Synthesis and biological evaluation of abietic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.01.014

A series of C18-oxygenated derivatives of abietic acid were synthesized and evaluated for their cytotoxic, antimycotic, and antiviral activities. In general, the introduction of an aldehyde group at C18 did improve the resultant bioactivity, while the presence of an acid or alcohol led to less active compounds.

Full text of DOI:10.1016/j.ejmech.2009.01.014

European Journal of Medicinal Chemistry 44 (2009) 2468–2472
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of abietic acid derivatives
a
,
b
b
b
b,
**
*
´
Miguel A. Gonzalez , Julieth Correa-Royero , Lee Agudelo , Ana Mesa , Liliana Betancur-Galvis
a
´
´
´
Departamento de Quımica Organica, Universidad de Valencia, Dr. Moliner 50, E-46100 Burjassot, Valencia, Spain
Grupo Infeccion y Cancer, Universidad de Antioquia, A.A1226 Medellın, Colombia
b
´
´
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 abietic acid were synthesized and evaluated for their cytotoxic,
antimycotic, and antiviral activities. In general, the introduction of an aldehyde group at C18 did improve
the resultant bioactivity, while the presence of an acid or alcohol led to less active compounds.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 9 December 2008
Received in revised form
12 January 2009
Accepted 15 January 2009
Available online 24 January 2009
Keywords:
Abietic acid
Cytotoxicity
Antimycotic activity
Antiviral activity
1. Introduction
functional groups at C18 and isomeric double bonds were tested.
All the compounds were easily obtained in good yield, by standard
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. Abietic acid, an
abietane diterpenoid, has shown antiallergic [3], anti-inflammatory
[4], phytoalexin-like [5], and anticonvulsant activities [6]. Despite
the diverse biological properties of this family of compounds, little
effort has been made for the biological study, specially of
derivatives.
or reported chemical procedures. Some of the synthesized
compounds have been isolated as natural products, in particular,
abietinol (3) and abietinal (4) [9], however, no reports have
appeared on their biological activities.
2. Results and discussion
2.1. Chemistry
As a part of our research program towards the discovery of
bioactive terpene compounds, we were interested in confirming
the results reported in a recent patent on the use of abietic acid and
derivatives as antitumor agents [7]. We describe here the syntheses
of a number of derivatives of commercially available (ꢀ)-abietic
acid (1) (Fig. 1) [8], 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 abietane skeleton. In this context,
simple and sequential modifications were performed in the mole-
cule of abietic acid 1. Compound 1 and 7 derivatives with different
The synthesis of the C18-functionalized abietanes used in this
work begins with the preparation of the required methyl ester 2
from commercial (ꢀ)-abietic acid, following a reported procedure
(Scheme 1) [10]. Thus, abietic acid 1 was esterified by treatment
with lithium hydroxide and methyl sulfate to give ester 2 in
quantitative yield. Reduction of ester 2 with LiAlH4 in dry tetra-
hydrofuran at reflux gave alcohol (abietinol) 3 in quantitative yield.
Oxidation of 3 was carried out by treatment with PCC on alumina
[11]. This oxidation afforded aldehyde (abietinal) 4 in 40% yield.
Next, the conjugated double bonds present in ester 2 were
isomerized using a known protocol [10] with few modifications
(Scheme 1). Thus, methyl abietate (2) was treated with 33% HBr in
acetic acid and the resulting bromo derivatives were heated in DMF
at 80 ꢁC in the presence of lithium hydroxide to afford compound 6
in 56% overall yield. Ester 6 was saponified with KOH in aqueous
methanol to give acid 5. Reduction of 6 with LiAlH4 in dry
* Corresponding author. Tel.: þ34 963543880; fax: þ34 963544328 (chemical
aspects).
** Corresponding author. Tel.: þ5745106059; fax: þ5745106062 (biological aspects).
E-mail addresses: miguel.a.gonzalez@uv.es (M.A. Gonza´lez), labeta@catios.
