Cytotoxic, immunomodulatory, antimycotic, and antiviral activities of semisynthetic 14-hydroxyabietane derivatives and triptoquinone C-4 epimers

Article abstract of DOI:10.1039/c3md00151b

A series of C14-hydroxy derivatives of dehydroabietic acid were synthesised from commercial abietic acid and evaluated for their cytotoxic, antimycotic, and antiviral activities. From these C14-hydroxy derivatives, triptoquinone C-4 epimers were obtained and their immunomodulatory activity was additionally evaluated. None of the tested compounds showed antiviral activity against herpes simplex virus type 1 (HHV-1), and nor did they display antimycotic activity against certain Aspergillus, spp. except for one compound, abieta-8,11,13-trien- 14,18-diol. Interestingly, two triptoquinone epimers showed cytotoxic activity, and one of them induced mitochondrial potential loss, DNA damage and cell cycle distribution alterations in Jurkat cells, but not in human peripheral blood mononuclear cells. In addition, these compounds inhibited monocyte's differentiation and production of pro-inflammatory cytokines, IL-1β and TNF-α, and the anti-inflammatory cytokine IL-10 in the presence of LPS. In conclusion, one of the triptoquinone molecules could be a promising scaffold for the development of novel anti-cancer agents, and two of them could be potential anti-inflammatory agents. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3md00151b

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Cytotoxic, immunomodulatory, antimycotic, and
antiviral activities of semisynthetic 14-hydroxyabietane
Cite this: DOI: 10.1039/c3md00151b
derivatives and triptoquinone C-4 epimers†
Bibiana Zapata,a Mauricio Rojas,b Liliana Betancur-Galvis,a Ana Cecilia Mesa-Arango,a
c
c
*
´
´
David Perez-Guaita and Miguel A. Gonzalez
A series of C14-hydroxy derivatives of dehydroabietic acid were synthesised from commercial abietic acid
and evaluated for their cytotoxic, antimycotic, and antiviral activities. From these C14-hydroxy derivatives,
triptoquinone C-4 epimers were obtained and their immunomodulatory activity was additionally
evaluated. None of the tested compounds showed antiviral activity against herpes simplex virus type 1
(HHV-1), and nor did they display antimycotic activity against certain Aspergillus, spp. except for one
compound, abieta-8,11,13-trien-14,18-diol. Interestingly, two triptoquinone epimers showed cytotoxic
activity, and one of them induced mitochondrial potential loss, DNA damage and cell cycle distribution
alterations in Jurkat cells, but not in human peripheral blood mononuclear cells. In addition, these
compounds inhibited monocyte's differentiation and production of pro-inflammatory cytokines, IL-1b
and TNF-a, and the anti-inflammatory cytokine IL-10 in the presence of LPS. In conclusion, one of the
triptoquinone molecules could be a promising scaffold for the development of novel anti-cancer agents,
and two of them could be potential anti-inflammatory agents.
Received 3rd June 2013
Accepted 1st July 2013
DOI: 10.1039/c3md00151b
www.rsc.org/medchemcomm
compounds, little effort has been made for their biological study,
especially the cytotoxicity of derivatives towards tumour and non-
1 Introduction
Natural abietane phenols and quinones, as well as other oxi-
dised related compounds, constitute an interesting group of
diterpene metabolites, due to the signicant biological activi-
ties exhibited by some of them. For example, ferruginol (1)
(Fig. 1) presents interesting biological activities, such as anti-
fungal and antitumor properties.1
tumour cells.
As a part of our research program towards the discovery of
bioactive terpene-based compounds, we were interested in the
biological study of some recently synthesised 14-hydrox-
yabietanes and their corresponding quinones obtained by
Another interesting phenol abietane diterpene is triptinine B
(2), which displays leukotriene D4 antagonism.2 Among the
abietane quinones, representative examples are the antitumor
and antimicrobial taxodione (3),3–5 the antileishmanial agent 12-
deoxyroyleanone (4),6 and the antifungal and cytotoxic crypto-
quinone (5).7 Other signicant quinones are a group of A-ring
functionalised diterpenoids such as triptoquinones A–F (6–11),
which are potent interleukin-1 inhibitors, and triptoquinone A,
which also suppresses inducible nitric oxide synthase mRNA
expression mediated by bacterial lipopolysaccharides.8–10
Despite the diverse biological properties of this family of
a
´
´
´
Grupo de Investigacion Dermatologica, Universidad de Antioquia, A.A1226, Medellın,
Colombia. E-mail: bibianazapata@gmail.com; Tel: +57 42196064
b
´
´
´
Grupo de Inmunologıa Celular e Inmunogenetica. Unidad de Citometrıa, Sede de
´
´
Investigacion Universitaria, Universidad de Antioquia, A.A1226, Medellın, Colombia
c
´
´
Departamento de Quımica Organica, Universidad de Valencia, E-46100 Burjassot,
Valencia, Spain. E-mail: miguel.a.gonzalez@uv.es; Fax: +34 963544328; Tel: +34
963543880
† Electronic supplementary information (ESI) available: Fig. 2–10 and
experimental details of the biological assays. See DOI: 10.1039/c3md00151b
Fig. 1 Bioactive oxidised abietane phenols and quinones.
