Catechols from abietic acid: Synthesis and evaluation as bioactive compounds

Article abstract of DOI:10.1016/S0968-0896(03)00063-4

Catechols from abietic acid were prepared by a short and good yielding chemical process and further evaluated for several biological activities namely, antifungal, antitumoral, antimutagenic, antiviral, antiproliferative and inhibition of nitric oxide. Th

Full text of DOI:10.1016/S0968-0896(03)00063-4

Bioorganic & Medicinal Chemistry 11 (2003) 1631–1638
Catechols from Abietic Acid:
Synthesis and Evaluation as Bioactive Compounds
B. Gigante,a,* C. Santos,a A. M. Silva,a M. J. M. Curto,a M. S. J. Nascimento,b
E. Pinto,b M. Pedro,b F. Cerqueira,b M. M. Pinto,b M. P. Duarte,c A. Laires,c
J. Rueff,c J. Goncalves,d M. I. Pegadod and M. L. Valdeirad
a
´
INETI, Departamento de Tecnologia de Indu´strias Quımicas, Estrada do Pac¸o do Lumiar, 22, 1649-038 Lisbon, Portugal
b
´
Centro de Estudos de Quımica Orgaˆnica, Fitoquımica e Farmacologia da Universidade do Porto, Faculdade de Farmacia,
´
´
´
Rua Anıbal Cunha, 164, 4050-047 Porto, Portugal
c
´
Faculdade de Cieˆncias Medicas, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisbon, Portugal
d
´
Faculdade de Farmacia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
Received 10 July 2002; accepted 23 January 2003
Abstract—Catechols from abietic acid were prepared by a short and good yielding chemical process and further evaluated for sev-
eral biological activities namely, antifungal, antitumoral, antimutagenic, antiviral, antiproliferative and inhibition of nitric oxide.
Their properties were compared with those of carnosic acid (6), a naturally occurring catechol with an abietane skeleton and known
to possess potent antioxidant activity, as well as anticancer and antiviral properties. From all the synthetic catechols tested com-
pound 2 showed the best activities, stronger than carnosic acid.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Phenols are an important class of compounds widely
used to inhibit the oxidation of materials of both com-
mercial and biological importance. In general, anti-
oxidants can intercept and react with free radicals at a
rate faster than the substrate, and since free radicals are
able to attack a variety of targets including lipids and
proteins, it is believed that they are implicated in a
number of important degenerative diseases.1 Naturally
occurring phenols with a tricyclic diterpenic structure
and a catechol moiety are amongst the most used and
studied compounds because of their antioxidant and
biological activities.2À10
Compounds 2–5 possess a skeleton and an aromatic moi-
ety similar to carnosic acid (6), one of the constituents of
extracts from rosemary and sage leaves,2,3 responsible for
the application as antioxidant.4À6,10 Anticancer4,5,7 and
antiviral properties5,6,8 of 6 were also documented.
The search for abietic acid (1) derivatives with potentially
interesting properties led to methyl 11,12-dihydroxy-
abietate-8,11,13-trien-15-oate (2), a catechol possessing an
dehydroabietane skeleton, previously described as an
intermediate in the hemisynthesis of sesquiterpene11 or
taxodione-type diterpene derivatives.12
In previously described procedures,11,12 2 and 5 were
obtained by a four-step procedure using, respectively,
methyl 12-hydroxyabieta-8,11,13-trien-18-oate (7)11 or
ferruginol12 (12-hydroxyabieta-8,11,13-triene) (9) as
starting materials, followed by nitration, reduction,
oxidation and hydrogenation.
Here, a shorter and high yielding procedure (Schemes 1
and 2) to obtain 2–5 is described. The catechols 2–5
*Corresponding author. Tel.: +351-217-165-141; fax: +351-217-168-
100; e-mail: barbara.gigante@ineti.pt
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00063-4

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B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
Scheme 1. (a) (PhCO)2O2, CH3Cl, Á, 2 h; (b) AlCl3, EtSH, CH2Cl2, 3- 5 ꢀC, 4 h for 2 or 48 h for 3; (c) Dibal-H, Et2O, À78 ꢀC, 10 min.
