Synthesis and biological evaluation of (-)-13,14-dihydroxy-8,11,13-podocarpatrien-7-one and derivatives from (+)-manool

Article abstract of DOI:10.1080/14786419.2014.942299

13,14-Dihydroxy-8,11,13-podocarpatrien-7-one (1) and a series of ring C aromatic diterpene derivatives were synthesised from (+)-manool (4) and evaluated for their cytotoxic, leishmanicidal and trypanocidal activities. Our results indicated that compound 1 and other podocarpane-type intermediates are cytotoxic. Cleavage of C6-C7 bond of compound 7 improved cytotoxic activity, indicating that, in particular, the 6,7-seco-podocarpane-type compound 20 might serve as a lead compound for further development.

Full text of DOI:10.1080/14786419.2014.942299

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Synthesis and biological evaluation
of ( - )-13,14-dihydroxy-8,11,13-
podocarpatrien-7-one and derivatives
from (+)-manool
María L. Novoaa, Yelisbeth Escalantea, Liliana Maldonadoa, Iván
Galindo-Castroc, Annamil Álvarezc, Katherine Figarellac, Sabrina
Marsiccobetrec, Irina Arochac, Jais Nievesc, Franklin Salazara,
Carlos Gámezab, Nieves Canudasb, Eleonora Troppera, Teresa
Gonzáleza & José E. Villamizara
Click for updates
a Centro de Química, Instituto Venezolano de Investigaciones
Científicas (I.V.I.C.), Apartado 21827, Caracas 1020-A, Venezuela
b Departamento de Química, Universidad Simón Bolívar, Apartado
89000, Caracas 1080-A, Venezuela
c Laboratorio de Genómica y Proteómica, Fundación IDEA,
Caracas, Venezuela
Published online: 30 Jul 2014.
To cite this article: María L. Novoa, Yelisbeth Escalante, Liliana Maldonado, Iván Galindo-Castro,
Annamil Álvarez, Katherine Figarella, Sabrina Marsiccobetre, Irina Arocha, Jais Nieves, Franklin
Salazar, Carlos Gámez, Nieves Canudas, Eleonora Tropper, Teresa González & José E. Villamizar
(2015) Synthesis and biological evaluation of ( - )-13,14-dihydroxy-8,11,13-podocarpatrien-7-one
and derivatives from (+)-manool, Natural Product Research: Formerly Natural Product Letters, 29:3,
207-212, DOI: 10.1080/14786419.2014.942299
To link to this article: http://dx.doi.org/10.1080/14786419.2014.942299
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Natural Product Research, 2015
Vol. 29, No. 3, 207–212, http://dx.doi.org/10.1080/14786419.2014.942299
Synthesis and biological evaluation of (2)-13,14-dihydroxy-8,11,13-
podocarpatrien-7-one and derivatives from (1)-manool
a
Marıa L. Novoa , Yelisbeth Escalante , Liliana Maldonado , Ivan Galindo-Castro ,
a
a
c
´
´
c
c
c
c
c
Annamil Alvarez , Katherine Figarella , Sabrina Marsiccobetre , Irina Arocha , Jais Nieves ,
´
a
Franklin Salazar , Carlos Gamez , Nieves Canudas , Eleonora Tropper , Teresa Gonzalez and
ab
b
a
a
´
´
a
Jose E. Villamizar *
´
a
´
Centro de Quımica, Instituto Venezolano de Investigaciones Cientıficas (I.V.I.C.), Apartado 21827,
Caracas 1020-A, Venezuela; Departamento de Quımica, Universidad Simon Bolıvar, Apartado 89000,
´
b
´
´
´
c
´ ´ ´
Caracas 1080-A, Venezuela; Laboratorio de Genomica y Proteomica, Fundacion IDEA, Caracas,
Venezuela
(Received 10 March 2014; final version received 3 July 2014)
13,14-Dihydroxy-8,11,13-podocarpatrien-7-one (1) and a series of ring C aromatic
diterpene derivatives were synthesised from (þ)-manool (4) and evaluated for their
cytotoxic, leishmanicidal and trypanocidal activities. Our results indicated that compound
1 and other podocarpane-type intermediates are cytotoxic. Cleavage of C6–C7 bond
of compound 7 improved cytotoxic activity, indicating that, in particular, the 6,7-
seco-podocarpane-type compound 20 might serve as a lead compound for further
development.
Keywords: podocarpane; abietane; cytotoxicity; leishmanicidal; trypanocidal
1. Introduction
Plants have an outstanding ability to synthesise aromatic substances, most of which are either
phenols or their oxygen-substituted derivatives. In many cases, these substances serve as plant
defence mechanisms against predation by microorganisms, insects and herbivores (Cowan
1999). Abietane-type and biosynthetically related polycyclic diterpenes are a major group of
ring C aromatic diterpenes (Nakano 1989). They exhibit interesting biological properties such as
antimalarial (Achenbach et al. 1992), antibiotic (Batista et al. 1995; Dellar et al. 1996) and
cytotoxic (Gao & Han 1997) activities. Moreover, podocarpane-type diterpenes are present only
in several genera, such as Azadirachta (Ara et al. 1988a, 1988b, 1990; Siddiqui et al. 1988),
Humirianther (Das et al. 1981), Micrandropsis (de Alvarenga et al. 1981) and Podocarpus
(Cambie & Mander 1962). In Kuo et al. (2000) isolated the podocarpane-type diterpene (2)-
13,14-dihydroxy-8,11,13-podocarpatrien-7-one (1) from the bark of Taiwaniacryptomerioides
and Lu et al. (2006) isolated this from the Chinese medicinal plant Celastruspaniculatus. Other
bioactive podocarpane-type catechols isolated were (þ)-deoxynimbidiol (2) (Xiong et al. 2006)
and (þ)-nimbidiol (3) (Majumder et al. 1987) (Figure 1).
To date, a number of synthetic approaches to these biologically active podocarpane-type
diterpenes have been reported via polyene cyclisation (Burnell & Caron 1992; Majetich et al.
1997; Tada et al. 2000; Ishihara et al. 2001, 2002; Ishibashi et al. 2004; Harring & Livinghouse
*Corresponding author. Email: jvillami@ivic.gob.ve
q 2014 Taylor & Francis

