Isolation, Derivative Synthesis, and Structure-Activity Relationships of Antiparasitic Bromopyrrole Alkaloids from the Marine Sponge Tedania brasiliensis

Article abstract of DOI:10.1021/acs.jnatprod.7b00876

The isolation and identification of a series of new pseudoceratidine (1) derivatives from the sponge Tedania brasiliensis enabled the evaluation of their antiparasitic activity against Plasmodium falciparum, Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) infantum, and Trypanosoma cruzi, the causative agents of malaria, cutaneous leishmaniasis, visceral leishmaniasis, and Chagas disease, respectively. The new 3-debromopseudoceratidine (4), 20-debromopseudoceratidine (5), 4-bromopseudoceratidine (6), 19-bromopseudoceratidine (7), and 4,19-dibromopseudoceratidine (8) are reported. New tedamides A-D (9-12), with an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxamide moiety, are also described. Compounds 4 and 5, 6 and 7, 9 and 10, and 11 and 12 have been isolated as pairs of inseparable structural isomers differing in their sites of bromination or oxidation. Tedamides 9+10 and 11+12 were obtained as optically active pairs, indicating an enzymatic formation rather than an artifactual origin. N¹²-Acetylpseudoceratidine (2) and N¹²-formylpseudoceratidine (3) were obtained by derivatization of pseudoceratidine (1). The antiparasitic activity of pseudoceratidine (1) led us to synthesize 23 derivatives (16, 17, 20, 21, 23, 25, 27-29, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51) with variations in the polyamine chain and aromatic moiety in sufficient amounts for biological evaluation in antiparasitic assays. The measured antimalarial activity of pseudoceratidine (1) and derivatives 4, 5, 16, 23, 25, 31, and 50 provided an initial SAR evaluation of these compounds as potential leads for antiparasitics against Leishmania amastigotes and against P. falciparum. The results obtained indicate that pseudoceratidine represents a promising scaffold for the development of new antimalarial drugs.