Scheme 2. (a) (PhCO)2O2, CH3Cl, Á, 2 h; (b) Dibal-H, Et2O, À78 ꢀC, 10 min.
were evaluated for antifungal activity, as well as for their
ability to interfere with the proliferation of human lym-
phocytes, production of nitric oxide and growth of human
cancer lines. The antimutagenic and antiviral activities of
compound 2 were also investigated. All the activities tes-
ted were compared with those of carnosic acid (6).2,3
The catechol 4 can be obtained from the benzoyloxy-
phenol 8 or the catechol 2, using diisobutylaluminium-
hydride (DIBAL-H).17 Other procedures for the clea-
vage of the ester groups had less success in terms of
selectivity or yield.18,19
The catechols 3 and 4 were disclosed by Ohtsuka and
Tahara11 but not characterized. As for all the catechols,
the ortho-diphenol moiety of 3 and 4 were first assigned
by the green coloured spots on the TLC plates used to
monitor the reaction, after spraying with a solution of
ferric chloride.20 These structures were established using
Results and Discussion
Synthesis of compounds
Methyl 12-hydroxyabieta-8,11,13-trien-18-oate (7)13
was converted into methyl 11,12-dihydroxyabieta-
8,11,13-trien-18-oate (2) by a two-step procedure. First,
7 was oxidized with benzoyl peroxide in refluxing
chloroform to give methyl 12-benzoyloxy-11-hydroxy-
abieta-8,11,13-trien-18-oate (8) as a major product.14
The benzoyloxy-phenol 8 is the key intermediate in the
synthesis of catechols 2–4 (Scheme 1).
FTIR, H and 13C NMR and mass spectrometry.
1
The above benzoyl peroxide oxidation was applied to
ferruginol (9),12 to give 12-benzoyloxyabieta-8,11,13-
trien-11-ol (10), which was then converted into 11,12-
dihydroxyabieta-8,11,13-triene (5) (Scheme 2). Both
cleavage methods previously mentioned could be used,
although the reductive cleavage with DIBAL-H pro-
vides a quantitative yield of 5, with spectral data in
agreement with the literature data.12
Catechol 2 was obtained from 8 by cleavage of the aro-
matic ester group at C-12 with an aluminium chloride-
ethanethiol system and dichloromethane as the co-sol-
vent, previously reported for the cleavage of benzyl and
methyl esters of aliphatic and aromatic carboxylic
acids15 and ethers.16 By controlling the reaction time, it
is possible to cleave selectively the aromatic ester group
leaving the methyl ester group at C-18 intact (4 h) to
give 2, or allowing the reaction to proceed for a longer
time (48 h) in order to cleave both groups and to afford
3, as detailed in the Experimental.
The structural characterization of the starting materials
6, 8, the intermediates 7, 9 and the target products 2–5
were made on the basis of the spectroscopic properties
and in the case of known compounds 2, 5–10 by com-
parison with previous data. The new compounds 3 and
4 were fully characterized.
Carnosic acid (6), used as reference, was extracted from
rosemary according to the literature.2,3,5

B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
1633
Biological Activity
acid (6) and the positive control drug l-NAME (N-nitro-
l-arginine methyl ester). Compounds 2 and 5 displayed
similar potency and were the most potent inhibitors.
Antifungal activity. Catechols 2–5 and carnosic acid (6)
were screened for antifungal activity against the yeasts
Candida albicans, Candida glabrata, Cryptococcus neofor-
mans, the mould Aspergillus fumigatus and the dermato-
phytes Microsporum canis, Trichophyton mentagrophytes
and Epidermophyton floccosum. No activity was detected
against C. albicans, C. glabrata, C. neoformans and A.
fumigatus even when the compounds were tested at 750
mM. However, an inhibition of the dermatophytes’
growth was observed with all the compounds. The
activities, expressed in minimum lethal concentration
(MLC) and defined as the lowest concentration showing
no visible fungal growth after the incubation time, are
shown in Table 1.
Effect on the growth of human cancer cell lines. The
effects of compounds 2–5 and of carnosic acid (6) on the in
vitro growth of five human cancer cell lines MCF-7
(breast), NCI-H460 (lung), SF-268 (CNS), TK-10 (renal)
and UACC-62 (melanoma) were evaluated and the results,
expressed as concentrations that were able to cause 50%
cell growth inhibition (GI50), are summarized in Table 3.
All these compounds (2–6) produce a dose-dependent
inhibition of the five human cancer cell lines. The inhi-
bitory effect presented by these compounds can not be
attributed to a toxic effect, as inferred from the sulfor-
hodamine B (SRB) assay (data not shown).
M. canis showed to be less sensitive to the tested cate-
chols than T. mentagrophytes and E. floccosum.
Catechols 2 and 5 were the most active compounds,
exhibiting a potent antifungal activity, greater than that
presented by the well-known antifungal agent flucona-
zole. Carnosic acid (6) showed less intense activity when
compared with these two catechols for the strains tested.
