Synthesis and anti-inflammatory activity of ent-kaurene derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.01.052

A series of kaurene derivatives (1-63) were prepared and evaluated for anti-inflammatory activity. Thirteen of the tested compounds were able to inhibit NO production with an IC₅₀ between 2 and 10 μM. Compounds 11, 12, 14 and 23 showed low perc

Full text of DOI:10.1016/j.ejmech.2011.01.052

European Journal of Medicinal Chemistry 46 (2011) 1291e1305
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-inflammatory activity of ent-kaurene derivatives
,
b
,
,
,b
Idaira Hueso-Falcón a 1, Irene Cuadrado c 1, Florencia Cidre d, Juan M. Amaro-Luis e, Ángel G. Ravelo a
,
,
b
,
c
,
d,
*
Ana Estevez-Braun a , Beatriz de las Heras , Sonsoles Hortelano
*
*
a Instituto Universitario de Bio-Orgánica “Antonio González”, Universidad de La Laguna, Avda. Astrofísico Fco. Sánchez 2, 38206 La Laguna, Tenerife, Spain
b Instituto Canario de Investigaciones del Cáncer (ICIC)2, Spain
c Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain
d Unidad de Inflamación y Cáncer, Área de Biología Celular y del Desarrollo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo, Km 2,200,
28220 Majadahonda, Madrid, Spain
e Departamento de Química, Facultad de Ciencias, Universidad de los Andes, Mérida, Venezuela
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of kaurene derivatives (1e63) were prepared and evaluated for anti-inflammatory activity.
Received 9 December 2010
Received in revised form
24 January 2011
Accepted 25 January 2011
Available online 3 February 2011
Thirteen of the tested compounds were able to inhibit NO production with an IC50 between 2 and 10
Compounds 11, 12, 14 and 23 showed low percentage of cell viability, whereas compounds 9, 10, 17, 28,
37, 48, 55, 61 and 62 were non-cytotoxic at the concentration up to 25 M. Some structureeactivity
relationships were outlined. Compounds 28, 55 and 62, were selected as representative compounds and
they potently inhibited the protein expression of NOS-2. We also determined that inhibition of NF-
activation might be the mechanism involved in anti-inflammatory effects of these kaurene derivatives.
As expected, cytokines IL-6, IL-1 , TNF- and IFN- were downregulated in the presence of compound
mM.
m
k
B
Keywords:
Inflammation
Kaurenes
NO
a
a
g
28, 55 and 62 after stimulation with LPS. These results indicate that kaurene derivatives might be used
for the design of new anti-inflammatory agents.
Ó 2011 Elsevier Masson SAS. All rights reserved.
NOS-2
NF-kB
Cytokines
1. Introduction
area. Thus, stimulation of TLR4 by lipopolysaccharide (LPS) triggers
the recruitment of the cytoplasmic adapter protein MyD88 and
Inflammation is an adaptive response that is triggered by
noxious stimuli and conditions, including infection and tissue
injury. If the acute inflammatory response fails to eliminate the
pathogen, the inflammatory process persists and acquires new
characteristics.
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
subsequently culminates in the activation of downstream signaling
pathways. These pathways induce the expression of various
inflammatory mediators, including nitric oxide (NO), prostaglan-
dins (PGs), chemokines and inflammatory cytokines that are well-
known to be involved in the pathogenesis of inflammatory
response [1e4].
One of the most important regulators of the inflammatory
response is the transcription factor NF-kB. NF-kB exists mainly as
a heterodimer comprised of subunits of the Rel family p50 and p65,
which is normally sequestered in the cytosol as an inactive complex
by association with the inhibitory proteins I
NF- B involves the phosphorylation of I Bs at two critical serine
residues (Ser32, Ser36) via the I B kinase (IKK) signalosome
complex [6,7]. Once I Bs have been phosphorylated, they are
ubiquitinated and degraded by 26S proteosome [8,9]. Degradation
of I B allows the NF- B to translocate to the nucleus, where it
regulates the transcription of all significant pro-inflammatory
chemokines/cytokines, many angiogenic growth factors, TNF-
kB [5]. The activation of
k
k
k
Abbreviations: NO, nitric oxide; NOS-2, inducible nitric oxide synthase; LPS,
lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PGs, prostaglandins; TLCK, Na-Tosyl-l-lysine chloromethyl ketone
hydrochloride; TLRs, Toll-like receptors.
k
k
k
* Corresponding authors. Tel.: þ34 918223291; fax: þ34 918223269.
E-mail addresses: aestebra@ull.es (A. Estevez-Braun), lasheras@farm.ucm.es
(B. de las Heras), shortelano@isciii.es (S. Hortelano).
a
,
1
inducible nitric oxide synthase (NOS-2), COX-2, ICAM-1, VCAM-1,
E-selectin and pathways of cell proliferation and survival [10e12].
Both authors contributed equally to this work.
http://www.icic.es.
2
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.052

1292
I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
Therefore, inhibition of NF-
k
B activation and the following
derivative 35 (68%) together the methyl ester 2 (26%). When
release of inflammatory mediators provide a promising strategy for
the development of potential anti-inflammatory agents. Indeed, the
anti-inflammatory effect of steroids, retinoids and a variety of anti-
rheumatic drugs act through a mechanism that involves inhibition
of NF-kB activation [13,14]. Moreover, recent evidence suggests that
anti-inflammatory drugs could also be beneficial in a number of
neurodegenerative disorders, including Alzheimer’s and Parkin-
compounds 9 and 25 were reacted with Ac2O in the presence of
pyridine the corresponding acetylated derivatives 36 and 37 were
quantitatively obtained. The dimer compound 38 was formed in
50% yield under treatment of compound 9 with 1 equiv of thionyl
chloride and 1 equiv of pyridine in DCM. Compound 39 with an
amide moiety was prepared by reaction of compound 26 with
2 equiv of oxalyl chloride and 3 equiv of aniline in DCM under
reflux. The reduction of compound 31 with LiAlH4/Et2O afforded
the corresponding dihydroxylated compound 40 in 54% yield.
Compounds (41e51) were obtained by transformations on func-
tional groups present in C and D rings of several kaurene deriv-
atives as it is shown in Scheme 2. The esterification of the
hydroxyl group at C-15 in compound 10 with lauroyl chloride or
pent-4-enoyl chloride in the presence of Et3N, DMAP in DCM
yielded the corresponding esters 41 and 42. Epoxidation of the
double bond in 33 using MCPBA produced the epoxide derivative
43 in 89% yield. The reaction of compound 11 with NH2OH$ HCl
provided the oxime derivative 44 in high yield (91%). From the
two double bonds present in compound 17 only the exocyclic
son’s diseases, which have
a prominent neuroinflammatory
component [15,16]. In this context, continuing our study in the
development of potential anti-inflammatory agents, the present
work describes the preparation and evaluation of anti-inflamma-
tory activity of kaurene derivatives (1e63).
2. Chemistry
Compounds 1e34 were synthesized as described in Ref. [17]
(Fig. 1). Compounds 35e40 were obtained by modifications at
carbon C-19 of several kaurene derivatives (Scheme 1). Thus, the
reaction of kaurenoic acid (1) with Me3SiCHN2 in Et2O yielded the
R3
R4
O
H
H
R2
R2
H
H
H
COOCH3
R1
R1
4 α
5 β
1
2
8
9
R1 = COOH; R2 = H
R1 = COOCH3; R2 = H
R1 = COOCH2Ph; R2 = H
R1 = CH2OH; R2 = H
3
6
7
R1 = COOCH3; R2 = R3 = H; R4 = CH3
R1 = COOCH3; R2 = R3 = H; R4 = CHO
R1 = COOCH3; R2 = R3 = H; R4 = CHNOH
N
N
14 R1 = COOCH3; R2 = H; R3 = OH; R4 = CH2OH
15 R1 = COOCH3; R2 = H; R3 = OCH3; R4 = CH3
10 R1 = COOCH3; R2 = OH
H
R1 = COOH; R2 = OAc
R1 = COOH; R2 = OH
26
27
O
31 R1 = COOH; R2 = H; R3 = OH; R4 = CH3
32 R1 = COOCH3; R2 = H; R3 = OH; R4 = CH3
34 R1 = COOCH3; R2 = H; R3 = Cl; R4 = CH3
H
COOCH3
30 R1 = COOCH3; R2 = OAc
29
R
H
H
O
O
R2
H
H
H
COOCH3
COOCH3
23
R
R1
11 R = CHO
33 R = CH3
12 R = COOCH3
28 R = COOH
16 R1 = COOH; R2 = H
17 R1 = COOCH3; R2 = H
19 R1 = COOCH3; R2 = OH
22 R1 = COOCH3; R2 = OAc
24 R1 = COOCH2Ph; R2 = H
25 R1 = CH2OH; R2 = H
HO
H
OH
OH
Ph
CHO
H
O
H
H
COOCH3
13
COOCH3
COOCH3
COOCH3
21
20
18
Fig. 1. Structures of kaurene derivatives (1e34).

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1293
Scheme 1. Obtention of derivatives (35e40) by modifications at C-19.
double bond was reduced under hydrogenation conditions and
compound 45 was consequently obtained. The spiro compounds
46 and 47 were obtained when the dihydroxyl derivative 14 was
treated with 3 equiv of SOCl2 and pyridine in DCM. Esterification
of compound 18 with 2-furoyl chloride afforded a mixture 1.2:1 of
the corresponding mono- (48) and di-furoate (49) compounds.
Epoxidation of the double bonds present in compound 17
produced the epoxide isomers (50) and (51). Compounds 52e63
were obtained by ozonolysis of the exocyclic double bond of
kaurene derivatives 2, 8, 10, 12, 30 and 35 (Scheme 3). With
compound 2 and 30 only the expected ketones (52) and (59) were
obtained. The treatment of compound 8 in dry DCM under N2

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I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
O
O
15
Cl
Cl
10
O
H
15
O
H
H
15
3'
O
Et3N / DCM
DMAP
1'
Et3N / DCM
DMAP
H
O
OH
5'
1'
H
H
2'
COOCH3
COOCH3
4'
12'
COOCH3
41 (54%)
42 (38%)
10
MCPBA
O
H
H
NaHCO3
DCM
H
H
COOCH3
COOCH3
33
4 3 (89%)
17
NOH
CHO
17
H
NH2OH·HCl
H
15
H
15
AcONa
reflux
H
H
COOCH3
COOCH3
11
44 (91%)
H2, Pd /C
THF
H
H
COOCH3
COOCH3
17
45 (37%)
O
O
OH
OH
O
16
O
S
S
O
O
SOCl2/py
DCM
17
H
H
H
+
H
H
H
COOCH321
COOCH231
COOCH3
14
47 (32%)
46 (31%)
5"
O
O
3"
1"
O
O
OH
OH
OH
O
16
Cl
+
O
1'
O
1'
17
17
3'
O
O
3'
H
H
H
Et3N /DMAP
toluene
O
O
COOCH3
COOCH3
COOCH3
5'
5'
reflux
48 (30%)
49 (25%)
18
O
O
O
O
11
MCPBA
16
17
NaHCO3
9
+
DCM
H
H
H
COOCH3
COOCH3
COOCH3
51 (15%)
17
50 (12%)
Scheme 2. Obtention of derivatives (41e51) by ring C and D modifications.
atmosphere at ꢀ78ꢁ with O3 for 30 s and then, with 2.7 equiv of
Me2S yielded after purification three compounds 53 (54%), 54
(21%) and 55 (20%). The structures of 54 and 55 were ratified by
the correlations detected in the HMBC spectrum. A plausible
explanation to the formation of the lactone moiety is based on
a BaeyereVilliger oxidation on the carbonyl compound formed in
the ozonolysis. Other tentative formation is shown in Scheme 4
according to the result published by Schank et al. [18].

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1295
Scheme 3. Derivatives (52e63) obtained by ozonolysis.
Derivative 55 apparently is formed by opening of the corre-
sponding lactone but 55 does not possess the corresponding
hydroxyl group at C-16. In the ozonolysis of compound 10
furthermore of the carbonyl derivative 56 another unexpected
derivative 57 was obtained in low yield (7%). Its structure was
determined on the basis of the following HMBC correlations H-17/
C-15, C-16 and H-7/C-15. The orientation of the hydrogen H-15
and the methylendioxy moiety was established by the NOE effects
detected between H-15 and the hydrogens H-5 and H-9. When
the a,b-unsaturated carbonyl compound (12) was ozonized only
the anhydride 58 was obtained in 39% yield. Finally four
compounds 60e62 were obtained from the reductive ozonolysis
of 35. The isolation of compound 63 having a dioxirane moiety
supports the proposed mechanism illustrated in Scheme 4.

