Antimalarial activity of abietane ferruginol analogues possessing a phthalimide group

Article abstract of DOI:10.1016/j.bmcl.2014.09.061

The abietane-type diterpenoid (+)-ferruginol, a bioactive compound isolated from New Zealand's Miro tree (Podocarpus ferruginea), displays relevant pharmacological properties, including antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and anticancer. Herein, we demonstrate that ferruginol (1) and some phthalimide containing analogues 2-12 have potential antimalarial activity. The compounds were evaluated against malaria strains 3D7 and K1, and cytotoxicity was measured against a mammalian cell line panel. A promising lead, compound 3, showed potent activity with an EC₅₀ = 86 nM (3D7 strain), 201 nM (K1 strain) and low cytotoxicity in mammalian cells (SI > 290). Some structure-activity relationships have been identified for the antimalarial activity in these abietane analogues.

Full text of DOI:10.1016/j.bmcl.2014.09.061

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5234–5237
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimalarial activity of abietane ferruginol analogues possessing
a phthalimide group
b
b
b,
Miguel A. González a, , Julie Clark , Michele Connelly , Fatima Rivas
⇑
⇑
a Departamento de Química Orgánica, Universidad de Valencia, 46100 Burjassot, Valencia, Spain
b Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The abietane-type diterpenoid (+)-ferruginol, a bioactive compound isolated from New Zealand’s Miro
tree (Podocarpus ferruginea), displays relevant pharmacological properties, including antimicrobial,
cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory
and anticancer. Herein, we demonstrate that ferruginol (1) and some phthalimide containing analogues
2–12 have potential antimalarial activity. The compounds were evaluated against malaria strains 3D7
and K1, and cytotoxicity was measured against a mammalian cell line panel. A promising lead, compound
3, showed potent activity with an EC50 = 86 nM (3D7 strain), 201 nM (K1 strain) and low cytotoxicity in
mammalian cells (SI > 290). Some structure–activity relationships have been identified for the antimalar-
ial activity in these abietane analogues.
Received 11 August 2014
Revised 19 September 2014
Accepted 22 September 2014
Available online 28 September 2014
Keywords:
Antimalarial
Abietane
Diterpene
Ferruginol
Ó 2014 Elsevier Ltd. All rights reserved.
Dehydroabietylamine
Parasitic diseases affect about 30% of the world’s population.1
Among parasitic diseases, malaria is one of the most devastating
infectious diseases claiming more lives than any other parasitic
infections. Malaria is endemic to more than 100 nations and
remains one of the main leading causes of death in children less
than five years of age worldwide.1 A drug cocktail regimen is rec-
ommended for the treatment of malaria to prevent the develop-
ment of resistance, particularly to artemisinin, one of the most
effective components of this protocol. The rise of resistance to arte-
misinin combination therapy is an eminent threat, and resistance
has already been reported in various Southeast Asian countries.2
Hence, there is urgency in identifying new antimalarial agents, par-
ticularly new molecular scaffolds with potential novel mechanisms
of actions.
products have a wide range of biological activities. Ferruginol (1)
(Fig. 1) was first isolated in 1939 from New Zealand’s Miro tree
(Podocarpus ferruginea), and has been extensively studied because
of intriguing chemical framework and promising biological proper-
ties.6 Ferruginol (1) occurs in plants belonging to the Podocarpa-
ceae, Cupressaceae, Lamiaceae and Verbenaceae families among
others. This diterpene exhibited important bioactivities, such as
antimicrobial,7 cardioactive,8 antioxidative,9 antileishmanial and
nematicidal,10 and antiulcer properties.11 In addition, ferruginol
was active against prostate cancer,12 and anti-inflammatory activ-
ity.13 Several reports on antimalarial activity have also been
described. In 2003, a report described that ferruginol (1) displayed
significant (IC50 < 1
lg/mL) in vitro antiplasmodial activity against
a
chloroquine-resistant (K1) and -sensitive (D10) strain of
Natural products have played a dominant role in the drug dis-
covery efforts for the treatment of human diseases.3 In fact, most
antimalarial chemotherapeutic agents are based on natural prod-
ucts, indicating the potential of identifying new therapeutic leads
from plant sources. Chemodiversity continues to be an important
task in the search for antimalarial drugs.4
Plasmodium falciparum, and low cytotoxicity (SI > 65) against two
mammalian cell lines (CHO and HepG2).14 In 2006, antimalarial
activity for ferruginol (1) was determined using the D6 (chloro-
quine-sensitive) clone of Plasmodium falciparum. Promising antima-
larial activity was shown by an IC50 of 1.95 l
g/mL.15 In 2008,
The abietane diterpenoids are widely produced by conifers
belonging to the families Araucariaceae, Cupressaceae, Pinaceae,
and Podocarpaceae, and also angiosperm species.5 These natural
⇑
Corresponding authors. Tel.: +34 96 3543880; fax: +34 96 3544328 (M.A.G.);
tel.: +1 901 595 6504; fax: +1 901 595 5715 (F.R.).
E-mail addresses: Miguel.A.Gonzalez@uv.es (M.A. González), Fatima.rivas@
stjude.org (F. Rivas).
Figure 1. Ferruginol (1).
http://dx.doi.org/10.1016/j.bmcl.2014.09.061
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