At concentrations above 20 mM, 3 and 4 as well as car-
nosic acid (6) showed a moderate effect on the inhibition
of cell growth. Compounds 2 and 5 exhibited the stron-
gest growth inhibitory effects, which were always higher
than those presented by carnosic acid for all the cancer
cell lines tested. Nevertheless compounds 2–6 are far to
be as active as doxorubicin.
Effect on lymphocyte proliferation and NO production.
Compounds 2–6 were evaluated for their effect on the
mitogenic response of human lymphocytes to PHA as
well as on the production of nitric oxide (NO) by the
murine macrophage cell line J774 stimulated with a
combination of lipopolysaccharide (LPS) and gamma-
interferon (IFN-g). The results, given in concentrations
that were able to produce 50% inhibition of lymphocyte
proliferation or inhibition of NO formation (IC50), are
summarized in Table 2.
Antimutagenic activity. The antimutagenic activity of
catechol 2 and carnosic acid (6) was also evaluated in
the Ames test using the strain Salmonella typhimurium
TA102, which is known to respond readily to active
oxygen-containing species. Based on the dose–response
curve of tert-butyl-hydroperoxide (t-BHP) (data not
shown) a dose of 0.44 mmol was used for the anti-
mutagenicity studies with 2 and carnosic acid (6). Com-
pound 2 dose-dependently reduced the t-BHP-induced
mutagenicity (Fig. 1a). The same was observed with
carnosic acid (6), which strongly inhibited the muta-
genicity of t-BHP (Fig. 1b). Carnosic acid (6) seems to
act as a powerful scavenger of hydroxyl radicals gener-
ated by the t-BHP.4 The chemical structure of 2 can
explain the similar inhibitory effect on the mutagenicity
of t-BHP when compared with 6.
All the catechols studied exhibited a dose-dependent
suppressor effect on the mitogenic response of human
lymphocytes to PHA. Catechols 2–5 showed an inter-
esting antiproliferative activity, stronger than carnosic
acid (6), inhibiting lymphocytes proliferation at con-
centrations below 10 mM. Compounds 2 and 5 were the
most potent compounds, exhibiting IC50 of 2.8 and 2.9
mM, respectively. It is noteworthy that their anti-
proliferative activity was only ten fold lower than that
of the well-known imunosuppressor cyclosporin A
(IC50=0.34 mM) which was used as positive control.
The present results on the inhibitory effects of com-
pound 2 on the mutagenicity of t-BHP in strain TA102
are similar to those obtained with carnosic acid (6) (Fig.
2), and better than those with carnosol, which showed a
weak inhibitory effect at the same concentrations.4
Additionally, the present results indicate that compounds
2 and 6 both exert strong antimutagenic effect, possibly by
Catechols 2–5 were also potent inhibitors of NO produc-
tion. Their effects were stronger than those of carnosic
Table 1. Effect of catechols 2–5 and carnosic acid (6) on the growth of dermatophytes
Compd
MLC (mM)
Microsporum canis
Trichophyton mentagrophytes
Epidermophyton floccosum
2
3
4
5
90.3Æ15.1
376.5Æ62.7
393.1Æ78.9
210.0Æ22.1
376.5Æ62.7
>208.9
22.6Æ2.4
94.1Æ9.9
22.6Æ2.4
94.1Æ15.7
98.3Æ16.4
13.1Æ2.2
196.5Æ39.4
26.2Æ5.5
6
Fluconazole
94.1Æ7.8
188.3Æ15.7
52.2Æ8.7
104.5Æ17.4
Fluconazole was used as positive control. Results show meansÆSEM of 4–6 independent experiments performed in duplicate.

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B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
Table 2. Effect of catechols 2–5 and of carnosic acid (6) on lympho-
cyte proliferation and NO production
uated for their antiviral activity against herpes simplex
viruses 1 (HSV-1), herpes simplex viruses 2 (HSV-2), vac-
cinia virus (VAC) and African swine fever virus (ASFV).
Compd
IC50 (mM)
Lymphocyte proliferation
NO production
The results obtained (Table 4) show that the synthetic
catechol 2 has a stronger effect on the viruses studied
when compared with carnosic acid (6), being HSV-1 and
HSV-2 the viruses most inhibited by 2.