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I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
Scheme 4. Plausible formation of compound 54.
3. Results and discussion
improved activity. The esterification of the hydroxyl group at C-15
of compound 10 with different acylation agents yielded inactive
compounds (compare 10 vs 30 and 41).
3.1. Lipopolysaccahride (LPS)-induced NO production
In the series of grandiflorenic acid (16) with a double bond at C-
To evaluate whether kaurene derivatives (1e63) regulate
inflammatory response, we investigated the anti-inflammatory
effects and underlying mechanisms of action of these derivatives
using LPS-induced inflammatory responses. In murine RAW 264.7
macrophage cells treatment with LPS induces NOS-2 and NO
release. NO production can be detected by measuring the accumu-
lation of nitrite with the Griess method [19]. RAW 264.7 cells
9eC-11, the methyl ester 17 presented an IC50 of 5 mM, and the
importance of the mentioned double bond for the activity is
evident when we compared the activity of 17 vs 2. The selective
hydrogenation of compound 17 afforded the inactive compound 45,
which indicated that the exocyclic double bond plays an important
role for the activity. The replacement of the two double bonds by
epoxides yielded the inactive compounds 50 and 51. Respect to the
nature of the substituents at C-19 the methyl ester (17) resulted to
be more active than the carboxylic acid (16) and the hydroxyl
derivative (25). The introduction of different groups at the ring D
led to derivatives less active and more cytotoxic (compare 17 vs 19
and 23). The tricyclic acids 55 and 62 obtained by ozonolisis
were pre-incubated with a range of concentrations (1e25 mM) of
compounds (Table 1) or the vehicle solution dimethyl sulfoxide
(DMSO) for 15 min before their stimulation with LPS (250 ng/ml).
Thirteen of the tested compounds were able to inhibit NO produc-
tion with an IC50 between 2 and 10 mM (Table 1). To discard that the
inhibitory effect on NO release was due to cytotoxicity, we analyzed
the percentage of cell viability by MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assays. Compounds 11, 12, 14
and 23 showed low percentage of cell viability, whereas compounds
9, 10, 17, 28, 37, 48, 55, 61 and 62 were non-cytotoxic at the
showed good inhibitory activity with an IC50 of 7 mM. With respect
to the unexpected lactones 54 and 61 formed also by ozonolisis,
only 61 showed inhibitory effect on NO release which indicated the
importance of the nature of esters at C-4.
In summary, these results indicate that compounds 10, 17, 28,
55, 61 and 62 can be pointed as the most active derivatives, exerting
their anti-inflammatory activity in a dose dependent manner
(Fig. 2).
concentration up to 25 mM.
From the obtained results some structureeactivity relationships
can be established. The higher inhibitory activities were exerted by
compounds 10, 12, 14, 17, 28, 55, 61 and 62. We observed two
different behaviors depending on the existence or not of the double
bond at C-9eC-11. In the series of kaurenoic acid (1), without the
mentioned double bond, only good activities were achieved with
the introduction of different groups at D ring. Thus, the introduc-
tion of a hydroxyl or a carbonyl group at C-15 led to an increased
activity (compare 2 vs 10 and 12). Compound (12) resulted be
cytotoxic but similar NO inhibitory activity was obtained with
compound 28 having a carboxylic acid instead of the methyl ester
and a 95% of cell viability. The replacement of the exocyclic double
bond at C-16eC-17 by epoxy (compounds 4 and 5), aldehyde
(compound 6), or oxime (compound 7) function did not produce an
3.2. Compounds 28, 55 and 62 inhibit NOS-2 protein and mRNA
expression
Although the six compounds exert inhibitory effects on NO
release with similar IC50 values, we selected compounds 28, 55 and
62 for further evaluation as they showed a more potent inhibition
at higher concentrations (Fig. 2). In order to discard cytotoxicity of
these compounds, we measured cell viability with MTT assay. As we
can observe in Fig. 3, none of them was toxic. To further analyze the
signaling pathways modulated by compounds 28, 55 and 62,
we studied the activation of the pro-inflammatory gene NOS-2.

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1297
Macrophages were pre-incubated with derivatives and stimulated
with LPS for 24 h. All compounds potently inhibited the protein
expression of NOS-2 (Fig. 4A). Inhibitory effects were exerted at the
transcriptional level as revealed analysis of NOS-2 mRNA by
quantitative PCR (Fig. 4B).
Table 1
IC50 values (mM) of compounds 1e63 on NO inhibition and cell viability.
Compound
NO inhibition
Cell viability (%)
1
2
3
4
5
6
7
8
>25 ꢂ 1.2
25 ꢂ 2.3
70
90
72
90
66
90
25
46
80
97
76
32
97
69
90
90
100
35
67
18
30
50
50
23
50
58
100
95
48
40
>25 ꢂ 1.7
>25 ꢂ 4.6
>25 ꢂ 3.1
>25 ꢂ 1.8
>25 ꢂ 1.2
>25 ꢂ 3.5
10 ꢂ 1.1
3.3. Anti-inflammatory effects of derivatives 28, 55 and 62 are
mediated by NF-kB inhibition
An important transcription factor in the regulation of inflam-
mation is NF- B. The binding of LPS to TLR4 on macrophages acti-
vates NF- B leading to the upregulation of pro-inflammatory
enzymes and cytokines such as NOS-2, COX-2, IL-1 , TNF- , MCP-1
and IL-6. Thus, NF- B signaling and pathways that regulate its
k
9
k
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
5 ꢂ 0.7
b
a
5 ꢂ 1.1
k
4 ꢂ 0.4
20 ꢂ 1.4
activity have become a focus for intense drug development and
application screening [20,21]. To investigate the mechanism of
action of kaurene derivatives on macrophage function, we exam-
ined the effect of compounds 28, 55 and 62 on the levels of cytosolic
5 ꢂ 1.2
>25 ꢂ 2.2
15 ꢂ 1.9
5 ꢂ 1.3
I
k
B
a
protein in LPS-activated macrophages. As we can observe in
Fig. 5, degradation of I was impaired in activated cells pre-
treated with compounds 28, 55 and 62. These results suggest that
inhibition of NF- B activation might be the mechanism involved in
anti-inflammatory effects of these kaurene derivatives.
Inhibition of NF- B activation can occur by different mecha-
nisms including (a) inhibiting the activation of IKK complex, (b)
targeting the proteasomal degradation of I Bs or (c) interfering the
translocation of NF- B to the nucleus, or the binding of NF- B to
DNA. Nevertheless, the most effective and selective approach for
the inhibition of NF- B activation is provided by inhibitors of the
>25 ꢂ 6.2
>25 ꢂ 4.2
25 ꢂ 1.7
kBa
>25 ꢂ 1.9
>25 ꢂ 2.3
10 ꢂ 1.9
k
>25 ꢂ 1.3
>25 ꢂ 6.9
>25 ꢂ 1.5
>25 ꢂ 2.6
2.5 ꢂ 0.3
>25 ꢂ 4.6
>25 ꢂ 3.5
Not tested
25 ꢂ 3.7
k
k
k
k
k
Not tested
52
100
82
95
99
90
99
IKK activity [20,21]. Since, we have previously described that
several kaurane diterpenes such as foliol and linearol specifically
targeting IKK kinase activity [22], we cannot rule out this mecha-
nism for the anti-inflammatory effects of compounds 28, 55 and 62.
Nonetheless, further experiments must be accomplished to deter-
>25 ꢂ 5.5
>25 ꢂ 1.2
>25 ꢂ 3.7
12 ꢂ 2.5
10 ꢂ 2.6
mine the degree of specificity on NF-kB inhibition of our
compounds.
>25 ꢂ 1.5
>25 ꢂ 2.8
>25 ꢂ 1.3
>25 ꢂ 4.7
Not tested
14 ꢂ 3.5
85
95
95
Not tested
95
99
95
95
60
99
Not tested
99
Not tested
98
95
50
95
95
3.4. Kaurene derivatives inhibit cytokine release
>25 ꢂ 3.2
>25 ꢂ 0.5
17 ꢂ 3.5
Macrophage activation is also associated with the release of
several pro-inflammatory cytokines and chemokines. In order to
explore whether other inflammatory mediators such as cytokines
were affected by treatment with kaurene derivatives, we analyzed
>25 ꢂ 4.1
10 ꢂ 0.5
Not tested
>25 ꢂ 1.3
Not tested
>25 ꢂ 1.2
12 ꢂ 0.7
levels of IL-6, IL-1a, TNF-a and IFN-g. As expected, all cytokines
were downregulated in the presence of compound 28, 55 and 62
after stimulation with LPS (Fig. 6). Interestingly, similar results have
been described for several IKK inhibitors that inhibit TNF-
>25 ꢂ 1.9
7 ꢂ 0.6
a
production upon LPS stimulation in murine models [20]. The
most potent inhibition was observed on IL-6 production; a pro-
inflammatory cytokine which is known to increase dramatically in
response to inflammation and brain injury, and it has been
described to play a key role in pathologies such as rheumatoid
arthritis. There are several binding sites for a number of tran-
>25 ꢂ 2.8
Not tested
>25 ꢂ 2.5
>25 ꢂ 3.8
Not tested
5 ꢂ 1.1
Not tested
95
99
Not tested
95
95
scription factors in the 50 region of the IL-6 gene, including NF-
kB,
CREB, NF-IL-6, and AP-1 box. Our results have shown that
7 ꢂ 0.9
Not tested
Not tested
compounds 28, 55 and 62 antagonized the increase of IL-6 induced
by LPS, indicating that NF-kB activation is the only transcription
factor involved on IL-6 release. These data are according with
recent studies on the IL-6 promoter that demonstrate that IL-6
induction by several transcription factors occurs in a highly stim-
IC50 values refer to the concentration needed to inhibit 50% of NO 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
1e25 mM in the presence of LPS (250 ng/ml) for 24 h. Supernatants were analyzed
for NO release by the Griess reaction and cell viability was determined by MTT assay.
IC50 values are means ꢂ SD of three independent experiments carried out by
triplicate.
ulus-specific or cell-specific manner. Thus, NF-kB has been shown
to regulate the induced transcription of IL-6 in murine macro-
phages [23].

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I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
10 20 30 40 50
0
10 20 30 40 50
0
10 20 30 40 50
Compound 10 (μM)
Compound 17 (μM)
Compound 28 (μM)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0 10 20 30 40 50
0 10 20 30 40 50
0
10 20 30 40 50
Compound 55 (μM)
Compound 61 (μM)
Compound 62 (μM)
Fig. 2. Dose-dependent effects of compounds 10, 17, 28, 55, 61 and 62 on NO release. RAW 264.7 cells were pre-treated with diterpenoids (1, 5, 10, 25 and 50 mM) for 15 min and
then stimulated with 250 ng/ml LPS for 24 h. The accumulation of nitrite in the culture medium was measured with the Griess reagent. Experiments were carried out in triplicate
and the results are the means ꢂ S.D. of three different experiments.
4. Conclusion
the presence of the ring D can further enhance the activity. In the
tetracyclic series the combination of methyl ester at C-19/hydroxyl
group at C-15 or carboxylic acid at C-19/carbonyl group at C-15 led
to an increase in the anti-inflammatory activity. Compounds 28,
In an attempt to discover novel anti-inflammatory agents,
a series of kaurene derivatives were synthesized and evaluated for
their ability to suppress NO production in LPS-stimulated RAW
264.7 macrophages. Thirteen compounds were especially potent
inhibitors of NO release, although the anti-inflammatory effects of
compounds 11, 12, 14 and 23 were attributable to their cytotoxicity
as assessed by MTT assay. Three of these analogs, compounds 28,
55 and 62 showed the most potent anti-inflammatory effect. The
existence of a carboxylic acid seems to play an important role for
NO inhibitory activity and cell survival, since it is present in the
three mentioned active compounds. The good activity of tricyclic
derivatives 55 and 62 obtained by ozonolysis demonstrates that
55 and 62 also demonstrated significant inhibitory effects on I
degradation. Therefore, the activity of these compounds may be at
least in part due to its NF- B inhibitory activity. In addition to the
kBa
k
inhibitory effects on NO production, compounds 28, 55 and 62
were able to inhibit several cytokines involved in the inflamma-
tory response after LPS stimulation such as IL-6, IL-1
b, TNF-a and
IFN-g
.
In summary, we have identified three novel compounds with
potential anti-inflammatory properties that might be use for the
future development of a new class of anti-inflammatory agents.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
5
10 15 20
0
5
10 15 20
0
5
10 15 20
Compound 28 (μM)
Compound 55 (μM)
Compound 62 (μM)
Fig. 3. Cell viability of compounds 28, 55 and 62. RAW 264.7 cells were pre-treated with diterpenoids (1, 5, 10, 25 and 50
mM) for 15 min and then stimulated with 250 ng/ml LPS for
24 h. Cell viability was determined by the MTT assay. Experiments were carried out in triplicate and the results are the means ꢂ S.D. of three different experiments.