M. A. González et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5234–5237
5235
Muhammad and co-workers reported antimalarial activity against
D6 (chloroquine-sensitive, IC50 = 4.2 g/mL) and W2 (chloro-
The compounds were obtained in enantiomerically pure form
from commercially available (+)-dehydroabietylamine as outlined
in Scheme 1.19,22 The synthesis starts with the introduction of
the phthalimide group on (+)-dehydroabietylamine to give
compound 2 in 96% yield. Then, Friedel–Crafts acylation of 2 gave
acetophenone 3 (88% yield), which was oxidized under Baeyer–
Villiger conditions to afford acetate 4 in 85% yield (Scheme 1).
Hydrolysis of the acetate in 4 gave phenol 5 in high yield, while
overall deprotection of 4 afforded the amino-phenol 6 in 75% yield.
Compound 6 was the intermediate of two separate approaches.
Firstly, oxidative deamination of 6 gave 18-oxoferruginol (7) in
moderate yield (50%), which was converted into ferruginol (1) by
Wolff–Kishner reduction (90% yield). Secondly, the treatment of 6
with tetrachlorophthalic anhydride (TCPA) afforded phenol 8 in
75% yield, which was acetylated with acetic anhydride in pyridine
to give acetate 9 in quantitative yield (Scheme 1). Acetate 9 was
oxidized at C-7 with excess of t-BuOOH as oxidant and CrO3/pyri-
dine mixture as a catalyst in DCM, the yield of ketone 10 was 66%.
Subsequently, the reaction of 10 with p-tosylhydrazide yielded the
corresponding p-tosylhydrazone 11 (77% yield). The fullerene–
terpenoid hybrid 12 was firstly reacted with NaOMe in anhydrous
l
quine-resistant, IC50 = 3.5 lg/mL) strains of P. falciparum for ferrug-
inol (1).10 Recently, an additional report confirmed significant
antimalarial activity against K1 strain (chloroquine-resistant,
IC50 = 0.9
lM), showing moderate selectivity index (SI = 15.6 (in
L6 cells)).16
To further explore the potential of ferruginol (1) as antimalarial
candidate, and in continuation of our research programs to dis-
cover bioactive terpenoids and new scaffolds as potential antima-
larial agents,17,18 we developed
a focused compound library
based on (+)-ferruginol (1) using a short, and efficient synthetic
strategy from commercially available (+)-dehydroabietylamine
(DHAA) (Scheme 1).19 With a limited number of atoms that could
be manipulated, it was decided to evaluate a series of phthali-
mides, which have been shown to possess antiparasitic proper-
ties.20
A fullerene terpenoid hybrid was also envisaged as
potential inhibitor of Plasmodium falciparum carbonic anhydrases,
which has been demonstrated that also inhibits the growth of
the pathogen.21 Herein, we report the evaluation of (+)-ferruginol
(1), and its analogues 2–12 against two malaria strains.
Scheme 1. Synthesis of ferruginol analogues 2–12.