2
3
4
5
2.8Æ0.9
8.3Æ0.2
10.6Æ1.0
2.9Æ0.7
35.4Æ3.1
0.34Æ0.04
—
5.2Æ1.2
7.9Æ0.9
12.7Æ1.3
5.3Æ0.4
18.5Æ2.1
—
6
The effect of carnosic acid (6) on HSV was previously
reported.5 These authors5 demonstrated that both, HSV-1
and HSV-2, were more inhibited in presence of the same
concentration of 6. However, their methodology was dif-
ferent. They studied the direct effect of 6 on the virus,
whereas in our work it was studied the effect of 6 on viral
infectivity (viral plaque formation) and on viral yield
(number of infectious extracellular virions produced).
Cyclosporin A
L-NAME
101.4Æ4.5
Cyclosporin A and l-NAME were used as positive controls; results
show meansÆSEM of 3–4 independent observations performed in
duplicate.
scavenging oxygen species. Since these active species are
involved in the process of carcinogenicity it is possible that
compound 2 could exhibit anticarcinogenic properties.
Activity against HIV. Compounds 2 and 6 were also
evaluated against Human Immunodeficiency Virus type
1 (HIV-1, NL4-3 strain). The results obtained indicate
Antiviral activity. Activity against HSV-1/2, vaccinia
virus and ASFV. Compounds 2 and 6 were also eval-
Table 3. Effects of catechols 2–5 and of carnosic acid (6) on the growth of human cancer cell lines
Compd
GI50 (mM)
MCF-7 (breast)
NCI-H460 (lung)
SF-268 (CNS)
TK-10 (renal)
UACC-62 (melanoma)
2
3
4
5
9.8Æ0.1
21.9Æ0.4
20.5Æ2.2
8.9Æ0.1
20.9Æ0.1
88.4Æ3.0
62.9Æ3.1
25.6Æ2.3
41.2Æ6.3
0.09Æ0.01
9.5Æ0.5
65.3Æ1.0
63.9Æ1.0
22.1Æ0.5
24.3Æ1.7
0.09Æ0.01
12.3Æ0.4
22.9Æ0.4
23.6a
12.9Æ1.1
47.2Æ9.9
0.55Æ0.06
17.7Æ0.7
59.5Æ1.4
44.8Æ2.0
19.9Æ0.4
21.6Æ1.1
0.09Æ0.01
6
Doxorubicin
24.2Æ0.3
0.04Æ0.01
Doxorubicin was used as positive control. Results show meansÆSEM of 3–6 independent experiments performed in duplicate.
aResult shows mean from one experiment performed in duplicate.
Figure 1. Antimutagenic effect of compound (2) and carnosic acid (6) on S. typhimurium TA 102 treated with t-BHP (0.44 mmol/plate). (a) n com-
pound (2), u t-BHP+compound (2); (b) l carnosic acid (6), s t-BHP+carnosic acid (6). The error bars represent the standard deviation of the mean
(SEM).

B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
1635
Figure 2. Inhibitory comparison of carnosic acid (6) (white bars) with compound (2) (black bars) on the mutagenicity of t-BHP, using S. typhi-
murium TA 102. The results are the average of three independent experiments for each inhibitory dose tested. The error bars represent the standard
deviation of the mean.
Table 4. Effect of catechols 2–5 and of carnosic acid (6) on viruses infectivity
Compd
Concentration (mM)
Virus tested
Virus yield inhibition (% control)
Plaque formation inhibition (% control)
2
15
HSV-1
HSV-2
VAC
ASFV
HSV-1
HSV-2
ASFV
80.01Æ1.72
79.21Æ3.00
29.98Æ0.92
0.01Æ0.05
29.89Æ4.30
35.00Æ3.74
nd
59.97Æ4.01
65.00Æ3.36
0.02Æ0.39
49.87Æ0.80
50.02Æ6.96
nd
6
15
40.11Æ2.51
For the effect on viral yield, the drug was present all the time after infection (1 pfu/cell), and the titres were determined by plaque assay as referred in
the Experimental. For the study of plaque formation the viruses were titrated in presence of the drug as referred in the Experimental. Each value
represents the averageÆSEM of three separate experiments performed in duplicate. All drugs were used at maximum concentration non cytotoxic.
nd, not done.
that the effective concentration to achieve 50% protec-
tion for carnosic acid (6) (EC50=458 mM) is higher than
for catechol 2 (EC50=275 mM) and AZT (EC50=50
mM). Although, 2 was more potent than 6, it is 7-fold
less potent than AZT.
fungal, antitumoral, antimutagenic, antiviral, anti-
proliferative and nitric oxide inhibition, in general
better than 6, used as the reference. Among the tested
catechols, compound 2 showed the best activities for all
the tests that are, nevertheless, lower than that of stan-
dards. Further tests and development of new com-
pounds within that class of molecules are under
investigation.