udea.edu.co (L. Betancur-Galvis).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.014

´
M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 44 (2009) 2468–2472
2469
Table 1
Cytotoxic activity of abietane diterpenes determined by the MTT technique
expressed as CC50
(m
g/mL).a
Compound
Cell linesb
HeLa
VERO
CC50
CC50
R2,c
R2
SId
1
2
3
4
5
6
7
8
14.9 ꢂ 0.6
3.6 ꢂ 1
0.99
0.87
0.97
0.95
0.95
1
0.91
0.81
0.95
52.5 ꢂ 5.6
49.4 ꢂ 3
0.92
0.98
0.76
0.80
0.94
0.92
0.95
0.99
0.95
3.5
13.7
2.4
5.6
0.8
0.4
1.0
1.6
22
5.2 ꢂ 0.5
5.6 ꢂ 0.5
53.1 ꢂ 4.6
46.6 ꢂ 1.3
19.7 ꢂ 2.1
33.4 ꢂ 6.6
0.05 ꢂ 0.01
12.7 ꢂ 2.4
31.5 ꢂ 6.5
43.9 ꢂ 4.7
19.2 ꢂ 1.8
20.2 ꢂ 1.5
55.3 ꢂ 2.2
1.1 ꢂ 0.2
Fig. 1. Chemical structures of tested abietanes.
Vincristine
Numbers in bold belong to the most potent compound.
a
tetrahydrofuran at reflux gave alcohol 7 in 86% yield. Finally,
oxidation with PCC on alumina [11] of 7 afforded aldehyde 8 in
40% yield.
50% Cytotoxic concentration in 48 h.
b
HeLa, human cervix epitheloid carcinoma ATCC CCL-2; Vero, Cercopithecus
aethiops African green monkey kidney ATCC CCL-81.
c
R2: coefficient of linear regression.
SI, selectivity index is defined as VERO CC50 over HeLa CC50.
d
2.2. Biological evaluation
the HeLa tumor line and the lowest cytotoxic on non-cancerous
cells (Vero cell line) was the abietane 2 with CC50 values of
Firstly, the abietanes 1–8 were tested in vitro for antimycotic
activity. All compounds did not show antimycotic activity against
Candida parapsilosis, Candida krusei, Candida tropicalis, Candida
albicans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,
3.6 ꢂ 1
m
g/mL and 49.4 ꢂ 3
mg/mL, respectively. This means that the
abietane with the highest selectivity index (SI) was methyl abietate
(2) (SI ¼ 13.7). Selectivity refers to the ability of a compound to
recognize its target without interacting with other targets. Nowa-
days SI values are evaluated for any biological activity because it can
be therapeutically relevant for designing and synthesizing new
drugs. Ideally a compound selectivity index of 100 to a 1000 or
more is ideal but in some instances lower values can provide
sufficient selectivity to derive a good therapeutic index. Recently,
an SI value of 14.3 on cell lines is indicative of potential antitumor
activity for biopharmaceutical use [12]. Specifically, according to SI
value the order of activity in the series abietanes (1–4) at position
C18 was found to be ester > aldehyde > acid > alcohol. On the other
hand, the antitumor activity data in the series of abietanes (5–8)
indicated a significant reduction of activity dependent of the
isomerization of the two double bonds.
and Aspergillus terreus in concentrations below 100
mg/mL except
abietane 4 with MIC value of 50 g/mL against A. fumigatus (data
m
not shown). The introduction of an aldehyde group at C18 led to
a compound with anti-A. fumigatus activity in the series of abie-
tanes 1–4. MIC values for the two reference antifungal drugs,
amphotericine B and itraconazole (Sigma, New Jersey, USA), used as
positive controls, were within the established values for the AFST-
EUCAST and CLSI-M38-A protocols.
Next, the abietanes 1–8 were tested in vitro for potential anti-
tumor and cytotoxic activities determining the concentration of the
compound that induces 50% killing (CC50) of the HeLa tumor line,
and the Vero cell line, respectively (Table 1). All these compounds
produced a dose-dependent inhibition on the growth of the HeLa
tumor cell line and Vero cell line, with R2 (coefficient of linear
regression) >0.8. The abietane 4 showed in vitro potential anti-
Gigante and co-workers [13] prepared different catechols from
abietic acid and evaluated their antitumoral activity. All compounds
produced a dose-dependent inhibition on growth of the tumor cells
evaluated. The order of activity in the catechol series with different
groups at position C18 was ester > acid ꢃ alcohol. The results
reported by Gigante and co-workers are consistent with our results
because introduction of a methyl ester group at C18 also enhances
the resultant antitumor activity.
