Evaluation of labdane derivatives as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2010.04.007

In the present study, a series of labdane derivatives (2-9) were prepared from labdanediol (1) and their potential as anti-inflammatory agents were evaluated on lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. All compounds were able to inhibit LPS-induced nitric oxide (NO), although compounds 1, 2, 5, 8 and 9 exhibited the most potent effects with a range of IC ₅₀ values of 5e15 mM. Similarly to the inhibitory effects on NO release, these labdane derivatives also inhibited prostaglandin E2 (PGE ₂) production. However, analysis of cell viability demonstrated that effects on NO release and (PGE₂) production of compounds 1, 8 and 9 were due to citotoxicity, whereas compound 2 and 5 did not show any effect in the survival of RAW 264.7 macrophages. In addition to these in vitro data, compound 5 also showed anti-inflammatory activity in vivo, when tested in mice. They prevented the extent of swelling in the TPA-induced ear edema model and inhibited MPO activity, showing similar potency to that of the widely used anti-inflammatory drug indomethacin. These results indicate that compound 2 and in particular compound 5 might be used for the design of new anti-inflammatory agents.

Full text of DOI:10.1016/j.ejmech.2010.04.007

European Journal of Medicinal Chemistry 45 (2010) 3155e3161
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of labdane derivatives as potential anti-inflammatory agents
Natalia Girón a, Elisa Pérez-Sacau b c, Raquel López-Fontal d,
**
,
,
f
,
g
,
c
,
a,
*
Juan M. Amaro-Luis e, Sonsoles Hortelano d , Ana Estevez-Braun
, Beatriz de las Heras
***
a Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain
b Centro Atlántico del Medicamento. Plaza Sixto Manchado No3. Sta. Cruz de Tenerife, Spain
c Instituto Canario de Investigacion del Cáncer (ICIC), Hospital Universitario La Candelaria, Carr. El Rosario, 38010 Tenerife, Spain
d Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain
e Departamento de Química, Facultad de Ciencias, Universidad de los Andes, Mérida, Venezuela
f Unidad de Inflamación y Cáncer, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain
g Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna. Avda. Astrofísico Fco. Sánchez 2. 38206. La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a series of labdane derivatives (2e9) were prepared from labdanediol (1) and their
potential as anti-inflammatory agents were evaluated on lipopolysaccharide (LPS)-treated RAW 264.7
macrophages. All compounds were able to inhibit LPS-induced nitric oxide (NO), although compounds 1,
Received 22 December 2009
Received in revised form
5 April 2010
Accepted 8 April 2010
Available online 14 April 2010
2, 5, 8 and 9 exhibited the most potent effects with a range of IC50 values of 5e15 mM. Similarly to the
inhibitory effects on NO release, these labdane derivatives also inhibited prostaglandin E2 (PGE2)
production. However, analysis of cell viability demonstrated that effects on NO release and (PGE2)
production of compounds 1, 8 and 9 were due to citotoxicity, whereas compound 2 and 5 did not show
any effect in the survival of RAW 264.7 macrophages. In addition to these in vitro data, compound 5 also
showed anti-inflammatory activity in vivo, when tested in mice. They prevented the extent of swelling in
the TPA-induced ear edema model and inhibited MPO activity, showing similar potency to that of the
widely used anti-inflammatory drug indomethacin. These results indicate that compound 2 and in
particular compound 5 might be used for the design of new anti-inflammatory agents.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
NOS-2
Inflammation
Labdanes
Prostaglandin E2
1. Introduction
Thus, acute inflammation is a limited beneficial process, particu-
larly in response to infectious pathogens, whereas chronic inflam-
Inflammation is the biological response of body to harmful
stimuli, such as pathogens, physical injury or damaged cells, and
ultimately leads to the restoration of a normal tissue structure and
function. Normal inflammatory responses are self-limited by
a process that involves the down-regulations of pro-inflammatory
proteins and the up-regulations of anti-inflammatory proteins.
mation is an undesirable persistent phenomenon that can lead to
the developments of inflammatory diseases.