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1299
A
LPS
B
Compound
NOS-2
28
55
62
500
400
300
200
100
-actin
80
60
40
20
0
**
**
***
0
Compound
28
55
62
LPS
Fig. 4. Compounds 28, 55 and 62 inhibit NOS-2 expression. A) Macrophages were pre-incubated for 15 min with compounds 28, 55 and 62 (5
250 ng/ml LPS for 24 h. NOS-2 protein was detected by Western blot.
m
M) followed by stimulation with
b
-Actin content was used as a loading control. Results show a representative experiment of three. B)
Macrophages were pre-incubated as in A and stimulated with 250 ng/ml LPS for 4 h. Relative expression of NOS-2 mRNA was determined by real-time quantitative PCR. Results
show the means ꢂ S.D. of three independent experiments carried out in triplicate. **p < 0.01, ***p < 0.001 vs LPS.
5. Experimental
Espeletia chardonii using the habitual previously described meth-
odology [24e28].
5.1. Chemistry
5.1.1. Preparation of compound 35
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 POLYGRAM SIL G/UV254 foils).
Purification by column flash-chromatography used Merk Kiesel 60-
H (0.063e0.2 mm) as adsorbent and different mixtures of hex-
aneseethylacetate as eluent. Pre-coated TLC plates SIL G-100 UV254
(Machery-Nagel) were used for preparative-TLC purification. 1H
NMR spectra were recorded in CDCl3 or C6D6 at 300 or 400 MHz,
using a Bruker AMX300 or Bruker AMX400 instruments. For 1H
spectra, chemical shifts are given in parts per million (ppm) and are
referenced to the residual solvent peak. The following abbrevia-
tions are used: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad. Proton assignments and stereochemistry are
supported by 1He1H COSY and ROESY where necessary. 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. 13C NMR spectra
were recorded at 75 and 100 MHz using a Bruker AMX300 or
Bruker AMX400 instruments. Carbon spectra assignments are
supported by DEPT-135 spectra, 13Ce1H (HMQC) and 13Ce1H
(HMBC) correlations where necessary. Chemical shifts are quoted in
ppm and are referenced to the appropriate residual solvent peak.
MS and HRMS were recorded on a VG Micromass ZAB-2F. IR spectra
were taken on a Bruker IFS28/55 spectrophotometer. Kaurenoic
To 489.5 mg of compound 1 (1.621 mmol) dissolved in Et2O was
added 1 ml of 2 M solution of Me2SiCHN2 in Et2O. The reaction
mixture was stirred at room temperature for 15 h. Then the solvent
was removed and the residue was purified by chromatotron using
hexanes/toluene (40%) to afford 30.1 mg of compound 2 (26%) as an
amorphous white solid and 424.9 mg of compound 35 (68%) as
yellow oil. 1H NMR (300 MHz, CDCl3)
d: 4.79 (1H, s, H-17a), 4.74 (1H,
s, H-17b), 3.75 (1H, d, J ¼ 14 Hz, H-21a), 3.57 (1H, d, J ¼ 14 Hz, H-
21b), 2.63 (1H, s, H-13), 2.03 (2H, s, H-15), 1.15 (3H, s, H-18), 0.80
(3H, s, H-20), 0.09 (9H, s, Si(CH3)3), 2.18e0.79 (18H, m). 13C NMR
(75 MHz, CDCl3) d: 178.1 (s, C-19), 155.4 (s, C-16), 102.8 (t, C-17),
56.8 (d, C-5), 56.6 (t, C-21), 54.8 (d, C-9), 48.7 (t, C-15), 43.9 (s, C-8),
43.7 (s, C-4), 43.5 (d, C-13), 41.1 (t, C-7), 40.5 (t, C-1), 39.3 (t, C-14),
39.2 (s, C-10), 38.0 (t, C-3), 32.8 (t, C-12), 28.7 (q, C-18), 21.8 (t, C-6),
18.9 (t, C-2), 18.1 (t, C-11), 15.2 (q, C-20), ꢀ3.13 (q, Si(CH3)3). EIMS
m/z (%): 388 ([M]þ, 92); 373 (95); 345 (29); 285 (28); 257 (100); 241
(34); 123 (52); 121 (26); 109 (33); 107 (25); 105 (27); 97 (23); 91
(30); 81 (38); 79 (23); 73 (42); 59 (35); 55 (29). HREIMS: 388.2794
(calcd for C24H40O2Si [M]þ 388.2798). IR nmax: 2931, 2853, 1722,
1462, 1250, 1227, 1147, 860 cmꢀ1. UV (EtOH) lmax (log
e): 202.0
(3.55) nm. [
a
]
20: ꢀ68.42 (c 0.9, CHCl3).
D
5.1.2. Preparation of compound 36
8.7 mg (0.030 mmol) of compound 9 dissolved in the minimum
amount of pyridine were treated with an excess of Ac2O (6.1 l,
m
3 equiv) in the presence of catalytic amount of DMAP. The reaction
mixture was stirred for 24 h, after elimination of solvent,
compound 36 (7.1 mg, 100%) was obtained as an amorphous white
acid (1), grandiflorenic acid (16), 15
and 16 -hydroxy kaurenoic acid (31) were used as starting material
to synthesize the diterpenes. These diterpenes were isolated from
a-acetoxy kaurenoic acid (26)
a
solid. 1H NMR (300 MHz, CDCl3)
d: 4.79 (1H, s, H-17a), 4.73 (1H, s,
Fig. 5. Kaurene derivatives inhibit NF-
k
B activity. Macrophages were pre-treated for 15 min with kaurene derivatives (5
mM) and then activated for the indicated times with
250 ng/ml LPS. The levels of I were determined by Western blot. b
k
B
a
-Actin was used as a loading control. Results show a representative experiment of three.

1300
I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
12000
10000
8000
6000
4000
2000
0
300
200
100
**
**
**
***
*
***
*
***
*
0
1400
1200
1000
800
5000
4000
3000
2000
1000
*
*
600
400
**
200
0
0
Compound
Compound
28
55
62
28
55
62
LPS
LPS
Fig. 6. Cytokine release is inhibited by kaurene derivatives. Macrophages were treated or untreated with compound 28, 55 and 62 (5
24 h with LPS 250 ng/ml. Production of IL-6, IL-1 , TNF- and IFN-
was determined in supernatant by ELISA. Results show the means ꢂ S.D. of three independent experiments
carried out in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs LPS.
mM) for 15 min followed by stimulation for
a
a
g
H-17b), 4.21 (1H, d, J ¼ 11 Hz, H-19a), 3.87 (1H, d, J ¼ 11 Hz, H-19b),
2.63 (1H, s, H-13), 2.04 (3H, s, OCOCH3), 1.02 (3H, s, H-18), 0.93 (3H,
s, H-20), 1.96e0.73 (20H, m). 13C NMR (75 MHz, CDCl3): 171.2 (s,
OCOCH3), 155.6 (s, C-16), 102.7 (t, C-17), 66.9 (t, C-19), 56.5 (d, C-5),
55.9 (d, C-9), 48.8 (t, C-15), 43.9 (s, C-8), 43.7 (d, C-13), 41.3 (t, C-7),
40.1 (t, C-1), 39.4 (t, C-14), 38.9 (s, C-4), 36.8 (s, C-10), 36.1 (t, C-3),
32.9 (t, C-12), 27.3 (q, C-18), 20.8 (q, OCOCH3), 20.2 (t, C-6), 18.0 (t,
C-2), 18.0 (t, C-11), 17.8 (q, C-20). EIMS m/z (%): 330 ([M]þ, 45); 315
(23); 270 (26); 257 (63); 255 (54); 227 (44); 167 (30); 149 (100);
147 (32); 145 (30); 135 (49); 133 (30); 123 (90); 121 (39); 109 (52);
107 (46); 105 (51); 95 (45); 93 (52); 91 (66); 81 (72); 79 (48); 67
(39); 57 (37); 55 (58). HREIMS: 330.2542 (calcd for C22H34O2 [M]þ
330.2559). IR nmax: 2927, 2855, 1740, 1448, 1370, 1239, 1032,
117 (29); 105 (47); 95 (22); 93 (28); 91 (54); 81 (33); 55 (44).
HREIMS: 328.2405 (calcd for C22H32O2 [M]þ 328.2402). IR nmax
:
2930, 2868,1738, 1659, 1460,1373,1240,1032, 983 cmꢀ1. UV (EtOH)
lmax (log
CHCl3).
e
): 202.0 (3.55) y 243.0 (3.11) nm. [
a
]
20: þ22.59 (c 1.1,
D
5.1.4. Preparation of compound 38
To 16.7 mg of compound 9 (0.058 mmol) dissolved in 5 ml of dry
CH2Cl2 were added 4.69 l of pyridine (1 equiv) and 4.22 l of SOCl2
m
m
(1 equiv) under N2 atmosphere. The reaction mixture was stirred at
room temperature for 4 h, until disappearance of the starting
material (4 h). Then H2O was poured and the mixture was extracted
several times with CH2Cl2. The organic phases were collected and
dried over anhydrous MgSO4, then were filtered, concentrated and
purified by preparative-TLC using hexanes/AcOEt (9.5:0.5). 9.1 mg
873 cmꢀ1. UV (EtOH) lmax (log
e
): 202.0 (3.58) nm. [
a
]
20: ꢀ40.13
D
(c 0.8, CHCl3).
of compound 38 (50%) were obtained. 1H NMR (300 MHz, CDCl3)
d:
5.1.3. Preparation of compound 37
Following the same procedure described above, 8.7 mg
(0.030 mmol) of compound 25 were treated with 8.63 l of Ac2O
4.80 (2H, s, H-17a, H-170a), 4.74 (2H, s, H-17b, H-170b), 4.15 (1H, d,
J ¼ 9.9 Hz, H-19a ó H-190a), 4.01 (1H, d, J ¼ 9.9 Hz, H-19a ó H-190a),
3.77 (1H, d, J ¼ 9.9 Hz, H-19b ó H-190b), 3.63 (1H, d, J ¼ 9.9 Hz, H-
19b ó H-190b), 2.64 (2H, s, H-13, H-130), 1.03 (3H, s, H-18, H-180),
0.98 (6H, s, H-20, H-200), 2.31e0.73 (41H, m). 13C NMR (75 MHz,
m
(3 equiv) to yield quantitatively 10.0 mg of compound 37 as
amorphous white solid. 1H NMR (300 MHz, CDCl3): 5.19 (1H, s, H-
11), 4.91 (1H, s, H-17a), 4.78 (1H, s, H-17b), 4.30 (1H, d, J ¼ 10.9 Hz,
H-19a), 4.07 (1H, d, J ¼ 10.9 Hz, H-19b), 2.73 (1H, s, H-13), 2.64 (1H,
d, J ¼ 15.6 Hz, H-15a), 2.17 (1H, d, J ¼ 15.6 Hz, H-15b), 2.05 (3H, s,
OCOCH3), 1.07 (3H, s, H-18), 0.93 (3H, s, H-20), 2.41e0.90 (15H, m).
13C NMR (75 MHz, CDCl3): 171.2 (s, OCOCH3), 158.4 (s, C-9), 156.8 (s,
C-16), 113.6 (d, C-11), 105.0 (t, C-17), 66.1 (t, C-19), 50.3 (t, C-15),
45.2 (d, C-5), 44.5 (t, C-14), 41.9 (s, C-4), 41.0 (d, C-13), 39.8 (t, C-1),
37.8 (s, C-8), 37.5 (t, C-3), 37.1 (s, C-10), 35.9 (t, C-12), 29.1 (t, C-7),
26.2 (q, C-18), 25.2 (q, C-20), 20.8 (q, OCOCH3), 18.5 (t, C-2), 17.6 (t,
C-6). EIMS m/z (%): 328 ([M]þ, 78); 313 (96); 255 (44); 253 (100);
185 (25); 183 (27); 173 (29); 159 (29); 147 (43); 143 (27); 129 (27);
CDCl3) d
: 158.2 (s, C-9, C-90), 156.6 (s, C-16, C-160), 113.7 (d, C-11, C-
110), 105.1 (t, C-17, C-170), 64.0 (t, C-19 ó C-190), 63.6 (t, C-19 ó C-190),
50.3 (t, C-15, C-150), 45.3 (d, C-5 ó C-50), 45.2 (d, C-5 ó C-50), 44.5 (t,
C-14, C-140), 41.9 (s, C-8, C-80), 41.0 (d, C-13, C-130), 39.7 (t, C-1, C-10),
37.8 (s, C-10, C-100), 37.5 (t, C-12, C-120), 37.4 (s, C-4 ó C-40), 37.4 (s,
C-4 ó C-40), 35.5 (t, C-3 ó C-30), 35.4 (t, C-3 ó C-30), 29.1 (t, C-7, C-70),
26.3 (q, C-18 ó C-180), 26.2 (q, C-18 ó C-180), 25.2 (q, C-20, C-200),
18.4 (t, C-2 ó C-20), 18.3 (t, C-2 ó C-20), 17.6 (t, C-6, C-60). EIMS m/z
(%): 622 ([M]þ, 5); 287 (79); 271 (54); 270 (50); 257 (79); 175 (23);
161 (63); 147 (47); 123 (47); 111 (100); 106 (37); 105 (64); 95 (49);
94 (59); 93 (43); 91 (38); 81 (61); 55 (35). HREIMS: 622.4421 (calcd