tumor activity with CC50 value of 5.6 ꢂ 0.5
mg/mL. However, the
abietane that showed the highest potential antitumor activity on
The antiviral activity of the abietanes against herpes simplex
virus type 1 (HSV-1) was determined using a modified end-point
titration technique (EPPT) (Table 2) [14]. The abietanes (2–5)
reduced the HSV-1 replication with values below 100
mg/mL. The
abietane that showed the highest antiviral activity was abietinol
(3). According to Vlietinck et al. [15], 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. Abietinol
(3) was found to be slightly active against HSV-1 over infected
confluent monolayers of HeLa cells with Rf value of 1 ꢄ101 at
a concentration of 6.25
the effect of the catechol methyl 11,12-dihydroxyabieta-
8,11,13-trien-18-oate on HSV-1 at concentration of 15 M [13].
mg/mL. Gigante and co-workers reported
m
These results are consistent with our results because the methyl
abietadien-18-oate (2) showed anti-HSV activity. However, the
replacement of the ester by a hydroxyl group at C18 enhances the
antiviral activity.
Scheme 1. Reagents and conditions: (a) LiOH, Me2SO4, DMF; (b) LiAlH4, THF; (c) PCC/
Al2O3, DCM; (d) HBr, AcOH; (e) LiOH, DMF; (f) KOH, MeOH, H2O.

´
2470
M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 44 (2009) 2468–2472
20
ꢀ92.0 (c 0.69); 1H NMR (300 MHz)
d 5.77 (1H, s), 5.36 (1H,
Table 2
[a
]
D
Cytotoxicity and anti-HSV-1 activity of abietane diterpenes on HeLa Cells deter-
mined by the end-point titration technique.
m), 3.62 (3H, s), 2.21 (1H, m), 1.25 (3H, s), 1.01 (3H, d, J ¼ 6.9), 0.99
(3H, d, J ¼ 6.9), 0.82 (3H, s); 13C NMR (75 MHz) dC 179.0 (s), 145.3 (s),
135.5 (s), 122.3 (d), 120.6 (d), 51.8 (d), 50.9 (d), 46.6 (s), 45.1 (d), 38.3
(t), 37.1 (t), 34.9 (q), 34.5 (s), 27.5 (t), 25.7 (t), 22.5 (t), 21.4 (q), 20.8
(q),18.1 (t), 17.0 (q),14.0 (q); HRMS (EI) m/z 316.2422 [M]þ, calcd for
C21H32O2: 316.2402.
Compound CC100 (m m
g/mL)a Viral reduction factorb Antiviral activity ( g/mL)c
1
2
3
4
5
6
>100
50
25
>50
>100
100
NA
–
6.25
6.25
12.5
100
–
100.5
101
100.5
101
NA
4.1.2. Abietadien-18-ol (abietinol, 3)
7
8
100
>100
>100 U.I./mL
>600
NA
–
–
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
NA
Heparin
Acyclovir
102
104
0.5 U.I./mL
6.0
a
Minimal toxic dose that detached 100% of the cell monolayer.
b
Ratio of the virus titer in the absence over virus titer in the presence of the tested
compound.
c
Maximal nontoxic dose that showed the highest viral reduction factor. NA: no
activity.
eluting with hexane-ethyl acetate (7:3) to give 3.3 g (100%) of pure
20
alcohol 3 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),
3. Conclusions
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.
In conclusion, we have prepared and tested several abietanes for
their antitumor, antifungal and antiviral activities in vitro. In
general, the isomerization of the double bonds present in abietic
acid led to less active compounds. In the series of abietic acid
derivatives (1–4), the compound with an aldehyde group presented
both antifungal and antitumor activities. Methyl abietate presented
the highest cytotoxicity and abietinol presented a weak antiviral
activity. These results encourage us to continue our research of this
series by synthesizing additional abietane-type derivatives with
the aim of obtaining biologically more potent compounds.
4.1.3. Abietadien-18-al (abietinal, 4)
A solution of alcohol 3 (400 mg, 1.39 mmol) in DCM (7 mL) was
treated with PCC on alumina [11] (4 g). After being stirred for 2 h,
the mixture was filtered through silica eluting with fresh DCM.
