z 300.2068 [M]þ, calcd for C20H28O2:
300.2089.
4.1.6. 8,11,13-Abietatrien-18-yl nicotinate (dehydroabietinol
nicotinate, 8)
A suspension of nicotinic acid (2.46 g, 20 mmol) in thionyl
chloride (10 mL) was refluxed for 2 h, then it was cooled and
concentrated under vacuum. Then, 500 mg of the resulting solid
was added to a solution of the alcohol 5 (300 mg, 1 mmol) and
dimethylaminopyridine (12 mg, 0.1 mmol) in triethylamine (8 mL)
at 0 ꢁC. The resulting mixture was stirred for 48 h. Then, it was
diluted ethyl acetate and washed with water and brine. The
aqueous phase was extracted twice with ethyl acetate and
the combined organic extracts were dried and concentrated.
The residue was purified by chromatography eluting with hexane-
ethyl acetate (7:3) to give 328 mg (80%) of nicotinate 8 as a slightly
4.1.3. 8,11,13-Abietatrien-18-ol (dehydroabietinol, 5)
To a solution of ester 3 (1.78 g, 5.7 mmol) in tetrahydrofuran
(25 mL), LiAlH4 (1.20 g, 31.5 mmol) was added in portions and the
resulting mixture was refluxed for 16 h. Then, it was cooled to 0 ꢁC
and 1.2 mL of H2O, 1.2 mL of 15% NaOH and 3.6 mL of H2O were
added sequentially and carefully. The resulting white solid was
filtered off and washed with ethyl acetate. The extract was
concentrated and purified by chromatography eluting with hexane-
yellow solid: 1H NMR (300 MHz)
d
9.21 (1H, br d, J ¼ 2.1), 8.75 (1H,
dd, J ¼ 4.8, 1.5), 8.26 (1H, ddd, J ¼ 7.8, 2.1, 2.1), 7.36 (1H, dd, J ¼ 8.1,
5.1), 7.19 (1H, d, J ¼ 8.1), 7.00 (1H, dd, J ¼ 8.1,1.8), 6.88 (1H, br s), 4.25
(1H, d, J ¼ 10.8), 4.01 (1H, d, J ¼ 10.8), 2.85 (1H, m), 1.25 (3H, s), 1.22
(6H, d, J ¼ 6.9), 1.04 (3H, s); 13C NMR (75 MHz) dC 165.1 (s), 153.3 (d),
ethyl acetate (6:4) to give 1.46 g (90%) of pure alcohol 5 as a slightly
20
yellow oil: [
a
]
D þ 43.5 (c 0.6); 1H NMR (300 MHz)
d 7.17 (1H, d,
J ¼ 8.1), 6.98 (1H, dd, J ¼ 8.1, 1.5), 6.88 (1H, br s), 3.45 (1H, d,

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M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 45 (2010) 811–816
815
150.7 (d), 146.7 (s), 145.5 (s), 136.9 (d), 134.4 (s), 126.8 (d), 126.1 (s),
124.2 (d), 123.8 (d), 123.2 (d), 73.2 (t), 44.8 (d), 38.2 (t), 37.4 (s), 37.1
(s), 35.7 (t), 33.3 (d), 30.3 (t), 25.4 (q), 23.8 (q), 23.8 (q), 19.0 (t), 18.4
(t), 17.5 (q); HRMS (EI) m/z 391.2526 [M]þ, calcd for C26H33NO2:
391.2511.