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Scheme 1 Reagents and conditions: (a) LiOH, Me2SO4, DMF; (b) OsO4, Me3NO, py, t-BuOH; (c) PhSeSePh, t-BuOH, CCl4; (d) TsOH, toluene, reflux; (e) KOH, MeOH, H2O;
(f) LiAIH4 and THF; (g) H2O2, RuCl3, AcOH.
oxidation.11,12 These quinones represent C-4 epimers of natural The oxidation of the primary alcohol in 18 and 21 with Dess–
triptoquinones the biological activity of which has not yet been Martin periodinane failed to yield the corresponding aldehydes.
studied. Here, we describe the syntheses of a number of abie-
tane derivatives from commercially available (ꢀ)-abietic acid
2.2 Biological evaluation
(12),13 and the evaluation of their cytotoxic, antimycotic, anti-
2.2.1 Antiviral activity. The antiviral activity of 14-hydrox-
viral and immunomodulatory activities. In this study, an
yabietane (phenols) (16–18) and triptoquinone epimers (19–21)
oxygenated moiety (such as methyl ester, alcohol, or acid) was
against herpes simplex virus type 1 (HHV-1) was determined
introduced into the lipophilic abietane skeleton. Compound 16
using a modied end-point titration technique (EPPT) accord-
and ve derivatives (17–21) with different functional groups at
ing to the protocol reported by us.15 None of the tested
C14 and C18 were tested (Scheme 1).
compounds reduced the HHV-1 replication at the evaluated
concentrations (data not shown).
2.2.2 Antimycotic activity. Compounds 16–21 were tested
in vitro for antimycotic activity. The compounds did not show
2 Results
2.1 Chemistry
antimycotic activity against Aspergillus fumigatus, A. avus, A.
The synthesis of the C18-functionalised dehydroabietanes used niger, and A. terreus in concentrations below 100 mg mLꢀ1
,
in this work begins with the preparation of the key intermediate except for the 14-hydroxyabietane derivative 18, which showed a
14-hydroxydehydroabietic acid or related compounds from MIC value of 25 mg mLꢀ1 against A. fumigatus and A. terreus, and
commercial (ꢀ)-abietic acid as outlined in Scheme 1. Firstly, 50 mg mLꢀ1 against A. niger (data not shown); however, it
abietic acid 12 was esteried by treatment with lithium showed cytotoxic activity against Vero cells. The MIC value for
hydroxide and methyl sulfate to yield ester 13 in a quantitative the reference antifungal drug, amphotericin B (Sigma, New
yield.14 Then, regioselective dihydroxylation of 13 gave diol 14 in Jersey, USA), used as a positive control, was within the estab-
a 70% yield. Oxidation of the allylic alcohol in 14 with PhSeSePh lished values for the CLSI-M38-A protocol.
and t-BuOOH in CCl4 gave the hydroxy ketone 15 in a 75% yield.
2.2.3 Cytotoxic activity: MTT assay. We determined the
Subsequent aromatisation of 15 with TsOH in reuxing concentration for each compound that inhibited 50% of Jurkat,
toluene provided the desired phenol (methyl 14-hydroxydehy- Vero and HeLa cells growth (IC50) at 48 h through a microcul-
droabietate) 16 in an 80% yield.
ture tetrazolium (MTT) assay.
With phenol 16 in hand, we carried out the change of
All the tested compounds (16–21) produced a dose-depen-
functionalization at C-18 and, nally, the reaction of oxidation dent inhibition of the growth of the Jurkat and HeLa tumor cell
with H2O2, and RuCl3 as a catalyst, to yield the corresponding lines and the Vero cell line, with an R2 (coefficient of linear
quinones. Thus, the methyl ester group in 16 was saponied regression) > 0.8. The triptoquinone epimers 20 (C-4 epimer of
with KOH in aqueous methanol to yield acid 17. Reduction of triptoquinone F) and 21 (C-4 epimer of triptoquinone D)
ester 16 with LiAlH4 in dry tetrahydrofuran at reux gave alcohol showed the lowest IC50 values on the Jurkat tumor cell line
18. Finally, phenol oxidation of 16–18 with H2O2, in the pres- corresponding to 14.4 ꢁ 2.4 mg mLꢀ1 and 14.6 ꢁ 3.8 mg mLꢀ1
ence of cat. RuCl3, supplied quinones 19–21 in a moderate yield. respectively (Table 1).
,
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Table
1
IC50 values (mg mLꢀ1
) of 14-hydroxyabietane and triptoquinone
cells at concentrations of 7.0 mg mLꢀ1 and 14.0 mg mLꢀ1 aer
72 h of treatment. In addition, the inhibition of proliferation of
CD4 and CD8 cells was higher aer treatment with the tripto-
quinone epimer 21 at the concentration of 14.0 mg mLꢀ1, cor-
responding to 88% and 77% of inhibition, respectively (Fig. 2b,
see ESI†). The results indicate that the effect on proliferation
inhibition of triptoquinone epimers 20 and 21 was not specic
for either CD4 or CD8 cells.