208
M.L. Novoa et al.
OH
OH
12
OH
OH
20
1
O
O
14
OH
9
8
H
H
5
3
O
R
7
H
H
H
H
19
18
5
4
2
3
R= H2
R= O
1
Figure 1. Structures of compounds 1–5.
2010). However, they generally require long reaction sequences, and furthermore, almost all of
them produce the racemic mixture of the natural substance.
(þ)-Manool (4) is a commercially available natural labdane-type diterpene with established
absolute stereochemistry that has been used as a starting material for the syntheses of drimane-
type sesquiterpenes (Salazar & Villamizar 2013a) and abietane-type and labdane-type
diterpenes (Salazar & Villamizar 2013b). Recently, we have reported a new synthetic route to
13-hydroxy-8,11,13-podocarpatriene compounds from compound 4, but this strategy involves
the use of potassium permanganate (Villamizar et al. 2010, 2011). Continuing our research
programme on the synthesis of bioactive diterpenes, the natural compound 1 and derivatives
were synthesised from compound 4 and screened for their in vitro cytotoxic activity against two
human cancer cell lines, and for antitrypanosomal and antileishmanial activities against
Trypanosoma cruzi and Leishmania mexicana.
2. Results and discussion
2.1. Chemistry
To synthesise podocarpane-type diterpenes from labdane-type diterpene, the construction of the
C-ring on the chiral podocarpane skeleton is necessary. This could be achieved by an
´
intramolecular aldol condensation of 14,15,17-trinorlabdan-8,13-dione (5) (Alvarez-Manzaneda
et al. 2007; Villamizar et al. 2010, 2011). Recently, Rogachev et al. (2012) reported that (þ)-
manool (4) was converted into compound 5 by ozonolysis in toluene in the presence of 10%
sodium hydroxide in low yield. In an attempt to increase this yield, the reaction was studied in
different solvents, resulting that the most favourable solvent to obtain compound 5 was toluene.
In addition, when toluene was used as solvent and catalytic amounts of TBAB were added,
compounds 5 and 6 were obtained in 64% and 10% yield, respectively. The approaches for the
synthesis of all derivatives are outlined in Scheme 1. In our ongoing effort to synthesise novel
bioactive podocarpane-type derivatives, we needed to prepare two key intermediates, natural
phenols 7 (Kuo et al. 2000) and 11 (Minh et al. (2012). We previously reported the synthesis of
compound 7 (Villamizar et al. 2010, 2011). Oxidation of compound 6 with sodium acetate and
air afforded compound 11 (91% yield) (Scheme 1). The oxime derivative 14 was obtained by
treatment of compound 13 with NH2OH/AcOK (93% yield). The a,b-unsaturated ketones 15
and 17 were obtained from compounds 11 and 13, respectively, by use of SeO2 (71% and 88%
yields, respectively). Compounds 12 and 13 can also be obtained from derivatives 8 and 9,
respectively, using different oxidation systems. Only oxidation with Na2CrO4 yielded compound
16 in low yield (Scheme 1).
To prepare 6,7-seco-podocarpane-type molecules, compound 10 was directly converted to
the olefin 18 with N-bromosuccinimide (84% yield). Ozonolysis of compound 18 was carried out
in CH2Cl2 and the reaction was quenched with PPh3 to afford dialdehyde 19 (89% yield).

