Manadoperoxides, a new class of potent antitrypanosomal agents of marine origin

Article abstract of DOI:10.1039/c2ob26124c

Chemical investigation of the marine sponge Plakortis cfr. lita afforded a library of endoperoxyketal polyketides, manadoperoxides B-K (3-5 and 7-13) and peroxyplakoric esters B₃ (6) and C (14). Eight of these metabolites are new compounds and some contain an unprecedented chlorine-bearing THF-type ring in the side chain. The library of endoperoxide derivatives was evaluated for in vitro activity against Trypanosoma brucei rhodesiense and Leishmania donovani. Some compounds, such as manadoperoxide B, exhibited ultrapotent trypanocidal activity (IC₅₀ = 3 ng mL^-1) without cytotoxicity. Detailed examination of the antitrypanosomal activity data and comparison with those available in the literature for related dioxane derivatives enabled us to draw a series of structure-activity relationships. Interestingly, it appears that minor structural changes, such as a shift of the methyl group around the dioxane ring, can dramatically affect the antitrypanosomal activity. This information can be valuable to guide the design of optimized antitrypanosomal agents based on the dioxane scaffold.

Full text of DOI:10.1039/c2ob26124c

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PAPER
Manadoperoxides, a new class of potent antitrypanosomal agents of marine
origin†
Giuseppina Chianese,a Ernesto Fattorusso,a Fernando Scala,a Roberta Teta,a Barbara Calcinai,b
Giorgio Bavestrello,c Henny A. Dien,d Marcel Kaiser,e,f Deniz Tasdemirg and Orazio Taglialatela-Scafati*a
Received 12th June 2012, Accepted 6th July 2012
DOI: 10.1039/c2ob26124c
Chemical investigation of the marine sponge Plakortis cfr. lita afforded a library of endoperoxyketal
polyketides, manadoperoxides B–K (3–5 and 7–13) and peroxyplakoric esters B3 (6) and C (14). Eight of
these metabolites are new compounds and some contain an unprecedented chlorine-bearing THF-type
ring in the side chain. The library of endoperoxide derivatives was evaluated for in vitro activity against
Trypanosoma brucei rhodesiense and Leishmania donovani. Some compounds, such as manadoperoxide
B, exhibited ultrapotent trypanocidal activity (IC50 = 3 ng mL−1) without cytotoxicity. Detailed
examination of the antitrypanosomal activity data and comparison with those available in the literature for
related dioxane derivatives enabled us to draw a series of structure–activity relationships. Interestingly, it
appears that minor structural changes, such as a shift of the methyl group around the dioxane ring, can
dramatically affect the antitrypanosomal activity. This information can be valuable to guide the design of
optimized antitrypanosomal agents based on the dioxane scaffold.
parasites invade the central nervous system they lead to neuro-
logical symptoms, including behavioural changes, coma, and
1. Introduction
African trypanosomiasis (also called sleeping sickness) is a
human disease caused by the single-celled protozoan parasites
Trypanosoma brucei gambiense (in Western and Central Africa)
and T. b. rhodesiense (in Eastern and Southern Africa) and trans-
mitted to humans by bites of tsetse flies (Glossina genus), which
have acquired their infection from human beings or from
animals harbouring the human pathogenic parasites.1 Once
injected into the humans, trypanosomes multiply in subcu-
taneous tissues, blood and lymph and, in a second stage, they
cross the blood-brain barrier and attack the central nervous
system. The process can take years with T. b. gambiense, while
T. b. rhodesiense is responsible for acute infections. When the
ultimately, if untreated, death. Disturbance of the sleep cycle,
which gives the disease its name, is a characteristic feature of
this second stage of the disease.
Recent figures have estimated that sleeping sickness threatens
at least 60 million people in 36 countries with the number of
infections ranging between 50 000 and 70 000 per year, thus
qualifying it as the world’s third most devastating tropical
disease.2 The rural populations living in regions where trans-
mission occurs and which depend on agriculture, fishing, animal
husbandry or hunting are the most exposed to the bite of the
tsetse fly and therefore to the disease. Socio-political instability
and the consequent population movements, combined with a
shortage of funds have largely contributed to determining the
recent spread of trypanosomiasis, which, together with other tro-
pical diseases, results in billions of dollars of lost productivity,
contributing to determine the poverty and the social isolation of
these regions.
Treatment of African trypanosomiasis is still today based on
very few molecules, almost all introduced about one century
ago. Pentamidine and suramin are the preferred options for the
treatment of the first stage, while to treat the second, cerebral
stage, eflornithine and the trivalent arsenic derivative melarso-
prol are practically the only therapeutic options.3 Melarsoprol
has many disadvantages since it can induce a reactive encephalo-
pathy (encephalopathic syndrome) which can be fatal, while
eflornithine is very expensive and ineffective against
T. b. rhodesiense. Moreover, an increase of cross-resistance to
aDipartimento di Chimica delle Sostanze Naturali, Università di Napoli
“Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy.
E-mail: scatagli@unina.it; Fax: +(0039) 081-678552;
Tel: +(0039) 081-678509
bDipartimento di Scienze del Mare, Università Politecnica delle Marche,
Via Brecce Bianche, 60131 Ancona, Italy
cDipartimento per lo Studio del Territorio e delle sue Risorse,
Università di Genova, Corso Europa 26, 16132 Genova, Italy
dFaculty of Fishery and Marine Science, Sam Ratulangi University,
Manado, Indonesia
eDepartment of Medical Parasitology and Infection Biology, Swiss
Tropical and Public Health Institute, CH-4002 Basel, Switzerland
fUniversity of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
gSchool of Chemistry, National University of Ireland, Galway, University
Road, Galway, Ireland
†Electronic supplementary information (ESI) available: 1D and 2D
NMR spectra for compounds 7–14. See DOI: 10.1039/c2ob26124c
This journal is © The Royal Society of Chemistry 2012
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Fig. 1 Chemical structures of known compounds: plakortin (1), manadoperoxides A–D (2–5) and peroxyplakoric ester B3 (6).
these drugs has been observed in recent times, especially in
central Africa and, thus, the few available drugs are rapidly
losing their effectiveness.4 In recent years, only one compound,
pafuramidine (a synthetic analogue of pentamidine), has been
clinically evaluated against African trypanosomiasis, but further
development was discontinued in 2008 due to liver toxicity.5
Recently, the nitroimidazole derivative fexinidazole, bearing
structural similarities with metronidazole and nifurtimox, has
entered Phase II/III clinical trials6 and is the most advanced drug
candidate against trypanosomiasis. However, there is still an
urgent need to find new, effective and, above all, affordable
alternatives to the existing options for treatment of sleeping
sickness.
We have been actively engaged in the field of tropical disease
treatment and discovered that some classes of natural products
are endowed with promising activity against Plasmodium falci-
parum and Leishmania parasites.7–9 Among them, plakortin (1,
Fig. 1), a simple 1,2-dioxane polyketide obtained from the Car-
ibbean sponge Plakortis simplex7 was demonstrated to possess
significant in vitro antimalarial activity against CQ-resistant
P. falciparum strains without cellular toxicity.7 By means of a
multidisciplinary approach (including computational and experi-
mental studies) on natural analogues10,11 and semisynthetic
derivatives,12 we also obtained valuable information about the
SARs needed for the antimalarial activity of these simple 1,2-
dioxanes. These results clearly indicated the crucial role of the
endoperoxide functionality, suggested the importance of the
alkyl side chain, and revealed conformation-dependent features
critical for antimalarial activity.13 We recently developed two
different synthetic approaches for the preparation of simplified
plakortin analogues and some of the prepared compounds suc-
ceeded in reaching the in vitro antimalarial potency of
plakortin.14,15
potency (5–10 μM), more than ten times lower than that exhib-
ited by plakortin (1).
As part of our ongoing screening of natural sources to find
new antiprotozoal lead compounds, we have undertaken the
chemical analysis of the sponge Plakortis cfr. lita de Laubenfels,
a species widely distributed in the Indo-West Pacific,18 which
was collected along the coasts of the Bunaken Marine Park of
Manado (North Sulawesi, Indonesia). Careful inspection of the
organic extract of this organism afforded the known manadoper-
oxides B–D (3–5) and peroxyplakoric ester B3 (6) (Fig. 1), along
with eight new peroxyketal polyketides, which we named mana-
doperoxides E–K (7–13) and peroxyplakoric ester C (14)
(Fig. 2). Herein we provide details about their isolation, stereo-
structural determination and SARs, as well as in vitro trypanoci-
dal and leishmanicidal effects. The present study provides the
first data on the antitrypanosomal activity of endoperoxyketal
derivatives.