Then, the filtrate was concentrated and chromatographed on silica
eluting with hexane-ethyl acetate (7:3) to give 160 mg of aldehyde
20
4. Experimental
4 as a colorless oil: [
a
]
D ꢀ55.0 (c 0.63); 1H NMR (300 MHz)
d 9.21
(1H, s), 5.78 (1H, s), 5.35 (1H, br s), 2.21 (1H, m), 1.14 (3H, s), 1.02
(3H, d, J ¼ 6.8), 1.00 (3H, d, J ¼ 6.8), 0.85 (3H, s); 13C NMR (75 MHz)
dC 206.2 (d), 145.5 (s), 135.6 (s), 122.2 (d), 120.0 (d), 50.5 (d), 49.0 (s),
42.4 (d), 38.3 (t), 34.8 (d), 33.8 (s), 32.9 (t), 27.4 (t), 25.4 (t), 22.5 (t),
21.3 (q), 20.8 (q), 17.2 (t), 14.3 (q), 14.1 (q); HRMS (EI) m/z 286.2320
[M]þ, calcd for C20H30O: 286.2297.
4.1. Chemistry
Optical rotations were determined using a 5-cm path-length
cell, using dichloromethane as solvent (concentration expressed in
g/100 mL). [a
]D-Values are given in 10ꢀ1 deg cm2/g. NMR spectra
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 hertz. 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 sulfate, filtered and concentrated under reduced pressure.
4.1.4. Methyl abieta-8,13(15)-dien-18-oate (6)
Methyl abietate (2) (45 g, 0.14 mol) was dissolved in a 33% HBr
solution in acetic acid (120 mL) and stirred for 20 h. Then, the
reaction was quenched with H2O and the supernatant was dec-
anted, the resulting solids were washed with additional H2O and
decanted again. The solid was dried under reduced pressure to
leave the corresponding crude dibromo derivative (66 g). The crude
bromo derivative was mixed with LiOH (10 g, 0.23 mol) and DMF
(200 mL) was added. The mixture was heated at 80 ꢁC for 16 h,
then, it was poured into H2O and extracted with hexane. The
combined extracts were washed with brine, dried, and concen-
trated. The resulting residue was chromatographed on silica (9:1
hexane–EtOAc as eluant) to give 25 g (56%) of the isomerized ester
6 as an orange oil. The 1H NMR and EIMS data agree with those
reported in the literature [10].
4.1.1. Methyl abietadien-18-oate (methyl abietate, 2)
4.1.5. 8,13(15)-Abietadien-18-oic acid (5)
A solution of abietic acid (1) (75%, 68.7 g, 0.17 mol) in DMF
(280 mL) was treated with LiOH (20 g, 2.8 equiv.) and stirred for
20 h. Then, Me2SO4 (110 mL, 7 equiv.) was added and stirred over-
night. The following day the mixture was poured into water and
extracted with hexane. The extract was washed with NaOH 0.1 N
and brine, dried, and concentrated. Purification of the residue by
flash chromatography, using hexane-ethyl acetate (from 7:3 to 6:4)
as eluent, provided methyl abietate 2 (54 g, 100%) as an orange oil:
A mixture of ester 6 (200 mg, 0.63 mmol), KOH (85%, 800 mg,
14 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 3 days more. After this time, the reaction mixture was
then cooled, poured into aqueous HCl (1.2 M, 20 mL) and extracted
three times with DCM. The organic extract was dried over MgSO4
and concentrated under reduced pressure to give the crude acid 5
20
(190 mg, 99%) as a foam: [
a
]
D þ 155.0 (c 0.2); 1H NMR (300 MHz)

´
M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 44 (2009) 2468–2472
2471
d
1.67 (3H, s), 1.64 (3H, s), 1.20 (3H, s), 0.99 (3H, s); 13C NMR
(GM) of triplicates of each compound tested three times against
each of the fungal species in different assays.
(75 MHz) dC 185.8 (s), 138.0 (s), 128.2 (s), 126.1 (s), 120.7 (s), 47.4 (s),
45.9 (d), 36.9 (s), 36.7 (t), 35.2 (t), 34.7 (t), 31.6 (t), 27.5 (t), 24.8 (t),
21.6 (t), 20.1 (q), 19.6 (q), 19.4 (q), 18.1 (t), 16.1 (q); HRMS (EI) m/z
302.2250 [M]þ, calcd for C20H30O2: 302.2246.
4.3. Antitumor activity and cytotoxicity
The cell lines used were human cervix epithelioid carcinoma
cells (HeLa cell line ATCC CCL-2) and Cercopithecus aethiops African
green monkey kidney cells (Vero cell line ATCC CCL-81). 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. Cells were grown in MEM sup-
plemented with 10% FBS, 100 units/mL of penicillin, 100 mg/mL of
streptomycin, 20 mg/mL of l-glutamine, 0.14% NaHCO3, and 1% each
of nonessential amino acids and vitamin solution. The cultures
were maintained at 37 ꢁC in humidified 5% CO2 atmosphere.