Natural Product Research
209
OR
O
O
OR
v
15 R= H
16 R= Ac
17 R= CH3
i
H
vi
R1
H
H
6
O
O
ix
11 R= H; R1= O
ii
viii
iii
12 R= Ac; R1= O
13 R= CH3; R1= O
14 R= CH3; R1= NOH
OR
iv
O
H
vii
H
OH
OH
7 R= H
8 R= Ac
9 R= CH3
ii
iii
x
O
10 R= TBDMS
H
xi
1
OR
OR
OH
xii
xiii
CHO
CHO
CHO
CHO
H
H
H
18 R= TBDMS
20
19 R= TBDMS
Scheme 1. Reagents and conditions: (i) AcONa, air, DMF, 1208C, 18 h; (ii) Ac2O, TEA, DMAP, acetone,
reflux, 12 h; (iii) K2CO3, MeI, acetone, reflux, 30 h; (iv) NH2OH, AcOK, MeOH, reflux, 1 h; (v) 13, SeO2,
AcOH, H2O, reflux, 2.5 h ! 17. For 11, same conditions, 17 h ! 15; (vi) IBX, CHCl3–MeOH (4:1), rt, 1 h;
(vii) then NaBH4, rt, 3 h; (viii) Villamizar et al. (2010, 2011); (ix) Method A: 8, CrO3, AcOH, rt,
120 h ! 12; Method B: 8, Na2CrO4, AcOH, Ac2O, benzene, reflux, 24 h ! 12 and 16. For 9, same
conditions ! 13; Method C: 8, PDC, TBHP, Celite, benzene, 108C, 15 min, then rt, 4 h ! 12; (x) Im,
TBDMSCl, DMAP, DMF, rt, 30 h; (xi) 10, NBS, BPO, CCl4, reflux, 2 h; (xii) (a) O3, CH2Cl2, 2788C,
10 min; (b) PPh3, CH2Cl2, 2788C, 1 h, then rt, 2 h; (c) H2O2, 08C, 1 h; (xiii) TBAF, THF, 08C, 5 min, then
rt, 40 min.
Dialdehyde 20 was then obtained in quantitative yield after treatment of compound 19 with
TBAF (Scheme 1). Even though the spectral data (1H NMR, 13C NMR and EI-MS) for
dialdehyde 20 were completely consistent with its structure, further confirmation was sought by
means of single crystal X-ray analyses. X-ray quality crystals of compound 20 were grown by
slow evaporation of a hexane–ethyl ether solution. An ORTEP depiction of compound 20 is
shown in Figure S25 (see Supplementary material). It is interesting to notice the cis position of
methyl C20 with respect to the aldehyde C7.
In order to obtain the desired catechol 1, compound 11 was oxidised according to the method
of Magdziak et al. (2002) and Pezzella et al. (2005) to afford compound 1 with 50% yield
(Scheme 1), whose physical and spectroscopic properties were identical with those reported
(Kuo et al. 2000).