2. Results
2.1 Chemistry
The sponge Plakortis cfr. lita de Laubenfels (order Homosclero-
phorida, family Plakinidae) was collected in January 2010 from
along the coasts of Bunaken Island (Manado, Indonesia). After
homogenization, the organism was exhaustively extracted
sequentially with MeOH and CHCl3. The combined extracts
were subjected to chromatography over reversed-phase silica
column eluting with a solvent gradient of decreasing polarity
from water to chloroform. Selected fractions were combined and
further fractionated by gravity column chromatography (CC) on
silica gel, followed by repeated open CC and HPLC (n-hexane–
EtOAc mixtures) to afford the new polyketide manadoperoxides
E–K (7–13) and peroxyplakoric ester C (14) in the pure state,
along with the known manadoperoxides B–D (3–5)17 and perox-
yplakoric ester B3 (6).19 The structures of the known compounds
3–6 were deduced by comparing their spectroscopic data with
those previously reported,17,19 while the structures of the new
compounds 7–14 have been deduced as follows.
In this context, we have recently reported the results of the
chemical investigation of the Indonesian sponge Plakortis cfr.
simplex,16 which resulted in the isolation of four endoperoxyke-
tal derivatives named manadoperoxides A–D (2–5, Fig. 1),17
These compounds were assayed in vitro against CQ-R and CQ-S
P. falciparum strains and showed relatively poor antimalarial
7198 | Org. Biomol. Chem., 2012, 10, 7197–7207
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Fig. 2 Chemical structures of the new compounds manadoperoxides E–K (7–13) and peroxyplakoric ester C (14). Asterisks indicate that the
configuration of the moiety is relative.
Compounds 7–14 have been subjected to a detailed spectro-
metric (ESI-MS) and spectroscopic (mainly 1D and 2D NMR)
analysis, which allowed the definition of their stereostructures
(Fig. 2) and the complete assignment of NMR resonances
(Tables 1 and 2 or Experimental section). In particular, the NMR
analysis revealed that both the 13C NMR resonances from C-1 to
H-11 (δH 5.45, δC 127.1) with an oxymethine at H-10 (δH 4.40,
δC 70.1), is identical to that present in the structure of 5, while
the second spin system included a second oxymethine (H-13)
attached to a –CH2CH3 moiety. The HMBC cross-peaks of 12-
Me with C-11, C-12, and C-13 allowed the connection of the
above moieties and defined the gross structure of the side chain
(moiety B) for manadoperoxide E, which is a homologue of
manadoperoxide D. The ROESY cross-peak of 12-Me with
H-10 indicated that also in this case the side chain double bond
possessed E geometry. Following the same procedure already
applied for manadoperoxide D, we determined the absolute
configuration at the two stereogenic carbons C-10 and C-13 of 7
through application of the Riguera method for 1,n-diols.20 Thus,
two aliquots of 7 were allowed to react with R- and S-MTPA
chloride obtaining the diesters 7a and 7b, respectively. Accord-
ing to the Riguera model,20 in 1,4 MTPA diesters, the combined
shielding of the two auxiliary reagents induces positive Δδ(S- R)
values for the carbinol protons and for all the protons between
them. Thus, the positive Δδ(S- R) values measured for protons
going from H-9 to H-14 (Fig. 3) indicated a 10S,13S
configuration.
The molecular formula of manadoperoxide F (8), determined
by HR-ESIMS, was identical with that of compound 7,
suggesting the same C10H19O2 composition for the side chain.
However, analysis of 1D NMR spectra (Tables 1 and 2), together
with 2D COSY and HSQC NMR, showed that the moiety B of 8
significantly differed from that of 7 and included a methoxy
group (δH 3.17, δC 49.3), an sp3 oxymethine (δH 3.60, δC 73.6)
and two mutually coupled sp2 methines (δH 5.67, δC 126.8; δH
5.49, δC 132.2). The COSY spectrum arranged all the proton
multiplets of the side chain of 8 within two spin systems: the
first spans from H2-7 to the double bond Δ,10,11 while the second
spin system includes only the oxymethine (H-13) coupled with a
1
C-6, including 4-Me and 6-OMe (moiety A), and the H NMR
resonances and coupling constants from H2-2 to H2-5 were prac-
tically identical within the entire series of compounds 3–5 and
7–13. This spectroscopic evidence clearly indicated that the new
compounds 7–13 must share with the co-occurring manadoper-
oxides B–D (3–5) the structure of the six-membered dioxyge-
nated ring, including the relative configuration at the stereogenic
carbons present in this moiety (2D NMR ROESY cross-peaks
fully supported this stereochemical assignment). Since we have
previously assigned the absolute configuration at all the stereo-
genic carbons of manadoperoxides A–D (2–5) through chemical
derivatization and application of the Mosher’s method,17 it is
biosynthetically reasonable to assume that the configurations at
C-3, C-4 and C-6 reported for compounds 7–13 in Fig. 2 are
also the absolute ones. Thus, the new compounds 7–13 differ
from manadoperoxides A–D exclusively by the structure of the
long (“western”) side chain (moiety B) and we will here describe
in detail only the determination of this part of their structures.
Manadoperoxide E (7) was isolated as a colorless amorphous
solid with molecular formula C19H34O7, which, taking into
account the structure of the dioxane ring, implied that moiety B
should have a C10H19O2 composition and include a single unsa-
1
turation degree. The H and 13C NMR spectra of 7 (Experimen-
tal section), analyzed with the help of COSY and HSQC data,
revealed the presence of two spin systems which closely paral-
leled those present in the structure of manadoperoxide D (5). In
particular, the first spin system, from H2-7 to the sp2 methine
This journal is © The Royal Society of Chemistry 2012
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Table 1 1H NMR (500 MHz) data of compounds 8–10 and 12–13 in CDCl3
8
9
10
12
13
Pos.
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
2a
2b
3
4
5a
5b
7a
7b
8a
8b
9a
9b
10
11
12
13a
13b
14a
14b
15
4-Me
12-Me
1-OMe
6-OMe
12-OMe
2.91, dd (15.6, 9.5)
2.52, dd (15.6, 4.2)
4.37, ddd (9.5, 4.2, 3.0)
2.50, m
2.92, dd (15.5, 9.4)
2.43, dd (15.5, 4.5)
4.46, ddd (9.4, 4.5, 3.0)
2.57, m
2.92, dd (15.5, 9.5)
2.43, dd (15.5, 3.8)
4.46, ddd (9.5, 3.8, 3.0)
2.56, m
1.71a
1.32a
1.58a
1.55a
1.30a
2.92, dd (15.7, 9.5)
2.42, dd (15.7, 3.5)
4.44, ddd (9.5, 3.5, 3.0)
2.57, m
1.70a
1.32a
1.69a
1.32a
1.26a
2.94, dd (15.6, 9.4)
2.44, dd (15.6, 3.8)
4.44, ddd (9.4, 3.8, 3.0)
2.57, m
1.68, dd (13.3, 4.3)
1.68a
1.70a
1.39a
1.30a
1.31a
1.66a
1.63a
1.59a
1.62a
1.60a
1.55a
1.46, m
1.46, m
2.14, q (7.1)
1.53, m
1.47, m
1.44a
1.32a
1.76, m
1.32a
1.53a
1.30a
1.72a
1.59a
1.51a
5.67, dt (15.9, 7.1)
5.49, d (15.9)
3.08, m
3.20, d (1.7)
1.24a
1.68a
2.50, m
1.66a
1.41a
1.33a
3.92, m
3.50, d (7.4)
4.55, bs
3.60, q (6.4)
1.08, d (6.4)
2.08, s
4.30, t (7.3)
3.74, t (7.3)
2.19, dq (14.2, 7.1)
1.97, m
1.00, t (7.1)
0.84, d (7.0)
5.10, bs
5.29, bs
3.73, s
1.50a
1.33a
0.88, t (7.0)
0.85, d (7.0)
9.79, d (2.5)
3.73, s
1.02, t (7.2)
0.85, d (7.0)
1.25, s
3.73, s
3.28, s
0.87, d (7.0)
1.20, s
3.71, s
3.23, s
3.17, s
0.87, d (7.1)
3.73, s
3.27, s
3.28, s
3.28, s
3.33, s
a Overlapped with other signals.