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1301
for C40H62O3S [M]þ 622.4420). IR nmax: 2932, 2868, 1461, 1189, 972,
C-4), 42.2 (d, C-13), 40.3 (t, C-1), 39.4 (s, C-10), 37.7 (t, C-3), 37.0
(t, C-7), 34.5 (t, C-14), 32.4 (t, C-12), 31.7 (t, C-20), 29.4 (t, C-30), 29.3
(t, C-40, C-50), 29.1 (t, C-60), 29.0 (t, C-70, C-80), 28.9 (t, C-90), 28.4 (t, C-
100), 24.9 (q, C-18), 22.4 (t, C-110), 20.6 (t, C-6),18.8 (t, C-2),þ18.1 (t, C-
11), 15.4 (q, C-20), 13.9 (q, C-120). EIMS m/z (%): 514 ([M] , 9); 332
(42); 315 (30); 314 (100); 299 (66); 273 (14); 255 (74); 254 (37); 239
(35); 183 (29); 147 (22); 121 (28); 107 (24); 71 (22); 57 (45); 55
20
925, 874, 756 cmꢀ1. UV (EtOH) lmax (log
þ46.75 (c 0.8, CHCl3).
e): 201.6 (3.29) nm. [a] :
D
5.1.5. Preparation of compound 39
To a solution of 33.5 mg of 26 (0.093 mmol) in dry DCM (5 ml) at
0 ꢁC were added 21.2
ml of oxalyl chloride (2 equiv) and one drop of
DMF. The reaction mixture was stirred for 26 h, then 4.5 equiv of
aniline were added and the reaction was left for 75 h at room
temperature. The solvent was removed and the residue was purified
by TC-preparative using hexanes/AcOEt (30%) to yield 18.8 mg of
(32). HREIMS: 514.4027 (calcd for C33H54O4 [M]þ 514.4022). IR nmax
:
2927, 2855, 1729, 1463, 1233, 1189, 1154 cmꢀ1. UV (EtOH) lmax
(log
e
): 202.0 (3.41) nm. [
a
]
20: ꢀ39.67 (c 0.9, CHCl3).
D
compound 39 (46%) as a yellow oil.1H NMR (300 MHz, CDCl3)
d
: 7.47
5.1.8. Preparation of compound 42
To a solution of 9.4 mg (0.028 mmol) of compound 10 in 4 ml of
dry DCM were added 10 ml of Et3N (2.5 equiv), 10 ml of 4-pentenoyl
(2H, d, J ¼ 7.5 Hz, H-20, H-60), 7.32 (2H, t, J ¼ 7.5 Hz, H-30, H-50), 7.10
(1H, t, J ¼ 7.5 Hz, H-40), 5.26 (1H, s, H-15), 5.09 (2H, s, H-17), 2.77 (1H,
s, H-13), 2.08 (3H, s, OCOCH3), 1.27 (3H, s, H-18), 0.90 (3H, s, H-20),
chloride (3.2 equiv) and catalytic amount of DMAP. The reaction
mixture was stirred for 42 h under N2 atmosphere. Then the solvent
was eliminated and the crude was purified by TLC using hexanes/
AcOEt 10% to yield 4.5 mg of compound 42 (38%) as an amorphous
2.19e0.85 (19H, m).13C NMR (75 MHz, CDCl3)
d: 174.8 (s, C-19),170.9
(s, OCOCH3), 155.0 (s, C-16), 137.7 (s, C-10), 128.8 (d, C-30, C-50), 124.0
(d, C-40), 120.0 (d, C-20, C-60), 109.8 (t, C-17), 82.7 (d, C-15), 56.9 (d,
C-5), 52.6 (d, C-9), 47.2 (s, C-8), 44.3 (s, C-4), 42.2 (d, C-13), 40.6 (t,
C-1), 39.6 (s, C-10), 38.0 (t, C-3), 36.8 (t, C-7), 34.7 (t, C-14), 32.3 (t, C-
12), 29.5 (q, C-18), 21.2 (t, C-6), 21.1 (q, OCOCH3),19.0 (t, C-2),18.2 (t,
C-11),15.8 (q, C-20). EIMS m/z (%): 435 ([M]þ, 25); 375 (12); 256 (21);
255 (100); 185 (11); 173 (28); 162 (17); 159 (15); 123 (15); 109 (12);
105 (11); 95 (11); 93 (87); 91 (12); 81 (17); 55 (12). HREIMS:
435.2754 (calcd for C28H37NO3 [M]þ 435.2773). IR nmax: 3398, 2932,
2866, 1729, 1667, 1597, 1521, 1435, 1370, 1307, 1243, 1022, 752,
white solid. 1H NMR (300 MHz, CDCl3) : 5.84 (1H, m, H-40), 5.27
d
(1H, s, H-17a), 5.08 (3H, sa, H-17b, H-15, H-50a), 5.01 (1H, dd, J ¼ 1.6
y 11.8 Hz, H-50b), 3.64 (3H, s, H-21), 2.78 (1H, s, H-13), 2.41 (4H, m,
H-20, H-30), 1.16 (3H, s, H-18), 0.83 (3H, s, H-20), 2.19e0.78 (18H, m).
13C NMR (75 MHz, CDCl3) : 178.2 (s, C-19), 172.9 (s, C-10), 155.3 (s,
d
C-16), 136.5 (d, C-40), 115.2 (t, C-50), 109.7 (t, C-17), 82.7 (d, C-15),
56.4 (d, C-5), 52.6 (d, C-9), 51.0 (q, C-21), 47.2 (s, C-8), 43.8 (s, C-4),
42.2 (d, C-13), 40.6 (t, C-1), 39.4 (s, C-10), 38.0 (t, C-3), 37.0 (t, C-20),
34.9 (t, C-30), 33.6 (t, C-7), 32.4 (t, C-14), 28.8 (t, C-12), 28.4 (q, C-18),
20.7 (t, C-6), 19.1 (t, C-2), 18.1 (t, C-11), 15.2 (q, C-20). EIMS m/z (%):
414 ([M]þ, 14); 332 (34); 315 (28); 314 (94); 299 (68); 255 (91); 254
(41); 239 (58); 211 (20); 147 (24); 121 (39); 109 (23); 107 (33); 105
(30); 93 (24); 91 (37); 83 (49); 81 (25); 79 (23); 55 (100). HREIMS:
414.2773 (calcd for C26H38O4 [M]þ 414.2770). IR nmax: 2929, 2855,
693 cmꢀ1. UV (EtOH) lmax (log
e): 202.4 (4.03) y 241.6 (3.68) nm.
[
a]
20: ꢀ96.80 (c 1.0, CHCl3).
D
5.1.6. Preparation of compound 40
To 13.3 mg of compound 31 dissolved in dry THF were added
6.3 mg of LiAlH4 (4 equiv) under N2 atmosphere. The reaction
mixture was stirred for six days. Then it was treated with a satu-
1727, 1458, 1232, 1155, 992, 908, 773 cmꢀ1. UV (EtOH) lmax (log
e):
rated solution of dipotassium salt of La
L
(þ)-tartaric acid and
201.6 (3.51) nm. [
a]
20: ꢀ41.11 (c 0.4, CHCl3).
D
extracted several times with AcOEt. The organic layers were sepa-
rated, dried over anhydrous MgSO4 and concentrated. The residue
was purified by preparative-TLC using hexanes/AcOEt 40% to yield
6.9 mg of compound 40 (54%) as an amorphous white solid. 1H
5.1.9. Preparation of compound 43
To 26.7 mg of compound 33 (0.225 mmol) dissolved in dry DCM
were added 42.3 mg (2 equiv) of MCPBA and 31.8 mg of NaHCO3
(4.5 equiv). The reaction mixture was left to room temperature for
4 h, then the organic phase was separated and treated with
a saturated solution of sodium thiosulfate. The organic phase was
separated again and dried over anhydrous magnesium sulfate, the
mixture was filtered, the solvent was removed under reduced
pressure and the crude was purified by preparative-TLC using
hexanes/AcOEt 20% to yield 25.1 mg of compound 43 (89%) as an
amorphous white solid.
NMR (300 MHz, CDCl3)
d, J ¼ 10.9 Hz, H-19b), 1.35 (3H, s, H-17), 1.01 (3H, s, H-18), 0.95 (3H,
s, H-20), 1.90e0.73 (23H, m). 13C NMR (75 MHz, CDCl3)
: 79.1 (s, C-
d
: 3.73 (1H, d, J ¼ 10.9 Hz, H-19a), 3.44 (1H,
d
16), 65.3 (t, C-19), 57.6 (t, C-15), 56.7 (d, C-5), 56.5 (d, C-9), 48.7 (d,
C-13), 45.0 (s, C-8), 42.2 (t, C-7), 40.2 (t, C-1), 39.0 (s, C-4), 38.4 (s, C-
10), 37.3 (t, C-3), 35.4 (t, C-14), 26.8 (q, C-17), 26.5 (t, C-12), 24.2 (q,
C-18), 20.4 (t, C-6), 18.0 (q, C-20), 18.0 (t, C-2), 17.8 (t, C-11). EIMS m/
z (%): 306 ([M]þ, 1); 302 (15); 288 (17); 275 (25); 258 (30); 257
(100); 161 (19); 147 (21); 135 (26); 123 (52); 121 (28); 109 (36); 107
(28); 105 (38); 95 (32); 94 (41); 91 (30); 81 (34); 79 (26); 55 (23).
1H NMR (300 MHz, CDCl3)
d: 3.62 (3H, s, H-21), 2.64 (1H, s, H-
15), 1.41 (3H, s, H-17), 1.16 (3H, s, H-18), 0.80 (3H, s, H-20),
2.18e0.83 (19H, m). 13C NMR (75 MHz, CDCl3): 177.7 (s, C-19), 67.8
(d, C-15), 61.1 (s, C-16), 56.5 (d, C-5), 50.9 (q, C-21), 49.3 (d, C-9),
43.6 (s, C-8), 43.5 (s, C-4), 40.6 (t, C-1), 39.0 (s, C-10), 38.8 (d, C-13),
37.8 (t, C-3), 35.5 (t, C-7), 31.9 (t, C-14), 28.5 (q, C-18), 26.7 (t, C-12),
20.4 (t, C-6), 18.8 (t, C-2), 18.0 (t, C-11), 14.9 (q, C-20), 14.4 (q, C-17).
EIMS m/z (%): 332 ([M]þ, 63); 317 (25); 314 (11); 289 (28); 273 (76);
257 (45); 255 (23); 173 (18); 159 (23); 149 (23); 147 (26); 135 (64);
133 (33); 123 (53); 121 (100); 109 (49); 107 (76); 105 (39); 95 (43);
93 (45); 91 (48); 81 (50); 79 (45); 67 (33); 55 (47). HREIMS:
332.2343 (calcd for C21H32O3 [M]þ 332.2351). IR nmax: 2947, 2848,
1724, 1446, 1232, 1194, 1153, 986, 845, 771 cmꢀ1. UV (EtOH) lmax
HREIMS: 306.2563 (calcd for C20H34O2 [M]þ 306.2559). IR nmax
:
.
3365, 2926, 2864, 2850, 1697, 1447, 1122, 1030, 1010, 756, 462 cmꢀ1
[
a]
20: ꢀ25.94 (c 0.7, CHCl3).
D
5.1.7. Preparation of compound 41
To a solution of 10.7 mg of compound 10 (0.032 mmol) in dry
DCM were added 11.2 l of lauroyl chloride (1.5 equiv), 13.5 l of
m
m
Et3N (3 equiv) and catalytic amount of DMAP. The reaction mixture
was stirred for 48 h under N2 atmosphere. Then the solvent was
removed and the crude was purified by preparative-TLC using
hexanes/AcOEt 10% to yield 9.0 mg of compound 41 (54%) as
a yellow oil. 1H NMR (300 MHz, CDCl3)
d
: 5.25 (1H, s, H-17a), 5.07
(log
e): 201.8 (2.91) nm. [
a]
20: ꢀ45.88 (c 1.0, CHCl3).
D
(1H, s, H-17b), 5.07 (1H, s, H-15), 3.63 (3H, s, H-21), 2.77 (1H, s, H-
13), 1.25 (20H, sa, H-20-110), 1.16 (3H, s, H-18), 0.89 (3H, m, H-120),
5.1.10. Preparation of compound 44
0.82 (3H, s, H-20), 2.33e0.85 (18H, m). 13C NMR (75 MHz, CDCl3)
d
:
To a solution of 19.8 mg of hydroxylamine hydrochloride
(7.2 mg, 3 equiv) and 19.8 mg sodium acetate (3.2 equiv) in 0.4 ml
of H2O, 29.8 mg of aldehyde 11 in 3 ml of EtOH was added. The
177.9 (s, C-19), 173.9 (s, C-10), 155.3 (s, C-16), 109.6 (t, C-17), 82.4 (d,
C-15), 56.3 (d, C-5), 52.6 (d, C-9), 50.9 (q, C-21), 47.2 (s, C-8), 43.5 (s,