Macrophages play a key role in the inflammatory response and
serve as an essential interface between innate and adaptive
immunity. On sensing the presence of pathogens through Toll-like
(TLRs) and other receptors, macrophages are stimulated to secrete
a battery of cytokines that recruit effector cells into the infected
area. Thus, stimulation of TLR4 by lipopolysaccharide (LPS) triggers
the recruitment of the cytoplasmic adaptor protein MyD88 and
subsequently culminates in the activation of downstream signalling
Abbreviations: NO, nitric oxide; NOS-2, nitric oxide synthase; LPS, lipopolysac-
charide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PGE2, prostaglandin E2; TLCK, Na-Tosyl-L-lysine chloromethyl ketone hydrochloride;
pathways: the transcription factor nuclear factor-kB (NF-kB)
pathway [1]. These pathways induce the expression of various
inflammatory mediators, including nitric oxide (NO), prostaglan-
dins (PGs), and inflammatory cytokines, that are well-known to be
involved in the pathogenesis of inflammatory response [2,3]. PGs
and NO are generated by the inducible isoforms of cyclooxygenase
(COX-2) and NO synthase (NOS-2), respectively [4].
TLRs, Toll-like receptors.
* Corresponding author. Tel.: þ34 91 3942276; fax: þ34 91 3941726.
** Corresponding author. Centro Nacional de Investigaciones Cardiovasculares
(CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
*** Corresponding author. Instituto Universitario de Bioorganica “Antonio Gon-
zález”, Universidad de La Laguna, Avda. Astrofísico Fco. Sánchez 2, 38206. La
Laguna. Tenerife, Spain.
There is considerable research interest in the identification of
new anti-inflammatory agents from plants used in traditional
E-mail addresses: shortelano@isciii.es (S. Hortelano), aestebra@ull.es (A. Estevez-
Braun), lasheras@farm.ucm.es (B. de las Heras).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.04.007

3156
N. Girón et al. / European Journal of Medicinal Chemistry 45 (2010) 3155e3161
O
b
a
OH
OR
R
OH
OH
OH
1
d
2 R=Ac
4 R=COpBrPh
6 R=CO(CH2)10CH3
3 R=OH
5 R=OMe
c
f
e
7 R=H
OH
OH
Complex mixture
of products
+
9
8
Scheme 1. Labdane derivatives (2e9) from labdanediol (1). (a) Et3N (2 equiv), ClCOR (1.2 equiv), dry CH2Cl2, 0 ꢀC, (6 h, 89% for 2), (overnight, 67% for 4), (15 min, 100% for 6);
(b) CrO3/H2SO4 (excess), acetone, 0 ꢀC, 3 h, 60%; (c) Me3SiCHN2 (excess), CH2Cl2/MeOH (9:1), rt, 15 min 84%; (d) PCC (excess), dry CH2Cl2, rt, overnight, 10%; (e) BBr3 (1.5 equiv), dry
CH2Cl2, 0 ꢀC-rt, overnight, 98%. (f) P2O5 (2.5 equiv), CH2Cl2, rt, 30 min, 10% for 9.
medicine. During our on-going screening program designed to
identify the anti-inflammatory potential of natural compounds, we
have prepared several labdane derivatives (2e9) from the natural
labdanediol (1) isolated from Oxylobus glanduliferus [5]. Diverse
labdane diterpenes with anti-inflammatory properties have been
described in the literature. Andalusol and hispanolone derivatives
[6e8], hedychilactones AeC from Hedychium coronarium [9] and
andrographolide from Andrographis paniculata [10] are represen-
tative examples. These labdane diterpenes show anti-inflammatory
activities by inhibition of NOS-2 and COX-2 expression, impaired
cytokine release or prevention of oxygen radical production. Thus,
to evaluate whether labdane derivatives (1e9) regulate inflam-
matory response, we investigated the anti-inflammatory effects of
these derivatives using LPS-induced inflammatory responses. Our
results show that two of these derivatives, the compounds 2 and 5
should be considered promising lead compounds for further
pharmacological studies.
compounds to decrease NOS-2 induction after LPS stimulation, by
measuring NO release with the Griess method. In murine macro-
phage RAW 264.7 cells, LPS induces NOS-2, and then NO produc-
tion. Therefore, this macrophage cell line provides an excellent
model for drug screening and for evaluation of potential inhibitors
on the pathway leading to the induction of NOS-2. RAW 264.7 cells
were pre-incubated for 30 min with a range of concentrations
(1e50 mM) of labdane derivatives following by stimulation with LPS
(250 ng/ml) for 24 h. Then culture media were harvested and nitrite
levels were measured. All derivatives were able to inhibit NO
production, being the most potent, compounds 1, 2, 5, 8 and 9 with
an IC50 between 5 and 15 mM (Table 1). Compounds 1 (Fig. 1A), 2, 5,
8 and 9 (Fig. 2) were found to reduce NO production in a dose-
dependent manner. Compounds 3, 4, 6 and 7 also exerted inhibitory
effects on NO release but higher doses (in the range of 50
required (Table 1).
