Synthesis and antimalarial activities of optically active labdane-type diterpenes

Article abstract of DOI:10.1080/14786410802164048

An efficient method for the synthesis of optically active labdane-type diterpenes from (+)-manool 8 is described. We prepared the natural labdane-type diterpene 5 via key intermediate peroxide 9, and synthetic hydroxybutenolides 6 and 7 via a furan photosensitised oxygenation reaction of labdafuran (14). Compounds 5, 6, 7 and 9 were evaluated as inhibitors of the β-haematin formation and globin proteolysis, and then were assayed in a malarial murine model. Compound 9 was the most promising compound, showing a positive correlation between in vitro and in vivo activities.

Full text of DOI:10.1080/14786410802164048

This article was downloaded by: [University of Hong Kong Libraries]
On: 21 September 2013, At: 15:49
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly
Natural Product Letters
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gnpl20
Synthesis and antimalarial activities
of optically active labdane-type
diterpenes
a
b
c
José Villamizar , Jean P. Pittelaud , Juan R. Rodrigues
,
c
b
a
Neira Gamboa , Nieves Canudas , Eleonora Tropper , Franklin
a
b
Salazar & Juan Fuentes
a
Centro de Química, Instituto Venezolano de Investigaciones
Científicas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela
b
Departamento de Química, Universidad Simón Bolívar, Caracas,
Venezuela
c
Facultad de Farmacia, Universidad Central de Venezuela,
Caracas, Venezuela
Published online: 29 Oct 2009.
To cite this article: José Villamizar , Jean P. Pittelaud , Juan R. Rodrigues , Neira Gamboa , Nieves
Canudas , Eleonora Tropper , Franklin Salazar & Juan Fuentes (2009) Synthesis and antimalarial
activities of optically active labdane-type diterpenes, Natural Product Research: Formerly Natural
Product Letters, 23:10, 891-902, DOI: 10.1080/14786410802164048
To link to this article: http://dx.doi.org/10.1080/14786410802164048
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“
Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Natural Product Research
Vol. 23, No. 10, 10 July 2009, 891–902
Synthesis and antimalarial activities of optically active labdane-type
diterpenes
a
b
c
c
Jose
´
Villamizar *, Jean P. Pittelaud , Juan R. Rodrigues , Neira Gamboa ,
b
a
a
Nieves Canudas , Eleonora Tropper , Franklin Salazar and Juan Fuentes
b
a
Centro de Quı´mica, Instituto Venezolano de Investigaciones Cientı ´f icas (IVIC), Apartado 21827,
Caracas 1020-A, Venezuela; Departamento de Quı ´m ica, Universidad Simo´n Bolı´var, Caracas,
b
c
Venezuela; Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela
(
Received 20 February 2006; final version received 30 April 2009)
An efficient method for the synthesis of optically active labdane-type diterpenes
from (þ)-manool 8 is described. We prepared the natural labdane-type diterpene
5
via key intermediate peroxide 9, and synthetic hydroxybutenolides 6 and 7 via a
furan photosensitised oxygenation reaction of labdafuran (14). Compounds 5, 6,
and 9 were evaluated as inhibitors of the ꢀ-haematin formation and globin
7
proteolysis, and then were assayed in a malarial murine model. Compound 9 was
the most promising compound, showing a positive correlation between in vitro
and in vivo activities.
Keywords: labdane-type diterpenes; Hedychium coronarium; synthesis;
antimalarial activities
1
. Introduction
Malaria is distributed throughout the tropical and sub-tropical regions of the world
and causes more than 300 million acute illnesses and at least 1 million deaths annually
(
WHO Report, 1999). Malaria parasites are developing unacceptable levels of resistance
and this can be attributed to the use of a single drug for treatment and the adaptation
of the malarial parasite by developing alternate pathways for survival. The present
strategy for new drug development is directed towards identifying the essential enzyme
systems in the parasite and developing molecules to inhibit them (Bodeker & Willcox,
1
996; Ridley et al., 1996). The evaluation of medicinal plants used in the preparation of
folk remedies has provided effective compounds with novel structures for the treatment
of diseases caused by protozoan parasites. In fact, the most effective antimalarials have
originated from plants: quinine from the bark of the Peruvian Cinchona tree, and
artemisinin from the Chinese antipyretic Artemisia annua L. (Bodeker & Willcox, 1996;
Ridley et al., 1996).
Labdane diterpenoids are among the most common types of diterpenes isolated from
plants and sponges (Hanson, 1999, 2000). These classes of compounds are interesting for
their cytotoxic, antifungal, anti-inflammatory, antiparasitic and analgesic properties
(
Dimas et al., 1998; Itokawa & Morita, 1988). However, as in the case of many other
*
Corresponding author. Email: jvillami@ivic.ve
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410802164048
http://www.informaworld.com