In order to study whether the proliferation inhibition
induced by triptoquinone epimers 20 and 21 was due to inhi-
bition of activation induced by PHA, the PBMCs were treated
one hour aer or one hour before adding PHA and then eval-
uated for the expression of the early lymphocyte activation
marker CD69.
Both compounds 20 and 21 did not induce reduction in the
percentage of activated CD4 or CD8 cells stimulated one hour
before or aer treatment at concentrations of 1, 7.0 and 14.0 mg
mLꢀ1, as compared to control cells (data not shown). These
results suggest that both compounds inhibited neither prolif-
eration nor stimulation induced by PHA.
epimers 16–21 on Jurkat, HeLa and Vero cell lines
Cell linesc
Vero
IC50
Jurkat
HeLa
SIb
IC50
SI
a
Compound IC50
16
17
18
19
20
21
14.2 ꢁ 4.2 2.59
18.6 ꢁ 4.0
1.90
36.8 ꢁ 6.2
34.0 ꢁ 6.9 3.09 136.4 ꢁ 2.1
0.77 105.2 ꢁ 11
8.2 ꢁ 1.2 2.30
18.5 ꢁ 4.3 2.34
14.4 ꢁ 2.4 8.96
14.6 ꢁ 3.8 5.46
26.6 ꢁ 5.8
35.2 ꢁ 1.9
27.2 ꢁ 5.0
0.71
1.23
19.0 ꢁ 4.6
43.3 ꢁ 5.2
4.74 129.0 ꢁ 6.3
79.5 ꢁ 16.0 1.00
79.7 ꢁ 15.6
a
Concentration of compounds that induces 50% growth inhibition in
b
48 h. SI, selectivity index is dened as Vero IC50 over either Jurkat or
HeLa IC50
HeLa, human cervix epitheloid adenocarcinoma cells ATCC CRL-1958;
Vero, Cercopithecus aethiops African green monkey kidney cells ATCC
CCL-81.
c
.
Jurkat, human acute T cell leukemia ATCC TIB-152;
Compounds 16 and 18 showed the lowest IC50 values on the
2.2.5 Evaluation of mitochondrial potential and integrity
HeLa cell line, corresponding to 18.6 ꢁ 4.0 mg mLꢀ1 and 26.6 ꢁ of the membrane. Considering that one of the reported effects
5.8 mg mLꢀ1, respectively (Table 1). However, the selectivity of antitumor quinone-based compounds, such as 2-methyl-
index (SI) of these compounds on HeLa was <5. Compounds 20 naphtho[2,3-b]furan-4,9-dione, is the induction of a loss in the
and 21 showed the lowest IC50 values and the highest selectivity mitochondrial potential in tumor cell lines,16,17 the effect of
index on the Jurkat cell line; therefore, their possible cytotoxic triptoquinone epimers 20 and 21 on Jurkat cells and PBMC
mechanism towards the Jurkat tumor cell line as well as the mitochondrial potential was evaluated using (DiOC6), and also
selectivity of their mechanism using PBMC as a non-tumor cell the membrane integrity was evaluated using PI. The triptoqui-
line control were evaluated.
none epimer 21 induced a loss of the mitochondrial membrane
2.2.4 Lymphoproliferative responses evaluation on PBMCs. potential in Jurkat cells at a concentration of 14.0 mg mLꢀ1 aer
Our preliminary data showed that compounds 20 and 21 could 15 h and 24 h of treatment, corresponding to 15% and 25%,
exhibit anti-tumour activity against acute T cell leukemia respectively (Fig. 3, see ESI†); this effect was not observed at a
(Jurkat), but it is necessary to prove the effect on non-tumor concentration of 7.0 mg mLꢀ1. In contrast, the triptoquinone
human cells. Therefore, the lymphoproliferative activity of epimer 20 did not induce a loss of the mitochondrial membrane
compounds 20 and 21 on PBMCs has also been evaluated. potential in Jurkat cells or PBMCs at the evaluated concentra-
Moreover, we evaluated if the cytotoxic effect of compounds 20 tions (Fig. 3a, see ESI†). In PBMCs, the triptoquinone epimer 21
and 21 on Jurkat cells is through the inhibition of cellular induced a loss of the mitochondrial membrane potential at a
proliferation. Lymphocyte proliferation by serial halving of the concentration of 14.0 mg mLꢀ1 aer 15 h or 24 h of treatment
uorescence intensity of the vital dye CFSE has become widely corresponding to 20% and 90%, respectively (Fig. 3b, see ESI†).
used. We calculated the percentage of inhibition of cell prolif- None of the compounds altered the membrane integrity in
eration caused by each compound, through the comparison of PBMCs or Jurkat cells (data not shown).
the CFSE uorescence intensity (MFI) of cells treated to the MFI
2.2.6 Evaluation of phosphatidylserine exposure. Taking
of untreated cells.