Table 2 13C NMR data (125 MHz) of compounds 8–10 and 12–13 in
CDCl3
pharmacological evaluation, we could not attempt any determi-
nation of the configuration at the two stereogenic carbons of the
side chain.
8
9
10
δH, mult.
12
δH, mult.
13
δH, mult.
The molecular formula of manadoperoxide G (9) (C16H26O7)
was indicative of a C7H11O2 side chain, implying two unsatura-
Pos.
δC, mult.
δH, mult.
1
tion degrees. H and 13C NMR data of 9 (Tables 1 and 2), ana-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
173.0, s
31.0, t
80.1, d
27.2, d
34.2, t
103.7, s
32.4, t
23.4, t
172.4, s
31.2, t
79.9, d
27.4, d
34.6, t
102.8, s
32.0, t
22.5, t
36.5, t
57.6, d
59.5, d
205.8, s
24.6, q
171.9, s
31.1, t
80.1, d
26.9, d
33.9, t
103.0, s
32.1, t
21.8, t
30.1, t
28.7, t
32.2, t
43.5, d
32.0, t
22.8, t
14.2, q
16.9, q
201.1, d
51.7, q
48.5, q
171.9, s
30.9, t
79.6, d
26.9, d
34.0, t
102.8, s
33.5, t
23.5, t
30.0, t
116.2, s
59.7, d
141.6, s
83.7, d
26.1, t
10.4, q
17.0, q
105.6, t
52.0, q
48.5, q
172.3, s
30.8, t
80.3, d
27.1, d
34.6, t
103.1, s
33.2, t
22.8, t
30.8, t
79.0, d
68.2, d
82.4, s
84.3, d
25.5, t
17.0, q
17.3, q
14.0, q
51.9, q
48.4, q
53.1, q
lyzed with the help of the 2D HSQC spectrum, sorted out the
seven carbon atoms of this moiety as three methylenes, two
methines resonating at δH 3.08 and 3.20, one methyl singlet (δH
2.08) and a ketone carbonyl (δC 205.8). The 2D NMR COSY
spectrum grouped all the proton multiplets of the side chain in a
single spin system terminating with the oxymethines H-10 and
H-11. The relatively high field 13C NMR resonances of C-10
and C-11 (δC 57.6 and 59.5, respectively) were strongly sugges-
tive of the presence of an epoxide group, which also accounted
for the remaining unsaturation degree.
The 2D HMBC cross-peaks of the methyl singlet at δH 2.08
with the ketone carbonyl (C-12) and with the epoxide carbon
C-11 allowed the connection between the two moieties and the
definition of the planar structure of manadoperoxide G (9),
which can be viewed as an epoxidated manadoperoxide C (4).
The ROESY correlation between H-11 (δH 3.20) and H2-9 indi-
cated the trans orientation of the epoxide protons. It should be
noted that this relative configuration at C-10 and C-11 has not
been connected with that of the remaining stereogenic carbons
of the molecule.
32.6, t
126.8, d
132.2, d
80.7, s
73.6, d
16.6, q
4-Me
12-Me
1-OMe
6-OMe
12-OMe
16.0, q
14.3, q
52.3, q
47.7, q
49.3, q
16.3, q
52.0, q
48.5, q
methyl doublet (Me-14). The HMBC cross-peaks of the methyl
singlet at δH 1.20 (12-Me) with C-11 and the oxygenated C-12
(δC 80.7) and C-13 (δC 73.6) and of the methoxy protons with
C-12, determined the connection for the above moieties, thus
defining the planar structure of the side chain of manadoperoxide
F as shown in Fig. 2. The value of JH-10,H-11 = 15.9 Hz indicated
the E geometry of Δ;10 while, due to the very small amounts of
Manadoperoxide H (10), C19H34O6 by HR-ESIMS, should
possess a C10H19O composition for moiety B. The presence of
an aldehyde carbonyl was suggested by the NMR resonances at
δH 9.79 and δC 201.1 (associated by means of the 2D HSQC
spectrum), and this group accounted for the single oxygen atom
manadoperoxide
F
isolated (∼1.0 mg), needed for
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Fig. 3 Application of the modified Riguera method to manadoperoxide E (7). Δδ(S–R) values are in ppm.
Fig. 4 COSY and key HMBC correlations for the side chains of manadoperoxides I–K.
and the single unsaturation degree implied for the side chain.
Accordingly, the 2D COSY spectrum revealed the presence of a
single spin system spanning from H2-7 to Me-15 (δH 0.88, t) and
including a methine resonating at δH 2.50 (H-12) coupled to the
doublet at δH 9.79. Thus, the aldehyde group should be attached
at C-12, as further confirmed by its HMBC cross-peaks with
C-11, C-12 and C-13. The configuration at the stereogenic
C-12 has been left undetermined.
Manadoperoxide J (12), C19H31ClO7 by HR-ESIMS, was
demonstrated to share with manadoperoxide I (11) the presence
of an oxygenated five-membered ring and an hemiketal function-
ality at C-10, in the moiety B. Accordingly, also in this case we
observed the presence of an equilibrating mixture (ca. 4 : 1 ratio)
1
of the two epimers at C-10. A detailed analysis of H and 13C
NMR data of the major epimer of 12 (Tables 1 and 2, respect-
ively), aided by 2D NMR spectra, revealed that manadoperoxide
J (12) actually differs from manadoperoxide I (11) by posses-
sing: (i) an ethyl group attached at the oxygenated C-13 (see the
COSY cross-peaks shown in Fig. 4) in place of the methyl
group; (ii) an sp2 methylene (δH 5.29 and 5.10, δC 105.6) instead
of the allylic methyl group (iii) a chlorine-bearing sp3 methine
(δH 4.55, δC 59.7) instead of the sp2 methine. The 2D HMBC
cross-peaks shown by H2-9 (with C-10 and C-11) and by the
hexomethylene protons (with C-11, C-12 and C-13), together
with the cross-peak H-13/C-10, proved to be crucial to join these
moieties as indicated in Fig. 4. The ROESY cross-peak H-11/
H-13 revealed the cis relative orientation of these protons, but it
was not possible to connect this relative configuration with that
of the stereogenic carbons of the dioxane ring.
Manadoperoxide K (13), C20H35ClO7 by HR-ESIMS, should
possess a C11H20ClO2 side chain with a single unsaturation
degree. The 1H NMR spectrum of 13 (Table 1) revealed, in
addition to the signals of moiety A, the resonances of a third
methoxy group (δH 3.33) and of three methines (δH 3.92, 3.50
and 3.74) in the midfield region of the spectrum. All the proton
signals were associated to those of the directly linked carbon
atoms by means of a 2D HSQC experiment, and then inspection
of the 2D COSY experiment disclosed the existence of two spin
systems, shown in bold in Fig. 4. The first spin system spans
from H2-7 to the chlorine-bearing methine H-11 (δH 3.50, δC
68.2) and includes the oxymethine at δH 3.92 (δC 79.0). The
second spin system spans from the oxymethine H-13 (δH 3.74)
Manadoperoxide I (11) was found to have the molecular
formula C18H30O7 by HR-ESIMS, thus implying a C9H15O2
1
side chain with two unsaturation degrees. Analysis of H and
13C NMR spectra (C6D6, see Experimental) of 11, guided by the
2D HSQC spectrum, revealed the presence of an sp2 methine
singlet (δH 5.24, δC 122.7), an sp3 oxymethine (δH 4.19, δC
77.6) and an hemiketal carbon resonating at δC 102.1. In the
COSY spectrum, two spin systems (highlighted in bold in
Fig. 4) were visible: the first includes a sequence of three
methylenes, while the second encompasses only the oxymethine
proton coupled to a methyl group. The HMBC cross-peaks of
H2-9 with the hemiketal carbon C-10 and with the sp2 methine
C-11 and those of the allylic methyl singlet 12-Me with C-11,
C-12 and with the oxymethine C-13 provided the information
needed to link the above moieties. Moreover, the HMBC cross-
peak H-13/C-10 indicated the presence of the oxygen bridge
between C-13 and C-10, thus accounting for the remaining unsa-
turation degree and completing the gross structure of manadoper-
oxide I (11). A series of signals attributable to a minor
1
compound were detectable both in H and 13C NMR spectra of
11 and all our initial attempts to further purify compound 11
failed. However, once the structure of manadoperoxide I was dis-
closed, these signals were easily rationalized with the presence
of an equilibrating mixture (ca. 4 : 1 ratio) of the two epimers at
1
C-10. Selected H NMR resonances of the minor epimer have
been reported in the Experimental section.