210
M.L. Novoa et al.
2.2. Biological evaluation
Compounds 1, 7, 9, 12–16, 19 and 20 were tested for cytotoxicity on human tumour cells, as well
as for leishmanicidal and trypanocidal activities. First, these compounds were tested in vitro for
potential cytotoxic activity to determine the concentration of the compound that induces 50%
growth inhibition (IC50) of human breast cancer-derived cells (MCF-7) or human prostate cancer-
derived cells (PC3). Primary cultures of human dermal fibroblast were used as reference for non-
malignant cells. Selectivity index (SI) for each compound was expressed as the ratio of IC50 on
fibroblasts to the IC50 on the corresponding cell type. Cell viability and specific cytotoxicity were
assessed by the MTT method (Denizot & Lang 1986). As can be seen in Table S1 (see
Supplementary material), the highest cytotoxic activity on both MCF-7 and PC3 cells was shown
by the oxime 14 with almost similar IC50 values, 19.9 ^ 2.7 and 20.5 ^ 4.3 mM, respectively.
However, the IC50 on fibroblasts showed similar values (18.0 ^ 3.2 mM). It is noteworthy that the
phenolic dialdehyde 20 exhibited the highest SI, with values greater than 3.5 and 2.8 for MCF-7
and PC3 cell lines, respectively. This compound also showed the lowest cytotoxicity on non-
cancerous cells (fibroblasts) with an IC50 value above 180 mM. By contrast, phenolic compounds
1, 7 and 15, all of them of podocarpane skeleton, were more cytotoxic to fibroblasts than to tumour
cells. This trend seems to be reversed when the skeleton is broken at C6–C7 bond, as seen in
compound 20. Compound 9 exhibited the highest SI with values greater than 2.8 and 3.1 for L.
mexicana and T. cruzi, respectively. According to these SI values, this molecule is the most
effective compound tested against the parasites studied. It is interesting that compound 7, although
very similar to compound 9, showed a lower SI value. This behaviour could be attributed to the
hydrophobicity of the methyl group in compound 9, which may facilitate the entry of the molecule
through the cytoplasmic membrane. Compound 20 showed the highest IC50 value on parasite cells
and phenolic compounds 1, 7 and 15were more cytotoxic against fibroblasts than to any parasite or
tumour cell tested (see Table S1 Supplementary material).
3. Conclusions
We reported the first enantiospecific synthesis of (2)-13,14-dihydroxy-8,11,13-podocarpatrien-
7-one (1), prepared several podocarpane-type derivatives and tested them for their in vitro
tumour-derived cell cytotoxicity, as well as antileishmanial and antitrypanosomal activities.
We found that compound 20 was the most effective on the tumour-derived cells tested (higher SI
values), implicating that cleavage of C6–C7 double bond in the phenol 7 is likely to enhance the
SI. We also demonstrated that compound 9 exhibited the highest SI value on both parasites
assayed, showing that some functional groups play a key role in compound bioactivity.
Supplementary material
Supplementary material relating to this article is available online, alongside Table S1 and
Figures S1–S25.
Acknowledgements
´
The authors thank the laboratories of Mass Spectrometry (Ana Angarita and Matilde Gomez), NMR (Sara
Pekerar, Alberto Fuentes, Liz Cubillan and Ligia Llovera) and Elemental Analyses (EleinneSeverino) of
IVIC for the research support.
Funding
This research was supported by FONACIT [grand number 2007000960], [grand number 2007001522],
Foundation IDEA (POA-2012), and FONDEN.

Natural Product Research
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