into account that the loss of the mitochondrial membrane
Flow cytometry analysis showed that the triptoquinone potential is an event that precedes several forms of cell death,
epimer 21 inhibited 77% and 82% of proliferation of Jurkat cells we veried whether apoptosis was occurring. To this end, Jurkat
at concentrations of 7.0 mg mLꢀ1 and 14.0 mg mLꢀ1, respectively. and PBMC cells were stained with Annexin V and counter-
In contrast, the triptoquinone epimer 20 did not inhibit Jurkat stained with PI, and then analyzed by means of ow cytometry.
cells proliferation at the evaluated concentrations (Fig. 2a, The viable cells, early apoptotic cells, late apoptotic cells and
see ESI†).
necrotic cells are shown in Fig. 4 and 5 (see ESI†). An increase
The CFSE assay has signicant advantages in terms of the of Annexin V+ Jurkat cells was observed aer 15 h and 24 h of
ability to gate specic populations of lymphocytes. Then, the treatment with compounds 20 and 21 at a concentration of
effect of triptoquinone epimers on proliferation of CD4 and 14.0 mg mLꢀ1. Additionally, the triptoquinone epimer 21
CD8 cells stimulated with PHA was evaluated. The percentage of induced the highest increase in the percentage of Annexin V+
proliferation of CD4 and CD8 cells was calculated comparing Jurkat cells, corresponding to 26% and 86% with respect to the
the percentage of proliferation of the treated CD4 or CD8 cells to control, aer treatment for 15 h and 24 h, respectively (Fig. 4,
the total untreated cells CD4 or CD8 (100%). Triptoquinone see ESI†). Similarly, aer the treatment of PBMCs with tripto-
epimers 20 and 21 inhibited proliferation of both CD4 and CD8 quinone epimers 20 and 21 at a concentration of 14.0 mg mLꢀ1
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for 15 h and 24 h, the percentage of positive Annexin cells reduced the production of IL-1b, TNFa and IL-10 in PBMCs
increased in comparison to the control. The triptoquinone stimulated with LPS aer treatment at concentrations of 5.0,
epimer 21 induced the highest increase in the percentage of 10.0 and 15.0 mg mLꢀ1 (Fig. 10, see ESI†), and there were no
Annexin V+ PBMCs corresponding to 75% and 90% in signicant differences in inhibition of cytokine production
comparison to the control, aer treatment for 15 h and 24 h, between compounds, except for IL-1b, which was more inhibi-
respectively (Fig. 5†). These results indicate that compounds 20 ted by the triptoquinone epimer 20 at concentrations of 5.0 and
and 21 induce phosphatidylserine exposure on Jurkat and 10.0 mg mLꢀ1. The level of cytokines did not differ in the
PBMC cells. Compound 21 induced the highest phosphati- absence of LPS aer treatment with both compounds (data not
dylserine exposure.
shown).
2.2.7 Evaluation of cell cycle distribution. We also investi-
gated whether compounds 20 and 21 induced either DNA
damage or alteration in the cell cycle distribution of Jurkat and
3 Discussion
PBMC cells, using ow cytometry analysis. Aer treating Jurkat The present study was performed to evaluate the antimycotic,
cells with the triptoquinone epimer 21 for 15 h and 24 h, there antiviral and cytotoxic activity of three semisynthetic tripto-
was a marked increase in the percentage of cells in the G2/M- quinone epimers and three semisynthetic 14-hydroxyabietane
phase of the cell cycle, corresponding to 13% and 15%, derivatives. None of the tested compounds showed relevant
respectively, accompanied by a decrease in the percentage of antiviral or antimycotic activity, however some of them showed
cells in the G0/G1-phase. In contrast, the triptoquinone epimer cytotoxic activity.
20 did not alter the cell cycle distribution of Jurkat cells (Fig. 6,
see ESI†).
Compound 16 showed the lowest IC50 value on the HeLa cell
line, but the selectivity index of this compound was <5. In the
The triptoquinone epimer 21 induced hypoploidy in Jurkat Jurkat tumor cell line, compounds 20 and 21 inhibited cellular
cells at a concentration of 14.0 mg mLꢀ1, aer 15 h and 24 h of growth and showed the highest selectivity index (SI) values >5,
treatment, corresponding to 12% and 35%, respectively, while corresponding to 8.96 and 5.46, respectively.
the triptoquinone epimer 20 did not induce hypoploidy at the
Triptoquinone epimers (19–21) were more selective to Jurkat
evaluated concentrations (Fig. 7, see ESI†). In stimulated than HeLa cells, especially compound 20, and this is possibly
PBMCs, triptoquinone epimers 20 and 21 did not induce due to the presence of a molecular target in Jurkat that is not
alteration of the cell cycle distribution, and did not cause present in HeLa cells.
hypoploidy at the tested concentrations (data not shown).