in one portion. After being stirred for 16 h, the mixture was filtered
through a pad of celite and concentrated. Then, the residue was
chromatographed on silica eluting with hexane-ethyl acetate (7:3)
20
to give 68 mg (60%) of ketone 12 as a colorless oil: [
a
]
D þ 15.0
(c 2.85); 1H NMR (300 MHz)
d
7.80 (1H, d, J ¼ 2.1), 7.36 (1H, dd,
J ¼ 8.1, 2.1), 7.28 (1H, d, J ¼ 8.1), 3.45 (1H, d, J ¼ 10.8), 3.13 (1H, d,
J ¼ 10.8), 2.88 (1H, sept., J ¼ 6.9), 1.24 (3H, s), 1.22 (3H, d, J ¼ 6.9),
1.21 (3H, d, J ¼ 6.9), 0.92 (3H, s); 13C NMR (75 MHz) dC 199.8 (s),
153.6 (s), 146.5 (s), 132.4 (d), 130.5 (s), 124.8 (d), 123.6 (d), 70.7 (t),
42.3 (d), 37.6 (s), 37.4 (t), 35.8 (t), 34.6 (t), 33.5 (d), 23.8 (q), 23.7 (q),
23.7 (q), 18.2 (t), 17.2 (q); HRMS (EI) m/z 300.2077 [M]þ, calcd for
C20H28O2: 300.2089.
4.1.7. 8,11,13-Abietatrien-18-al (dehydroabietinal, 9)
A solution of alcohol 5 (1.1 g, 3.84 mmol) in DCM (11 mL) was
treated with Dess–Martin periodinane [19] (2.4 g, 5.7 mmol). After
being stirred for 16 h, the mixture was diluted with diethyl ether,
saturated sodium thiosulphate solution, and saturated sodium
hydrogen carbonate solution. The resulting mixture was stirred
vigorously for 1 h and then extracted with ethyl acetate. The
combined organic extracts were washed with brine, dried and
concentrated. Then, the residue was chromatographed on silica
4.1.11. 7
(7 -hydroxydehydroabietinal, 13)
A solution of diol 11 (275 mg, 0.91 mmol) in DCM (3 mL) was
a-Hydroxyabieta-8,11,13-trien-18-al
a
eluting with hexane-ethyl acetate (7:3) to give 1.0 g (90%) of alde-
20
hyde 9 as a slightly yellow semisolid: [
(300 MHz)
a]
D þ 55.0 (c 0.63); 1H NMR
treated with Dess–Martin periodinane [19] (390 mg, 0.91 mmol).
After being stirred for 4 h, the mixture was diluted with diethyl
ether, saturated sodium thiosulphate solution, and saturated
sodium hydrogen carbonate solution. The resulting mixture was
stirred vigorously for 1 h and then diluted with ethyl acetate. The
aqueous phase was extracted with ethyl acetate twice and the
combined organic extracts were washed with brine, dried and
concentrated. Then, the residue was chromatographed on silica
eluting with hexane-ethyl acetate (7:3) to give 232 mg (85%) of
d
9.25 (1H, s), 7.18 (1H, d, J ¼ 8.4), 6.99 (1H, dd, J ¼ 8.1,
1.8), 6.89 (1H, br s), 2.85 (1H, m), 1.22 (3H, s), 1.22 (6H, d, J ¼ 6.9),
1.15 (3H, s); 13C NMR (75 MHz) dC 206.0 (d),146.1 (s), 145.8 (s), 134.3
(s), 127.0 (d), 124.1 (d), 123.9 (d), 49.7 (s), 42.7 (d), 37.8 (t), 36.2 (s),
33.4 (d), 31.9 (t), 29.7 (t), 25.1 (q), 23.9 (q), 23.9 (q), 21.3 (t), 17.7 (t),
13.9 (q); HRMS (EI) m/z 284.2125 [M]þ, calcd for C20H28O: 284.2140.
4.1.8. Abietadien-18-ol (abietinol, 10)
A suspension of LiAlH4 (3.10 g, 82 mmol) in tetrahydrofuran
(100 mL) was stirred as abietic acid (1) (70%, 5 g, 11.6 mmol) in
tetrahydrofuran (50 mL) was added. The mixture was refluxed for
15 h, then it was cooled to 0 ꢁC and 3.1 mL of H2O, 3.1 mL of 15%
NaOH and 9.3 mL of H2O were added sequentially and carefully. The
resulting white solid was filtered off and washed with ethyl acetate.