8
92
J. Villamizar et al.
natural products, they can be isolated only in minute amounts, limiting the study of
biological activities. Hedychium coronarium Koeng has been cultivated in Japan, China,
India and Brazil. The rhizome of H. coronarium has been used for the treatment of
headache, sharp pain and rheumatism (Dimas et al., 1998; Itokawa & Morita, 1988).
Several labdane-type diterpenes have been isolated from this herbal medicine. In 1988,
Itokawa et al. isolated the labdane-type diterpenes coronarin A (1), B (2), C (3) and D (4)
from the rhizomes of H. coronarium cultivated in Brazil, showing cytotoxic activity against
Chinese hamster V-79 cells. In 2002, Matsuda, Morikawa, Sakamoto, Toguchida and
Yoshikawa isolated the labdane-type diterpene labda-8(17),13(14)-dien-15,16-olide (5)
from the methanolic extract of a fresh rhizome of H. coronarium cultivated in Japan,
showing anti-inflammatory activity (Morikawa et al., 2002). In 2003, Scio et al. isolated
labdane-type diterpenes from Alomia myriadenia, showing cytotoxic and trypanocidal
activity. Previously, Scio et al. (2002) have reported that some labdane-type diterpenes
isolated from A. myriadenia showed strong activity against Plasmodium falciparum, one of
the ethiological agents of human malaria.
OH
O
O
CHO
O
15
13
O
16
1
4
O
OH
1
2
1
1
O
20
O
1
7
1
4
9
6
2
3
10
8
OH
5
7
OH
H
H
H
H
19
18
1
2
3
4
O
OH
O
O
O
O
O
HO
H
H
H
5
6
7
To date, a number of semi-syntheses of these biologically active labdane-type
diterpenoids have been reported by employing (ꢀ)-sclareol as a starting material (Jung,
¨
¨
Ko, & Lee, 1998a; Jung et al., 1998b; Muller, Schroder, Magg, & Seifert, 1998). In 1982,
Nakano, Martin and Rojas reported the synthesis of some labdane-type diterpenes, such
as lactone 5 from (þ)-manool 8, but with poor yield. Recently, we have developed a new
highly efficient synthesis of optically active labdane-related natural products. The key
reaction consists of the dehydration of commercially available (þ)-manool 8 and the

Natural Product Research
893
photooxidation reaction of the resulting diene to the peroxide 9 (Villamizar, Fuentes,
Salazar, Tropper, & Alonso, 2003). Continuing with our research into the synthesis of the
labdane-type diterpenes, we have been interested in the development of synthetic routes to
the natural labdane-type diterpene 5 and synthetics 6 and 7, and a screening search of new
antimalarial agents with novel structures.
2
2
. Results and discussion
.1. Synthesis of labdane-type diterpenes
The first step of the synthetic sequence involves reduction of peroxide 9, whose efficient
synthesis from (þ)-manool 8 has been previously reported by the present authors
(
Villamizar et al., 2003). Reduction of compound 9 with LiAlH afforded the labda-
4
8
1
(17),13(Z)-diene-15,16-diol (10). In order to synthesise the title compound 5, compound
0 was submitted to oxidative lactonisation. A satisfactory completion of the synthesis of
lactone 5 required a selective oxidation of the C-15 hydroxymethyl group of the diol 10.
Oxidation with tetra-n-propylammonium perruthenate (TPAP) (Ley, Norman, Griffith, &
1
Marsden, 1994) yielded a mixture of lactones 5 and 11, as evidenced from the H NMR
spectrum. Separation of this mixture over silica gel failed. It has been found that silver
carbonate absorbed on celite (Fe
neutral oxidising agent which selectively transforms primary diols to lactones. Oxidation
of compound 10 with Fetizon’s reagent (Fetizon et al., 1969) afforded the desired lactone 5
tizon’s reagent) (Fetizon, Golfier, & Louis, 1969) is a
´ ´
´
´
in good yield, whose physical and spectroscopic properties were identical with those
reported (Matsuda et al., 2002; Morikawa et al., 2002; Nakano et al., 1982), and only a
small amount of isomeric lactone 11 (Scheme 1).
Compound 14 was prepared recently by the present authors by different methods
Villamizar et al., 2003). In an attempt to increase the yield of the compound 14, we first
(
prepared the alcohol 12 (Villamizar et al., 2003). Bromination of alcohol 12 with carbon
tetrabromide and triphenylphosphine (Ph P) under neutral conditions gave the
3
corresponding bromide 13. The nucleophilic addition of the organolithium compound,
derived from 3-bromofuran, to the bromide (13) afforded the desired compound 14, whose
physical and spectroscopic properties were identical with those reported (Villamazir et al.,
2
003). With compound 14 in hand, we continued the oxidation of furan ring to obtain the
desired hydroxybutenolides. The photooxidation of 3-substituted furans is not regiospe-
cific, yielding both the 2-alkyl-4-hydroxy- and the 3-alkyl-4-hydroxybutenolide regioi-
somers (Feringa, 1987; Kernan & Faulkner, 1988). Irradiation of labdafuran (14) in THF
(external 150 W halogen–tungsten lamps, Pyrex well) in the presence of oxygen and a
catalytic amount of Rose Bengal afforded a mixture of two regioisomeric hydroxybute-
nolides, 6 and 7. Both lactones were separated by crystallisation from hexane in a 32 and
2
1
1% yield, respectively (Scheme 2). The H NMR spectrum of 6 (Table 1) was consistent
with a ꢁ-alkyl-substituted-15-hydroxybutenolide, similar to natural coronarin C 3
Dimas et al., 1998; Itokawa & Morita, 1988). The presence of signals at 6.81 ppm,
which must be assigned to proton H-14, placed at the ꢀ-position of an ꢁ,ꢀ-unsaturated
(
1
butenolide, and the signal at 6.08 ppm assigned to H-15. The H NMR spectrum of
regioisomeric hydroxybutenolide derivative 7, the most deshielded signal appeared at
5
.95 ppm attributable to H-16, and the signal at 5.83 ppm assigned to H-14, therefore the
structure of ꢁ,ꢀ-alkyl-substituided-16-hydroxidebutenolide has been assigned to this
compound. The formation of compounds 6 and 7 was deduced by thermal decomposition