According to the National Cancer Institute (USA), a crude
2.2.8 TUNEL assay. To prove that the epimer 20 did not extract is cytotoxic when its IC50 on normal cells is below 30 mg
induce DNA damage in Jurkat and PBMC cells in comparison to mLꢀ1
;
therefore compounds 20 and 21, with IC50 values of
18
the epimer 21, we carried out a TUNEL assay. The triptoquinone 129.0 ꢁ 6.3 and 79.7 ꢁ 15.6, respectively, are not cytotoxic in
epimer 21 induced an increase of positive TUNEL Jurkat cells at Vero cells. So they were selected to evaluate their possible
a concentration of 14.0 mg mLꢀ1, aer 15 h and 24 h of treat- mechanism of cell death induction.
ment, corresponding to 10% and 35%, respectively, but did not
Here, it was demonstrated that only the triptoquinone
induce an increase in PBMCs at the evaluated concentrations epimer 21 induced proliferation inhibition in the Jurkat cell line
(Fig. 8b†). In contrast, the triptoquinone epimer 20 did not when it was evaluated for the vital dye carboxyuorescein
induce any DNA damage in Jurkat or PBMC cells at the evalu- diacetate succinimidyl (CFSE) assay. In addition, in order to
ated concentrations (Fig. 8, see ESI†). These results are in identify the selectivity on proliferation inhibition of compounds
agreement with the results of staining with PI that were 20 and 21 in tumor-lines, their effect on cell proliferation in
described above (Fig. 7, see ESI†).
PBMCs was determined. Both compounds 20 and 21 affected
2.2.9 Evaluation of mononuclear phagocyte differentia- CD4 or CD8 cells proliferation aer 72 h of treatment, but not
tion. As triptoquinone D and triptoquinone F are potent inter- their activation (CD69 expression), which is very important
leukin-1a and interleukin-1b inhibitors,8 we evaluated the anti- considering that CD69 is a co-stimulatory molecule for T-cell
inammatory activity of their C-4 epimers, compounds 20 and activation aer stimulation through the T cell antigen receptor
21 respectively, in lipopolysaccharide (LPS)-stimulated mono- (TCR) as well as a signal-transmitting receptor for synthesis of
cyte-derived macrophages. Initially we investigated the effect cytokines (IL-2, INF-a, and TNF-a), expression of the IL-2R
of triptoquinone epimers 20 and 21 on the differentiation of subunit, rise of intracellular calcium concentration and cell
monocytes into macrophages measuring the expression of proliferation necessary for immune response.19
HLA-DR and uorescein diacetate labeling (FDA) aer 120 h
Desmond et al. (2005) have reported the ability of the fur-
of culture.
anonaphthoquinones to cause growth arrest and apoptosis in a
Both compounds induced a reduction in HLA-DR expression variety of human leukemia and multiple myeloma cell lines;16
(Fig. 9a, see ESI†) and MFI of FDA (Fig. 9b, see ESI†) aer these effects were associated with a decrease in mitochondrial
treatment at concentrations of 5.0, 10.0 and 15.0 mg mLꢀ1, as function that mediates the release of some proapoptotic
compared to control cells.
proteins. In this study, it was found that only the triptoquinone
2.2.10 Cytokine production by monocytes. The effect of epimer 21 induced a loss of the mitochondrial membrane
triptoquinone epimers 20 and 21 on cytokine production, in the potential in both Jurkat cells and PBMCs at a concentration of
presence or absence of LPS, was evaluated. Both compounds 14 mg mLꢀ1 but not at a concentration of 7 mg mLꢀ1, suggesting
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that at lower concentrations, it can be more selective to Jurkat
The triptoquinones A–F (6–11)(Fig. 1) are principal compo-
cells. Furthermore, none of the triptoquinone epimers altered nents of Triptergium wilfordii and have been found to inhibit
the membrane integrity in PBMCs or Jurkat cells. This suggests production or release of IL-1 by human monocytes stimulated
that triptoquinone epimers 20 and 21 do not induce necrosis, by endotoxin.9,27,28 Then, the effects of compounds 20 and 21 on
which is important because pharmacological-induced necrosis differentiation of monocytes were investigated and it was found
might lead to tissue damages.20
Alteration of cell cycle distribution was only observed with as assessed by HLA-DR expression and FDA labeling.