The extract was concentrated and purified by chromatography
aldehyde 13 as a slightly yellow oil: 1H NMR (300 MHz)
d 9.28 (1H,
s), 7.19 (1H, d, J ¼ 8.4), 7.17 (1H, br s), 7.12 (1H, dd, J ¼ 8.1, 2.1), 4.74
(1H, br d, J ¼ 2.7), 2.85 (1H, m), 1.22 (6H, d, J ¼ 6.9), 1.16 (3H, s), 1.14
(3H, s); 13C NMR (75 MHz) dC 206.3 (d), 146.6 (s), 145.9 (s), 135.7 (s),
128.0 (d), 126.5 (d), 124.2 (d), 67.6 (d), 49.1 (s), 37.4 (t), 37.2 (d), 36.5
(s), 33.4 (d), 31.8 (t), 30.9 (t), 24.2 (q), 23.9 (q), 23.8 (q), 17.7 (t), 13.9
(q); HRMS (EI) m/z 300.2058 [M]þ, calcd for C20H28O2: 300.2089.
eluting with hexane-ethyl acetate (7:3) to give 3.3 g (100%) of pure
20
alcohol 10 as a white solid: mp 81–83 ꢁC; [
a
]
D ꢀ 132.0 (c 0.33); 1H
NMR (300 MHz)
d
5.75 (1H, s), 5.37 (1H, br s), 3.31 (1H, d, J ¼ 11.0),
4.2. Biological assays
3.07 (1H, d, J ¼ 11.0), 0.99 (3H, d, J ¼ 6.8), 0.98 (3H, d, J ¼ 6.8), 0.84
(3H, s), 0.80 (3H, s); 13C NMR (75 MHz) dC 144.9 (s), 135.4 (s), 122.4
(d), 120.9 (d), 71.8 (t), 50.7 (d), 43.4 (d), 38.8 (t), 37.3 (s), 35.6 (t),
34.7 (d), 34.5 (s), 27.4 (t), 23.7 (t), 22.6 (t), 21.3 (q), 20.7 (q), 18.1 (t),
17.6 (q), 14.1 (q); HRMS (EI) m/z 288.2440 [M]þ, calcd for C20H32O:
288.2453.
Stock solutions of compounds were prepared in dimethyl sulf-
oxide (DMSO, Sigma) and frozen at ꢀ70 ꢁC until required. The
concentration of DMSO in biological assays was of 0.05%. Cell
controls with DMSO at 0.05% were used.
4.1.9. 7
(7 -hydroxydehydroabietinol, 11)
A suspension of selenium dioxide (3.3 g, 29.7 mmol) in DCM
a,18-Dihydroxyabieta-8,11,13-triene
a
4.3. Antifungal assay
(40 mL) was treated with 70% tert-butyl hydroperoxide (8.8 mL,
66 mmol) and stirred for 15 min. Then, a solution of the alcohol 10
(1.9 g, 6.6 mmol) in DCM (20 mL) was added and stirring continued
for 20 h. Then, the reaction was diluted with DCM and washed with
brine. The aqueous phase was extracted with ethyl acetate and the
combined organic extracts were dried and concentrated. The
resulting residue was chromatographed on silica (8:2–4:6 hexane-
The antifungal activity of dehydroabietanes was evaluated
following the standard method proposed by the Antifungal
Susceptibility Testing Subcommittee of the European Committee
on Antibiotic Susceptibility Testing (AFST-EUCAST) [23] for
fermentative yeasts. C. parapsilosis (ATCC 22019), C. krusei (ATCC
6258), C. tropicalis (CECT 11901), C. albicans (ATCC 10231) were used
to evaluate antifungal activity. Briefly, seven serial dilutions of the
compounds were dispensed into 96-well flat-bottom microdilution
EtOAc as eluant) to give 1.3 g (65%) of the aromatic diol 11 as an
20
yellow solid: mp 85–86 ꢁC; [
d
a
]
D þ 10.1 (c 5.7); 1H NMR (300 MHz)
plates in duplicate at final concentrations between 100