When viral particles were exposed to catechols 2 and 6
before infection of cells no inhibition was detected in
viral replication. An effect in viral infectivity was detec-
ted only when the cells were previously incubated with
these compounds. This leads to the conclusion that the
effect of these compounds is intracellular.
Experimental
FTIR spectra were recorded on Perkin-Elmer 1725 and
UV–VIS spectra on Hitachi 150-20 spectrophotometers.
NMR spectra were run on a General Electric QE-300
spectrometer with resonance frequence of 300.65 MHz
Furthermore, a quantification of proliferation and viabi-
lity was performed on cells exposed to the inhibitory con-
centrations of compounds 2 and 6, by a colorimetric assay
based on the cleavage of the tetrazolium salt WST-1 by
mitochondrial dehydrogenases of viable cells. Cells in the
presence of compounds have similar growth curves when
compared with non-exposed cells (data not shown).
1
for H and 75.6 MHz for 13C, with appropriate solvent.
The chemical shifts (d) are reported in ppm, and cou-
pling constants (J) in Hz. EI mass spectra were deter-
mined on a Kratos MS 25RF instrument at 70 eV.
Microanalyses were performed on a Carlo Erba 1106R
microanalyser. Silica gel for TLC refers to Merck silica
gel GF254 and for flash chromatography to Merck
silica gel 60, 230–400 mesh. Organic extracts were dried
over anhydrous sodium sulfate.
Conclusions
From this work, we have shown that the catechols (2–
5), analogues to carnosic acid (6), can be easily obtained
from abietic acid (1), an abundant natural product.
They show promising biological activities namely, anti-
All the reagents were of the highest quality available.
Solvents for the reactions were reagent grade, dried and

1636
B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
1
distilled prior to use following standard procedures. The
solvents for extraction and chromatography were of
technical grade, freshly distilled before use.
mp 180–182 ꢀC; FTIR (KBr) nmax 3450, 1691 cmÀ1; H
NMR (CDCl3/TMS) d 1.23 (3H, d, J=6.3, 16-H or 17-
H), 1.25 (3H, d, J=6.3, 16-H or 17-H), 1.30 (3H, s, 19-
H), 1.37 (3H, s, 20-H), 1.42–1.49 (1H, m, 1-Ha), 1.6–1.9
(6H, m, 2-H, 3-H and 6-H), 2.23 (1H, d, J5a,6a=12, 5-
H), 2.75 (1H, dd, J7e,7a=16.5 and J7e,6a=4.8, 7-He),
2.91 (1H, ddd, J7a,7e=16.5, J7a,6a=12.3 and J7a,6e=6.3,
7-Ha), 2.93 (1H, sept, J=6.9, 15-H), 3.16 (1H, dt,
J1e,1a=13.5, 1-He), 4.49 (1H, brs, 12-OH, D2O
exchange), 5.73 (1H, s, 11-OH, D2O exchange), 6.43
(1H, s, 14-H) ppm; 13C NMR (CDCl3/TMS) d 16.65 (C-
19), 18.60 (C-2), 20.28 (C-20), 22.35 (C-6), 22.49 (C-16
or C-17), 22.76 (C-16 or C-17), 27.31 (C-15), 31.81 (C-
7), 35.80 (C-1), 36.55 (C-3), 38.47 (C-10), 46.71 (C-5),
48.08 (C-4), 117.23 (C-14), 129.66 (C–Ar), 131.66 (C–
Ar), 132.35 (C–Ar), 137.87 (C–Ar), 143.10 (C–Ar),
183.31 (C-18) ppm; FABMS m/z 332(M+,100), 317(21),
271(36), 229(20), 165(93). Elemental analysis (C 72.26,
H 8.49%, calcd for C20H28O4): C 72.20, H 8.83%.
Methyl 12-benzoyloxy-11-hydroxyabieta-8,11,13-trien-
18-oate (8). Methyl 12-hydroxyabieta-8,11,13-trien-18-
oate (7) (5.0 g) (mp 160–162 ꢀC) obtained as described
in the literature,13 was oxidized with benzoyl peroxide
(3.7 g) in chloroform (60 mL) at reflux for 2 h under
nitrogen. After the workup the residue was chromato-
graphed to obtain the major of four products, methyl
12-benzoyloxy-11-hydroxyabieta-8,11,13-trien-18-oate
(8), 60% yield, mp 155–156 ꢀC (methanol) (lit.:14 155–
157 ꢀC; from methanol).