compound 21, which was an increase in the percentage of cells The inhibition of production or release of IL-1b in rat thoracic
in the G2/M-phase and a decrease in the percentage of cells in aorta aer LPS treatment has been shown by the triptoquinone A.8
the G0/G1-phase. In addition, T. wilfordii extracts and their active compounds,
that both compounds interfered with monocyte differentiation
Gomathinayagam et al. (2008) observed that “Plumbagin”, a tryptoquinones D and F, have also showed inhibition of IL-1b
naphthoquinone, present in plants from the Droseraceae and production in human monocytes28 and IL-1b activity in human
Plumbaginaceae families, caused an increase in the percentage peripheral mononuclear cells.9 Takaishi et al. (1997) observed that
of cells in the G2/M-phase in human lung cancer cells (H460) by triterpenoids isolated from T. wilfordii var. Regelii have inhibitory
down-regulating G2/M regulatory proteins (cyclinB1 and activity against IL-1a and IL-1b release from lipopolysaccharide-
Cdc25B), and it also induced a decrease in the percentage of stimulated human peripheral mononuclear cells.27
cells in the G0/G1-phase.21
Several studies have reported that Triptolide, a puried
Likewise, only compound 21 induced selective DNA damage diterpenoid component of T. wilfordii, is able to suppress the
in Jurkat cells at the tested concentrations. It is noteworthy to production of inammatory mediators such as IL-1b and TNF-a
say that this has been observed with another diterpenoid induced by LPS in various cell types including bronchial
quinone, “Salvicine”, which induces DNA damage through epithelial cells,24,29 peripheral blood mononuclear cells,30,31 and
double-strand breaks by inhibition of topoisomerase II and macrophages.32
glutathione depletion (Cai et al., 2008).22 In contrast, diterpe-
Several studies suggest that the T. wilfordii bioactive
noid quinones isolated from the plant Peltodon longipes and compounds (triptolide, celastrol and tripchlorolide) exert their
that possess a para-quinone structure such as 7-a-acetoxyr- immunosuppressive and anticancer activities by modulating the
oyleanone, horminone, royleanone, and 7-ketoroyleanone have transcriptional activity of the nuclear factor-NF-kB. NF-kB activates
shown cytotoxic activity against the human pancreatic cancer the enhancer region of various genes, including the pro-inam-
cell line MIA PaCa-2, mainly through the inhibition of the matory cytokines tumour necrosis factor (TNF)-a and oncogenic
relaxation activity of human topoisomerase I (induced by DNA genes, such as survivin, cyclinD1 and c-myc.24 Additionally, several
strand breaks) at concentrations lower than or similar to those clinical trials have shown the efficacy of Triptolide in the T. wil-
of the positive control, camptothecin.23 As said by Fronza et al. fordii extracts in the treatment of rheumatic diseases, including
(2012),23 it is possible that the structural properties of para- rheumatoid arthritis and systemic lupus erythematosus.24,33–37
quinone diterpenes can inuence or determine their molecular
Based on this evidence, the anti-inammatory effect of trip-
mode of producing cell death. Therefore, these different modes toquinone epimers 20 and 21was evaluated. Both compounds not
of action suggest that cell death induced by abietane quinone only inhibited the production of IL-1b and TNF-a, pro-inam-
diterpenes may not follow a single mechanism, but several matory cytokines, but also IL-10 an anti-inammatory cytokine in
mechanisms instead.
the presence of LPS. Therefore the compounds did not possess a
Apoptotic cell death is a process of programmed cell death differential effect on the production of pro-inammatory and
that includes several biochemical events, among others, protein anti-inammatory cytokines.
cleavage, protein cross-linking, DNA breakdown and loss of the
mitochondrial membrane potential. Wong et al. (2012) have
observed that Tanshinone IIA (Tan IIA), a diterpenoid naph-
4 Conclusions
thoquinone derived from Radix salvia miltiorrhiza, induces In summary, the triptoquinone epimers 20 and 21 showed
apoptosis in lung cancer cells (A549) and human colon adeno- different effects and selectivity, which could be due to the
carcinoma cells (Colo-205) through a mitochondrial mediated difference in their chemical structure; both compounds differ
intrinsic cell death pathway.24–26 Tan IIA has shown cytotoxic only in one of their substitutions on carbon 18. Compound 20
activity against human prostate cancer cells (LNCaP) at a has a carboxylic acid as the functional group and compound 21
concentration of 35 mM and induces a reduction of the mito- has an alcohol. In addition, the triptoquinone epimer 20
chondrial membrane potential at a concentration of 50 mM.24
induced only phosphatidylserine exposure on Jurkat cells,
In this study, the externalization of phosphatidylserine therefore, it was not clear if this epimer induces cell death. On
residues in the outer plasma membrane, an early biochemical the other hand, the triptoquinone epimer 21 induced phos-
event that occurs in apoptotic cells, was also evaluated. It was phatidylserine exposure and proliferation inhibition in PBMCs
observed that both compounds 20 and 21 induced phosphati- but it did not affect their activation. Moreover, it induced a loss
dylserine (Anexin V) exposure on both PBMCs and Jurkat cell of the mitochondrial potential at a concentration of 7.0 mg
line at a tested concentration of 14.0 mg mLꢀ1 and compound 21 mLꢀ1, DNA damage and alteration of cell cycle distribution in
induced the highest percentage of positive Annexin cells in both Jurkat cells but not in PBMCs. These results suggest that this
PBMCs and Jurkat cell line.