1302
I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
reaction mixture was stirred under reflux for 23 h. Then the EtOH
was removed, and the residue was extracted with DCM (3 ꢃ 10 ml).
The organic phases were collected and dried over anhydrous
MgSO4, filtered and concentrated. The residue was purified by
preparative-TLC using hexanes/AcOEt (4:1) and 23.5 mg of
compound 44 (91%) were obtained as an amorphous yellow solid.
396.1970). IR nmax: 2946, 2875, 1727, 1467, 1212, 1161, 962, 819, 762,
681 cmꢀ1. [
a
]
D
20: ꢀ64.49 (c 1.2, CHCl3). Compound 47: 1H RMN
(300 MHz, CDCl3)
d
: 4.71 (1H, d, J ¼ 8.6 Hz, H-17a), 4.33 (1H, d,
J ¼ 8.6 Hz, H-17b), 3.64 (3H, s, H-21), 2.60 (1H, s, H-13), 1.16 (3H, s,
H-18), 0.81 (3H, s, H-20), 2.29e0.72 (20H, m). 13C NMR (75 MHz,
CDCl3) d: 177.7 (s, C-19), 98.6 (s, C-16), 71.1 (t, C-17), 56.5 (d, C-5),
1H NMR (300 MHz, CDCl3)
3.64 (3H, s, H-21), 2.94 (1H, d, J ¼ 3 Hz, H-13), 1.17 (3H, s, H-18), 0.86
(3H, s, H-20), 2.18e0.76 (19H, m). 13C NMR (75 MHz, CDCl3)
: 177.8
d: 7.85 (1H, s, H-17), 5.82 (1H, s, H-15),
55.3 (t, C-15), 54.8 (d, C-9), 51.0 (q, C-21), 46.1 (d, C-13), 44.5 (s, C-8),
43.5 (s, C-4), 40.9 (t, C-7), 40.4 (t, C-1), 39.1 (s, C-10), 38.0 (t, C-3),
37.7 (t, C-14), 28.4 (q, C-18), 26.6 (t, C-12), 21.7 (t, C-6), 18.8 (t, C-2),
18.8 (t, C-11), 15.3 (q, C-20). EIMS m/z (%): 396 ([M]þ, 4); 337 (100);
315 (37); 273 (49); 255 (20); 123 (43); 121 (45); 109 (37); 107 (29);
95 (23); 91 (24); 81 (27); 79 (23); 55 (24). HREIMS: 396.1988 (calcd
for C21H32O5S [M]þ 396.1970). IR nmax: 2948, 2875, 1726, 1467, 1209,
d
(s, C-19), 147.3 (d, C-17), 147.0 (d, C-15), 139.4 (s, C-16), 56.4 (d, C-5),
51.0 (q, C-21), 49.7 (s, C-8), 46.4 (d, C-9), 43.6 (s, C-4), 43.0 (t, C-7),
40.5 (t, C-1), 39.6 (d, C-13), 39.5 (s, C-10), 38.5 (t, C-14), 37.8 (t, C-3),
28.5 (q, C-18), 24.8 (t, C-12), 20.4 (t, C-6), 18.8 (t, C-2), 18.6 (t, C-11),
15.0 (q, C-20). EIMS m/z (%): 345 ([M]þ, 18); 330 (11); 329 (51); 328
(100); 312 (32); 268 (79); 267 (54); 252 (35); 162 (21); 123 (66);
121 (29); 117 (24); 109 (47); 108 (24); 107 (36); 91 (35); 81 (38); 79
(29); 67 (32); 55 (40). HREIMS: 345.2301 (calcd for C21H31NO3 [M]þ
345.2304). IR nmax: 3421, 3286, 2939, 2851, 1723, 1464, 1446, 1233,
1161, 963, 822, 685 cmꢀ1. [
a]
20: ꢀ44.84 (c 1.2, CHCl3).
D
5.1.13. Preparation of compounds 48 and 49
To 9.3 mg of compound 18 (0.027 mmol) dissolved in 3 ml of
toluene were added 8.5 ml of 2-furoyl (3 equiv), Et3N (15 ml, 4 equiv)
1191, 1159, 970, 950, 738 cmꢀ1. UV (EtOH) lmax (log
e): 248.4 (3.83)
and catalytic amount of DMAP. The reaction mixture was stirred
under reflux for 18 h. The solvent was removed and the residue was
purified by preparative-TLC using hexanes/AcOEt 40%, to yield
10.0 mg of compound 48 (30%) and 3.6 mg of compound 49 (25%)
as amorphous white solids. 49 resulted be unstable and decom-
nm. [
a
]
D
20: ꢀ107.94 (c 1.0, CHCl3).
5.1.11. Preparation of compound 45
72.6 mg (0.231 mmol) of compound 17 dissolved in 4 ml of dry
THF were hydrogenated in the presence of catalytic amount of Pd/C
10%. The reaction mixture was stirred for 1 h until disappearance of
the starting material. After elimination of solvent the resulting
residue was purified by preparative-TLC using hexanes/AcOEt (20%)
to yield 27.1 mg of compound 45 (37%) as a white amorphous solid.
poses with the time. Compound 48: 1H NMR (300 MHz, CDCl3)
d:
7.59 (1H, d, J ¼ 1.0 Hz, H-50), 7.21 (1H, d, J ¼ 3.5 Hz, H-30), 6.53 (1H,
dd, J ¼ 1.0, 3.5 Hz, H-40), 5.16 (1H, s, H-11), 4.33 (1H, d, J ¼ 11.3 Hz, H-
17a), 4.27 (1H, d, J ¼ 11.3 Hz, H-17b), 3.65 (3H, s, H-21), 1.17 (3H, s,
H-18), 0.90 (3H, s, H-20), 2.48e0.83 (16H, m). 13C NMR (75 MHz,
1H NMR (300 MHz, CDCl3)
H-21), 1.16 (3H, s, H-18), 1.00 (3H, d, J ¼ 8 Hz, H-17), 0.89 (3H, s, H-
20), 2.41e1.04 (19H, m). 13C NMR (75 MHz, CDCl3)
: 177.9 (s, C-19),
d: 5.18 (1H, t, J ¼ 3 Hz, H-11), 3.64 (3H, s,
CDCl3) d
: 177.7 (s, C-19), 158.5 (s, C-9), 157.4 (s, C-10), 146.3 (d, C-50),
144.1 (s, C-20), 118.1 (d, C-11), 113.3 (d, C-30), 111.7 (d, C-40), 82.6 (s,
C-16), 70.3 (t, C-17), 54.7 (t, C-15), 51.1 (q, C-21), 46.4 (d, C-5), 44.6
(d, C-13), 44.6 (s, C-4), 42.7 (s, C-8), 42.6 (t, C-14), 40.7 (t, C-1), 38.4
(s, C-10), 38.1 (t, C-3), 30.2 (t, C-12), 29.6 (t, C-7), 27.9 (q, C-18), 23.1
(q, C-20), 19.9 (t, C-2), 18.2 (t, C-6). EIMS m/z (%): 424 ([Mþ ꢀ 18]
12); 312 (67); 297 (36); 273 (25); 271 (17); 255 (23); 253 (37); 237
(43); 213 (26); 197 (34); 173 (20); 172 (25); 157 (23); 145 (27); 144
(26); 143 (27); 131 (40); 129 (25); 117 (26); 112 (33); 109 (26); 107
(19); 105 (39); 95 (100); 91 (50); 81 (18); 79 (19); 55 (29). HREIMS:
424.2264 (calcd for C26H32O5 ([M]þ ꢀ 18) 424.2250). IR nmax: 3501,
2931, 2858, 1720, 1469, 1397, 1297, 1226, 1179, 1151, 1122, 971,
d
158.1 (s, C-9), 114.8 (d, C-11), 51.0 (q, C-21), 49.7 (t, C-15), 46.5 (d, C-
5), 45.8 (t, C-14), 44.6 (s, C-4), 42.2 (s, C-8), 41.0 (t, C-1), 38.3 (s, C-
10), 38.2 (t, C-3), 37.5 (d, C-13), 36.8 (d, C-16), 30.1 (t, C-12), 29.6 (t,
C-7), 27.9 (q, C-18), 23.1 (q, C-20), 20.1 (t, C-2), 18.6 (q, C-17), 18.4 (t,
C-6). EIMS m/z (%): 316 ([M]þ, 38); 301 (77); 271 (41); 257 (49); 241
(100); 197 (63); 159 (36); 134 (40); 121 (53); 109 (94); 107 (62); 95
(53); 93 (46); 91 (98); 81 (54); 79 (37); 57 (47); 55 (55). HREIMS:
316.2390 (calcd for C21H32O2 [M]þ 316.2402). IR nmax: 2933, 2867,
1723, 1460, 1376, 1220, 1149, 978, 757 cmꢀ1. [
CHCl3).
a]
20: þ37.33 (c 0.9,
D
762 cmꢀ1. UV (EtOH) lmax (log
e): 202.0 (3.51) y 251.0 (3.60) nm.
[
a
]
20: þ7.60 (c 0.5, CHCl3). Compound 49: 1H NMR (300 MHz,
D
5.1.12. Preparation of compounds 46 and 47
CDCl3)
d
: 7.55 (2H, s, H-50, H-500), 7.12 (1H, d, J ¼ 3.4 Hz, H-30 ó H-300),
To 33.8 mg of compound 14 (0.097 mmol) dissolved in 5 ml of
dry CH2Cl2 were added 23.5 l of pyridine (3 equiv) and 21.1 l of
7.08 (1H, d, J ¼ 3.4 Hz, H-30 ó H-300), 6.47 (2H, m, H-40, H-400), 5.28
(1H, J ¼ 12.3 Hz, H-17a), 5.23 (1H, s, H-11), 4.37 (1H, d, J ¼ 12.3 Hz,
H-17b), 3.64 (3H, s, H-21), 2.77 (1H, s, H-13), 1.17 (3H, s, H-18), 0.91
(3H, s, H-20), 2.48e0.83 (17H, m). 13C NMR (75 MHz, CDCl3): 177.7
(s, C-19), 158.0 (s, C-9), 157.0 (s, C-10, C-100), 146.2 (d, C-50 ó C-500),
145.9 (d, C-50 ó C-500), 145.1 (s, C-20 ó C-200), 144.1 (s, C-20 ó C-200),
117.8 (d, C-11), 117.8, 117.4 y 113.8 (4C, d, C-30, C-300, C-40, C-400), 93.2
(s, C-16), 65.6 (t, C-17), 53.6 (t, C-15), 51.1 (q, C-21), 46.2 (d, C-5),
44.6 (s, C-4), 42.7 (t, C-14), 42.4 (d, C-13), 42.3 (s, C-8), 40.7 (t, C-1),
38.4 (s, C-10), 38.0 (t, C-3), 29.9 (t, C-12), 29.5 (t, C-7), 27.8 (q, C-18),
23.2 (q, C-20), 19.9 (t, C-2), 18.1 (t, C-6). EIMS m/z (%): 424
([M]þ ꢀ 112(C5H4O3), 7); 312 (44); 297 (30); 253 (16); 237 (24); 197
(16); 145 (15); 111 (23); 105 (18); 97 (11); 95 (100); 91 (21); 57 (21);
55 (24). HREIMS: 424.2262 (calcd for C26H32O5 ([M]þ ꢀ 112
(C5H4O3)) 424.2250).
m
m
SOCl2 (3 equiv) under N2 atmosphere. The reaction mixture was
stirred at room temperature for 1 h, until disappearance of the
starting material (4 h). Then H2O was poured and the mixture was
extracted several times with CH2Cl2. The separated organic phases
were dried over anhydrous MgSO4, filtered and concentrated. The
residue was purified by preparative-TLC using toluene to yield
11.8 mg of compound 46 (31%) and 12.2 mg of compound 47 (32%)
as amorphous white solids. Compound 46: 1H NMR (300 MHz,
CDCl3)
d
: 4.58 (1H, d, J ¼ 8.7 Hz, H-17a), 4.42 (1H, d, J ¼ 8.7 Hz, H-
17b), 3.63 (3H, s, H-21), 2.28 (1H, d, J ¼ 15.2 Hz, H-15a), 2.16 (1H, d,
J ¼ 15 Hz, H-14a), 2.14 (1H, s, H-13), 1.16 (3H, s, H-18), 0.81 (3H, s, H-
20), 2.05e0.74 (18H, m). 13C NMR (75 MHz, CDCl3)
d: 177.7 (s, C-19),
98.9 (s, C-16), 69.7 (t, C-17), 56.5 (d, C-5), 54.7 (d, C-9), 53.1 (t, C-15),
51.0 (q, C-21), 44.9 (d, C-13), 44.6 (s, C-8), 43.5 (s, C-4), 40.7 (t, C-7),
40.3 (t, C-1), 39.1 (s, C-10), 37.8 (t, C-3), 37.7 (t, C-14), 28.4 (q, C-18),
26.8 (t, C-12), 21.7 (t, C-6), 18.8 (t, C-2), 18.1 (t, C-11), 15.2 (q, C-20).
EIMS m/z (%): 396 ([M]þ, 9); 364 (14); 337 (100); 336 (35); 315 (67);
314 (37); 273 (60); 255 (59); 147 (34); 135 (23); 123 (67); 121 (90);
109 (59); 107 (50); 105 (30); 95 (35); 93 (37); 91 (42); 81 (41); 79
(39); 67 (33); 55 (39). HREIMS: 396.1966 (calcd for C21H32O5S [M]þ
5.1.14. Epoxidation of compound 17 to obtain compounds 50 and 51
To 70.7 mg of compound 17 (0.225 mmol) dissolved in 15 ml of
dry DCM were added 222.6 mg (4 equiv) of MCPBA and 153.3 mg of
NaHCO3 (8.1 equiv). The reaction mixture was left to room
temperature for 4 h, then the organic phase was separated and
treated with a saturated solution of sodium thiosulfate. The organic