mM) were
To discard that the inhibitory effect on NO release was due to
cytotoxicity, we analyzed the percentage of cell viability by MTT
assays. As we can observe in Fig. 1B, compound 1 showed toxicity
2. Chemistry
effects at concentrations of 10
affected by compounds
compound 8 and 9 caused a reduction in cell viability at 25
(Fig. 3). In view of these data, compound 5 showed the most potent
m
M. In contrast, cell viability was not
2
and 5, whereas treatment with
From labdanediol (1) isolated from O. glanduliferus [5] we have
carried out several transformations on the hydroxyl functions in
order to study their role in the pharmacological activity. The acyl
derivatives 2, 4 and 6 were obtained under treatment of 1 with
1.2 equiv of the corresponding acyl chlorides in CH2Cl2 in the
presence of Et3N (2 equiv). Compound 3 was obtained by oxidation
of 1 with Jones reagent. The methyl ester (5) was obtained from
methylation of the carboxylic acid derivative (3) with an excess of
Me3SiCHN2. When 1 was oxidised with PCC the aldehyde 7 was
achieved in low yield. When 1 was treated with 1.5 equiv of BBr3 in
CH2Cl2 at 0 ꢀC overnight, the rearranged compound 8 was quanti-
tatively obtained. The treatment of 1 with the dehydrating agent
P2O5 yielded compound 9 together with a complex mixture of
inseparable products (Scheme 1).
m
M
effects with an IC50 of 5
mM and a 97% of cell viability. It is
Table 1
IC50 valuesa
(mM) of compounds 1e9 on NO and PGE2 production and cell viability.
Compound
NO inhibition
Cell viability
(%)
PGE2 inhibition
Cell viability
(%)
1
2
3
4
5
6
7
8
9
15 ꢁ 1.1
15 ꢁ 0.85
>50
50
90
80
80
97
80
80
80
80
25 ꢁ 3.2
25 ꢁ 1.35
>50
40
90
80
80
95
80
80
75
75
>50
>50
5 ꢁ 0.4
10 ꢁ 1.3
>50
>50
3. Results and discussion
>50
>50
10 ꢁ 1.2
25 ꢁ 2.6
15 ꢁ 1.6
20 ꢁ 2.4
Continuing with our interest in the evaluation of the biological
potential of natural diterpenes, we have studied the effects of
several labdane derivatives (1e9) in the inflammatory response in
an attempt to search novel anti-inflammatory agents from natural
products. To analyse the potential anti-inflammatory activity of the
labdane derivatives (1e9), we assayed the ability of these
a
IC50 values refer to the concentration needed to inhibit 50% of NO release or
PGE2 release after LPS stimulation in the presence of the compounds.
RAW 264.7 cells were treated with the compounds in a range of concentrations of
1e50 mM in the presence of LPS (250 ng/ml) for 24 h. Supernatants were analyzed
for NO release by the Griess reaction and for PGE2 release with ELISA. IC50 values are
an average of three independent experiments carried out in triplicate.

N. Girón et al. / European Journal of Medicinal Chemistry 45 (2010) 3155e3161
3157
Compound 1 ( M)
µ
Compound 1 (µM)
B
A
20
15
10
5
120
100
80
60
40
20
0
*
*
**
**
***
***
***
***
0
-
-
1
2
5
10 25 1400W
-
-
1
2
5 10 25 1400W
LPS
LPS
C
Control
LPS
LPS + compound 1
Fig. 1. Dose-dependent inhibitory effect on NO release and cell viability of compound 1. RAW 264.7 cells were pre-incubated for 30 min with different concentrations of compound
1 (1, 2, 5, 10, and 25 M) or the NOS-2 inhibitor, 1400 W (50 M) followed by stimulation with 250 ng/ml LPS for 24 h. A) The accumulation of nitrite in the culture medium was
measured with the Griess reagent. B) Cell viability was determined by MTT assay. C) Phase-contrast micrographs showing toxicity of compound 1 (25 M) in RAW 264.7 cells.