compound induces cell death in the Jurkat tumoral cell line at a
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concentration of 7.0 mg mLꢀ1, however, in vivo studies will be and the mixture was reuxed for 15 h. Then, the solvent was
necessary to dene its possible clinical usefulness. evaporated to yield a residue, which was chromatographed on
In addition, taking into account that both compounds silica, eluted with hexane–ethyl acetate (8 : 2) to yield phenol 16
inhibited monocyte differentiation, they could be potential (1.5 g, 80%) as an orange oil: [a]2D5 +36.0 (c 4.5) [(ref. 8) +51.7
immunosuppressive and anti-inammatory agents that can be (c 0.5, CHCl3); (ref. 9) +7.7 (c 1.0, CHCl3)]. 1H NMR (300 MHz) d
useful in treatment of autoimmune diseases such as rheuma- 7.00 (1H, d, J ¼ 8.2), 6.82 (1H, d, J ¼ 8.2), 3.64 (3H, s), 3.16 (1H,
toid arthritis and non-infectious inammatory processes.38
sept., J ¼ 6.9), 2.77 (1H, dd, J ¼ 16.8, 6.3), 2.67 (1H, m), 2.27 (1H,
br d, J ¼ 12.9), 2.21 (1H, dd, J ¼ 12.6, 2.1), 1.27 (3H, s), 1.21 (6H,
d, J ¼ 6.9), 1.20 (3H, s); 13C NMR (75 MHz) dC 179.0 (s), 150.3 (s),
148.0 (s), 130.3 (s), 123.1 (d), 120.7 (s), 115.9 (d), 51.7 (d), 47.4 (s),
44.0 (d), 37.9 (t), 36.7 (s), 36.4 (t), 26.5 (q), 24.8 (q), 23.9 (t), 22.6
5 Materials and methods
5.1 Chemistry
Optical rotations were determined with a 5 cm path-length cell, (q), 22.4 (q), 20.9 (t), 18.4 (t), 16.3 (q); HRMS (EI) m/z 330.2204
using dichloromethane as a solvent (concentration expressed in [M]+, calcd for C21H30O3: 330.2195.
g per 100 mL). [a]D-values are given in 10ꢀ1 deg cm2 gꢀ1. NMR
5.1.4 14-Hydroxyabieta-8,11,13-trien-18-oic acid (17). A
spectra were recorded on a 300 MHz spectrometer with tetra- mixture of ester 16 (200 mg, 0.61 mmol), KOH (85%, 1.5 g, 23
methylsilane as an internal standard. All spectra were recorded mmol), H2O (2 mL) and methanol (12 mL) was reuxed for 48 h.
in CDCl3 as the solvent unless otherwise described. Complete Aer this time, the reaction mixture was cooled, poured into
assignments of 13C NMR multiplicities were made on the basis aqueous HCl (1.2 M, 30 mL) and extracted three times with
of DEPT experiments. J values are given in Hz. Mass spectra DCM. The organic extract was dried over MgSO4 and concen-
(MS) were run by electron impact (EI) at 70 eV. Reactions were trated under reduced pressure to yield the crude acid 17, which
monitored by means of thin-layer chromatography (TLC) using was chromatographed on silica, eluted with hexane–ethyl
Merck silica gel 60 F-254 in 0.25 mm thick plates. Compounds acetate (6 : 4) to yield acid 17 (144 mg, 75%) as a yellow foam:
on TLC plates were detected under UV light at 254 nm and [a]2D5 +46.4 (c 1.2) [(ref. 8) +58.9 (c 0.5, CHCl3)]. 1H NMR
visualized by immersion in a 10% sulfuric acid solution and (300 MHz) d 7.01 (1H, d, J ¼ 8.1), 6.84 (1H, d, J ¼ 8.1), 3.14 (1H,
heating on a hotplate. Purications were performed by ash sept., J ¼ 6.9), 2.76 (1H, dd, J ¼ 16.8, 6.6), 2.68 (1H, m), 2.28 (1H,
chromatography on Merck silica gel (230–400 mesh). All non- d, J ¼ 12.6), 2.21 (1H, d, J ¼ 11.1), 1.27 (3H, s), 1.22 (6H, d, J ¼
aqueous reactions were carried out in an argon atmosphere in 6.9), 1.20 (3H, s); 13C NMR (75 MHz) dC 185.2 (s), 150.1 (s), 148.1
oven-dried glassware. Commercial reagent grade solvents and (s), 130.4 (s), 123.3 (d), 120.7 (s), 116.2 (d), 47.3 (s), 43.9 (d), 37.9
chemicals were used as received unless otherwise noted. (t), 36.8 (s), 36.6 (t), 26.7 (q), 25.0 (q), 23.9 (t), 22.7 (q), 22.5 (q),
Combined organic extracts were washed with brine, dried over 21.0 (t), 18.5 (t), 16.1 (q); HRMS (EI) m/z 316.2045 [M]+, calcd for
anhydrous sodium sulphate, ltered and concentrated under
C
20H28O3: 316.2038.
reduced pressure.