8
94
J. Villamizar et al.
OH
O
O
OH
OH
i
H
H
H
8
10
9
ii or iii
O
O
O
O
+
H
H
11
5
Scheme 1. Reagents and conditions: (i) LiAlH
N-oxide, CH Cl (mixture of 5 and 11); (iii) Ag
4
, THF, reflux (96%); (ii) TPAP, N-methylmorpholine
CO -Celite, Benzene, r.t. (96% of 5; 3% of 11).
2
2
2
3
of the unstable endoperoxide, which result from [4 þ 2] addition of singlet oxygen to
labdafuran (14) (Feringa, 1987; Kernan & Faulkner, 1988). In an attempt to increase the
yield of the photooxidation reaction, we irradiated labdafuran (14) in CHCl (external
3
1
50 W halogen–tungsten lamp) in the presence of oxygen, 2,6-lutidine and a catalytic
amount of meso-tetraphenylporphine, which afforded a mixture of two regioisomeric
hydroxybutenolides 6 and 7 (Scheme 2). The one which formed in larger amounts (54%)
was found to be hydroxybutenolide 7, and the other (5%) was found to be 6. The major
formation of compound 7 was deduced by regiospecific removal of the hydrogen at C-15
on the intermediate endoperoxide, with a hindered base at low temperature in order to
favour base-catalysed decomposition rather than thermal decomposition (Feringa, 1987;
Kernan & Faulkner, 1988).
1
As none of the signals were doublets in the H NMR spectrum of compounds 6 and 7,
1
3
while in the C NMR spectrum no additional signals were observed, only one of the two
possible C-15 or C-16 epimers in compounds 6 and 7 respectively was present. However,
the configuration at C-15 or C-16 could not be determined.
2
.2. Biological activity
Plasmodium parasite proteases degrade the host haemoglobin in order to get the
aminoacids for its protein synthesis (Goldberg, 1992). The free haem moieties produced

Natural Product Research
895
O
O
O
O
CH OH
CH Br
2
2
i
ii
iii or iv
H
H
H
H
1
2
13
14
OH
O
O
O
O
HO
+
H
H
6
7
Scheme 2. Reagents and conditions: (i) CBr
ꢁ
4
ꢁ
, Ph
3
P, THF, r.t.; (ii) 3-bromofuran, n-BuLi, THF,
ꢀ
78 C; (iii) O
tetraphenylporphine, 2,6-lutidine, CHCl
2
, hv, Rose Bengal, THF, ꢀ0 C, 2 h (32% of 6; 21% of 7); (iv) O
2
, hv, meso-
ꢁ
3
, ꢀ78 C, 2 h (5% of 6; 54% of 7).
Table 1. Biological results of the labdane-type diterpenes assayed.
Compound
% IHF
% IGP
% P
a
5
6
7
9
77.68 ꢂ 2.93
55
44.85 ꢂ 2.97
65.01 ꢂ 1.33
95.6 ꢂ 0.42
23.89 ꢂ 1.85
NA
26.92 ꢂ 1.85
93.77 ꢂ 0.67
90.0 ꢂ 0.69
24.2 ꢂ 4.85
19.06 ꢂ 1.67
18.89 ꢂ 2.23
4.4 ꢂ 1.91
20 ꢂ 2.86
3.66 ꢂ 0.66
NA
b
55
c
50.81 ꢂ 3.61
Saline
CQ
PEP
LEP
NA
76.82 ꢂ 3.92
NA
NA
NA
Notes: CQ: chloroquine; LEP: leupeptin (cysteine protease inhibitor); PEP: pepstatin (aspartic
protease inhibitor); IHF: inhibition of ꢀ-haematin formation; IGP: inhibition of globin
proteolysis; P: parasitaemia. The results are expressed by the mean ꢂ standard error of the mean.
a
c
b
p40.05 compared to CQ. p50.0001 and p50.05 compared to LEP and PEP, respectively.
p50.01 compared to saline and p40.05 compared to CQ-treated mice. NA: not applicable.
spontaneously crystallise in the acid environment of the digestive vacuole. This mechanism
guarantees the parasites’ survival due to the fact that free haem is highly toxic to these
parasites because of its oxidant properties (Fitch et al., 1982; Orijh & Fitch, 1993).
Compounds which inhibit the ꢀ-haematin formation and the haemoglobin degradation
might be potential antimalarials.