g/mL. Amphotericine B (Sigma Chemical Co, MO, USA) and
itraconazole (Sigma Chemical Co, MO, USA) were used as positive
controls at a range of 0.031–16 g/mL. The plates were frozen at
mg/mL and
7.17 (1H, d, J ¼ 8.4), 7.16 (1H, s), 7.09 (1H, dd, J ¼ 8.1, 1.8), 4.68 (1H,
2 m
t, J ¼ 2.4), 3.47 (1H, d, J ¼ 11.7), 2.90 (1H, d, J ¼ 11.4), 2.86 (1H, m),
1.23 (6H, d, J ¼ 7.2), 1.11 (3H, s), 0.74 (3H, s); 13C NMR (75 MHz) dC
147.2 (s), 145.9 (s), 135.8 (s), 127.8 (d), 126.2 (d), 124.2 (d), 70.2 (t),
68.0 (d), 38.0 (t), 37.5 (s), 37.2 (s), 37.0 (d), 34.4 (t), 33.4 (d), 27.8 (t),
24.3 (q), 24.0 (q), 23.8 (q), 18.6 (t), 17.6 (q); HRMS (EI) m/z 302.2265
[M]þ, calcd for C20H30O2: 302.2246.
m
ꢀ70 ꢁC until required. The inoculum size for microdilution plates
were 0.5–2.5 ꢄ105 CFU/mL for yeast. For the AFST-EUCAST method,
the Minimal Inhibitory Concentrations (MICs) were determined
after 24 h of incubation and is defined as the lowest concentration
that resulted in a 90% reduction of growth. MICs results were
expressed as range and geometric mean (GM) of triplicates of each
compound tested three times against each of the fungi species in
different assays.
4.1.10. 7-Oxoabieta-8,11,13-trien-18-ol (7-oxodehydroabietinol, 12)
To a solution of diol 11 (115 mg, 0.38 mmol) in DCM (4 mL)
activated manganese dioxide (85%, 970 mg, 9.5 mmol) was added

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M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 45 (2010) 811–816
816
´
4.4. Antitumor activity and cytotoxicity
to ‘‘Grupo Infeccion y Cancer’’ from Medical School-Antioquia
University for their collaboration.
The cell lines used were human cervix epitheloid carcinoma
cells (HeLa cell line ATCC CCL-2), acute T cell leukemia human cells
(Jurkat cell line ATCC TIB-152) and Cercopithecus aethiops african
green monkey kidney cells (Vero cell line ATCC CCL-81). HeLa and
Vero cells were grown in MEM supplemented with 10% FBS,
References
[1] D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann, Plant Physiol. 129 (2002)
1003–1018.
100 units/mL of penicillin, 100 mg/mL of streptomycin, 20 mg/mL of
glutamine, 0.14% NaHCO3, and 1% each of nonessential amino acids
and vitamin solution. Jurkat cells was grown in RPMI-1640 (Sigma
Chemical Co, St Louis, MI, USA) supplemented with 10% FBS,
100 units/mL of penicillin and 100 mg/mL of streptomycin. The
cultures were maintained at 37 ꢁC in humidified 5% CO2
atmosphere.
[2] A. San Feliciano, M. Gordaliza, M.A. Salinero, J.M. Miguel del Corral, Planta
Med. 59 (1993) 485–490.
[3] H. Wada, S. Kodato, M. Kawamori, T. Morikawa, H. Nakai, M. Takeda, S. Saito,
Y. Onoda, H. Tamaki, Chem. Pharm. Bull. 33 (1985) 1472–1487.
[4] (a) S. Savluchinske Feio, J. Carlos Roseiro, B. Gigante, M.J. Marcelo-Curto, J.