Experiments were carried out in triplicate and results show the mean ꢁ S.D. of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, with respect to the LPS condition.
m
m
m
20
15
10
5
20
15
10
5
µ
Compound 5 ( M)
µ
Compound 2 ( M)
**
**
**
**
**
**
**
**
***
***
***
***
***
***
-
0
0
-
-
1
2
5
10 25 1400W
-
1
2
5
10 25 1400W
LPS
LPS
20
15
10
5
20
15
10
5
µ
Compound 9 ( M)
µ
Compound 8 ( M)
*
*
*
**
**
**
***
***
***
***
0
0
-
-
1
2
5
10 25 1400W
-
-
1
2
5
10 25 1400W
LPS
LPS
Fig. 2. Dose-dependent inhibitory effect of compounds 2, 5, 8 and 9 on NO release. RAW 264.7 cells were pre-incubated for 30 min with different concentrations of compounds 2, 5,
8 and 9 (1, 2, 5, 10, and 25 M) or the NOS-2 inhibitor, 1400 W (50 M) followed by stimulation with 250 ng/ml LPS for 24 h. The accumulation of nitrite in the culture medium was
m
m
measured with the Griess reagent. Experiments were carried out in triplicate and results show the mean ꢁ S.D. of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,
with respect to the LPS condition.

3158
N. Girón et al. / European Journal of Medicinal Chemistry 45 (2010) 3155e3161
Compound 2 (µM)
Compound 5 (µM)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
-
-
1
2
5
10 25 1400W
-
-
1
2
5 10 25 1400W
LPS
LPS
Compound 9 (µM)
120
100
80
60
40
20
0
Compound 8 (µM)
120
100
80
60
40
20
0
*
*
-
-
1
2 5 10 25 1400W
LPS
-
-
1
2 5 10 25 1400W
LPS
Fig. 3. Cell viability in the presence of compounds 2, 5, 8 and 9. RAW 264.7 cells were pre-incubated for 30 min with different concentrations of compounds 2, 5, 8 and 9 (1, 2, 5, 10,
and 25 M) or the NOS-2 inhibitor, 1400 W (50 M) followed by stimulation with 250 ng/ml LPS for 24 h. Cell viability was determined by MTT assay. Experiments were carried out
in triplicate and results show the mean ꢁ S.D. of three independent experiments. *P < 0.05 with respect to the LPS condition.
m
m
interesting to note that inhibitory effects exerted by compound 5 at
25 M are similar to those elicited by the NOS-2 inhibitor, 1400 W
(50
(compounds
compound 1 but the percentages of cell viability increased. The
oxidation of the primary hydroxyl of to carboxylic acid
8 and 9) produced similar NO inhibition than
m
m
M) (Fig. 2).
1
Moreover, a large body of evidence suggests that PGs are
(compound 3) or aldehyde (compound 7) led to inactive
compounds. The most effective compound 5 was achieved by
methylation of the inactive labdanolic acid (3). These results indi-
cates that tertiary hydroxyl group at C-8 is not essential for the
involved in various pathophysiological processes, including
inflammation and carcinogenesis, and inducible COX-2 is mainly
responsible for the production of large amounts of these mediators
[11]. Then, we also tested the effects of labdane derivatives on PGE2
production. Similarly to the inhibitory effects on NO release,
compounds 1, 2, 5, 8 and 9 also inhibited PGE2 production, being
120
100
the compound 5 the most potent with an IC50 of 10 mM (Table 1).
Indeed, compound 5 was found to reduce PGE2 production in
a dose-dependent manner (Fig. 4). These inhibitory effects were
compared with those of the commercially available anti-inflam-
matory drug indomethacin. Results showed that compound 5 at
80
**
**
25 mM inhibited PGE2 release in a similar way than indomethacin.
To understand the physiological relevance of our results, the in vivo
anti-inflammatory activity of compound 5 was tested in the
TPA-induced mouse ear edema model. Topical application of
compound 5 significantly reduced the extent of swelling induced
by TPA (Fig. 5A). This topical anti-inflammatory activity was also
detected by measuring myeloperoxidase (MPO) activity in ear
biopsies. TPA-induced high levels of MPO activity detected at 4 h
after application, and this activity was significantly attenuated by
pretreatment with compound 5 (Fig. 5B). The inhibitory potency of
compound 5 was comparable with that of the widely used
anti-inflammatory drug indomethacin. These results indicated that
TPA-induced inflammation was attenuated in the presence of
compound 5 and are consistent with the in vitro data described
above.
60
40
20
0
**
***
***
-
-
-
+
-
+
1
-
+ +
+
25
-
+
-
LPS
Compound5 (µM)
5
-
10
Indo
-
-
+
Fig. 4. Inhibitory activity of compound 5 on the production of PGE2 in LPS-stimulated
RAW 264.7 cells. RAW 264.7 cells were pre-incubated for 30 min with different
concentrations of compound 5 or indomethacin (20 mM) followed by stimulation with
250 ng/ml LPS for 24 h. PGE2 release was determined by an ELISA kit. Experiments
were carried out by triplicate and results show the mean ꢁ S.D. of three independent
experiments. **P < 0.01, ***P < 0.001, with respect to the LPS condition.