8
96
J. Villamizar et al.
To evaluate the biological activities of the compounds, we test 5, 6, 7 and 9 to inhibit
the haem polymerisation and the globin proteolysis and we evaluated the effects on
a malarial murine model (Table 1).
Compound 5 inhibited the ꢀ-haematin formation without significant differences
compared to chloroquine control (p40.05), while 9 showed a moderate inhibition. This
result may suggest the relevance of a lactone group to interfere with this process.
In addition, all of the compounds tested were active as inhibitors of the globin proteolysis.
Compound 7 was the most effective: even better than leupeptin (p50.0001) and pepstatin
(p50.05). On the other hand, the importance of the regioisomery arrangement might be
suggested by the lowered activity showed by 6.
Particular attention must be paid to 9, which was able to decrease the parasitemia
levels as well as chloroquine did (p40.05). We can assume that this antimalarial
activity obeys a different mechanism of actions, as our results showed moderate inhibition
of ꢀ-haematin formation and globin proteolysis; in fact, it is noticeable that this
compound is an endoperoxide-labdane and it is well known that endoperoxide-
related structures, such as artemisinin and its derivatives, are potent antimalarials
(Meshnick, Taylor, & Kamchonwongpaisan, 1996; Olliaro, Haynes, Meunier, &
Yuthavong, 2001).
In summary, we have synthesised and identified new active labdane-type diterpenes as
antimalarial compounds, which act by inhibiting the ꢀ-haematin formation and/or the
globin proteolysis. Some of them showed a significant inhibition of one process or a
moderate inhibition of both. Compound 9 was the most interesting compound, showing
a positive correlation between in vitro and in vivo activities.
3
3
. Experimental
.1. General
Melting points were measured with a Kofler hot-stage apparatus and were uncorrected.
NMR spectra were recorded with Bruker Avance-300 and Avance-500 spectrometers.
IR spectra were recorded using a Nicolet Magna 560 FT-IR spectrometer. High-resolution
mass spectra (HRMS) were obtained on a JEOL JMS-AX505WA mass spectrometer. The
intensity of each peak in the mass spectrum relative to the base peak is reported in
parentheses. Optical rotations were obtained for CHCl solutions on a Perkin-Elmer 341
3
polarimeter, and their concentrations are expressed in g/100 mL. Manool resin was
2
4
D
purchased from Westchem Industries, Ltd. and purified to obtain (þ)-Manool, ½ꢁꢃ þ 28
(
c 1.5, CHCl ). THF, ether, DME and benzene were freshly distilled from
3
Na-benzophenone before use. All other solvents and reagents were obtained from
commercial suppliers and used without further purification. Merck silica gel (70–230
mesh ASTM) was used for column chromatography. TLC was performed on Analtech
silica gel 60 G254 and the spots were observed either by exposure to iodine or by UV light.
All organic extracts were dried over Na SO and evaporated under reduced pressure
2
4
ꢁ
below 60 C.
3
.2. Labda-8(17),13(Z)-diene-15,16-diol (10)
To a suspension of LiAlH (37.43 mg, 0.98 mmol) in dry THF (5 mL), peroxide 9 (0.250 g,
4
ꢁ
0
.82 mmol) in THF (4 mL) was added dropwise at 0 C. This mixture was refluxed for 2 h,
then water was added and extracted with ether. The solvent was evaporated under pressure

Natural Product Research
897
and the product was chromatographed over silica gel and when eluted with 50% ether in
ꢁ
hexane, which afforded diol 3 (0.241 g, 96%) as white crystals (hexane): m.p. 119–120 C;
2
4
D
ꢁ
ꢁ
½
ꢁꢃ ¼ þ40 (c ¼ 1.0, CHCl ), (Matsuda et al., 2002; Morikawa et al., 2002) [ꢁ] ¼ 42
3
1
D
ꢀ
(
c ¼ 0.39, CHCl ); ꢂ /cm (KBr) 3602, 3079, 1642 and 900; ꢃ (300 MHz; CDCl ) ꢃ 0.65,
3
max
H
3
0
1
.77, 0.84 (3H each, s, CH ), 4.15 (1H, d, J ¼ 12.9 Hz, H-16), 4.17 (1H, d, J ¼ 12.9 Hz, H-
3
6), 4.18 (2H, bd, H-15), 4.49 (1H, bs, H-17), 4.80 (1H, bt, H-17) and 5.57 (1H, bt,
J ¼ 6.9 Hz, H-14); ꢃ (75.45 MHz; CDCl ) 14.47 (C-20), 19.34 (C-11), 21.69 (C-19), 22.09
C
3
(
(
C-2), 24.41 (C-6), 33.58 (C-4), 33.58 (C-18), 34.63 (C-12), 38.31 (C-7), 39.06 (C-1), 39.67
C-10), 42.11 (C-3), 55.48 (C-5), 56.39 (C-9), 58.50 (C-16), 60.80 (C-15), 106.29 (C-17),
1
26.05 (C-14), 144.47 (C-13) and 148.56 (C-8); m/z (EI) 306.2558 (C H O requires
20 34 2
3
06.2560), 288 (20), 205 (75), 177 (65), 137 (100), 98 (86).
3
.3. Labda-8(17),13(14)-dien-15,16-olide (5)
Method A: Diol 3 (0.110 g, 0.36 mmol) was dissolved in dichloromethane (3 mL)
˚
containing both 4 A molecular sieves (0.200 g) and N-methylmorpholine N-oxide
(
63.27 mg, 0.54 mmol). After stirring the mixture for 10 min., tetra-n-propylammonium
perruthenate (6.3 mg, 0.018 mmol) was added and the reaction was followed by TLC until
complete. After the usual work-up, the crude product (0.105 g) was obtained. The NMR
spectrum indicated that it consisted of a mixture of lactones 5 and 11. Further purification
over silica gel failed.
Method B: To a suspension of silver carbonate-celite (37.43 mg, 0.98 mmol) in
dry benzene (3 mL) was added diol 10 (0.100 g, 0.32 mmol) in benzene (2 mL) at
room temperature. The reaction mixture was filtered through silica gel and the filtrate
was evaporated. The resulting crude product was chromatographed over silica gel and
eluted with 50% ether in hexane, which afforded lactone 5 (95 mg, 96%), which after
ꢁ
24
D
ꢁ
recrystallisation from hexane showed: m.p. 76–78 C; ½ꢁꢃ ¼ 39 (c ¼ 1.4, CHCl ),
3
ꢁ
ꢀ1
(
and 1630; ꢃ (300 MHz; CDCl ) 0.67, 0.78, 0.85 (3H each, s, CH ), 4.42 (1H, br s, H-17),
Nakano et al., 1982)] [ꢁ] ¼ þ41 (c ¼ 1.2, CHCl ); ꢂ /cm (KBr) 1775, 1740, 1640
D
3
max
H
3
3
4
.66 (1H, dd, J ¼ 17.3 and 1.7 Hz, H-16), 4.72 (1H, dd, J ¼ 17.3 and 1.7 Hz, H-16), 4.84
(
1H, br s, H-17), 5.81 (1H, t, J ¼ 1.6 Hz, H-14); ꢃ (75.45 MHz; CDCl ) 14.36, 19.24, 21.20,
C
3
2
1
1.63, 24.32, 27.45, 33.51, 33.51, 38.14, 39.09, 39.70, 41.93, 55.42, 56.06, 73.08, 106.37,
15.07, 147.84, 171.06 and 174.17; m/z (EI) 302.2258 (C H O requires 302.2255), 287
2
0
30
2
(
15), 206 (34), 137 (71), 109 (100), 98 (92).
3
.4. 5-(2-Bromo-ethyl)-1,1,4a-trimethyl-6-methylene-decahydro-naphthalene (13)
To a solution of alcohol 12 (0.2 g, 0.84 mmol) in THF (6 mL) was added PPh (0.88 g,
3
3
.36 mmol) and CBr (0.83 g, 2.50 mmol), and the whole mixture was stirred for 20 min at
4
room temperature. The reaction mixture was diluted with brine and extracted with ether.
The solvent was evaporated under reduced pressure and the product was chromato-
graphed over silica gel and eluted with hexane, which afforded bromide 13 (0.21 g, 83%) as
colourless oil; ꢃH (300 MHz; CDCl ) 0.67, 0.78, 0.86 (3H each, s, CH ), 2.38 (1H, ddd,
3
3
J ¼ 13, 4, 2 Hz), 3.25 (1H, ddd, J ¼ 8.6, 8, 7 Hz, H-12), 3.51 (1H, ddd, J ¼ 8.6, 8, 4 Hz,
H-12), 4.45 (1H, br s, H-17), 4.82 (1H, br s, H-17); ꢃ (75.45 MHz; CDCl ) 14.81, 19.4,
C
3
2
1
1.8, 24.42, 28.06, 33.61, 33.61, 33.75, 38.22, 39.10, 39.64, 42.11, 55.33, 55.42, 106.20 and
47.71; m/z (EI) 298.1299 (C H Br requires 298.1296).
1
6
27