Methyl 11,12-dihydroxyabieta-8,11,13-trien-18-oate (2).
Anhydrous aluminum chloride (650 mg) was added
slowly, under nitrogen, to a stirred solution of methyl
12-benzoyloxy-11-hydroxyabieta-8,11,13-trien-18-oate (8)
(300 mg) and ethanethiol (0.51 mL) in dry dichloro-
methane (10 mL) at 0 ꢀC in a ice-water bath during a 15
min period. After stirring at this temperature for 15 min
and at room temperature for 4 h, the mixture was
poured into ice-diluted hydrochloric acid and extracted
with ether. The extract was washed with brine, dried
over sodium sulfate and evaporated in vacuum. The
residue (230 mg, 90%) was chromatographed on silica
gel, using dichloromethane as eluent, and the product
recrystallized as white crystals, mp 155–157 ꢀC from
chloroform/hexane (lit.:11 145–146.5 ꢀC from ether/pet-
11,12-Dihydroxyabieta-8,11,13-trien-18-ol (4). To
a
solution of methyl 11,12-dihydroxyabieta-8,11,13-trien-
18-oate (2) or methyl 12-benzoyloxy-11-hydroxyabieta-
8,11,13-trien-18-oate (8) (100 mg) in dry ether (20 mL),
at À78 ꢀC and under N2, was slowly added diisobutyl-
aluminum hydride (Dibal-H) (1 mL). After stirring at
this temperature for 10 min, the mixture was worked up
with a saturated solution of sodium bisulfide and
allowed to warm up to room temperature. Then the
product was extracted with ether, dried over anhydrous
sodium sulfate and the solvent was evaporated in
vacuum. The product (90 mg, 98%) was recrystallized
from a mixture of chloroform and hexane as white
crystals of 4: mp 161–163 ꢀC; FTIR (KBr) nmax 3526,
3505, 3289 cmÀ1; 1H NMR (CDCl3/TMS) d 0.88 (3H, s,
19-H), 1.22 (3H, d, J=6.3, 16-H or 17-H), 1.24 (3H, d,
J=6.3, 16-H or 17-H), 1.27–1.35 (1H, m, 5-H), 1.37
(3H, s, 20-H), 1.45 (1H, dd, J1a,1e=12.9 and J1a,2e=4.2,
1-Ha), 1.5–1.9 (6H, m, 2-H, 3-H and 6-H), 2.7–2.9 (2H,
m, 7-H), 2.99 (1H, sept, J=6.9, 15-H), 3.09 (1H, dt,
J1e,1a=13.2 and J1e,2e=3.3, 1-He), 3.21 and 3.49 (2H,
2d, J=10.9, 18-H), 3.48 (1H, sh, 18-OH, D2O
exchange), 4.50 (1H, brs, 12-OH, D2O exchange), 5.70
(1H, s, 11-OH, D2O exchange), 6.43 (1H, s, 14-H) ppm;
13C NMR (CDCl3/TMS) d 17.93 (C-19), 18.72 (C-2),
19.04 (C-6), 20.62 (C-20), 22.51 (C-16 or C-17), 22.77
(C-16 or C-17), 27.29 (C-15), 32.10 (C-7), 34.76 (C-1),
36.22 (C-3), 38.01 (C-10), 39.07 (C-4), 46.48 (C-5), 72.43
(C-18), 117.24 (C-14), 129.65 (C–Ar), 131.61 (C–Ar),
132.89 (C–Ar), 138.10 (C–Ar), 143.05 (C–Ar) ppm;
FABMS m/z 318 (M+, 100), 285(22), 217(27), 205(62),
191(46), 165(63). Elemental analysis (C 75.43, H 9.50%,
calcd for C20H30O3): C 75.45, H 9.28%.