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1303
phase was separated again and dried over anhydrous magnesium
sulfate, the mixture was filtered, the solvent was removed under
reduced pressure and the crude was purified by preparative-TLC
using hexanes/AcOEt 20% to yield 9.4 mg of compound 50 (12%)
and 11.6 mg of compound 51 (15%) as amorphous white solids.
amorphous white solids. Compound 53: 1H NMR (300 MHz, CDCl3)
: 7.35 (5H, m, H-22eH-26), 5.14 (1H, d, J ¼ 12.4 Hz, H-20a), 5.07
(1H, d, J ¼ 12.4 Hz, H-20b), 2.37 (1H, s, H-13), 1.21 (3H, s, H-17), 0.82
(3H, s, H-19), 2.37e0.76 (21H, m). 13C NMR (75 MHz, CDCl3)
: 222.6
d
d
(s, C-16), 176.9 (s, C-18), 135.9 (s, C-21), 128.3 (d, C-23, C-25), 128.0
(d, C-22, C-26),127.9 (d, C-24), 68.8 (t, C-20), 56.7 (d, C-5), 54.7 (t, C-
15), 53.7 (d, C-9), 47.5 (d, C-13), 43.7 (s, C-8), 42.2 (s, C-4), 40.8 (t,
C-7), 40.4 (t, C-1), 39.4 (s, C-10), 37.7 (t, C-3), 36.9 (t, C-14), 29.2
(t, C-12), 28.6 (q, C-17), 20.6 (t, C-6), 18.8 (t, C-2), 18.5 (t, C-11), 15.8
(q, C-19). IEMS m/z (%): 394 ([M]þ, 14); 303 (30); 285 (11); 259 (15);
257 (22); 149 (15); 121 (11); 109 (12); 107 (13); 95 (12); 93 (11); 91
(100); 81 (14); 79 (12); 55 (13). HREIMS: 394.2522 (calcd for
Compound 50: 1H NMR (300 MHz, CDCl3)
d: 3.63 (3H, s, H-21), 3.08
(1H, dd, J ¼ 3.7, 9.8 Hz, H-11), 2.83 (1H, d, J ¼ 5.4 Hz, H-17a), 2.79
(1H, d, J ¼ 5.2 Hz, H-17b), 1.19 (3H, s, H-18), 0.65 (3H, s, H-20),
2.27e0.85 (18H, m). 13C NMR (100 MHz, CDCl3)
d: 177.7 (s, C-19),
69.1 (s, C-9), 66.0 (s, C-16), 54.7 (t, C-17), 53.9 (d, C-11), 51.1 (q, C-
21), 48.5 (d, C-5), 46.0 (t, C-15), 44.0 (s, C-4), 42.1 (s, C-8), 39.3 (t,
C-1), 39.0 (t, C-3), 37.9 (s, C-10), 37.6 (t, C-14), 36.0 (d, C-13), 31.9
(t, C-12), 28.4 (q, C-18), 26.6 (t, C-7), 19.6 (t, C-2), 18.9 (t, C-6), 14.6
(q, C-20). EIMS m/z (%): 346 ([M]þ, 10); 330 (8); 287 (19); 286 (13);
258 (19); 257 (12); 173 (23); 150 (26); 149 (100); 147 (27); 136 (70);
135 (57); 133 (27); 123 (71); 122 (74); 121 (60); 109 (49); 107 (65);
105 (53); 95 (39); 93 (46); 91 (55); 81 (41); 79 (42); 67 (32); 55 (56).
C26H34O3 [M]þ 394.2508). IR nmax: 2947, 2872, 1723, 1455, 1232,
20
1146, 752, 699 cmꢀ1. UV (EtOH) lmax (log
e
): 202.0 (3.53) nm. [
a
]
D
:
:
ꢀ40.19 (c 1.1, CHCl3). Compound 54: 1H NMR (300 MHz, CDCl3)
d
7.36 (5H, m, H-22eH-26), 5.14 (1H, d, J ¼ 12.4 Hz, H-20a), 5.06 (1H,
d, J ¼ 12.4 Hz, H-20b), 4.76 (1H, sa, H-13), 2.25 (2H, m, H-15), 1.21
(3H, s, H-17), 0.76 (3H, s, H-19), 2.27e0.80 (18H, m). 13C NMR
HREIMS: 346.2137 (calcd for C21H30O4 [M]þ 346.2144). IR nmax
:
2929, 2855, 1722, 1458, 1226, 1148, 1036, 983, 893, 757 cmꢀ1. UV
(75 MHz, CDCl3) d: 176.8 (s, C-18), 172.1 (s, C-16), 135.7 (s, C-21),
(EtOH) lmax (log
e): 201.2 (3.10) nm. [
a
]
20: ꢀ0.64 (c 0.47, CHCl3).
128.3 (d, C-23, C-25), 128.0 (d, C-22, C-26), 127.9 (d, C-24), 75.6 (d,
C-13), 65.9 (t, C-20), 56.6 (d, C-5), 52.8 (d, C-9), 47.9 (t, C-15), 43.6 (s,
C-4), 42.8 (t, C-7), 40.9 (t, C-1), 39.2 (s, C-10), 37.5 (t, C-3), 33.6 (s, C-
8), 32.8 (t, C-14), 28.6 (q, C-17), 28.5 (t, C-12), 19.8 (t, C-6), 18.9 (t, C-
2), 16.1 (t, C-11), 16.0 (q, C-19). IEMS m/z (%): 410 ([M]þ, 4); 319 (16);
273 (17); 259 (11); 121 (8); 107 (10); 91 (100); 81 (11); 55 (10).
D
Compound 51: 1H NMR (300 MHz, CDCl3)
d: 3.64 (3H, s, H-21), 3.07
(1H, d, J ¼ 3.4 Hz, H-11), 2.85 (1H, d, J ¼ 4.5 Hz, H-17a), 2.80 (1H, d,
J ¼ 4.5 Hz, H-17b), 2.23 (1H, d, J ¼ 13.5 Hz, H-15a), 1.21 (3H, s, H-18),
0.67 (3H, s, H-20), 2.10e0.98 (17H, m). 13C NMR (75 MHz, CDCl3)
d:
177.7 (s, C-19), 69.6 (s, C-9), 68.8 (s, C-16), 53.5 (d, C-11), 51.1 (q, C-
21), 50.5 (t, C-17), 48.5 (d, C-5), 45.4 (t, C-14), 44.1 (s, C-4), 43.2 (s,
C-8), 39.3 (t, C-1), 38.7 (t, C-3), 38.7 (d, C-13), 37.9 (s, C-10), 37.7
(t, C-12), 29.5 (t, C-7), 28.9 (t, C-15), 28.4 (q, C-18), 19.6 (t, C-2), 18.8
(t, C-6), 14.8 (q, C-20). EIMS m/z (%): 346 ([M]þ, 49); 328 (35); 317
(37); 288 (72); 269 (32); 257 (35); 253 (53); 225 (40); 211 (57); 173
(71); 159 (45); 149 (79); 147 (52); 145 (51); 135 (57); 133 (54); 129
(43); 121 (86); 118 (78); 109 (61); 107 (84); 105 (96); 95 (53); 93
(60); 91 (100); 81 (60); 79 (67); 67 (48); 55 (81). HREIMS: 346.2144
(calcd for C21H30O4 [M]þ 346.2144). IR nmax: 2926, 2855, 1722, 1459,
HREIMS: 410.2458 (calcd for C26H34O4 [M]þ 410.2457). IR nmax
:
2951, 2930, 1725, 1458, 1227, 1143, 996, 697 cmꢀ1. UV (EtOH) lmax
(log
e): 207.6 (3.43) nm. [
a]
20: ꢀ61.00 (c 0.4, CHCl3). Compound 55:
D
1H NMR (300 MHz, CDCl3)
d
: 7.34 (5H, m, H-22eH-26), 5.15 (1H, d,
J ¼ 12.4 Hz, H-20a), 5.04 (1H, d, J ¼ 12.4 Hz, H-20b), 2.72 (1H, d,
J ¼ 13.0 Hz, H-15a), 2.18 (1H, d, J ¼ 13.0 Hz, H-15b), 1.20 (3H, s, H-
17), 0.83 (3H, s, H-19), 2.54e0.65 (21H, m). 13C NMR (75 MHz,
CDCl3) d: 177.2 (s, C-18), 176.9 (s, C-16), 135.9 (s, C-21), 128.2 (d, C-
23, C-25),127.9 (d, C-22, C-26),127.8 (d, C-24), 65.8 (t, C-20), 56.9 (d,
C-5), 50.9 (d, C-9), 44.9 (t, C-15), 43.6 (s, C-4), 41.8 (t, C-7), 40.7 (t, C-
1), 39.5 (s, C-10), 37.6 (t, C-3), 36.7 (s, C-8), 30.3 (t, C-12, C-14), 28.7
(q, C-17), 21.8 (t, C-6 ó C-13), 20.3 (t, C-6 ó C-13), 19.7 (t, C-2), 19.1 (t,
C-11), 18.1 (q, C-19). EIMS m/z (%): 412 ([M]þ, 1); 394 (2); 352 (21);
303 (10); 277 (16); 275 (21); 261 (14); 135 (10); 121 (10); 109 (14);
107 (15); 95 (13); 91 (100); 81 (14); 55 (8). HREIMS: 412.2628 (calcd
1383, 1226, 1147, 1038, 981, 775 cmꢀ1. UV (EtOH) lmax (log
e): 201.0
(2.95) nm. [
a]
20: ꢀ6.92 (c 0.5, CHCl3).
D
5.1.15. Preparation of compound 52
A solution of 24.4 mg of compound 2 (0.077 mmol) in 30 ml of
dry CH2Cl2 at ꢀ78 ꢁC was ozonized until the color of the solution
changed to dark blue-gray (30 s). The reaction mixture was then
for C26H36O4 [M]þ 412.2614). IR nmax: 2934, 2870, 1721, 1461, 1225,
20
quenched with 10
ml of dry Me2S (1.76 equiv) and concentrated
1140, 698 cmꢀ1. UV (EtOH) lmax (log
ꢀ40.19 (c 1.1, CHCl3).
e): 205.4 (3.52) nm. [a] :
D
under vacuum. The residue was purified by preparative-TLC using
hexanes/AcOEt (30%) to afford 4.3 mg of compound 52 (18%) as an
amorphous white solid. 1H NMR (300 MHz, CDCl3)
d
: 3.66 (3H, s, H-
20), 2.39 (1H, s, H-13), 1.16 (3H, s, H-17), 0.88 (3H, s, H-19),
2.30e0.82 (20H, m). 13C NMR (75 MHz, CDCl3)
: 222.5 (s, C-16),
5.1.17. Preparation of compounds 56 and 57
A solution of 44.0 mg of compound 10 (0.133 mmol) in 30 ml of
d
dry CH2Cl2 at ꢀ78ꢁ C was ozonized until the color of the solution
177.7 (s, C-18), 56.5 (d, C-5), 54.7 (t, C-15), 53.7 (d, C-9), 51.0 (q, C-
20), 47.5 (d, C-13), 43.5 (s, C-4), 42.2 (s, C-8), 40.8 (t, C-7), 40.4 (t,
C-1), 39.3 (s, C-10), 37.7 (t, C-3), 37.0 (t, C-14), 29.3 (t, C-12), 28.5 (q,
C-17), 20.5 (t, C-6), 18.8 (t, C-2), 18.5 (t, C-11), 15.7 (q, C-19). EIMS
m/z (%): 318 ([M]þ, 26); 300 (4); 286 (16); 259 (100); 149 (12); 133
(10); 123 (17); 121 (23); 109 (23); 107 (19); 95 (15); 93 (12); 79 (15);
67 (12); 55 (15). HREIMS: 318.2185 (calcd for C20H30O3 [M]þ
changed to dark blue-gray (2 min). The reaction mixture was then
quenched with 20 ml of dry Me2S (2.0 equiv) and concentrated
under vacuum. The residue was purified by preparative-TLC using
hexanes/AcOEt (15%) to afford 16.8 mg of compound 56 (38%) and
3.2 mg of compound 57 (7%) as amorphous white solids. Compound
56: 1H NMR (300 MHz, CDCl3)
d
: 3.65 (3H, s, H-20), 3.63 (1H, s, H-
15), 3.35 (1H, d, J ¼ 1.92 Hz, H-13), 1.20 (3H, s, H-17), 0.90 (3H, s, H-
19), 2.52e0.82 (19H, m). 13C NMR (75 MHz, CDCl3)
: 221.8 (s, C-16),
318.2195). IR nmax: 2945, 2870, 1726, 1463, 1235, 1192, 1150 cmꢀ1
.
d
[
a]
20: ꢀ67.07 (c 0.4, CHCl3).
177.7 (s, C-18), 80.8 (d, C-15), 56.5 (d, C-5), 53.5 (d, C-9), 51.0 (q,
C-20), 46.4 (d, C-13), 44.7 (s, C-8), 43.5 (s, C-4), 40.4 (t, C-1), 39.4
(s, C-10), 37.7 (t, C-3), 33.9 (t, C-7), 33.6 (t, C-14), 29.4 (t, C-12), 28.5
(q, C-17), 19.7 (t, C-6), 18.8 (t, C-2), 18.8 (t, C-11), 16.0 (q, C-19). EIMS
m/z (%): 334 ([M]þ, 96); 319 (10); 302 (18); 291 (15); 274 (100); 259
(23); 247 (17); 215 (16); 175 (11); 147 (17); 135 (22); 123 (47); 121
(82); 109 (55); 107 (53); 95 (37); 93 (35); 81 (44); 79 (35); 55 (44);
53 (12). HREIMS: 334.2153 (calcd for C20H30O4 [M]þ 334.2144). IR
D
5.1.16. Preparation of compounds 53, 54 and 55
A solution of 19.7 mg of compound 8 (0.050 mmol) in 30 ml of
dry CH2Cl2 at ꢀ78ꢁ C was ozonized until the color of the solution
changed to dark blue-gray (30 s). The reaction mixture was then
quenched with 10 ml of dry Me2S (2.7 equiv) and concentrated
under vacuum. The residue was purified by preparative-TLC using
hexanes/AcOEt (20%) to afford 10.6 mg of compound 53 (54%),
4.4 mg of compound 54 (21%) and 4.2 mg (20%) of compound 55 as
nmax: 3467, 2934, 2854, 1725, 1464, 1234, 1155, 1002, 736 cmꢀ1
.
[a
]
20: ꢀ23.91 (c 0.2, CHCl3). Compound 57: 1H NMR (300 MHz,
D