8
98
J. Villamizar et al.
3
.5. 15,16-Epoxy-8(17),13(16),14-labdadiene (14)
ꢁ
To a cooled solution of the 3-bromofuran (0.163 g, 1.1 mmol) in dry THF (3 mL) at ꢀ78 C
was added n-butyllithium (0.6 mL, 1.6 M in hexane). The resulting brown solution was
ꢁ
stirred for 10 min at ꢀ78 C and then a solution of bromide 13 (0.121 g, 0.51 mmol) in THF
ꢁ
(
H O was added at room temperature with additional stirring for 30 min. The product was
2 mL) was added dropwise. After this mixture had been stirred for 2 h at ꢀ78 C, excess
2
extracted with ether, dried, and the solvent was evaporated under reduced pressure and the
product was chromatographed over silica gel and eluted with 5% diethyl ether in hexane,
2
D
4
ꢁ
which afforded furanolabdane 14 (0.192 g, 93%) as colourless oil; ½ꢁꢃ ¼ þ23 (c ¼ 2.0,
ꢁ
CHCl ), (Dimas et al., 1998; Itokawa et al., 1988) [ꢁ] ¼ 22 (c ¼ 0.14, CHCl ); ꢂ /
max
cm (KBr) 3050, 1635, 1495, 870 cm ; ꢃ (300 MHz; CDCl ) 0.67, 0.78, 0.85 (3H each, s,
3
D
3
ꢀ
1
ꢀ1
H
3
CH ), 2.23 (1H, m, H-12), 2.39 (1H, m, H-7), 2.54 (1H, m, H-17), 4.55 (1H, bs, H-17), 4.84
3
(
(
1H, bs, H-17), 6.25 (1H, bs, H-14), 7.18 (1H, bs, H-16) and 7.33 (1H, t, H-15); ꢃC
75.45 MHz; CDCl ) 14.47 (C-20), 19.36 (C-11), 21.70 (C-19), 23.59 (C-12), 24.05 (C-2),
3
2
5
4.42 (C-6), 33.55 (C-4), 33.55 (C-18), 38.30 (C-7), 38.98 (C-1), 39.56 (C- 10), 42.10 (C-3),
5.42 (C-5), 56.05 (C-9), 106.23 (C-17), 110.94 (C-14), 125.59 (C-13), 138.62 (C-16), 142.58
þ
(
(
(
C-15) and 148.51 (C-8); GC/MS m/z 286 (M , 31), 271 (9), 253 (3), 191 (27), 135 (26), 95
100), 67 (18) and 41 (13); m/z (EI) 286.2290 (C H O requires 286.2299), 271 (9), 191
27), 137 (80), 95 (100), 67 (18).
2
0
30
3
.6. Labda-8(17),13-diene-15-hydroxy-15,16-olide 6 and Labda-8(17),13-diene-16-
hydroxy-15,16-olide (7)
Method A: A solution of labdafuran (14) (0.105 g; 0,37 mmol) in THF (10 mL), containing
ꢁ
Rose Bengal (1 mg), was irradiated at 0 C with an external 150 W halogen–tungsten lamp
for 2 h, during which time oxygen was bubbled through the reaction mixture. The solvent
was evaporated under reduced pressure and the residue chromatographed over silica gel
and eluted with 15% ethyl acetate in hexane, which afforded a mixture of compounds 6
and 7, as a colourless oil (as evidenced by the NMR spectrum). Crystallisation from
ꢁ
24
D
ꢁ
hexane gave pure compound 7 (25 mg; 21%): m.p. 89–91 C; ½ꢁꢃ ¼ þ48 (c ¼ 0.7; CHCl ).
3
ꢀ
/cm (KBr) 3364, 3015, 1645, 1762; ꢃH (300 MHz; CDCl ) 0.67 (3H, s, CH ); 0.78
3 3
1
ꢂ
max
(
3H, s, CH ); 0.85 (3H, s, CH ); 1.31 (1H, m, H-6); 1.72 (1H, m, H-6); 1.93 (1H, m, H-7);
3 3
2
bs, H-16); ꢃ (75.45 MHz; CDCl ) 14.41 (C-20); 19.31 (C-11); 20.82 (C-2); 21.69 (C-19);
.37 (1H, m, H-7); 4,45 (1H, bs, H-17); 4.84 (1H, bs, H-17); 5.83 (1H, s, H-14); 5.95 (1H,
C
3
2
4
4.39 (C-6); 26.76 (C-12); 33.57 (C-4); 33.57 (C-18); 38.20 (C-7); 39.14 (C-10); 39.78 (C-1);
2.04 (C-3); 55.49 (C-5); 56.33 (C-9); 99.02 (C-16); 106.51 (C-17); 117.14 (C-14); 147.91
(
C-8); 170.41 (C-13); 171.57 (C-15); m/z (EI) 318.1970 (C H O requires 318.2193) (23),
20 30 3
3
00 (15), 204 (19), 177 (25), 137 (100), 95 (37).
Chromatography of the mother-liquid of compound 6 over silica gel with 15% ethlyl
2
4
ꢁ
acetate in hexane afforded pure compound 6 (37.6 mg, 32%) as an oil; ½ꢁꢃ ¼ þ30
D
ꢀ1
(
s, CH ); 0.77 (3H, s, CH ); 0.84 (3H, s, CH ); 4.51 (1H, bs, H-17); 4.83 (1H, bs, H-17); 6.08
c ¼ 0.6; CHCl ); ꢂ /cm (KBr) 3426, 3015, 1643, 1763; ꢃ (300 MHz; CDCl ) 0.65 (3H,
3
max
H
3
3
3
3
(
(
(
(
1H, bs, H-15); 6.81 (1H, d, J ¼ 1.5 Hz, H-14); ꢃ (75.45 MHz; CDCl ) 14.41 (C-20); 19.32
C
3
C-2); 21.47 (C-12); 21.69 (C-19); 24.38 (C-6); 24.48 (C-11); 33.56 (C-4); 33.56 (C-18); 38.21
C-7); 39.11 (C-1); 39.70 (C-10); 42.08 (C-3); 55.49 (C-5); 56.45 (C-9); 97.08 (C-15); 106.56
C-17); 139.02 (C-13); 142.85 (C-14); 147.97 (C-8); m/z (EI) 318.2284 (C H O requires
2
0
30
3
3
18.2193) (20), 303 (21), 177 (23), 137 (100), 95 (37).