The antitumor activity on HeLa 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 [18], which was used with a few modifi-
cations. Cell monolayers were trypsinized 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. Vincristine was
used as positive control. The CC50 for each compound were
obtained from dose–effect curves for linear regression methods and
CC50 values are expressed as the mean ꢂ S.E.M. of at least four
dilutions by quadruplicate.
4.1.6. 8,13(15)-Abietadien-18-ol (7)
A suspension of LiAlH4 (2.10 g, 58 mmol) in tetrahydrofuran
(45 mL) was stirred as ester 6 (3.1 g, 9.8 mmol) in tetrahydrofuran
(15 mL) was added. The mixture was refluxed for 15 h, then it was
cooled to 0 ꢁC and 2.1 mL of H2O, 2.1 mL of 15% NaOH and 6.3 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 eluting with
hexane-ethyl acetate (5:5) to give 2.4 g (85%) of pure alcohol 7 as
20
a colorless oil: [
a
]
D þ 55 (c 4.3); 1H NMR (300 MHz)
d 3.42 (1H, d,
J ¼ 11.0), 3.16 (1H, d, J ¼ 11.0), 1.66 (3H, s), 1.64 (3H, s), 1.01 (3H, s),
0.80 (3H, s); 13C NMR (75 MHz) dC 138.7 (s), 128.3 (s), 125.8 (s),
120.6 (s), 72.2 (t), 45.2 (d), 37.5 (s), 37.4 (s), 35.7 (t), 35.0 (t), 34.7 (t),
31.8 (t), 27.5 (t), 25.0 (t), 20.1 (q), 19.6 (q), 19.6 (q), 18.6 (t), 18.2 (t),
17.4 (q); HRMS (EI) m/z 288.2460 [M]þ, calcd for C20H32O:
288.2453.
4.1.7. 8,13(15)-Abietadien-18-al (8)
A solution of alcohol 7 (600 mg, 2.1 mmol) in DCM (8 mL) was
treated with PCC on alumina [11] (6 g). After being stirred for 2 h,
the mixture was filtered through silica eluting with fresh DCM and
ethyl acetate. Then, the filtrate was concentrated and chromato-
graphed on silica eluting with hexane-ethyl acetate (8:2) to give
20
240 mg of aldehyde 8 as a colorless oil: [
NMR (300 MHz)
a]
D þ 155.0 (c 0.8); 1H
d
9.22 (1H, s), 1.67 (3H, s), 1.65 (3H, s), 1.08 (3H, s),
4.4. Antiviral assays
1.02 (3H, s); 13C NMR (75 MHz) dC 206.5 (d), 138.0 (s), 128.0 (s),
126.2 (s), 120.8 (s), 49.6 (s), 44.0 (d), 36.3 (s), 35.1 (t), 34.6 (t), 31.9
(t), 31.3 (t), 27.4 (t), 24.8 (t), 21.2 (t), 20.0 (q), 19.6 (q), 19.4 (q), 17.3
(t), 13.8 (q); HRMS (EI) m/z 286.2250 [M]þ, calcd for C20H30O:
286.2297.
The antiviral activity against HSV-1 has been carried out on HeLa
cells using end-point titration technique, according to the protocol
reported by us [14], which was used with a few modifications.
Twofold 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 incu-
bated again at 37 ꢁC in humidified 5% CO2 atmosphere for 36 h.
Acyclovir and Heparin sodium salt were used as positive controls.
4.2. Biological assays
4.2.1. Antifungal assay
The antifungal activity of abietanes 1–8 was evaluated following
the Clinical and Laboratory Standards Institute M38-A protocol
(CLSI) [16] for filamentous fungi and the standard method
proposed by the Antifungal Susceptibility Testing Subcommittee of
the European Committee on Antibiotic Susceptibility Testing (AFST-
EUCAST) [17] for fermentative yeasts. C. parapsilosis (ATCC 22019),
C. krusei (ATCC 6258), C. tropicalis (CECT 11901), C. albicans (ATCC
10231), A. flavus (ATCC 204304) A. fumigatus (ATCC 204305),
A. terreus (CDC 317), and A. niger (ATCC 10124), were used to
evaluate antifungal activity. Briefly, seven serial dilutions of the
compounds were dispensed into 96-well flat-bottomed micro-
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 and COLCIENCIAS, Bogota, Colombia (Grant
´
´
RC 432-2004).