1304
I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
CDCl3)
d
: 5.08 (1H, s, H-17a), 5.02 (1H, s, H-17b), 3.66 (1H, s, OH),
9.8 mg of compound 61 (12%), 9.5 mg (20%) of compound 62 and
9.3 mg of compound 63 as yellow oils. Compound 60: 1H NMR
3.64 (3H, s, H-21), 3.64 (1H, s, H-15), 1.18 (3H, s, H-18), 0.84 (3H, s,
H-20), 2.20e0.87 (19H, m). 13C NMR (75 MHz, CDCl3)
d: 177.9 (s, C-
(300 MHz, CDCl3)
d
: 3.74 (1H, dd, J ¼ 4.0, 14.2 Hz, H-20a), 3.59 (1H,
19), 111.5 (s, C-16), 93.9 (t, C-17), 93.7 (d, C-15), 56.6 (d, C-5), 52.6 (d,
C-9), 51.0 (q, C-21), 45.7 (s, C-8), 43.5 (s, C-4), 42.1 (d, C-13), 40.3 (t,
C-1), 39.2 (s, C-10), 37.8 (t, C-3), 34.8 (t, C-7), 34.5 (t, C-12), 28.5 (q,
C-18), 25.7 (t, C-14), 20.4 (t, C-6), 18.8 (t, C-2), 17.9 (t, C-11), 15.2 (q,
C-20). EIMS m/z (%): 364 ([M]þ, 1); 334 (84); 274 (97); 259 (29); 247
(19); 149 (22); 133 (29); 123 (57); 121 (100); 109 (66); 107 (66); 105
(34); 95 (48); 93 (45); 91 (48); 81 (56); 79 (46); 67 (43); 55 (62).
dd, J ¼ 4.0, 14.2 Hz, H-20b), 2.39 (1H, s, H-13), 1.17 (3H, s, H-17), 0.86
(3H, s, H-19), 0.10 (9H, s, Si(CH3)3), 2.28e0.80 (20H, m). 13C NMR
(75 MHz, CDCl3) d: 222.5 (s, C-16), 178.1 (s, C-18), 56.8 (t, C-20), 56.7
(d, C-5), 54.7 (t, C-15), 53.6 (d, C-9), 47.5 (d, C-13), 43.8 (s, C-8), 42.2
(s, C-4), 40.8 (t, C-7), 40.5 (t, C-1), 39.3 (s, C-10), 37.8 (t, C-3), 37.0 (t,
C-14), 29.2 (t, C-12), 28.7 (q, C-17), 20.7 (t, C-6), 18.8 (t, C-2), 18.5 (t,
C-11),15.7 (q, C-19), ꢀ3.1 (q, Si(CH3)3). EIMS m/z (%): 390 ([M]þ, 47);
375 (100); 287 (15); 259 (82); 199 (19); 163 (20); 123 (33); 121 (24);
109 (34); 107 (32); 105 (21); 97 (34); 95 (41); 93 (33); 91 (32); 81
(52); 79 (38); 73 (61); 67 (35); 59 (49); 55 (59). HREIMS: 390.2586
(calcd for C23H38O3Si [M]þ 390.2590). IR nmax: 2951, 2871, 1741,
HREIMS: 364.2252 (calcd for C21H32O5 [M]þ 364.2250). IR nmax
:
3439, 2943, 2854, 1724, 1464, 1234, 1159, 1097, 1064, 1002,
977 cmꢀ1. UV (EtOH) lmax (log
e
): 202.0 (3.23) nm. [
a
]
20: ꢀ24.48
D
(c 0.3, CHCl3).
1720, 1249, 1147, 858 cmꢀ1. UV (EtOH) lmax (log
e
): 201.2 (2.75) nm.
5.1.18. Preparation of compound 58
[a
]
20: ꢀ44.06 (c 1.0, CHCl3).
D
A solution of 40.0 mg of compound 12 (0.121 mmol) in dry
CH2Cl2 at ꢀ78ꢁ C was ozonized until the color of the solution
changed to dark blue-gray (2 h). The reaction mixture was then
Compound 61: 1H NMR (300 MHz, CDCl3)
d: 4.79 (1H, s, H-13),
3.76 (1H, d, J ¼ 14 Hz, H-20a), 3.59 (1H, d, J ¼ 14 Hz, H-20b), 1.18
(3H, s, H-17), 0.84 (3H, s, H-19), 0.10 (9H, s, Si(CH3)3), 2.24e0.84
quenched with 20
ml of dry Me2S (2.2 equiv) and concentrated
(20H, m). 13C NMR (75 MHz, CDCl3)
d: 178.0 (s, C-18), 172.1 (s, C-16),
under vacuum. The residue was purified by preparative-TLC using
hexanes/AcOEt (40%) to afford 16.3 mg of compound 58 (39%) as an
75.6 (d, C-13), 56.9 (t, C-20), 56.6 (d, C-5), 52.9 (d, C-9), 47.9 (t, C-15),
43.6 (s, C-4), 42.8 (t, C-7), 40.9 (t, C-1), 39.2 (s, C-10), 37.6 (t, C-3),
33.7 (s, C-8), 32.8 (t, C-14), 28.8 (q, C-17), 28.8 (t, C-12), 19.9 (t, C-6),
18.9 (t, C-2), 16.2 (t, C-11), 16.0 (q, C-19), ꢀ3.1 (q, Si(CH3)3). EIMS m/z
(%): 406 ([M]þ, 27); 391 (100); 303 (36); 275 (45); 215 (27); 193
(22); 121 (20); 107 (22); 95 (31); 81 (24); 73 (33); 59 (23); 55 (26).
amorphous white solid. 1H NMR (300 MHz, CDCl3)
d
: 3.65 (3H, s, H-
20), 2.75 (1H, s, H-13), 1.16 (3H, s, H-17), 0.86 (3H, s, H-19),
2.43e0.77 (18H, m). 13C NMR (75 MHz, CDCl3)
: 184.7 (s, C-15),
d
182.3 (s, C-16), 177.8 (s, C-18), 55.6 (d, C-5), 51.1 (q, C-20), 44.9 (d, C-
9), 44.1 (s, C-8), 43.4 (s, C-4), 41.4 (t, C-7), 39.1 (t, C-1), 38.6 (s, C-10),
37.6 (t, C-3), 36.9 (d, C-13), 31.3 (t, C-14), 28.5 (q, C-17), 21.8 (t, C-12),
19.6 (t, C-6), 19.0 (t, C-2), 18.4 (t, C-11), 17.5 (q, C-19). EIMS m/z (%):
348 ([M]þ, 15); 330 (6); 276 (36); 261 (38); 217 (60); 180 (34); 135
(33); 121 (100); 109 (60); 107 (50); 93 (43); 91 (41); 81 (42); 79
(59); 67 (39); 55 (52). HREIMS: 348.1925 (calcd for C20H28O5 [M]þ
HREIMS: 406.2556 (calcd for C23H38O4Si [M]þ 406.2539). IR nmax
:
2954, 2874, 1724, 1223, 1146, 858 cmꢀ1. UV (EtOH) lmax (log
e):
201.4 (2.79) nm. [
NMR (300 MHz, CDCl3)
a
]
20: ꢀ58.37 (c 0.9, CHCl3). Compound 62: 1H
D
d
: 3.76 (1H, d, J ¼ 14 Hz, H-20a), 3.76 (1H, d,
J ¼ 14 Hz, H-20b), 2.75 (1H, d, J ¼ 12.8 Hz, H-15a), 2.17 (1H, d,
J ¼ 12.8 Hz, H-15b), 1.17 (3H, s, H-17), 0.87 (3H, s, H-19), 0.10 (9H, s,
348.1937). IR nmax: 2948, 2875, 1712, 1457, 1239, 1151, 755 cmꢀ1
.
Si(CH3)3), 2.22e0.69 (19H, m). 13C NMR (75 MHz, CDCl3)
d: 178.4 (s,
[
a]
20: ꢀ21.43 (c 0.6, CHCl3).
C-16), 177.6 (s, C-18), 56.8 (d, C-5), 56.8 (t, C-20), 51.0 (d, C-9), 45.0
(t, C-15), 43.7 (s, C-4), 41.9 (t, C-7), 40.7 (t, C-1), 39.5 (s, C-10), 37.8 (t,
C-3), 36.8 (s, C-8), 30.3 (t, C-14, C-12), 28.8 (q, C-17), 21.9 (t, C-6 or C-
13), 20.3 (t, C-6 or C-13), 19.8 (t, C-2), 19.1 (t, C-11), 18.1 (q, C-19),
ꢀ3.1 (q, Si(CH3)3). EIMS m/z (%): 408 ([M]þ, 9); 393 (51); 349 (41);
305 (19); 277 (100); 245 (21); 217 (79); 173 (14); 147 (21); 135 (40);
123 (27); 121 (33); 109 (52); 107 (28); 95 (38); 93 (24); 81 (37); 79
(20); 73 (36); 67 (22); 59 (32); 55 (28). HREIMS: 408.2679 (calcd for
D
5.1.19. Preparation of compound 59
A solution of 32.5 mg of compound 30 (0.087 mmol) in dry
CH2Cl2 at ꢀ78ꢁ C was ozonized until the colour of the solution
changed to dark blue-gray (2 h). The reaction mixture was then
quenched with 15 ml of dry Me2S (2.3 equiv) and concentrated
under vacuum. The residue was purified by preparative-TLC using
Hexanes/AcOEt (20%) to afford 11.1 mg of compound 59 (34%) as an
C23H40O4Si [M]þ 408.2696). IR nmax: 2952, 2872, 1703, 1250, 1141,
20
amorphous white solid. 1H NMR (300 MHz, CDCl3)
d: 4.80 (1H, d,
858, 757 cmꢀ1. UV (EtOH) lmax (log
e
): 201.8 (2.89) nm. [
a]
:
:
D
J ¼ 1.89 Hz, H-15), 3.65 (3H, s, H-20), 2.56 (1H, s, H-13), 2.10 (3H, s,
ꢀ34.11 (c 0.9, CHCl3). Compound 63: 1H NMR (300 MHz, CDCl3)
d
OCOCH3), 1.17 (3H, s, H-17), 0.89 (3H, s, H-19), 2.27e0.82 (18H, m).
3.75 (1H, d, J ¼ 14.2 Hz, H-20a), 3.57 (1H, d, J ¼ 14.2 Hz, H-20b), 3.10
13C NMR (75 MHz, CDCl3)
d
: 217.5 (s, C-16), 177.6 (s, C-18), 169.9
(1H, s, H-13), 1.15 (3H, s, H-17), 0.83 (3H, s, H-19), 0.10 (9H, s, Si
(s, OCOCH3), 80.3 (d, C-15), 56.2 (d, C-5), 53.1 (d, C-9), 51.1 (q, C-20),
46.8 (d, C-13), 44.5 (s, C-8), 43.5 (s, C-4), 40.2 (t, C-1), 39.4 (s, C-10),
37.6 (t, C-3), 34.6 (t, C-7), 33.8 (t, C-14), 29.4 (t, C-12), 28.4 (q, C-17),
20.4 (q, OCOCH3), 19.6 (t, C-6), 18.7 (t, C-2, C-11), 15.9 (q, C-19). EIMS
m/z (%): 376 ([M]þ, 51); 361 (29); 335 (21); 334 (100); 317 (29); 316
(43); 275 (42); 274 (65); 257 (36); 256 (27); 123 (26); 121 (67); 109
(34); 107 (34); 105 (22); 95 (29); 93 (25); 91 (26); 81 (34); 79 (28);
67 (24); 55 (32). HREIMS 376.2235 (calcd for C22H32O5 [M]þ
(CH3)3), 2.32e0.79 (20H, m). 13C NMR (75 MHz, CDCl3)
d: 178.1 (s, C-
18), 117.5 (s, C-16), 56.7 (t, C-20), 56.5 (d, C-13), 54.9 (d, C-5), 50.4 (t,
C-15), 43.8 (s, C-4), 42.8 (s, C-8), 41.0 (t, C-7), 40.4 (t, C-1), 39.5 (s, C-
10), 39.3 (d, C-9), 37.9 (t, C-3), 36.9 (t, C-14), 28.7 (q, C-17), 25.6 (t, C-
12), 21.5 (t, C-6), 18.8 (t, C-2), 17.8 (t, C-11), 15.1 (q, C-19), ꢀ3.1 (q, Si
(CH3)3). EIMS m/z (%): 406 ([M]þ, 8); 391 (76); 375 (35); 347 (40);
303 (20); 275 (44); 259 (33); 231 (21); 215 (60); 199 (30); 157 (30);
149 (29); 135 (54); 133 (41); 123 (46); 121 (74); 109 (62); 107 (73);
105 (39); 95 (67); 93 (55); 91 (50); 81 (87); 79 (51); 73 (100); 67
(45); 59 (67); 55 (63). HREIMS: 406.2545 (calcd for C23H38O4Si [M]þ
406.2539). IR nmax: 2950, 2850, 1721, 1250, 1154, 857, 757 cmꢀ1. UV
376.2250). IR nmax: 3487, 2947, 2870, 1754, 1725, 1464, 1371, 1232,
1155, 1023, 756 cmꢀ1. UV (EtOH) lmax (log
ꢀ11.39 (c 1.1, CHCl3).
e): 200.6 (2.55) nm. [a] :
20
D
(EtOH) lmax (log
e
): 201.4 (3.04) nm. [
a
]
20: ꢀ63.23 (c 0.9, CHCl3).
D
5.1.20. Preparation of compounds 60e63
A solution of 77.3 mg of compound 35 (0.199 mmol) in 25 ml of
dry CH2Cl2 at ꢀ78ꢁ C was ozonized until the colour of the solution
changed to dark blue-gray (30 s). The reaction mixture was then
5.2. Cell culture
quenched with 30
ml of dry Me2S (2 equiv) and concentrated under
The murine macrophage cell line RAW 264.7 was maintained in
vacuum. The residue was purified by preparative-TLC using
hexanes/AcOEt (20%) to afford 22.3 mg of compound 60 (29%),
RPMI 1640 medium supplemented with 10% FCS,
antibiotics, as previously described [29].
L-glutamine and