From the obtained results some structureeactivity relationships
can be established. When 1 was converted into the corresponding
acyl derivatives 2, 4 and 6, only the introduction of the acetyl group
did not lead a loss of activity. The lost of the hydroxyl group at C-8

N. Girón et al. / European Journal of Medicinal Chemistry 45 (2010) 3155e3161
3159
PGE2 production in macrophage cells and provided a useful starting
point for the rational design of anti-inflammatory agents.
120
100
80
5. Experimental
5.1. Chemistry
**
All solvents and reagents were purified by Standard techniques
reported in Perrin, D.D.; Amarego, W.L.F. Purification of Laboratory
Chemicals, 3rd edition, Pergamon Press, Oxford, 1988 or used as
supplied from commercial sources as appropriate. Reactions were
monitored by TLC (on silica gel POLYGRAMÒ SIL G/UV254 foils).
Purification by column chromatography used Merk Kiesel 60-H
(0.063e0.2 mm) as adsorbent and different mixtures of hexanes-
ethylacetate as eluent. 1H NMR and 13C NMR spectra were recorded
in CDCl3 using a Bruker AMX300 instrument. Chemical shifts are
given in parts per million (ppm) and are referenced to the residual
solvent peak. The following abbreviations are used: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet and data are reported in
the following manner: chemical shift (multiplicity, coupling
constant if appropriate, integration). Coupling constants (J) are
given in Hertz (Hz) to the nearest 0.5 Hz. MS and HRMS were
recorded at VG Micromass ZAB-2F. IR spectra were taken on
a Bruker IFS28/55 spectrophotometer.
60
***
***
40
***
20
0
120
100
80
60
40
20
0
**
**
5.1.1. Formation of labdan-8a-ol-15-acetate (2)
67 mg (0.22 mmol) of labdanediol (1) in 10 mL of dry CH2Cl2
were treated with 2 equiv of triethylamine and 1.2 equiv of acetyl
chloride at 0 ꢀC for 10 min. The reaction was monitored by TLC until
disappearance of the starting labdane. Then the reaction mixture
was neutralized with diluted HCl (1%) and extracted several times
with CH2Cl2. The organic layers were dried over anhydrous MgSO4,
filtered and the solvent eliminated under reduced pressure. The
crude was purified by preparative TLC using hexanes/EtOAc (20%)
as eluent to yield 50.1 mg of 2 (89.3%). 2 showed identical spec-
troscopic data to those reported [12,13].
***
***
-
-
-
+
-
+
0.25
-
+
0.5
-
+
1
-
+
-
TPA
Compound 5 (mg)
Indo (mg)
-
1
Fig. 5. Compound 5 exerts anti-inflammatory activity in vivo. Ears of male mice were
treated with the indicated amounts of compound 5 or indomethacin (1 mg/ear), fol-
5.1.2. Formation of labdanic acid (3)
lowed by application of TPA (2.5 mg). After 4 h, animals were killed and the percentage
To 54 mg (0.174 mmol) of labdanediol (1) in 5 mL of acetone was
added 0.4 mL of Jones reagent at 0 ꢀC. The reaction was followed by
TLC and it was quenched after 2 h with 0.5 mL of isopropanol. The
reaction mixture was filtered through florisil and washed several
times with AcOEt. Then the solvent was removed and the residue
was purified by preparative TLC using hexanes/EtOAc (40%) as
eluent to yield 34 mg of 3 (60.3%). 3 showed identical spectroscopic
data to those reported [14e16].
of edema was determined from the ratio of the weights of identical ear tissue sections
from the test and control (vehicle-treated) ears. Myeloperoxidase (MPO) activity was
determined in tissue homogenates. Results show the means ꢁ S.D. of three indepen-
dent experiments carried out in triplicate. **P < 0.01, ***P < 0.001 vs. treatment with
TPA alone.
activity and the presence of a terminal acetate group (2) or meth-
ylcarboxy group (5) resulted in good inhibitory effects on NO
release, being more effective the methylcarboxy group.