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Table 3 In vitro antiprotozoal activity (IC50) and calculated logP
values of compounds 3–6, 8–11, and 13–14a
to methyl triplet Me-15. The remaining unprotonated carbon
atom (δC 82.4) present in the 13C NMR spectrum of 13 was
attributed to a methoxy-bearing carbon (C-12) located between
Compounds
T. b. rhodesiense L. donovani logPb
2,3
the two above described moieties on the basis of the
J
C,H
Manadoperoxide B (3)
Manadoperoxide C (4)
Manadoperoxide D (5)
Peroxyplakoric ester B3 (6)
Manadoperoxide F (8)
Manadoperoxide G (9)
Manadoperoxide H (10)
Manadoperoxide I (11)
Manadoperoxide K (13)
0.003
0.678
36.7
3.61
0.792
1.84
0.375
0.062
0.087
0.589
3.24
19.2
12.0
5.73
3.22
2.44
0.633
1.89
4.84
2.52
2.33
4.53
2.82
1.75
4.25
3.00
3.63
2.55
HMBC cross-peaks of the methyl singlet at δH 1.24 with C-12,
C-11 and C-13 and of the cross-peak of the methoxy group (12-
OMe) with C-12. The presence of an ether bridge between C-10
and C-13 to define a THF-type ring was inferred by the HMBC
cross-peak H-13/C-10 and accounted for the single unsaturation
degree present in the side chain of 13. This THF-ring includes
four stereogenic carbons, whose relative configuration was
deduced by the ROESY cross-peaks 12-Me/H2-9, 12-Me/H-11,
H-11/H-13, and H2-9/H-13. As for compounds 9 and 12, this
relative configuration has not been connected with that of the
stereogenic carbons of the dioxane ring.
Peroxyplakoric ester C (14) 30.9
Reference compound
43.4
0.003c
0.174d
—
a IC50 values are in μg mL−1 and mean values from at least two
replicates (the variation is max. 20%). b Calculated by Virtual
Computational Chemistry Laboratory software. c Melarsoprol.
d Miltefosine.
Peroxyplakoric ester C (14), C17H28O6 by HR-ESIMS, proved
to belong to the structural series of peroxyplakoric esters,19
which differ from manadoperoxides by the substituents attached
at positions 2 and 4 of the dioxane ring (see Fig. 1). A detailed
analysis of its 1H and 13C NMR spectra (see Experimental
section), accomplished by inspection of 2D COSY and HSQC
NMR spectra, revealed that compound 14 shared with peroxy-
plakoric ester B3 (6)19 all the carbon and proton resonances,
along with the proton–proton coupling constants, for the pos-
itions C-1 to C-6 (including also 2-Me and 6-OMe). On the
manadoperoxide B (3) (IC50 = 3 ng mL−1) proved to be as active
as the reference compound and, to our knowledge, it can be
viewed as the most potent trypanocidal marine natural product
reported to date. Also remarkably active agents are manadoper-
oxides I (11) and K (13) (IC50 = 60–90 ng mL−1) and manado-
peroxides H (10), C (4) and F (8) (IC50 = 300–800 ng mL−1),
although their potencies are, respectively, about 30 and 200
times lower than that of manadoperoxide B. The remaining
manadoperoxides and the two peroxyplakoric esters proved to be
much less active, with IC50s in the low–medium μg mL−1 range.
The tested compounds displayed moderate to good activity
against L. donovani. The most active compounds of the series
were manadoperoxide B (IC50 = 0.589 μg mL−1) and manado-
peroxide I (IC50 = 0.633 μg mL−1) with comparable activities to
the reference compound miltefosine (IC50 = 0.174 μg mL−1).
The remaining compounds showed activity in the μg mL−1
range. All the test compounds proved to be non-toxic (IC50
>10 μg mL−1) towards the human cell line HMEC-1 (human
microvascular endothelial cell line, data not shown). The calcu-
lated partition coefficient (logP) values of tested metabolites
vary between 1.75 to 4.84 (Table 3), hence they comply with
Lipinski’s rule of five.22 This also indicates that manadoperox-
ides will be able to penetrate through lipid cellular membranes
and potentially through the blood brain barrier (BBB), which is
critical for the treatment of cerebral stage of Trypanosoma
infections.
1
other hand, H and 13C NMR resonances of the side chain of 14
were practically superimposable to those of manadoperoxide
C (4), including an α,β-unsaturated ketone group (δC 198.7). The
2D HMBC spectrum provided key information to support these
assignments and to join the above partial moieties, thus fully
confirming the structure of peroxyplakoric ester C (14).
Thus, analysis of the organic extract of P. cfr. lita afforded
twelve endoperoxyketal-containing compounds, eight of which
are being described for the first time. These compounds, with the
exception of 6 and 14, share the same stereostructure of the
dioxane ring while they differ by the length and/or the functiona-
lization of the “western” side chain. Recently, Garson and co-
workers have reported the isolation of a series of dioxane
derivatives from the sponge Plakinastrella clathrata differing by
modifications in the side chain,21 evidencing the existence of an
extraordinary enzymatic activity in Plakinidae sponges,
especially dedicated to the functionalization of side chains.
However, some of the metabolites isolated in the course of this
study are unique in many extents: three of them show the for-
mation of an unprecedented five-membered oxygenated ring
which, in two cases, bears also a chlorine atom. To the best of
our knowledge this has never been reported for marine
endoperoxides.
3. Discussion
As part of our ongoing screening of natural sources to find new
antiprotozoal lead compounds,7–15 we have now discovered that
the endoperoxyketal derivatives manadoperoxides constitute a
new class of potent antitrypanosomal agents. The structural
variability present within the library of endoperoxides tested in
this study clearly defines some structure–activity relationships.
The comparison of the activities of manadoperoxides with
those of the closely related peroxyplakoric esters provides valu-
able information. Indeed, in addition to a negligible difference in
the side chain, manadoperoxide B (3) and peroxyplakoric ester
2.2 Antiprotozoal activity
The library of endoperoxide derivatives obtained from the
chemical investigation of P. cfr. lita have been evaluated in vitro
for their antiprotozoal activity against Trypanosoma brucei rho-
desiense and Leishmania donovani. Results are reported in
Table 3.
Some of the tested compounds showed an ultrapotent activity
against T. b. rhodesiense at low ng mL−1 range. In particular,
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Fig. 5 Comparison of the structures and the antiprotozoal activities of manadoperoxide B (3) and peroxyplakoric ester B3 (6).
B3 (6) differ only by the position of a methyl group on the ring
moiety. In 3 a methyl group is present on the dioxane ring (at
C-4), while in 6 it is attached on the short ester-containing side
chain (at C-2) (Fig. 5). Interestingly, this minor structural differ-
ence is able to induce dramatic effects both in the antimalarial
and in the antitrypanosomal activities, but in opposite directions.
Indeed, while 6 has been demonstrated to be about 60-fold more
active as an antimalarial agent,17 manadoperoxide B (3) is more
than 1000-fold more potent as an antitrypanosomal agent. This
trend is fully supported by the antitrypanosomal activity of man-
adoperoxide C (4, IC50 = 0.678 μg mL−1) (Fig. 1) and peroxy-
plakoric ester C (14, IC50 = 30.9 μg mL−1) (Fig. 2). These two
compounds share the same structure of the “western” side chain
and, thus, they differ exclusively by the shift of a methyl group
from C-4 (in 4) to C-2 (in 14). As observed for the pair 3/6, this
structural change induces a marked reduction of the antitrypano-
somal activity in 14.