Natural Product Research
899
Method B: A solution of labdafuran (14) (0.1 g; 0.35 mmol) in CH Cl (10 mL),
2
2
containing meso-tetraphenylporphine (1 mg) and diisopropylamine (10 eq), was
ꢁ
irradiated at ꢀ78 C with an external 150 W halogen–tungsten lamp for 2 h, during
which time oxygen was bubbled through the reaction mixture. The solution was
ꢁ
warmed to 23 C, and saturated aqueous oxalic acid (3 mL) was added. After 30 min of
vigorous stirring, water (15 mL) and CH Cl –methanol (3 : 1, 50 mL) were added to the
2
2
colourless mixture, and the aqueous portion was extracted with CH Cl –methanol (3 : 1,
2
2
2
ꢄ 50 mL). The solvent was evaporated under reduced pressure and the residue was
chromatographed over silica gel. Crystallisation from hexane gave pure compound 7
60.1 mg, 54%) as white crystals. Chromatography of the mother-liquid of compound
over silica gel with 15% ethlyl acetate in hexane afforded the pure compound 6
5 mg, 5%).
(
6
(
3
.7. Determination of the antimalarial activity
.7.1. Inhibition of the b-haematin formation
3
The inhibition of the ꢀ-haematin formation was followed according to a slightly modified
method of Baelmans, Deharo, Munoz, Sauvain and Gisnburg (2000). Briefly, a solution of
˜
ꢀ
1
hemin chloride (50 mL; 5.2 mg mL ) was distributed in 96-well micro plates. Different
concentrations of the compounds dissolved in DMSO (5–100 mM), were added in
triplicate in test wells (50 mL) to a final concentration of 25–0.25 mM. Controls contained
either water (50 mL), DMSO (50 mL) or chloroquine (50 mL, 2.5 mM). ꢀ-Haematin
formation was initiated by the addition of acetate buffer (100 mL 0.2 M, pH 4.4).
ꢁ
Plates were incubated (37 C ꢄ 48 h) to allow completion of the reaction and centrifuged
(
4000 RPM ꢄ 15 min, IEC-CENTRA, MP4R). After discarding the supernatant, the pellet
was washed twice with DMSO (200 mL) and finally dissolved in NaOH (200 mL, 0.2 N).
The solubilised aggregates were further diluted 1 : 2 with NaOH (0.1 N) and absorbance
recorded at 405 nm (Microplate Reader, BIORAD-550). The results were expressed as a
percentage of inhibition of ꢀ-haematin formation.
3
.7.2. Experimental host and strain maintenance
Male albino mice (National Institute of Hygiene strain), weighing 18–22g, were
maintained on a commercial pellet diet and housed under conditions approved by the
Ethics Committee of the Faculty of Pharmacy, Central University of Venezuela.
Plasmodium berghei (ANKA strain), a rodent malarial parasite, was used for infection.
7
Mice were infected by i.p. passage of 10 infected erythrocytes diluted in phosphate
buffered saline solution (PBS 10 mM, pH 7.4, 0.1 mL). Parasitemia was monitored by
microscopic examination of Giemsa-stained smears.
3
.7.3. Parasite extracts
Blood of infected animals at high levels of parasitemia (30–50%) was collected by
cardiac puncture with a heparinised syringe, and the blood was centrifuged
ꢁ
(
(
500 g ꢄ 10 min, 4 C). Plasma and buffy coat were removed, and the red blood cell
RBC) pellet was washed twice with chilled PBS–glucose (5.4%). The washed RBC
pellet was centrifuged on a discontinuous percoll gradient (80–70% percoll in
ꢁ
PBS–glucose, 20,000 g ꢄ 30 min, 4 C). The upper band (mature forms) was removed