5.1.3. Formation of labdan-8a-ol-15-p-bromobenzoate (4)
51 mg (0.16 mmol) of labdanediol (1) in 10 mL of dry CH2Cl2
were treated with 2 equiv of triethylamine and 1.5 equiv of
p-bromobenzoyl chloride at 0 ꢀC for 24 h. The reaction was moni-
tored by TLC until disappearance of the starting labdane. Then the
solvent was removed under reduced pressure and the crude was
purified by silica gel column chromatography with hexanes/EtOAc
(20%) as eluent to provide 53 mg of 4 (67%). 1H NMR (300 MHz,
4. Conclusion
The present results show the pharmacological evaluation of
a series of labdane derivatives which were prepared from labda-
nediol (1). All compounds were evaluated for their ability to
suppress NO and PGE2 production in LPS-stimulated RAW 264.7
macrophages. Compounds 1, 2, 5, 8 and 9 were especially potent
inhibitors of NO release and PGE2 production, although the anti-
inflammatory effects of compound 1 were attributable to their
cytotoxicity as assessed by MTT assay. One of these analogues
(compound 5) showed the most potent anti-inflammatory effect
indicating that methylation of the inactive labdanolic acid is
a critical factor for NO inhibitory activity and cell survival.
CDCl3):
d
¼ 7.89 (d, J ¼ 6.7 Hz, 2H), 7.57 (d, J ¼ 6.7 Hz, 2H), 4.41e4.29
(m, 2H), 1.89e1.75 (m, 2H), 1.59 (m, 6H), 1.38e1.22 (m, 9H), 1.14 (s,
3H), 1.01e0.87 (m, 2H), 0.94 (d, J ¼ 6.7 Hz, 3H), 0.85 (s, 3H), 0.78 (s,
6H) ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 165.7 (C), 2 ꢂ 131.4 (CH),
2 ꢂ 130.8 (CH), 129.1 (C), 127.6 (C), 74.0 (C), 63.5 (CH2), 62.2 (CH),
55.9 (CH), 44.2 (CH2), 41.7 (CH2), 40.8 (CH2), 39.5 (CH2), 38.9 (C),
35.3(CH2), 33.1 (CH3), 32.9 (C), 30.6 (CH), 23.7 (CH3), 22.7 (CH2),
21.2 (CH3), 20.3 (CH2), 19.3 (CH3), 18.2 (CH2), 15.2 (CH3) ppm; IR
(CHCl3): nmax ¼ 3521, 2927, 2867, 1721, 1591, 1461, 1390, 1274, 1106,
1012, 938, 848, 758 cmꢃ1; MS (EI, 70 eV): m/z (%): 494 (2) [M þ 2]þ,
In summary, the findings of the present study suggest that some
labdane derivatives are potent inhibitors of LPS-induced NO and

3160
N. Girón et al. / European Journal of Medicinal Chemistry 45 (2010) 3155e3161
492 (2) [M]þ, 474 (20) [M ꢃ H2O]þ, 461 (31), 293 (3), 191 (100);
HRMS: m/z [M]þ calcd. for C27H41O3Br: 492.2239, found 492.2265.
1160, 1082, 936, 758 cmꢃ1; MS (EI, 70 eV): m/z (%): 308 (2) [M]þ,
291 [M ꢃ OH]þ,17), 275 (18), 191 (65), 137 (100). HRMS m/z
[M]þcalcd. for C20H36O2: 308.2715, found: 308.2733.
5.1.4. Formation of labdanic acid methyl ester (5)
40 mg (0.12 mmol) of labdanic acid 3 in 20 mL of CH2Cl2/MeOH
were treated with 15 equiv of trimethylsilyl diazomethane (10 M
in diethyl ether) at rt for 15 min. Then the solvent was evaporated
under reduced pressure and the crude was purified by preparative
TLC using hexanes/EtOAc (30%) as eluent to yield 35 mg of 5 (84%).
5.1.7. Formation of compound (8)
76 mg of labdanodiol (0.245 mmol) in 10 mL of dry CH2Cl2 were
treated with 35
m
L (1.5 equiv) of BBr3 at 0 ꢀC and the reaction
mixture was left overnight at room temperature. Then 5 ml of H2O
were added, the aqueous phase was extracted several times with
CH2Cl2. The organic layers were dried over anhydrous MgSO4
filtered and the solvent eliminated under reduced pressure. The
crude was purified by preparative TLC with hexanes/EtOAc (40%) as
eluent to yield 70 mg of 8 (98%). 8 showed identical spectroscopic
data to those reported [17].