These results seem to exclude a non-specific antiprotozoal
activity for these peroxyketal derivatives, strongly suggesting
their molecular interaction with a defined target(s). In this
regard, our studies on plakortin derivatives provided indications
about the mechanism of the antimalarial action of simple 1,2-
dioxanes13 which, upon reaction with Fe(II) (most likely the
heme iron), should generate an oxygen radical, simultaneously
transferred to a side chain carbon. The so formed C-centered
radical should then represent the toxic species responsible for the
antimalarial activity. In the case of manadoperoxides and peroxy-
plakoric esters we already demonstrated that the different pattern
of substitution around the dioxane ring can induce different con-
formational preferences of this ring and, consequently, different
accessibilities for the interaction with heme iron.17 Intriguingly,
the situation for the antitrypanosomal activity seems to be com-
pletely different. In this case, the different pattern of substitution
around the dioxane ring and the consequent different confor-
mational behaviour appear to play in favour of the antitrypanoso-
mal activity of manadoperoxides. It is also interesting to notice
that the above data suggest, indirectly, that the target(s) of these
endoperoxyketal compounds might be different in the two differ-
ent protozoan parasites.
diene system, while the least active compound is manadoperox-
ide D (5), where the presence of a diol group provokes an almost
complete loss of activity. The negative effects of an increase in
the side chain polarity is fully supported by the trend of activities
measured for the other manadoperoxides. Manadoperoxides I
(11) and K (13), bearing a five-membered ring in the side chain,
retain very good antitrypanosomal activity, while the carbonyl-
containing manadoperoxides H (10, saturated C9 alkyl chain
with an aldehyde group in place of the methyl group) and C (4,
conjugated ketone group in side chain) evidence an about 100-
fold lower activity compared to manadoperoxide B. An even
lower activity was exhibited by manadoperoxides F (8, hydroxy
group in side chain) and G (9, epoxy-ketone group in side
chain).
An almost linear relationship between the antitrypanosomal
activity and the lipophilicity of the moiety B is clearly indicated
by the logP values calculated for the members of the manado-
peroxide series (Table 3). This relationship suggests that this
moiety is probably not directly involved in the interaction with
the target(s), but is responsible for the pharmacokinetic
behaviour of these compounds, thus modulating their activities.
Manadoperoxides represent the first endoperoxide derivatives
bearing a ketal functionality on the dioxane ring to be evaluated
against Trypanosoma. A comparison with data available in the
literature for the antitrypanosomal activity of related 1,2-diox-
anes seems to indicate a positive effect for the alkoxy group at
C-6. Indeed, all the natural23 and synthetic24 analogues showing
an ethyl group or an hydrogen atom in place of the methoxy
group at C-6 proved to be less active than manadoperoxides.
However, an unambiguous evaluation of this relationship is pre-
vented by the differences in the side chain structures.
Endoperoxide-containing compounds have been reported to
target several protozoan parasites and to be potential lead com-
pounds against different tropical diseases. However, while some
members of this class show a broad spectrum activity against
Plasmodium, Trypanosoma, Leishmania, etc., other endoperox-
ide-bearing derivatives show a specific action against selected
protozoa. Probably the most remarkable example of this second
type of antiprotozoal agents is given by artemisinin, a well
known antimalarial compound (activity against P. falciparum in
the low nM range),25 displaying a very modest activity (about
25 μg mL−1) against T. b. rhodesiense.25 As we have shown
before, peroxyplakoric ester B3 (6) and its analogue 14 follow
the same behaviour as artemisinin. On the other hand, manado-
peroxides show an opposite behaviour, possessing moderate
The complete homogeneity of the stereostructure of the
dioxane ring (moiety A) in the series of manadoperoxides B–K
can allow a clear evaluation of the impact on the trypanocidal
activity of modifications on the alkyl side chain (moiety B). The
most active compound of the series, manadoperoxide B (3),
shows a methyl branched C9 side chain including a conjugated
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(Phenomenex) SI60 or C18 (250 × 4 mm) columns. The logP
values of manadoperoxides were estimated using VCCLAB,
Virtual Computational Chemistry Laboratory, online server
(http://www.vcclab.org).32
4.2 Animal material, extraction, isolation
A specimen of Plakortis lita de Laubenfels (order Homosclero-
phorida, family Plakinidae) was collected in January 2010 from
along the coasts of the Bunaken island in the Bunaken Marine
Park of Manado. Avoucher sample (Man/10/02) has been depos-
ited at the Dipartimento di Chimica delle Sostanze Naturali, Uni-
versità di Napoli Federico II. The sponge is massive, roundish;
the colour in vivo and in the preserved state is light brown exter-
nally and internally. The sponge is very soft and easy to cut, but
it contracts after preservation. Surface smooth in the living speci-
men, but after collection, especially in the dried state, the surface
became irregular and wrinkled. Ostia are not detectable on the
living specimens. Oscules with a very low rim, about 1.5 mm in
diameter. The ectosomal skeleton is an irregular reticulation of
rounded or elliptical meshes. In some area a disorganized
arrangement of tangential spicules is present. Choanosome with
a confused skeleton of scattered spicules. Spicules are straight or
bent, thin diods (98 × 2.5 μm on average); rare triods (actines
33 μm on average); irregular strongyloid microrhabds
(2.5–25 μm) and smooth ovoid spheres (about 2 μm in diameter).
The studied material is very similar to P. lita de Laubenfels in
the spicular complement, which consists of diods, triods and in
irregular strongyloid microrhabds, comparable in size and
similar in shape. A skeletal organisation of the ectosome with
rounded meshes, typical of P. lita de Laubenfels, is also present.
This species differs from P. lita de Laubenfels by the presence of
tangential spicules irregularly arranged, never reported in P. lita,
and by the colour which in this last species may be black, dark
brown, or grey-brown. Spheres, detected in the present material,
were not described in P. lita. The present sample has been com-
pared also to Plakortis hooperi Muricy18 from Papua New
Guinea. P. hooperi possesses a spicule complement, comparable
to that of the present material, of diods, triods, microrhabds and
spheres; it is characterized by a brown colour, similar to that of
P. cfr. lita and by a confused ectosomal skeleton, without trace of
reticulation. P. hooperi differs from P. cfr. lita in lacking an ecto-
somal reticulation of rounded meshes, in the shape and size of
the diods, relatively thick and without a well-marked centre, in
the shape and size of microrhabds (shorter and irregular in
P. hooperi), and of the spheres (more spherical and larger).
Pending deeper morphological studies, the present sample is pro-
visionally assigned to P. cfr. lita on the basis of the numerous
shared morphological characters.
Fig. 6 Schematic view of the structure–activity relationships for anti-
trypanosomal 1,2-dioxanes.
activity against Plasmodium falciparum (3–10 μM), which is ca.
1000 times lower than the trypanocidal activity described herein
(Fig. 5).
Since the cell organization and the biochemical composition
of trypanosomes differ in numerous aspects from those of mam-
malian cells, several parasite-specific processes may provide
selective targets for drug development and, among these, purine
salvage, fatty acid elongation, cytochrome-independent oxidase
and trypanothione reductase have been suggested.26,27 This latter
flavoenzyme is of key importance to maintain the redox balance
within the parasite acting as regulator of the levels of trypa-
nothione, a molecule composed by glutathione and spermidine
and involved in the reduction of hydroperoxides and ribonucleo-
tides.27 Although a shortage of material prevented us from inves-
tigating the mechanism of action of manadoperoxides, an
interference with the finely regulated redox balance within the
parasite could be anticipated.
The analysis of our library of natural compounds and compari-
son with data available in the literature have demonstrated unam-
biguously that the endoperoxy group does not confer per se
activity against Trypanosoma (and other protozoan parasites
responsible for tropical diseases), while the contribution of the
carbon skeleton is of pivotal importance. We have summarized
in Fig. 6 the most important structural requirements for the class
of simple 1,2-dioxanes as they have emerged from the present
study.
4. Experimental section
4.1 General experimental procedures
Low and high resolution ESI-MS spectra were performed on a
LTQ OrbitrapXL (Thermo Scientific) mass spectrometer. 1H
(500 MHz) and 13C (125 MHz) NMR spectra were measured on
Varian INOVA spectrometers. Chemical shifts were referenced to
the residual solvent signal (CDCl3: δH 7.26, δC 77.0; C6D6: δH
7.16, δC 128.1). Homonuclear 1H connectivities were determined
1
by the COSY experiment. Through-space H connectivities were
After homogenization, the organism was exhaustively
extracted, in sequence, with methanol and chloroform. The com-
bined organic extracts (8.16 g) were subjected to chromato-
graphy over C18 silica column (200–400 mesh) eluting with a
solvent gradient of decreasing polarity from water to chloroform.