Microbiol. Methods 28 (1997) 201–206;
(b) B. Gigante, A.M. Silva, M.J. Marcelo-Curto, S. Savluchinske Feio, J. Roseiro,
L.V. Reis, Planta Med. 68 (2002) 680–684;
The antitumor activity on HeLa and Jurkat cells, and cytotoxic
activity on Vero cells have been carried out using in vitro assay on
cell growth and tetrazolium- dye (MTT) cytotoxicity assay,
according to the protocol reported by us [24], which was used with
a few modifications. HeLa and Vero cell monolayers were trypsi-
nized and washed with culture medium and then plated at
1.5 ꢄ104 cells per well for HeLa and 1.25 ꢄ104 cells per well for
Vero cells in a 96-well flat-bottomed plate. After 24 h of incubation,
each diluted compound was added to the appropriate wells and the
plates were incubated for further 48 h at 37 ꢁC in a humidified
incubator with 5% CO2. Jurkat cells that grow in suspension were
plated at 3.00 ꢄ 104 cells per well and diluted compounds were
added to plates and incubated for 48 h at 37 ꢁC. Vincristine was
used as positive control. The IC50 for each compound were
obtained from dose–effect curves for linear regression methods and
IC50 values are expressed as the mean ꢂ S.E.M. of at least four
dilutions by quadruplicate.
(c) G.M. Woldemichael, G. Wa¨chter, M.P. Singh, W.M. Maiese, B.N. Timmermann,
J. Nat. Prod. 66 (2003) 242–246;
(d) S. Savluchinske Feio, L. Nunes, P. Tavares Pereira, A.M. Silva, J.C. Roseiro,
B. Gigante, M.J. Marcelo-Curto, J. Microbiol. Methods 70 (2007) 465–470.
[5] I.A. Tolmacheva, A.V. Tarantin, A.A. Boteva, L.V. Anikina, Y.B. Vikharev,
V.V. Grishko, A.G. Tolstikov, Pharm. Chem. J 40 (2006) 489–493.
[6] J.R. Tagat, D.V. Nazareno, M.S. Puar, S.W. McCombie, A.K. Ganguly, Bioorg. Med.
Chem. Lett. 4 (1994) 1101–1104.
[7] (a) Y. Kinouchi, H. Ohtsu, H. Tokuda, H. Nishino, S. Matsunaga, R. Tanaka, J. Nat.
Prod. 63 (2000) 817–820;
(b) R. Tanaka, H. Tokuda, Y. Ezaki, Phytomedicine 15 (2008) 985–992.
[8] (a) S. Prinz, U. Mu¨llner, J. Heilmann, K. Winkelmann, O. Sticher, E. Haslinger,
A. Hu¨fner, J. Nat. Prod. 65 (2002) 1530–1534;
´
´
˜
(b) B. Sepulveda, L. Astudillo, J.A. Rodrıguez, T. Yanez, C. Theoduloz,
G. Schmeda-Hirschmann, Pharmacol. Res. 52 (2005) 429–437;
(c) S.F.H. Zaidi, S. Awale, S.K. Kalauni, Y. Tezuka, H. Esumi, S. Kadota, Planta
Med. 72 (2006) 1231–1234;
(d) X. Rao, Z. Song, L. He, W. Jia, Chem. Pharm. Bull. 56 (2008) 1575–1578.
[9] T. Ohwada, T. Nonomura, K. Maki, K. Sakamoto, S. Ohya, K. Muraki,
Y. Imaizumi, Bioorg. Med. Chem. Lett. 13 (2003) 3971–3974.
[10] S.D. Bang, S.K. Johnson, J.C.S. Park, U.S. Patent 5,248,696; 1993.
[11] C-H. Lin, H-S. Chuang, U.S. Patent 7,015,248 B2; 2006.
[12] M.A. Gonza´lez, J. Correa-Royero, L. Agudelo, A. Mesa, L. Betancur-Galvis, Eur. J.