9
00
J. Villamizar et al.
by aspiration, collected in Eppendorf tubes and washed twice with chilled PBS–glucose,
and the infected RBC were lysed with the non-ionic detergent saponin (0.1% in
PBS ꢄ 10 min). Cold PBS (1 mL) was added, and the samples were centrifuged
ꢁ
(
cyte haemoglobin). The free parasites were mixed with PBS–glucose (5.4%) and
13,000 g ꢄ 5 min, 4 C) to remove erythrocyte cytoplasmic content (including erythro-
ꢁ
ꢁ
subjected to three freeze–thaw cycles (ꢀ70 C/þ37 C). The final homogenate was used
in the haemoglobin proteolysis assay.
3
.7.4. Mice native haemoglobin
Native haemoglobin from non-infected mice was obtained by treating one volume of pellet
erythrocytes with two volumes of water. The resulting lysate was used as the substrate in
the haemoglobin proteolysis assay.
3
.7.5. Inhibition of globin proteolysis
To assay the proteolytic activity of a trofozoite-rich extract, we incubated in a 96-well
tissue culture plate (Greiner Bio-One): mice native haemoglobin (10 mL), parasite
extract (50 mL), GSH (10 mL, 10 mM), and acetate buffer (0.2 M, pH 5.4) to a final
volume of 100 mL. The compounds (5 mM) were incorporated in the incubation mixture
ꢁ
dissolved in DMSO. After incubation (at 37 C ꢄ 18 h) the reactions were stopped by
the addition of reduced sample buffer (upper buffer 25%, SDS 2%, ꢀ-mercaptoethanol
5
%, glicerol 20%, bromophenol blue 0.0025%). The degree of digestion was
evaluated electrophoretically by 15% SDS-PAGE (Rosenthal, 1995) followed by
densitometric comparison of globin bands. DMSO, leupeptin and pepstatin controls
were electrophoresed at the same time.
3
.7.6. Four day suppressive test
The compounds were also tested in a malaria murine model according to Peters and
7
Robinson (1999). Briefly, NIH male albino mice (18–23 g) were infected i.p. with 10 red
blood cells infected with P. berghei (n ¼ 6). Compounds were dissolved in DMSO (0.1 M),
diluted with Saline–Tween 20 solutions (2%). Two hours after infection, each compound
ꢀ
1
was administered once i.p. (20 mg kg ) for four consecutive days. On day 4, the
parasitemia was counted by examination of Giemsa-stained smears. Chloroquine
ꢀ
1
25 mg kg ) was used as a positive control. The results were expressed as the percentage
(
of parasitemia at the fourth day post-infection and were compared to the values reported
by non-treated mice.
Data were statistically analysed using unpaired t-tests for specific group comparisons;
assuming 95% confidence according GraphPad Prism 3.02.
Acknowledgements
The authors thank to CDCH (PG-CDCH-06.30.5125-2003) and Ms Diana Lourenco for support in
the antimalarial evaluation. Decanato de Estudios Profesionales and Decanato de Investigacio
Desarrollo of Universidad Simon Bolıvar for the financial support. We thank Professor Julio
Herrera (Universidad Simon Bolıvar) for the measurement of mass spectra.
´
n y
´
´
´
´