Fractions eluted with H2O–MeOH 2 : 8 and H2O–MeOH 1 : 9
were combined (1.35 g) and further fractionated by gravity
column chromatography on silica gel using a n-hexane–EtOAc
gradient. Fractions eluted with n-hexane–EtOAc 9 : 1 were
evidenced using a ROESY experiment with a mixing time of
500 ms. One-bond heteronuclear 1H–13C connectivities were
determined by the HSQC experiment; two- and three-bond
1H–13C connectivities by gradient-HMBC experiments opti-
mized for a 2,3J of 8 Hz. Medium pressure liquid chromato-
graphy was performed on a Büchi apparatus using a silica gel
(230–400 mesh) column. HPLC were achieved on a Knauer
apparatus equipped with a refractive index detector and LUNA
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4.5 Manadoperoxide F (8)
subjected to repeated column and HPLC chromatographies
(n-hexane–EtOAc 95 : 5) affording manadoperoxides B (3,
15.1 mg), J (12, 0.9 mg) and I (11, 1.3 mg) in the pure state.
Fractions eluted with n-hexane–EtOAc 8 : 2 were re-chromato-
Colorless amorphous solid; [α]2D5 −12.1 (c 0.1, CHCl3); 1H
NMR (CDCl3, 500 MHz): Table 1; 13C NMR (CDCl3,
125 MHz): Table 2; (+) ESI-MS m/z 375 [M + H]+, 397
[M + Na]+. HR-ESIMS (positive ions): found m/z 397.2197,
C19H34NaO7 requires m/z 397.2202.
graphed by HPLC (n-hexane–EtOAc 8 : 2, flow 0.8 mL min−1
)
to give manadoperoxide K (13, 2.3 mg), F (8, 1.1 mg) and G (9,
1.3 mg). Fractions eluted with n-hexane–EtOAc 7 : 3 were re-
chromatographed by HPLC (n-hexane–EtOAc 75 : 25) affording
pure manadoperoxide C (4, 1.1 mg), H (10, 1.3 mg) and another
fraction, further purified by RP-HPLC (MeOH–H2O 85 : 15,
flow 0.8 mL min−1) to yield peroxyplakoric ester C (14,
1.3 mg). Fractions eluted with n-hexane–EtOAc (1 : 9) were re-
chromatographed by RP-HPLC (MeOH–H2O 6 : 4, flow 0.8 mL
min−1) affording manadoperoxide D (5, 2.1 mg) and E (7,
2.5 mg).
4.6 Manadoperoxide G (9)
Colorless amorphous solid; [α]2D5 −13.5 (c 0.2, CHCl3); 1H
NMR (CDCl3, 500 MHz): Table 1; 13C NMR (CDCl3,
125 MHz): Table 2; (+) ESI-MS m/z 331 [M + H]+, 353
[M + Na]+. HR-ESIMS (positive ions): found m/z 353.1510;
C16H26NaO7 requires m/z 353.1506.
4.3 Manadoperoxide E (7)
4.7 Manadoperoxide H (10)
Colorless amorphous solid; [α]2D5−13.0 (c 0.2, CHCl3); 1H NMR
(CDCl3, 500 MHz): δH 5.45 (1H, d, J = 8.7 Hz, H-11), 4.43
(1H, ddd, J = 9.5, 4.3, 3.0 Hz, H-3), 4.40 (1H, m, H-10), 4.26
(1H, m, H-13), 3.72 (3H, s, 1-OMe), 3.26 (3H, s, 6-OMe), 2.97
(1H, dd, J = 15.5, 9.5 Hz, H-2a), 2.57 (1H, m, H-4), 2.44 (1H,
dd, J = 15.5, 4.3 Hz, H-2b), 1.71 (3H, bs, H-17), 1.69 (1H, over-
lapped, H-5a), 1.64 (1H, overlapped, H-9a), 1.62 (1H, over-
lapped, H-7a), 1.61 (2H, overlapped, H2-14), 1.55 (1H,
overlapped, H-9b), 1.42 (1H, overlapped, H-8a), 1.35 (1H, over-
lapped, H-8b), 1.34 (1H, overlapped, H-7b), 1.00 (3H, t, J = 7.1
Hz, Me-15), 0.86 (3H, d, J = 7.1 Hz, 4-Me); 13C NMR (CDCl3,
125 MHz) δC 172.5 (s, C-1), 142.0 (s, C-12), 127.1 (d, C-11),
103.0 (s, C-6), 79.7 (d, C-3), 73.4 (d, C-13), 70.1 (d, C-10), 52.2
(q, 1-OMe), 48.7 (q, 6-OMe), 38.4 (t, C-9), 34.5 (t, C-5), 31.7 (t,
C-2), 31.4 (t, C-7), 27.9 (t, C-14), 27.2 (d, C-4), 22.0 (t, C-8),
17.0 (q, C-7), 10.1 (q, C-15). (+) ESI-MS m/z 375 [M + H]+,
397 [M + Na]+. HR-ESIMS (positive ions): found m/z
397.2200, C19H34NaO7 requires m/z 397.2202.
Colorless amorphous solid; [α]2D5 −18.1 (c 0.1, CHCl3); 1H
NMR (CDCl3, 500 MHz): Table 1; 13C NMR (CDCl3,
125 MHz): Table 2; (+) ESI-MS m/z 359 [M + H]+, 381
[M + Na]+. HR-ESIMS (positive ions): found m/z 381.2259;
C19H34NaO6 requires m/z 381.2253.
4.8 Manadoperoxide I (11)
Colorless amorphous solid; [α]2D5 −19.3 (c 0.1, CHCl3); 1H
NMR (C6D6, 500 MHz): δH 5.24 (1H, bs, H-11), 4.50 (1H, m,
H-3), 4.19 (1H, q, J = 6.9 Hz, H-13), 3.34 (3H, s, 1-OMe), 3.20
(3H, s, 6-OMe), 2.88 (1H, dd, J = 15.5, 9.3 Hz, H-2a), 2.50 (1H,
m, H-4), 2.14 (1H, dd, J = 15.5, 4.4 Hz, H-2b), 1.68 (1H, m,
H-7a), 1.66 (1H, m, H-9a), 1.62 (1H, m, H-9b), 1.59 (1H, m,
H-7b), 1.44 (1H, overlapped, H-5a), 1.38 (1H, m, H-8a), 1.36
(3H, bs, 12-Me), 1.33 (1H, m, H-8b), 1.17 (3H, d, J = 6.9 Hz,
Me-14), 0.88 (1H, overlapped, H-5b), 0.38 (3H, d, J = 7.0 Hz
1
4-Me); H NMR data for the minor epimer (only resonances dif-
fering from those of the major epimer are reported): δH 4.15
(1H, q, J = 6.9 Hz, H-13), 1.19 (3H, d, J = 6.9 Hz, H-14), 1.69
(1H, m, H-9a), 1.58 (1H, m, H-9b); 13C NMR (C6D6,
125 MHz): δC 171.8 (s, C-1), 135.8 (s, C-12), 122.7 (d, C-11),
102.9 (s, C-6), 102.1 (s, C-10), 79.7 (d, C-3), 77.6 (d, C-13),
52.2 (q, 1-OMe), 48.8 (q, 6-OMe), 40.8 (t, C-9), 34.3 (t, C-5),
33.8 (C-7), 31.7 (t, C-2), 27.1 (d, C-4), 22.5 (t, C-8); 18.5 (q,
C-14), 17.2 (q, 12-Me), 15.8 (q, 4-Me); (+) ESI-MS: m/z 359
[M + H]+, 381 [M + Na]+. HR-ESIMS (positive ions): found
m/z 381.1892; C18H30NaO7 requires m/z 381.1889.