Med. Chem. 44 (2009) 2468–2472.
[13] M.A. Gonza´lez, M.J. Gil-Gimeno, A.J. Blake, Acta Cryst. E 62 (2006) 3346–3347.
[14] A. Ulubelen, G. Topcu, Phytochemistry 31 (1992) 3949–3951.
[15] B.M. Fraga, T. Mestres, C.E. Diaz, J.M. Arteaga, Phytochemistry 35 (1994) 1509–
1512.
4.5. Antiviral assays
Herpes simplex virus type 1 (HSV-1) was obtained from the
Center for Disease Control (Atlanta, GA). The virus stock was
prepared from HSV-1-infected HeLa cell cultures. The antiviral
activity against HSV-1 have been carried out on HeLa and Vero cells
using end-point titration technique, according to the protocol
reported by us [24], which was used with a few modifications. Two-
fold dilutions of the compounds and viral suspension (one infection
dose, 1 D.I) were mixed and incubated for 0.5 h at 37 ꢁC before they
were added on confluent monolayer cells and incubated again at
37 ꢁC in humidified 5% CO2 atmosphere for 36 h. Acyclovir and
Heparin sodium salt were used as positive controls.
´
˜
¨
[16] (a) A.F. Barrero, J.F. Sanchez, E.J. Alvarez-Manzaneda, M. Munoz, A. Haıdour,
Phytochemistry 31 (1992) 615–620;
(b) M. Dakir, F. El Hanbali, F. Mellouki, M. Akssira, A. Benharref, J.F. Quilez del
Moral, A.F. Barrero, Nat. Prod. Res. 19 (2005) 719–722.
[17] (a) A.H. Conner, B.A. Nagasampagi, J.W. Rowe, Phytochemistry 19 (1980) 1121–
1131;
(b) R. Tanaka, H. Ohtsu, S. Matsunaga, Phytochemistry 46 (1997) 1051–1057;
(c) A.M.L. Seca, A.M.S. Silva, I.L. Bazzocchi, I.A. Jimenez, Phytochemistry 69
(2008) 498–505.
[18] N.J. Halbrook, R.V. Lawrence, J. Org. Chem. 31 (1966) 4246–4247.
[19] D.B. Dess, J.C. Martin, J. Am. Chem. Soc. 113 (1991) 7277–7287.
[20] S.N. Suryawanshi, A. Rani, T.S. Dhami, D.S. Bhakuni, Synth. Commun. 19 (1989)
2927–2937.
[21] B. Gigante, C. Santos, A.M. Silva, M.J.M. Curto, M.S.J. Nascimento, E. Pinto,
M. Pedro, F. Cerqueira, M.M. Pinto, M.P. Duarte, A. Laires, J. Rueff, J. Gonçalves,
M.I. Pegado, M.L. Valdeira, Bioorg. Med. Chem. 11 (2003) 1631–1638.
[22] A.J. Vlietinck, L. Van Hoof, J. Totte´, A. Lasure, D. Vanden Berghe, P.C. Rwangabo,
J. Mvukiyumwami, J. Ethnopharm. 46 (1995) 31–47.
[23] National Committee for Clinical Laboratory Standards, Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi M38-A.
Approved standard. National Committee for Clinical Laboratory Standards,
Wayne, PA, 2002.
Acknowledgments
Financial support from the Spanish Ministry of Science and
Education, under a ‘‘Ramon y Cajal’’ research grant, and also from
the Generalitat Valenciana (project GV/2007/007) is gratefully
acknowledged. L. B-G. thanks the financial support from Antioquia
University of Colombia CODI-UdeA (Grant Mediana Cuantı´a 2009)
´
[24] M. Arno´, L. Betancur-Galvis, M.A. Gonza´lez, J. Sierra, R.J. Zaragoza´, Bioorg. Med.
Chem. 11 (2003) 3171–3177.
´
and COLCIENCIAS, Bogota, Colombia (Grant RC 432-2004). Thanks