I. Hueso-Falcón et al. / European Journal of Medicinal Chemistry 46 (2011) 1291e1305
1305
5.3. Determination of NO synthesis
5.9. Statistical analysis
NO release was determined with Griess reagent as previously
described [30]. Briefly, NO release was determined spectrophoto-
metrically by the accumulation of nitrite in the medium. The
absorbance at 548 nm was compared with a standard of NaNO2.
The data shown are the mean ꢂ S.D. Statistical significance was
estimated with Student’s t test for unpaired observations using the
InStart program (GraphPad Software). Values of *p < 0.05 were
considered significant.
5.4. MTT assay for cell viability
Acknowledgments
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 concentra-
tions 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 sulfoxide (DMSO) and the absorbance was read at 540 nm.
Assays were performed in triplicate, and results are expressed as the
percent reduction in cell viability compared to untreated control
cultures for at least three independent experiments.
This work was supported by grants PI08.0070 from FIS, MPY
1410/09 from ISCIII and Fundación Mutua Madrileña to S.H., by
a Santander-Complutense grant to S.H. and B. de las H. We also
thank the financial support from the Spanish MICINN (SAF2009-
13296-C02-01) and ICIC (Instituto Canario de Investigación del
Cáncer) to A.E.B. IHF thanks MICINN for a predoctoral fellowship.
We thank Sandra Herranz for her excellent technical assistance.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.01.052.
5.5. Preparation of cytosolic and nuclear extracts
Cells were washed twice with ice-cold buffer A (10 mM Hepes,
pH 7.9; 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM DTT, 0.5 mM
References
PMSF, 2
mg/ml aprotinin, 10 mg/ml leupeptin, 2 mg/ml TLCK (Na-
[1] D.J. Berg, J. Zhang, J.V. Weinstock, H.F. Ismail, K.A. Earle, H. Alila, R. Pamukcu,
S. Moore, R.G. Lynch, Gastroenterology 123 (2002) 1527e1542.
[2] J.W. Rose, K.E. Hill, H.E. Watt, N.G. Carlson, J. Neuroimmunol. 149 (2004)
40e49.
[3] P.F. Gomez, M.H. Pillinger, M. Attur, N. Marjanovic, M. Dave, J. Park,
C.O. Bingham III, H. Al Mussawir, S.B. Abramson, J. Immunol. 175 (2005)
6924e6930.
[4] K.H. Kwon, A. Murakami, R. Hayashi, H. Ohigashi, Biochem. Biophys. Res.
Commun. 337 (2005) 647e654.
[5] P.A. Baeuerle, Cell 95 (1998) 729e731.
Tosyl- -lysine chloromethyl ketone hydrochloride), 5 mM NaF,
L
1 mM NaV04, 10 mM Na2MoO4) containing 120 mM NaCl and
scraped off the plate. Cells were lysed at 4 ꢁC with 0.2 ml of buffer A
supplemented with 0.5% Nonidet P-40 and under continuous
shaking. After centrifugation of the cell lysate the supernatant was
stored at ꢀ80 ꢁC (cytosolic extract) and the pellets were resus-
pended in 50 ml of buffer A supplemented with 20% glycerol-0.4 M
KCl and gently shaken for 30 min at 4 ꢁC. Protein content was
assayed using the Bio-Rad protein reagent. All cell fractionation
steps were carried out at 4 ꢁC.
[6] K. Brown, S. Gerstberger, L. Carlson, G. Franzoso, U. Siebenlist, Science 267
(1995) 1485e1488.
[7] M.A. O’Connell, B.L. Bennett, F. Mercurio, A.M. Manning, N. Mackman, J. Biol.
Chem. 273 (1998) 30410e30414.
[8] J. DiDonato, F. Mercurio, C. Rosette, J. Wu-Li, H. Suyang, S. Ghosh, M. Karin,
Mol. Cell Biol. 16 (1996) 1295e1304.
[9] I. Sanchez-Perez, S.A. Benitah, M. Martinez-Gomariz, J.C. Lacal, R. Perona, Mol.
Biol. Cell. 13 (2002) 2933e2945.
5.6. Western blot analysis
[10] Y. Kim, S.M. Fischer, J. Biol. Chem. 273 (1998) 27686e27694.
[11] S. Ghosh, M. Karin, Cell 109 (S81eS96) (2002).
[12] M. Karin, Nature 441 (2006) 431e436.
[13] M.L. Handel, Inflamm. Res. 46 (1997) 282e286.
[14] I. Tegeder, E. Niederberger, E. Israr, H. Guhring, K. Bruce, C. Euchenhofer,
S. Grosch, G. Geisslinger, FASEB J. 15 (2001) 2e3.
Western blot of cell extracts were prepared and incubated with
anti-NOS-2, anti-I
Blots were developed by ECL according to the manufacturer’s
instructions (GE Healthcare). -Actin was used as a loading control.
kBa and anti-b-actin (Santa Cruz Biotechnology).
b
[15] H.M. Gao, J.S. Hong, Trends Immunol. 29 (2008) 357e365.
[16] A. Klegeris, P.L. McGeer, Curr. Alzheimer Res. 2 (2005) 355e365.
[17] I. Hueso-Falcon, N. Giron, P. Velasco, J.M. Amaro-Luis, A.G. Ravelo, B. de las
Heras, S. Hortelano, A. Estevez-Braun, Bioorg. Med. Chem. 18 (2010)
1724e1735.
[18] K. Schank, H. Beck, S. Pistorius, Helv. Chim. Acta 87 (2004) 2025e2049.
[19] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok,
S.R. Tannenbaum, Anal. Biochem. 126 (1982) 131e138.
[20] S. Uwe, Biochem. Pharmacol. 75 (2008) 1567e1579.
[21] B. de las Heras, S. Hortelano, Inflamm. Allergy Drug Targets 8 (2009) 28e39.
[22] A. Castrillo, B. de las Heras, S. Hortelano, B. Rodriguez, A. Villar, L. Bosca, J. Biol.
Chem. 276 (2001) 15854e15860.
[23] B.-C. Chen, C.-C. Liao, M.-J. Hsu, Y.-T. Liao, C.-C. Lin, J.-R. Sheu, C.-H. Lin,
J. Immunol. 177 (2006) 681e693.
[24] R.D. Torrenegra, A. Tellez, N. Alba, G. Garcia, Rev. Colomb. Quim. 23 (1994)
29e35.
[25] A. Usubillaga, J. De Hernandez, N. Perez, M. Kiriakidis, Phytochemistry 12
(1973) 2999.
5.7. RNA analysis and quantitative PCR
Total RNA was isolated from cells using TRIzol reagent (Invi-
trogen) according to the manufacturer’s instruction. Quantitative
PCR (SYBRgreen) analysis was performed with an ABI 7700
sequence detector as described [30]. Each sample was run in
duplicate, and all samples were analyzed in parallel for the
expression of the housekeeping gene 36B4 (acidic ribosomal
phosphoprotein P0), used as an endogenous control to normalize
the expression level of target genes. Fold induction was determined
from average replicate values. Primer sequences are available on
request.
[26] A. Usubillaga, A. Morales, Phytochemistry 11 (1972) 1856e1857.
[27] J. Cuatrecasas, Phytologia 35 (1976) 43e61.
5.8. Assay of cytokines
[28] A. Usubillaga, M. Romero, R. Aparicio, Acta Hortic. 597 (2003) 129e130.
[29] N. Girón, P.G. Traves, B. Rodriguez, R. Lopez-Fontal, L. Bosca, S. Hortelano, B. de
las Heras, Toxicol. Appl. Pharmacol. 228 (2008) 179e189.
[30] M. Zeini, P.G. Traves, R. Lopez-Fontal, C. Pantoja, A. Matheu, M. Serrano,
L. Bosca, S. Hortelano, J. Immunol. 177 (2006) 3327e3336.
Cytokine production in the cell culture was measured with
ELISA kits from R&D systems according to the manufacturer’s
instructions.