4.4 Reaction of manadoperoxide E (7) with R- and S-MTPA
chloride
Compound 7 (1.0 mg, 2.8 μmol) was treated with R-MTPA
chloride (30 μL) in 400 μL of dry pyridine with a catalytic
amount of DMAP overnight at rt. Then, the solvent was removed
and the product was purified by HPLC (n-hexane–EtOAc, 97 : 3)
to obtain the S-MTPA diester 7a (1.2 mg, 60% yield). When
compound 7 (1.0 mg, 2.8 μmol) was treated with S-MTPA chlor-
ide, following the same procedure, 1.5 mg (69% yield) of
R-MTPA diester 7b was obtained. S-MTPA diester 7a: amor-
phous solid; ESI-MS (positive ions) m/z 807 [M + H]+; 1H
NMR (CDCl3): selected values δ 5.61 (1H, m, H-10), 5.45 (1H,
d, J = 8.7 Hz, H-11), 5.43 (1H, m, H-13), 1.77 (3H, s, 12-Me),
1.60 (1H, m, H-9a), 1.55 (2H, m, H-7), 1.40 (1H, m, H-9b),
1.64 (2H, overlapped, H2-14). R-MTPA diester 7b: amorphous
solid; ESI-MS (positive ions) m/z 807 [M + H]+; 1H NMR
(CDCl3): selected values δ 5.59 (1H, m, H-10), 5.43 (1H, d,
J = 8.7 Hz, H-11), 5.38 (1H, m, H-13), 4.37 (1H, m, H-3), 1.75
(3H, s, 12-Me), 1.59 (1H, m, H-9a), 1.55 (2H, m, H-7), 1.39
(1H, m, H-9b), 1.62 (2H, overlapped, H2-14).
4.9 Manadoperoxide J (12)
Colorless amorphous solid; [α]2D5 −11.9 (c 0.1, CHCl3); 1H
1
NMR (CDCl3, 500 MHz): Table 1; H NMR data for the minor
epimer (only resonances differing from those of the major
epimer are reported): δH 4.55 (1H, bs, H-11), 4.38 (1H, m,
H-13), 1.78 (1H, overlapped, H-14a), 1.56 (1H, overlapped,
H-14b), 1.00 (3H, t, J = 7.0 Hz, H-15); 13C NMR (CDCl3,
125 MHz): Table 2; (+) ESI-MS m/z 407 and 409 (about 3 : 1
ratio) [M + H]+, 429, 431 (about 3 : 1 ratio) [M + Na]+.
This journal is © The Royal Society of Chemistry 2012
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HR-ESIMS found m/z 429.1663; C19H3135ClNaO7 requires m/z
429.1656.
without a serial drug dilution were seeded in 96-well microtitre
plates. Serial drug dilutions covering a range from 90 to
0.001 μg mL−1 were prepared. After 72 h of incubation the
plates were inspected under an inverted microscope to assure
growth of the controls and sterile conditions. 10 μL of a resa-
zurin solution (12.5 mg resazurin dissolved in 100 mL double-
distilled water) was then added to each well and the plates incu-
bated for another 2 h. Then the plates were read in a Spectramax
Gemini XS microplate fluorometer using an excitation wave-
length of 536 nm and an emission wavelength of 588 nm. Data
were analyzed using the software Softmax Pro. Decrease of flu-
orescence (i.e. inhibition) was expressed as percentage of the flu-
orescence of control cultures and plotted against the drug
concentrations. The IC50 values were calculated from the sigmoi-
dal inhibition curves. Miltefosine was used as a reference drug.
4.10 Manadoperoxide K (13)
Colorless amorphous solid; [α]2D5 −11.5 (c 0.3, CHCl3); 1H
NMR (CDCl3, 500 MHz): Table 1; 13C NMR (CDCl3,
125 MHz): Table 2; (+) ESI-MS: m/z 423 and 425 (about 3 : 1
ratio) [M + H]+, 445 and 447 (about 3 : 1 ratio) [M + Na]+.
HR-ESIMS
(positive
ions):
found
m/z
445.1972;
C20H3535ClNaO7 requires m/z 445.1969.
4.11 Peroxyplakoric ester C (14)
1
Colorless amorphous solid; [α]2D5 −3.0 (c 0.3, CHCl3); H NMR
(CDCl3, 500 MHz): δH 6.77 (1H, dt, J = 16.5, 6.5 Hz, H-10),
6.18 (1H, d, J = 16.5 Hz, H-11), 4.19 (1H, m, H-3), 3.75 (3H, s,
1-OMe), 3.27 (3H, s, 6-OMe), 3.07 (1H, dq, J = 9.5, 7.0 Hz,
H-2), 2.25 (2H, m, H2-9), 2.24 (3H, s, Me-13), 2.10 (1H,
m, H-4a), 1.75 (1H, m, H-5a), 1.69 (1H, m, H-7a), 1.65 (1H, m,
H-5b), 1.53 (2H, m, H2-8), 1.40 (1H, m, H-4b), 1.30 (1H, m,
H-7b), 1.12 (3H, d, J = 7.0 Hz, 2-Me); 13C NMR (CDCl3,
125 MHz): δC 198.7 (s, C-12), 172.5 (s, C-1), 147.0 (d, C-10),
132.1 (d, C-11), 104.5 (s, C-6), 81.0 (d, C-3), 52.0 (q, 1-OMe),
48.8 (6-OMe), 41.6 (t, C-2), 32.9 (t, C-7), 32.6 (t, C-9), 27.3 (q,
C-13), 26.5 (t, C-5), 21.8 (t, C-8), 20.5 (t, C-4), 17.0 (q, 2-Me).
(+) ESI-MS m/z 351 [M + Na]+. HR-ESIMS (positive ions):
found m/z 351.1790, C17H28NaO6 requires m/z 351.1784.
4.14 Cell cytotoxicity assay
HMEC-1 cell line immortalized by SV 40 large T antigen was
maintained in MCDB 131 medium supplemented with 10% fetal
calf serum, 10 ng ml−1 of epidermal growth factor, 1 μg ml−1 of
hydrocortisone, 2 mM glutamine, 100 U ml−1 of penicillin,
100 μg ml−1 of streptomycin, and 20 mM Hepes buffer. Cells
were treated with serial dilutions of test compounds and cell pro-
liferation evaluated using the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay.15
Conclusions
In the last decade, the urgent need for viable alternatives to the
existing antiquated and unsatisfactory therapeutic options has
stimulated a remarkable increase in the efforts towards finding
new antitrypanosomal agents, often as a result of a series of
public–private partnerships. A number of different classes of
active compounds have been proposed and, among them, purine
nitriles,28 thiosemicarbazones,29 quinolones,30 and chalcone–
benzoxaborole31 appear to display better trypanocidal potential
and better selectivity. Somewhat surprisingly, in spite of the
incredible chemical diversity associated with the secondary
metabolites elaborated by terrestrial plants and marine invert-
ebrates, the contribution of natural products chemistry to the
fight against trypanosomiasis has been until now modest. As
matter of fact, the above reported classes of recently developed
trypanocidal compounds include only synthetically produced
molecules.
Data presented in this paper have disclosed that manadoperox-
ides constitute one of the most potent classes of natural products
acting against sleeping sickness isolated to date and strongly
suggest that endoperoxyketal-containing molecules are of
promise for further development as lead structures against
African trypanosomiasis. Moreover, the structure–activity
relationships deduced have revealed that minor structural
changes around the dioxane ring can dramatically affect the
pharmacological activity, thus suggesting the interaction of this
moiety with a specific target(s).
4.12 Activity against Trypanosoma brucei rhodesiense
Minimum Essential Medium (50 μL) supplemented with 25 mM
HEPES, 1 g L−1 additional glucose, 1% MEM non-essential
amino acids (100×), 0.2 mM 2-mercaptoethanol, 1 mM Na-pyru-
vate and 15% heat inactivated horse serum was added to each
well of a 96-well microtitre plate. Serial drug dilutions of eleven
3-fold dilution steps covering a range from 100 to 0.002 μg
mL−1 were prepared. Then 104 bloodstream forms of STIB 900
strain of T. b. rhodesiense in 50 μL was added to each well and
the plate incubated at 37 °C under a 5% CO2 atmosphere for
72 h. 10 μL of a resazurin solution (12.5 mg resazurin dissolved
in 100 mL double-distilled water) was then added to each well
and incubation continued for a further 2–4 h. Then the plates
were read in a Spectramax Gemini XS microplate fluorometer
(Molecular Devices Cooperation, Sunnyvale, CA, USA) using
an excitation wavelength of 536 nm and an emission wavelength
of 588 nm. The IC50 values were calculated by linear regression
(Huber 1993)33 from the sigmoidal dose inhibition curves using
SoftmaxPro software (Molecular Devices Cooperation, Sunny-
vale, CA, USA). Melarsoprol was the standard drug.