1H NMR (300 MHz, CDCl3):
d
¼ 3.66 (s, 3H), 2.33 (dd, J ¼ 5.8 and
14.6 Hz, 1H), 2.15 (dd, J ¼ 8.0 and 14.6 Hz, 1H), 1.94 (m, 1H), 1.85
(dt, J ¼ 12.2 and 3.0 Hz, 1H), 1.66e1.53 (m, 3H), 1.49e1.20 (m, 8H),
1.14 (m, 3H), 1.01e0.89 (m, 4H), 0.95 (d, J ¼ 6.7 Hz, 3H), 0.86 (s,
3H), 0.78 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 173.8 (C),
74.2 (C), 62.2 (CH), 56.1 (CH), 51.4 (CH3), 44.2 (CH2), 41.9 (CH2),
41.4 (CH2), 40.63 (CH2), 39.7 (CH2), 39.1 (C), 33.4 (CH3), 33.2 (C),
31.2 (CH), 24.0 (CH3), 22.8 (CH2), 21.4 (CH3), 20.5 (CH2), 19.9 (CH3),
18.4 (CH2), 15.4 (CH3) ppm; IR (CHCl3) nmax ¼ 3514, 2944, 2870,
1728, 1462, 1385, 1294, 1263, 1200, 1160, 1085, 937, 757 cmꢃ1; MS
(EI, 70 eV): m/z (%) 338 (2) [Mþ], 320 (38) [M ꢃ H2O]þ, 305 (76),
191 (100); HRMS: m/z [M]þ calcd. for C21H38O3: 338.2821, found:
338.2810.
5.1.8. Formation of compound (9)
230 mg (0.74 mmol) of labdanodiol (1) in 50 mL of dry CH2Cl2
were treated with 2.5 equiv of P2O5 (263 mg). The reaction was
monitored by TLC until disappearance of the starting labdane
(30 min). The reaction mixture was filtered and the solvent elimi-
nated. The crude was purified by column chromatography with
hexanes/EtOAc (20%) as eluent to yield 38 mg of 9 (10%) together
with an inseparable mixture of products. 9 showed identical
spectroscopic data to those reported [12,13].
5.1.5. Formation of labdan-8a-ol-15-lauroate (6)
110 mg (0.35 mmol) of labdanediol (1) in 30 mL of dry CH2Cl2
were treated with 2 equiv of triethylamine and 1.2 equiv of lauroyl
chloride at 0 ꢀC for 10 min. The reaction was monitored by TLC
until disappearance of the starting labdane. Then the reaction
mixture was neutralized with diluted HCl (1%) and extracted
several times with CH2Cl2. The organic layers were dried over
anhydrous MgSO4, filtered and the solvent eliminated under
reduced pressure. The crude was purified by silica gel column
chromatography with hexanes/EtOAc (from 5% to 20%) as eluent to
5.2. Pharmacological activity
5.2.1. Cell culture conditions
The murine macrophage cell line RAW 264.7 was maintained in
RPMI 1640 medium supplemented with 10% FCS, L-glutamine and
antibiotics in a humidified 5% CO2 atmosphere, as previously
described [8].
yield 171 mg of
6
(97.9%); 1H NMR (300 MHz, CDCl3):
d
¼ 4.09e4.01 (m, 2H), 2.23 (t, J ¼ 6.7 Hz, 2H), 1.80 (d, J ¼ 12.0 Hz,
5.2.2. Determination of NO synthesis
NO release was determined spectrophotometrically by the
accumulation of nitrite in the medium. Nitrite levels in culture
media were determined using the Griess reaction. Briefly, 200
of cell culture medium was mixed with 200 l of Griess reagent
[equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric
acid and 0.1% (w/v) naphtylethylenediamineeHCl], and incu-
bated at room temperature for 10 min. Absorbance was then
measured at 540 nm using a microplate reader (Perkin Elmer
Cetus, Foster City, CA, USA) and compared with a standard of
NaNO2.