5.1.5 Abieta-8,11,13-trien-14,18-diol (18). To a solution of
5.1.1 Methyl 13b,14b-dihydroxyabieta-7-en-18-oate (14). To ester 16 (500 mg, 1.51 mmol) in dry THF (10 mL) LiAlH4
a solution of methyl abietate (13) (Gonzalez et al., 2009)14 (6.0 g, (500 mg, 13 mmol) was added in portions, and then reuxed for
´
0.018 mol) in t-BuOH (30 mL) and pyridine (1.4 mL), Me3NO 15 h. Then, the mixture was cooled to 0 ꢂC, and 0.5 mL of H2O,
(2.8 g, 1.3 equiv.) and a 4% solution of OsO4 (2.0 mL) were 0.5 mL of 15% NaOH and 1.5 mL of H2O were added sequen-
added. The reaction mixture was stirred under an Ar atmo- tially and carefully. The resulting white solid was ltered off and
sphere at reux for one week. Then, NaHSO3 (5 mL) was added washed with ethyl acetate. The extract was concentrated and
and the solvent was evaporated. The residue was dissolved in puried by means of chromatography, eluted with hexane–ethyl
AcOEt and washed with brine. The organic layer was dried and acetate (6 : 4) to yield 380 mg (83%) of pure alcohol 18 as a
concentrated to yield the crude product which was puried by yellowish foam: [a]2D5 +32.6 (c 2.1) [(ref. 8) +51.4 (c 0.75, CHCl3)].
chromatography on silica, eluted with hexane–ethyl acetate 1H NMR (300 MHz) d 6.99 (1H, d, J ¼ 8.1), 6.83 (1H, d, J ¼ 8.1),
(from 7 : 3 to 6 : 4) to yield diol 14 (4.6 g, 70%) as an orange oil 5.03 (1H, s, OH), 3.45 (1H, d, J ¼ 10.8), 3.18 (1H, d, J ¼ 10.8), 3.14
1
that solidies upon standing. The MS, H and 13C data are in (1H, sept., J ¼ 6.9), 2.76 (1H, dd, J ¼ 16.2, 5.7), 2.62 (1H, m), 2.25
agreement with the literature data.39
(1H, br d, J ¼ 12.9), 1.23 (3H, d, J ¼ 6.9), 1.21 (3H, s), 1.20 (3H, d,
5.1.2 Methyl 13b-hydroxy-14-oxoabieta-7-en-18-oate (15). J ¼ 6.9), 0.85 (3H, s); 13C NMR (75 MHz) dC 150.2 (s), 148.6 (s),
Diphenyl diselenide (6.2 g, 20 mmol) and 5.5 M t-BuOOH in 130.2 (s), 123.0 (d), 120.8 (s), 116.2 (d), 71.9 (t), 42.9 (d), 38.4 (t),
decane (7.3 mL, 40 mmol) were added to a stirred solution of 14 37.6 (s), 37.2 (s), 34.9 (t), 26.7 (q), 25.2 (q), 23.9 (t), 22.7 (q), 22.5
(5.0 g, 14.3 mmol) in dry CCl4 (100 mL), and the mixture was (q), 18.6 (t), 18.1 (t), 17.3 (q); HRMS (EI) m/z 302.2257 [M]+, calcd
reuxed under an Ar atmosphere for 4 h. Then, the solvent was for C20H30O2: 302.2246.
evaporated under vacuum, and the residue was puried by
5.1.6 Methyl 11,14-dioxoabieta-8,12-dien-18-oate (19). To a
chromatography on silica, eluted with hexane–diethyl ether solution of phenol 16 (90 mg, 0.27 mmol) in AcOH (1 mL) at
(7 : 3) to yield ketone 15 (3.7 g, 75%) as a colourless oil. The MS, 10 ꢂC, a catalytic amount of RuCl3$3H2O (14 mg, 0.2 equiv.) and
1H and 13C data are in agreement with the literature data.40
30% H2O2 (150 mL, 1.42 mmol) were added. The mixture was
5.1.3 Methyl 14-hydroxyabieta-8,11,13-trien-18-oate (16). allowed to warm to room temperature and stirred for 15 h.
To a solution of the ketone 15 (2.0 g, 5.7 mmol) in toluene Then, the reaction mixture was carefully poured into saturated
(100 mL), p-toluenesulfonic acid (1.1 g, 5.7 mmol) was added NaHCO3 and extracted with ethyl acetate. The organic layer was
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washed with brine, dried and concentrated. The residue was the Antioquia University of Colombia, CENIVAM, COLCIENCIAS
´
chromatographed on silica, eluted with hexane–ethyl acetate (Patrimonio Autonomo del Fondo Nacional de Financiamiento
´
´
´
(8 : 2) to yield quinone 19 (56 mg, 60%) as a yellow-orange oil: para la Ciencia, la Tecnologıa y la Innovacion, Francisco Jose de
[a]2D5 ꢀ43.6 (c 2.2) [(ref. 8) ꢀ69.7 (c 0.5, CHCl3)]. 1H NMR Caldas) Grant RC RC-245-2011 and RC 366-2011.
(300 MHz) d 6.32 (1H, d, J ¼ 1.2), 3.68 (3H, s), 2.98 (1H, m), 2.78
(1H, br d, J ¼ 11.1), 2.65 (1H, dd, J ¼ 20.1, 6.0), 2.40 (1H, dd, J ¼
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1.11 (3H, d, J ¼ 6.9), 1.09 (3H, d, J ¼ 6.9); 13C NMR (75 MHz) dC
187.8 (s), 178.6 (s), 152.9 (s), 150.0 (s), 142.6 (s), 131.8 (d), 52.0
(d), 47.7 (s), 45.7 (d), 37.8 (s), 36.4 (t), 35.5 (t), 26.3 (q), 25.4 (t),
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