roleum ether), FTIR (KBr) nmax 3500, 3392, 1700 cmÀ1
;
1H NMR (CDCl3/TMS) d 1.22 (3H, d, J=6, 16-H or
17-H), 1.24 (3H, d, J=6, 16-H or 17-H), 1.28 (3H, s, 19-
H), 1.35 (3H, s, 20-H), 1.42 (1H, dd, J1a,1e=13.2 and
J1a,2e=3.6, 1-Ha), 1.5–1.9 (2H, m, 3-H), 1.62 (1H, dt,
J2e,2a=9.9 and J2e,1a=3.9, 2-He), 1.65–1.75 (1H, m, 6-
Ha), 1.69 (1H, dd, J6e,6a=12.6 and J6e,7a=6, 6-He), 1.78
(1H, d, J2a,2e=10.2, 2-Ha), 2.21 (1H, d, J5a,6a=12, 5-
H), 2.72 (1H, dd, J7e,7a=16.2 and J7e,6a=4.8, 7-He),
2.85 (1H, ddd, J7a,7e=16.2, J7a,6a=12.3 and
J7a,6e=6.3, 7-Ha), 2.99 (1H, sept, J=6, 15-H), 3.15
(1H, dt, J1e,1a=12.6 and J1e,2e=2.1, 1-He), 3.67 (3H, s,
18-H), 4.85 (1H, brs, 12-OH, D2O exchange), 5.81 (1H,
s, 11-OH, D2O exchange), 6.41 (1H, s, 14-H) ppm; 13C
NMR (CDCl3/TMS) d 16.82 (C-19), 18.62 (C-2), 20.26
(C-20), 22.29 (C-6), 22.51 (C-16 or C-17), 22.77 (C-16 or
C-17), 27.17 (C-15), 31.82 (C-7), 35.82 (C-1), 36.47 (C-
3), 38.52 (C-10), 46.96 (C-5), 48.39 (C-4), 51.97 (C-21),
117.13 (C-14), 129.55 (C–Ar), 131.93 (C–Ar), 132.38
(C–Ar), 137.93 (C–Ar), 143.25 (C–Ar), 179.64 (C-18)
ppm; FABMS m/z 346 [M+,100], 331 (11), 285 (28), 271
(66), 229 (16), 205 (27), 165 (82). Elemental analysis (C
72.80, H 8.73%, calcd for C21H30O4): C 72.65, H 9.08%.
12-Benzoyloxy-11-hydroxyabieta-8,11,13-triene
(10).
Ferruginol [12-hydroxyabieta-8,11,13-triene (9)] (400
mg) obtained as described in the literature,12 was oxi-
dized with benzoyl peroxide (400 mg) as described
above. After the workup and column chromatography
(Et2O/hexane, 5–40%), the major of four products was
isolated, 12-benzoyloxy-11-hydroxyabieta-8,11,13-triene
(10), 347 mg, 61% yield, mp 130–132 ꢀC from petroleum
ether (lit.:14 132.5–133 ꢀC; from petroleum ether).
11,12-Dihydroxyabieta-8,11,13-trien-18-oic acid (3).
This was obtained from 8 using the same procedure as
for 2 with stirring at room temperature for 48 h. After
flash chromatography on silica gel, using hexane/diethyl
ether (7:3) as an eluent, 3 was obtained as a yellowish
powder (40%). Recrystallization from a mixture of
chloroform and hexane gave pale yellow crystals of 3:

B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
1637
11,12-Dihydroxyabieta-8,11,13-triene (5). This was
obtained from 10 (100 mg) using either the cleavage
system AlCl3/EtSH or Dibal-H. With Dibal-H as
described above for 4, after the workup and column
chromatography (Et2O/hexane) a white gum (63.5 mg;
drug screening that use the sulforhodamine B (SRB)
assay to assess growth inhibition.25 Five human tumor
cell lines were used, MCF-7 (breast cancer), TK-10
(renal cancer), UACC-62 (melanoma), NCI-H460 (non-
small cell lung cancer) and SF-268 (CNS cancer). Cells
were routinely maintained as adherent cell cultures in
RPMI-1640 medium containing 5% heat-inactivated
fetal bovine serum, 2 mM glutamine and 50 mg/mL
gentamicin at 37 ꢀC in an humidified air incubator con-
taining 5% CO2. Doxorubicin was used as the positive
control. Growth inhibition of 50% (GI50) as well as
toxicity of compounds was calculated as described else-
where.26
1
86%) was obtained. The IR and H NMR spectra were
identical to literature.12
Carnosic acid (6). This was extracted from dry rosemary
leaves (22 g).5 Column chromatography of the extract
(1.19 g) on polyamide with CH2Cl2/hexane gave a frac-
tion from which, after repeated recrystallization from
CHCl3/hexane, pale crystals of 6 (46 mg, 3.9%) mp
192–197 ꢀC (lit.:6 189–192 ꢀC, from hexane; lit.:2 192–
197 ꢀC, from hexane/C6H6) were obtained. H and 13C
Mutagenicity experiments. The antimutagenicity of
compound 2 and carnosic acid (6) was evaluated using
the Ames test. Salmonella typhimurium strain TA 102
was used as tester strain in the pre-incubation assay.27
This strain has been shown to be highly sensitive to
reactive oxygen species, including those generated by
t-BHP.27 Mutagenic activity was expressed as His+
revertants per plate (Rev/plate). Dose–response curves
were generated and three independent experiments were
performed for t-BHP. A concentration of 0.44 mmol
t-BHP was used for the inhibition experiments with the
compound 2 and the carnosic acid (6). This dose of 0.44
mmol t-BHP was chosen from the linear portion of the
dose–response curve of this compound using strain TA
102 (data not shown).