1H), 1.58 (m, 6H), 1.47e1.29 (m, H), 1.20 (s, H), 1.01 (s, 3H), 0.86
(d, J ¼ 6.3 Hz, 3H), 0.82 (t, J = 6.9 Hz, 3H), 0.81 (s, 3H), 0.73 (s, 6H)
ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 176.8 (C), 74.1 (C), 62.6 (CH2),
ml
62.2 (CH), 56.0 (CH), 44.2 (CH2), 41.9 (CH2), 40.9 (CH2), 39.6 (CH2),
39.0 (C), 35.4(CH2), 34.3 (CH2), 33.3 (CH3), 33.1 (C), 31.8 (CH2), 30.6
(CH), 29.5 (CH2), 29.4 (CH2), 29.2 (CH2), 29.2 (CH2), 29.1 (CH2), 29.0
(CH2), 24.9 (CH2), 23.7 (CH3), 22.8 (CH2), 22.6 (CH2), 21.4 (CH3),
20.4 (CH2), 19.4 (CH3), 18.3 (CH2), 15.4 (CH3), 14.01 (CH3) ppm; IR
(CHCl3): nmax ¼ 2930, 2859, 1740, 1466, 1390, 1179, 1114, 1084, 942,
726 cmꢃ1 MS (EI, 70 eV): m/z (%) 492 (Mþ, 1), 474 (Mþ ꢃ H2O, 18),
459 (MþꢃH2OeCH3, 32), 191 (100). HRMS: m/z [Mþ] calcd. for
C32H60O3: 492.4542, found: 492.4543.
m
5.2.3. MTT assay for cell viability
RAW 264.7 cells were plated at a density of 105 cells/well in
96-well plates. To determine the appropriate concentration not
toxic to cells, cells were incubated in the presence of different
concentrations of derivatives for 24 h, before they were then
reacted with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) at 37 ꢀC for 4 h. The reaction product,
formazan, was extracted with dimethyl sulphoxide (DMSO) and the
absorbance was read at 540 nm. Assays were performed in tripli-
cate, and results are expressed as the percent reduction in cell
viability compared to untreated control cultures for at least three
independent experiments. IC50 values refer to the concentration
needed to inhibit 50% of cell growth in the presence of the
compounds.
5.1.6. Formation of labdan-8a-ol-15-al (7)
102 mg (0.33 mmol) of labdanediol (1) in 30 mL of dry CH2Cl2
were treated with 2.5 equiv of PCC at room temperature for 24hrs.
Then the crude was diluted with 50 mL of CH2Cl2 and washed with
water several times. The organic layers were dried over anhydrous
MgSO4, filtered and then the solvent eliminated under reduced
pressure. The crude was purified by silica gel column chroma-
tography with hexanes/EtOAc (10%) as eluent to yield 10 mg of 7
(10%) and 51 mg (50%) of unreacted 1. 1H NMR (300 MHz, CDCl3):
d
¼ 9.70 (s, 1H), 2.36 (m, 1H), 2.19 (m, 1H), 1.98 (m, 1H), 1.80
(m, 1H), 1.56 (m, 2H), 1.36e1.18 (m, 12H), 1.08 (s, 3H), 0.92
(d, J ¼ 6.5 Hz, 3H), 0.87 (s, 2H), 0.81 (s, 3H), 0.73 (s, 6H) ppm; 13C
NMR (75 MHz, CDCl3):
d
¼ 203.1 (CH), 74.0 (C), 62.0 (CH), 55.9
(CH), 50.9 (CH2), 44.3 (CH2), 41.7 (CH2), 40.7 (CH2), 39.5 (CH2), 38.9
(C), 33.2 (CH3), 33.0 (C), 28.9 (CH), 23.7 (CH3), 22.6 (CH2), 21.2
(CH3), 20.3 (CH2), 19.8 (CH3), 18.2 (CH2), 15.2 (CH3) ppm; IR
(CHCl3): nmax ¼ 3435, 2929, 2870, 1712, 1462, 1387, 1294, 1217,
5.2.4. Assay of PGE2 release
RAW 264.7 cells were pretreated with different concentrations
of labdane derivatives for 30 min and then stimulated with LPS
(250 ng/ml) for 24 h. Levels of PGE2 in the culture media were

N. Girón et al. / European Journal of Medicinal Chemistry 45 (2010) 3155e3161
3161
quantified per duplicate using EIA kits (R&D Systems, Minneapolis,
MN. USA).
the Spanish Ministry of Science and Innovation and the Pro-CNIC
Foundation. This work has been also partly funded by the Spanish
MICINN (SAF2009-13296-C02-01) and ICIC (Instituto Canario de
Investigación del Cáncer) to AEB.
5.2.5. Mouse ear edema
All experiments were approved by the institutional Animal Care
and Use Committee and were carried out in accordance with the
Declaration of Helsinki and EC guidelines for the handling and use
of laboratory animals. 12-O-tetradecanoylphorbol acetate (TPA)
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Acknowledgements
This work was supported by grants from PI05.0050, PI080070
and the Fundación Mutua Madrileña to S.H, and by a Santander-
Complutense grant to S.H. and B.de las H. The CNIC is supported by