References
[1] D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann, Plant Physiol. 129 (2002)
1003–1018.
[2] A. San Feliciano, M. Gordaliza, M.A. Salinero, J.M. Miguel del Corral, Planta
Med. 59 (1993) 485–490.
[3] N.N. Ulusu, D. Ercil, M.K. Sakar, E.F. Tezcan, Phytother. Res. 16 (2002) 88–90.
[4] (a) M.A. Ferna´ndez, M.P. Tornos, M.D. Garcı´a, B. de las Heras, A.M. Villar,
dilution plates in duplicate at final concentrations between 100
mL and 2 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
mg/
m
m
frozen at ꢀ70 ꢁC until required. The inoculum size for microdilution
plates were 0.5–2.5 ꢄ105 and 0.4–5 ꢄ104 CFU/mL for yeast and
filamentous fungi, respectively. 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. For the CLSI M38-A method,
the MICs were determined after 48 h of incubation and are defined
as the lowest dilution that resulted in total inhibition of visible
growth. MIC results were expressed as range and geometric mean
´
M.T. Saenz, J. Pharm. Pharmacol. 53 (2001) 867–872;
(b) N. Takahashi, T. Kawada, T. Goto, C.-S. Kim, A. Taimatsu, K. Egawa,
T. Yamamoto, M. Jisaka, K. Nishimura, K. Yokota, R. Yu, T. Fushiki, FEBS Lett. 550
(2003) 190–194.
[5] G.O. Spessard, D.R. Matthews, M.D. Nelson, T.C. Rajtora, M.J. Fossum,
J.L. Giannini, J. Agric. Food Chem. 43 (1995) 1690–1694.
[6] A. Talevi, M.S. Cravero, E.A. Castro, L.E. Bruno-Blanch, Bioorg. Med. Chem. 17
(2007) 1684–1690.
[7] C-H. Lin, H-S. Chuang, U.S. Patent 7,015,248 B2, 2006.
[8] M.A. Gonza´lez, M.J. Gil-Gimeno, A.J. Blake, Acta Crystallogr. E62 (2006)
3346–3347.

´
M.A. Gonzalez et al. / European Journal of Medicinal Chemistry 44 (2009) 2468–2472
2472
[9] (a) A.F. Barrero, J.F. Quı´lez del Moral, M. Mar Herrador, M. Akssira,
A. Bennamara, S. Akkad, M. Aitigri, Phytochemistry 65 (2004) 2507–2515 and
references therein;
[14] L. Betancur-Galvis, C. Zuluaga, M. Arno´, M.A. Gonza´lez, R.J. Zaragoza´, J. Nat.
Prod. 65 (2002) 189–192.
[15] A.J. Vlietinck, L. Van Hoof, J. Totte´, A. Lasure, D. Vanden Berghe, P.C. Rwangabo,
J. Mvukiyumwami, J. Ethnopharmacol. 46 (1995) 31–47.
´
(b) A.F. Barrero, J.F. Quılez del Moral, M. Mar Herrador, J.F. Arteaga, M. Akssira,
A. Benharref, M. Dakir, Phytochemistry 66 (2005) 105–111;
(c) H. Erdtman, L. Westfelt, Acta Chem. Scand. 17 (1963) 1826–1827.
[16] 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.
´
´
[10] A. Abad, M. Arno, L.R. Domingo, R.J. Zaragoza, Tetrahedron 41 (1985)
4937–4940.
[11] Y.-S. Cheng, W.-L. Liu, S.-H. Chen, Synthesis (1980) 223–224.
[12] P. Prayong, S. Barusrux, N. Weerapreeyakul, Fitoterapia 79 (2008) 598–601.
[13] 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.
[17] M. Cuenca-Estrella, C.B. Moore, F. Barchiesi, E. Chryssanthou, D.W. Denning,
J.P. Donnelly, F. Dromer, B. Dupont, J.H. Rex, M.D. Richardson, B. Sancak,
P.E. Verweij, J.L. Rodrı´guez-Tudela, Clin. Microbiol. Infect. 9 (2003) 467–474.
[18] M. Arno´, L. Betancur-Galvis, M.A. Gonza´lez, J. Sierra, R.J. Zaragoza´, Bioorg. Med.
Chem. 11 (2003) 3171–3177.