4.13 Activity against Leishmania donovani
Amastigotes of L. donovani (strain MHOM/ET/67/L82) were
grown in axenic culture at 37 °C in SM medium at pH 5.4 sup-
plemented with 10% heat-inactivated fetal bovine serum under
an atmosphere of 5% CO2 in air. One hundred μL of culture
medium with 105 amastigotes from axenic culture with or
Since trypanosomiasis affects the poorest countries, the cost of
the treatment is one of the main concerns in the design of new
chemotherapeutics and, although effective, too expensive drugs
7206 | Org. Biomol. Chem., 2012, 10, 7197–7207
This journal is © The Royal Society of Chemistry 2012

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13 O. Taglialatela-Scafati, E. Fattorusso, A. Romano, F. Scala, V. Barone,
P. Cimino, E. Stendardo, B. Catalanotti, M. Persico and C. Fattorusso,
Org. Biomol. Chem., 2010, 8, 846–856.
14 C. Fattorusso, M. Persico, N. Basilico, D. Taramelli, E. Fattorusso,
F. Scala and O. Taglialatela, Bioorg. Med. Chem., 2011, 19, 312–320.
15 M. Persico, A. Quintavalla, F. Rondinelli, C. Trombini, M. Lombardo,
C. Fattorusso, V. Azzarito, D. Taramelli, S. Parapini, Y. Corbett,
G. Chianese, E. Fattorusso and O. Taglialatela-Scafati, J. Med. Chem.,
2011, 54, 8526–8540.
16 In our paper cited in ref. 17 this sponge was tentatively attributed to
Plakortis cfr. simplex on the basis of its morphological relationships with
the Plakortis simplex species complex. However, on the basis of a
comparison with the detailed morphological data later published (ref. 18),
we consider conspecific the specimens analyzed both in this study and
in our previous work (ref. 17) and attribute them to the species Plakortis
cfr. lita.
should be discarded. In this regard, the natural product state of
manadoperoxides should not be considered as a drawback on the
way to their development as antitrypanosomal drugs; indeed
their relatively simple structures should guarantee the feasibility
of a total synthesis. They are small, lipophilic and appear to have
good pharmacokinetic properties, so they offer significant drug-
likeness to become bioavailable lead compounds. A number of
syntheses of the six-membered endoperoxyketal scaffold have
been already published in the literature, very recently also by our
group.15 These should allow the preparation of large libraries of
compounds and the development of this new class of antitrypa-
nosomal agents.
17 C. Fattorusso, M. Persico, B. Calcinai, C. Cerrano, S. Parapini,
D. Taramelli, E. Novellino, A. Romano, F. Scala, E. Fattorusso and
O. Taglialatela-Scafati, J. Nat. Prod., 2010, 73, 1138–1145.
18 G. Muricy, J. Mar. Biol. Assoc. UK, 2011, 91, 303–319.
19 M. Kobayashi, K. Kondo and I. Kitagawa, Chem. Pharm. Bull., 1993,
41, 1324–1326.
20 F. Freire, J. M. Seco, E. Quinoa and R. Riguera, J. Org. Chem., 2005, 70,
3778–3790.
21 K. W. L. Yong, L. K. Lambert, P. Y. Hayes, J. J. De Voss and M.
J. Garson, J. Nat. Prod., 2012, 75, 351–360.
Acknowledgements
This work was supported by MIUR (PRIN2008,
20084MMXNM: Leads ad Attività Antimalarica di Origine Nat-
urale, Isolamento, Ottimizzazione e Valutazione Biologica). MS
and NMR spectra were registered by Authors at CSIAS, Centro
Interdipartimentale di Analisi Strumentale, Faculty of Pharmacy,
University of Naples.
22 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Adv. Drug
Delivery Rev., 2001, 46, 3–26.
23 M. H Kossuga, A. M. Nascimento, J. Q. Reimao, A. G. Tempone,
N. N. Taniwaki, K. Veloso, A. G. Ferreira, B. C. Cavalcanti, C. Pessoa,
M. O. Moraes, A. M. S. Mayer, E. Haidu and R. G. S. Berlinck, J. Nat.
Prod., 2008, 71, 334–339.
24 H. Holla, M. Labaied, N. Phom, I. D. Jenkins, K. Stuart and R. J. Quinn,
Bioorg. Med. Chem. Lett., 2011, 21, 4793–4797.
25 M. Kaiser, S. Wittlin, A. Nehrbass-Stuedli, Y. Dong, X. Wang,
A. Hemphill, H. Matile, R. Brun and J. L. Vennerstrom, Antimicrob.
Agents Chemother., 2007, 51, 2991–2993.
References
1 C. Burri, S. Nkunku, A. Merolle, T. Smith, J. Blum and R. Brun, Lancet,
2000, 355, 1419–1425.
2 E. M. Fèvre, B. V. Wissmann, S. C. Welburn and P. Lutumba, PLoS Negl.
Trop. Dis., 2008, 2, e333.
3 N. M. El-Sayed, P. J. Myler and D. C. Bartholomeu, Science, 2005, 309,
409–415.
26 A. Cavalli and M. L. Bolognesi, J. Med. Chem., 2009, 52, 7339–
7359.
4 M. P. Barrett, I. M. Vincent, R. J. S. Burchmore, A. J. N. Kazibwe and
E. Matovu, Fut. Microbiol., 2011, 6, 1037–1047.
27 R. L. Krauth-Siegel, H. Bauer and R. H. Schirmer, Angew. Chem., Int.
Ed., 2005, 44, 690–715.
5 P. G. Kennedy, Ann. Neurol., 2008, 64, 116–126.
6 http://www.dndi.org/portfolio/fexinidazole.html
28 Z. Qiao, Q. Wang, F. Zhang, Z. Wang, T. Bowling, B. Nare, R. T. Jacobs,
J. Zhang, D. Ding, Y. Liu and H. Zhou, J. Med. Chem., 2012, 55, 3553–
3557.
29 J. P. Mallari, A. A. Shelat, T. Obrien, C. R. Caffrey, A. Kosinski,
M. Connelly, M. Harbut, D. Greenbaum, J. H. McKerrow and R. K. Guy,
J. Med. Chem., 2008, 51, 545–552.
30 J. P. Mallari, A. A. Shelat, A. Kosinski, C. R. Caffrey, M. Connelly,
F. Zhu, J. H. McKerrow and R. K. Guy, Bioorg. Med. Chem. Lett., 2008,
18, 2883–2885.
31 G. Hiltensperger, N. G. Jones, S. Niedermeier, A. Stich, M. Kaiser,
J. Jung, S. Puhl, A. Damme, H. Braunschweig, L. Meinel, M. Engstler
and U. Holzgrabe, J. Med. Chem., 2012, 55, 2538–2548.
32 I. V. Tetko, J. Gasteiger, R. Todeschini, A. Mauri, D. Livingstone, P. Ertl,
V. A. Palyulin, E. V. Radchenko, N. S. Zefirov, A. S. Makarenko,
V. Y. Tanchuk and V. V. Prokopenko, J. Comput. Aided Mol. Des., 2005,
19, 453–463.
7 E. Fattorusso, S. Parapini, C. Campagnuolo, N. Basilico, O. Taglialatela-
Scafati and D. Taramelli, J. Antimicrob. Chemother., 2002, 50, 883–888.
8 G. Chianese, S. R. Yerbanga, L. Lucantoni, A. Habluetzel, N. Basilico,
D. Taramelli, E. Fattorusso and O. Taglialatela-Scafati, J. Nat. Prod.,
2010, 73, 1448–1452.
9 F. Scala, E. Fattorusso, M. Menna, O. Taglialatela-Scafati, M. Tierney,
M. Kaiser and D. Tasdemir, Mar. Drugs, 2010, 8, 2162–2174.
10 C. Campagnuolo, E. Fattorusso, A. Romano, O. Taglialatela-Scafati,
N. Basilico, S. Parapini and D. Taramelli, Eur. J. Org. Chem., 2005,
5077–5083.
11 E. Fattorusso, O. Taglialatela-Scafati, A. Ianaro and M. Di Rosa, Tetra-
hedron, 2000, 56, 7959–7967.
12 C. Fattorusso, G. Campiani, B. Catalanotti, M. Persico, N. Basilico,
S. Parapini, D. Taramelli, C. Campagnuolo, E. Fattorusso, A. Romano
and O. Taglialatela-Scafati, J. Med. Chem., 2006, 49, 7088–7094.
33 W. Huber and J. C. Koella, Acta Trop., 1993, 55, 257–261.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7197–7207 | 7207