Natural Product Research
901
References
Baelmans, R., Deharo, E., Mun
˜
oz, E., Sauvain, M., & Gisnburg, H. (2000). Experimental conditions
for testing the inhibitory activity of chloroquine on the formation of ꢀ-hematin. Experimental
Parasitology, 96, 243–248.
Bodeker, G., & Willcox, M. (2000). New research initiative on plant-based antimalarials. Lancet,
3
55, 761.
Dimas, K., Demetzos, C., Marsellos, M., Sotiriadou, R., Malamas, M., & Kokkinopoulos, D.
1998). Cytotoxic activity of labdane type diterpenes against human leukemic cell lines in vitro.
(
Planta Medica, 64, 208–211.
Feringa, B.L. (1987). Photooxidation of furans. Recueil des Travaux Chimiques des Pays-Bas, 106,
469–488.
Fe
´
tizon, M., Golfier, M., & Louis, J.-M. (1969). Oxidations by silver carbonate on celite XIII.
Preparations of lactones. Tetrahedron, 31, 171–176.
Fitch, C.D., Chevli, R., Banyal, H.S., Phillips, G., Pfaller, M.A., & Krogstad, D.J. (1982). Lysis of
Plasmodium falciparum by ferriprotoporphyrin IX and a chloroquine-ferriprotoporphyrin IX
complex. Antimicrobial Agents and Chemotherapy, 21, 819–822.
Goldberg, D.E. (1992). Plasmodial hemoglobin degradation: An ordered pathway in a specialized
organelle. Infectious Agents and Disease, 1, 207–211.
Hanson, J.R. (1999). Diterpenoids. Natural Product Reports, 16, 209–219.
Hanson, J.R. (2000). Diterpenoids. Natural Product Reports, 17, 165–174.
Itokawa, H., & Morita, H. (1988). Cytotoxic and antifungal diterpenes from the seeds of Alpinia
galanga. Planta Medica, 54, 117–120.
Itokawa, H., Morita, H., Katou, T., Takeya, K., Cavalheiro, A.J., de Oliveira, R.C.B., et al. (1988).
Cytotoxic diterpenes from the rhizomes of Hedychium coronarium. Planta Medica, 54,
3
11–315.
Jung, M., Ko, I., & Lee, S. (1998a). A concise conversion of (-)-sclareol into (þ)-coronarin E and
-)-7-epi-coronarin A. Journal of Natural Products, 61, 1394–1396.
(
Jung, M., Ko, I., Lee, S., Choi, S.J., Youn, B.H., & Kim, S.K. (1998b). A concise synthesis and
in vitro cytotoxicity of new labdane diterpenes. Bioorganic & Medicinal Chemistry Letters, 8,
3295–3298.
Kernan, M.R., & Faulkner, D.J. (1988). Regioselective oxidation of 3-alkylfurans to 3-alkyl-4-
hydroxybutenolides. Journal of Organic Chemistry, 53, 2773–2776.
Ley, S.V., Norman, J., Griffith, W., & Marsden, S. (1994). Tetrapropylammonium perruthenate,
þ
ꢀ
Pr
Matsuda, H., Morikawa, T., Sakamoto, Y., Toguchida, I.,
4
N RuO , TPAP: A catalytic oxidant for organic synthesis. Synthesis, 7, 639–665.
&
4
Yoshikawa, M. (2002).
Antiinflammatory principles and three new labdane-type diterpenes, hedychilactones A, B,
and C, from the rhizome of Hedychium coronarium Koeng. Heterocycles, 56, 45–50.
Meshnick, S.R., Taylor, T.E., & Kamchonwongpaisan, S. (1996). Artemisinin and the antimalarial
endoperoxides: From herbal remedy to targeted chemotherapy. Microbiological Review, 60,
301–315.
Morikawa, T., Matsuda, H., Sakamoto, Y., Ueda, K., Toguchida, I., & Yoshikawa, M. (2002). New
farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, and inhibitors of degranula-
tion in RBL-2H3 cells from the rhizome of Hedychium coronarium. Chemical and
Pharmaceutical Bulletin, 50, 1045–1049.
Mu
¨
ller, M., Schro
Letters, 39, 4655–4656.
Nakano, T., Martin, A., & Rojas, A. (1982). Synthesis of labda-8(17),13(14)-dien-15,16-olide and
5,16-epoxylabda-8(17),13(16),14-triene and their rearrangements to clerodane derivatives.
Tetrahedron, 38, 1217–1219.
¨
der, J., Magg, Ch., & Seifert, K. (1998). Synthesis of (þ)-coronarin E. Tetrahedron
1

9
02
J. Villamizar et al.
Olliaro, P., Haynes, R., Meunier, B., & Yuthavong, Y (2001). Possible modes of action of the
artemisinin-type compounds. Trends in Parasitology, 14, 122–126.
Orijh, A.U., & Fitch, C.S. (1993). Hemozoin production by Plasmodium falciparum: Variation with
strain and exposure to chloroquine. Biochimica et Biophysica Acta, 1157, 270–274.
Peters, W., & Robinson, B.L. (1999). Chapter 92 – Malaria. In O. Zack & M. Sande (Eds.),
Handbook of animal models of infection (pp. 757–773). London: Academic Press.
Ridley, R.G., Hofheinz, W., Matile, H., Jaquet, C., Dorn, A., Masciadri, S., et al. (1996).
4
-aminoquinoline analogs of chloroquine with shortened side chains retain activity against
chloroquine-resistant Plasmodium falciparum. Antimicrobial Agents and Chemotherapy, 40,
846–1854.
1
Rosenthal, P.J. (1995). Plasmodium falciparum: Effects of proteinase inhibitors on globin hydrolysis
by cultured malaria parasites. Experimental Parasitology, 80, 272–281.
Scio, E., Alves, T.M.A., Kretli, A.U., Soares, L., Shin, G.Y., Cordell, G.A., et al. (2002).
Antiplasmodial and antitumoral activity of labdanes from Alomia miryadenia (Asteraceae).
In 22nd IUPAC Symposium on the Chemistry of Natural Products, Sao Carlos, CP, Brazil,
4–8 September.
Scio, E., Ribeiro, A., Alves, T.M.A., Romahna, A.J., de S. Filho, J.D., Cordell, G., et al. (2003).
Diterpenes from Alomia myriadenia (Asteraceae) with cytotoxic and trypanocidal activity.
Phytochemistry, 64, 1125–1131.
Villamizar, J., Fuentes, J., Salazar, F.E., Tropper, E., & Alonso, R. (2003). Facile access to optically
active labdane-type diterpenes from (þ)-manool. Synthesis of (þ)-coronarin E, (þ)-15,16-
epoxy-8(17),13(16),14-labdatriene, and (þ)-labda-8(17),13(Z)-diene-15,16-diol. Journal of
Natural Products, 66, 1623–1627.
WHO Report (1999). Rolling back malaria (pp. 49–63). World Health Organization: Geneva,
Switzerland.