1
NMR spectra of 6 were identical to data reported for
carnosic acid.3
Biological activity
Stocks solutions of catechols were prepared in DMSO
and stored at À20 ꢀC, providing uniform samples for re-
tests. These frozen concentrates were then diluted to the
desired final concentrations with the appropriate sol-
vents just prior to the different assays. Final concentra-
tions of DMSO showed no interference with the
biological activities tested.
Antifungal activity. The antifungal activity of com-
pounds was evaluated as described elsewhere.21 Seven
clinical fungi strains were used, the yeasts C. albicans, C.
glabrata, C. neoformans, the mould A. fumigatus and the
dermatophytes M. canis, T. mentagrophytes and E. floc-
cosum. They were grown on Sabouraud chlor-
amphenicol agar at 30 ꢀC for 48–72 h in the case of
yeasts and moulds and on Sabouraud Chloramphenicol
cycloheximide at 30 ꢀC for 7 days just up to 15 days for
dermatophytes. Fluconazole was used as the positive
control.
Anti-HSV, VA, ASFV assays. Vero cells were grown
as monolayers in Dulbecco’s modified Eagle’s med-
ium (DME), supplemented with 10% newborn calf
serum (NCS) and 50 mg/mL gentamycin. HSV-1
strain SC 16, HSV-2 strain HD, VAC strain WR
from ATCC (USA) and the Lisbon 60 strain of
African swine fever virus (ASFV)28 were used. All
viruses were grown in Vero cells, as described.29 The
cytotoxicity of catechols 2 and 6 was previously eval-
uated in order to determine their maximum concen-
tration non cytotoxic (MCNC) to Vero cells by dye
uptake assay.30
Lymphocyte proliferation assay. The effects of com-
pounds on the mitogenic response of human lympho-
cytes to PHA (10 mg/mL) were evaluated using a
modified colorimetric MTT assay.22 This assay was
previously described by us.23 Cyclosporin A was used as
positive control.
The effect of 2 and 6 on viral plaque formation and on
viral replication were done as described previously.31,32
Anti-HIV-1 assays. Suspension culture of Jurkat cells
was incubated for 30 min at 37 ꢀC with the compounds
in predetermined concentrations. The cells were washed
with RPMI supplemented with 10% FBS at the same
temperature. Jurkat cells were then infected with HIV-1
at a multiplicity of infection (m.o.i.) of 1 (1000
TCID50/1Â106) and in the presence of compounds 2
and 6. After adsorption of the viruses for 2 h at 37 ꢀC
the cells were again washed and cultured in same
media. As a control, cells with no HIV-1 infection were
also incubated with the same concentrations of those
compounds. Cells with or without infection were
maintained in culture for 7 days at 37 ꢀC, with media
change at day 4. At day 9 cells were harvested and
HIV p24 was quantified. The 50% inhibitory concen-
tration of the drug was determined using the median
effect equation.
NO production assay. The effects of catechols on the
production of nitric oxide (NO) were evaluated quanti-
fying nitrite accumulation in cell culture supernatant by
the Griess reaction.24 The murine macrophage cell line
J774 was cultured in RPMI-1640 supplemented with 2
mM l-glutamine, 50 mg/mL gentamicine and 10% foe-
tal bovine serum. Cells were plated in 96-well culture
plates at a density of 0.5Â106 cells/well and allowed to
adhere for 2 h at 37 ꢀC in a 5% CO2 humidified atmo-
sphere. To induce nitric oxide synthase, culture medium
was replaced by fresh media containing the LPS (1 mg/
mL) and IFN-g (100 U/mL).
Cancer cell growth assay. The effects of catechols on the
growth of cancer cell lines were evaluated according to
the procedure adopted in the NCI’s in vitro anticancer

1638
B. Gigante et al. / Bioorg. Med. Chem. 11 (2003) 1631–1638
16. Matsumoto, T.; Tanaka, Y.; Terao, H.; Takeda, Y.;
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