5236
M. A. González et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5234–5237
Table 1
Antiplasmodial activity and cytotoxicity (EC50
-l
M) of ferruginol (1) and compounds 2–12 on P. falciparum 3D7 strain, K1 strain, and HepG2, RAJI, BJ, and HEK293 cell lines
Compound
P. falciparuma
Cell linesb
3D7
K1
HepG2
RAJI
EC50
BJ
HEK293
c
c
c
c
c
c
EC50
SId
EC50
SId
EC50
EC50
EC50
1
2
3
4
5
6
7
8
9
10
11
12
2.47
1.36
0.086
2.09
15–50
6.67
15–50
15–50
15–50
15–50
15–50
15–50
0.023
—
4.6
1.33
8.6
n.c.
>124
n.c.
>6.3
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
<2.0
<2.0
11.49
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.24
<2.0
19.57
>25
>25
>25
>25
17.43
>25
>25
>25
>25
>25
>25
>25
0.21
<2.0
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.21
<2.0
>18.4
>290
>12.0
n.c.
2.6
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
—
15–50
0.201
15–50
4.0
15–50
15–50
15–50
15–50
15–50
15–50
15–50
0.69
Chloroquine
Gambogic acid
Staurosporine
—
—
—
—
—
—
a
b
c
3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant).
BJ, normal human foreskin fibroblast; HEPG2, human liver carcinoma cell line; HEK293, human embryonic kidney cell line; RAJI, Burkitt lymphoma cell line.
EC50 M): concentration corresponding to 50% growth inhibition of the parasite or cells.
Selectivity index (SI): EC50 lower values of cytotoxic activity/EC50 values of antimalarial activity. N.C.: not calculated.
(l
d
pyridine for 20 min at room temperature, then, a solution of C60 in
chlorobenzene was added and the mixture was stirred for 24 h at
70 °C. This gave compound 12 in ca. 30% yield. Attempts to hydro-
lyze the acetate group in 12 in acidic conditions were unsuccessful.
The analogues can be divided into three groups: (a) precursors
such as N,N-phthaloyldehydroabietylamine (2) and 12-acetyl-N,
N-phthaloyldehydroabietylamine (3); (b) compounds possessing
a hydroxy group as ferruginol (1) itself and 12-hydroxy-N,N-phtha-
loyldehydroabietylamine (5), 12-hydroxy-dehydroabietylamine
(18-aminoferruginol, 6), 18-oxoferruginol (7), and 12-hydroxy-N,
N-(tetrachloro)phthaloyldehydroabietylamine (8); (c) compounds
possessing a acetate group such as 12-acetoxy-N,N-phthaloylde-
of outcome. The presence of an acetyl group at C-12 led to the most
active compound of the series. The presence of an acetate group at
C-12 led to a decrease in activity in the chloroquine-resistant strain
K1, however, it interestingly increased the activity in the chloro-
quine-sensitive strain 3D7. When the phthalimide group is chlori-
nated, the analogues showed EC50 values between 15 and 50 lM.
Also, if the amino group was left unprotected, there was a decrease
in activity against the chloroquine-resistant strain K1. Gratifyingly,
the dehydroabietylamine analogue 3 showed higher potency than
the parent compound, with an EC50 of 86 nM (3D7 strain) and
201 nM (K1 strain). This compound represents a new scaffold for
the development of antimalarial agents. These encouraging findings
warrant further structure activity relationship and target identifica-
tion studies.
hydroabietylamine
(4),
12-acetoxy-N,N-(tetrachlorophthaloyl)
dehydroabietylamine (9), 12-acetoxy-7-oxo-N,N-(tetrachloroph-
thaloyl)dehydroabietylamine (10), N,N-(tetrachlorophthaloyl)
dehydroabietylamine-p-tosylhydrazone (11), and 12-acetoxy-7-
C60-N,N-(tetrachlorophthaloyl)dehydroabietylamine adduct (12).
The antimalarial and cytotoxic activities of (+)-ferruginol (1)
and its synthetic analogues 2–12 (Scheme 1) were evaluated
against malaria strains 3D7 and K1, utilizing a previously described
assay.23 The effectiveness of the compounds on Plasmodium falcipa-
rum in culture were measured as the minimum drug concentration
(EC50) required to inhibit parasite growth by more than 50% during
72 h incubation period with test compounds.
Acknowledgments
Financial support from the Spanish Ministry of Science and
Education, under a ‘Ramón y Cajal’ research Grant, and also from
the Generalitat Valenciana is gratefully acknowledged. We are also
gratefully to the financial support of the American Lebanese Syrian
Associated Charities (ALSAC) at St. Jude Children’s Research
Hospital.
The compounds were also evaluated against a panel of mamma-
lian cell lines to evaluate cytotoxic properties. Only those com-
pounds with significant selectivity index are considered as
potential lead candidates. The results are shown in Table 1.
The inhibitory values against P. falciparum strains ranged from
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