Full text of DOI:10.1021/acs.jnatprod.7b00876

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Isolation, Derivative Synthesis, and Structure−Activity Relationships
of Antiparasitic Bromopyrrole Alkaloids from the Marine Sponge
Tedania brasiliensis
Lizbeth L. L. Parra,† Ariane F. Bertonha,
,‡,#
†,§,#
Ivan R. M. Severo, Anna C. C. Aguiar,
†
⊥
⊥
⊥
⊥
∥
ata R. Costa,∥
Guilherme E. de Souza, Glaucius Oliva, Rafael V. C. Guido, Nathalia Grazzia, Tab
Danilo C. Miguel, Fernanda R. Gadelha, Antonio G. Ferreira, Eduardo Hajdu, Daniel Romo,
and Roberto G. S. Berlinck*
́
∥
∥
▽
△
§
,†
†
Instituto de Química de Sao
̃
Carlos, Universidade de Sao
̃
Paulo, CP 780, CEP 13560-970, Sao
̃
Carlos, SP, Brazil
§
Department of Chemistry & Biochemistry, Baylor University, Waco, Texas 76706, United States
⊥
∥
Instituto de Física de Sao
̃
Carlos, Avenida Joao Dagnone, 1100, Jardim Santa Angelina, Sao
̃
Carlos, SP, 13563-120, Brazil
Departamento de Biologia Animal e Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade Estadual
de Campinas, CEP 13083-862, Campinas, SP, Brazil
▽
Departamento de Química, Universidade Federal de Sao Carlos, Rod. Washington Luiz, km 235 - SP-310, CEP 13565-905, Sao
̃ ̃
Carlos, SP, Brazil
△
Museu Nacional, Universidade Federal do Rio de Janeiro, Quinta da Boa Vista, s/n, CEP 20940-040, Rio de Janeiro, RJ, Brazil
S Supporting Information
*
ABSTRACT: The isolation and identification of a series of
new pseudoceratidine (1) derivatives from the sponge
Tedania brasiliensis enabled the evaluation of their antipar-
asitic activity against Plasmodium falciparum, Leishmania
(
Leishmania) amazonensis, Leishmania (Leishmania) infantum,
and Trypanosoma cruzi, the causative agents of malaria,
cutaneous leishmaniasis, visceral leishmaniasis, and Chagas
disease, respectively. The new 3-debromopseudoceratidine
(
4), 20-debromopseudoceratidine (5), 4-bromopseudocerati-
dine (6), 19-bromopseudoceratidine (7), and 4,19-dibromop-
seudoceratidine (8) are reported. New tedamides A−D (9−
1
2), with an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxamide moiety, are also described.
Compounds 4 and 5, 6 and 7, 9 and 10, and 11 and 12 have been isolated as pairs of inseparable structural isomers differing in
their sites of bromination or oxidation. Tedamides 9+10 and 11+12 were obtained as optically active pairs, indicating an
1
2
12
enzymatic formation rather than an artifactual origin. N -Acetylpseudoceratidine (2) and N -formylpseudoceratidine (3) were
obtained by derivatization of pseudoceratidine (1). The antiparasitic activity of pseudoceratidine (1) led us to synthesize 23
derivatives (16, 17, 20, 21, 23, 25, 27−29, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51) with variations in the polyamine chain
and aromatic moiety in sufficient amounts for biological evaluation in antiparasitic assays. The measured antimalarial activity of
pseudoceratidine (1) and derivatives 4, 5, 16, 23, 25, 31, and 50 provided an initial SAR evaluation of these compounds as
potential leads for antiparasitics against Leishmania amastigotes and against P. falciparum. The results obtained indicate that
pseudoceratidine represents a promising scaffold for the development of new antimalarial drugs.
romopyrrole alkaloids isolated from marine sponges
encompass a remarkable chemical diversity of potently
maxinella (Suberitidae, Hadromerida), Hymeniacidon (Hal-
ichondriidae, Halichondrida), and Stylissa (Halichondriidae,
B
bioactive compounds. These metabolites range from oroidin-
related archetypal motifs to structurally complex oroidin-
1
−7
Halichondrida).
Therefore, past assumptions on the
1
−3
tetramers, the stylissadines.
Such alkaloids are prevalent in
character of bromopyrrole alkaloids as chemotaxonomical
sponges belonging to the order Agelasida (particularly within
markers of these sponges4−7 are now challenged by their more
1
−3
the genus Agelas, family Agelasidae).
Nevertheless, several
widespread occurrence. Such is also the case for our present
of these alkaloids have also been found in sponges of the
genera Axinella (Axinellidae, Halichondrida), Axinyssa (Hal-
ichondriidae, Halichondrida), Callyspongia (Callyspongiidae,
Haplosclerida), Eurypon (Raspailiidae, Poecilosclerida), Ho-
Received: October 20, 2017
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00876
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
1
Table 1. H NMR Data (δ, ppm) for Pseudoceratidines 2−8 in DMSO-d
6
b
b
a
a
b
b
b
position
2
(J in Hz)
3
(J in Hz)
4
(J in Hz)
5
(J in Hz)
6
(J in Hz)
7
(J in Hz)
8
(J in Hz)
N−H
12.60, br s
12.63/12.61, s
12.18, br s
12.63, d (2.2)
n.o.
n.o.
n.o.
2
3
4
6.72, dd (2.5, 2.7)
6.11, dd (2.5, 3.7)
6.86, m
6.88/6.87, s
6.89, d (2.2)
6.89, s
5
6
N−H
8
9
8.06, t (5.6)/8.04, m
3.19, m
1.47/1.41, m
1.53/1.45, m
3.24/3.21, m
n.o.
8.10, m
8.03, t (5.7)
3.20, m
1.52, m
1.57, m
2.90, br m
n.o.
8.14, t (5.7)
3.20, m
1.52, m
1.57, m
2.90, br m
n.o.
7.76, t (6.1)
3.25, m
1.53, m
1.58, m
2.90, m
n.o.
8.14, t (5.6)
3.21, m
1.52, m
1.58, m
2.89, m
n.o.
7.34, br t
3.16, m
1.48, m
1.57, m
2.88, t (7.4)
n.o.
3.21/3.17, m
1.40/1.37, m
1.49/1.45, m
3.24/3.20, m
n.o.
1
1
0
1
N−H
1
1
1
3
4
5
3.25/3.24, m
1.73/1.64, m
3.17, m
3.23/3.22, m
1.68/1.64, m
3.16/3.15, m
8.10, m
2.90, br m
1.77, m
3.25, m
2.90, br m
1.77, m
3.25, m
2.90, m
1.79, m
3.29, m
8.24, t (5.6)
2.86, m
1.76, m
3.25, m
7.72, t (6.0)
2.83, t (7.4)
1.73, m
3.20, q (6.2)
7.61, t (6.0)
N−H
8.09, t (5.6)/8.04, m
8.24, t (6.0)
8.15, t (5.7)
1
1
1
2
2
7
8
9
0
1
6.89, m
6.88/6.87, s
6.90, d (2.2)
6.13, dd (2.5, 3.7)
6.71, dd (2.5, 2.7)
6.90 s
n.o.
N−H
2
12.60, br s
12.63/12.61, s
8.00/7.99, s
12.67, d (2.2)
12.13, br s
n.o.
n.o.
3
2
4
1.97/1.94, s
a
b
1
−
4
00 MHz. 600 MHz; n.o.: not observed; H signals for the HCO counterion observed between δH 8.12 and 8.33 for all compounds.
2
isolation of pseudoceratidine (1) derivatives from the marine
sponge Tedania brasiliensis (Tedaniidae, Poecilosclerida).
Pseudoceratidine (1) was previously isolated solely from the
sponge Pseudoceratina purpurea (Pseudoceratinidae, Verongi-
falciparum, Leishmania (Leishmania) amazonensis, L. (L.)
infantum, and Trypanosoma cruzi.
Malaria, caused by Plasmodium spp., is among the most
devastating vector-born human infectious diseases. It is
estimated that over 3.4 billion people worldwide are at risk
of infection. Malaria is prevalent in tropical and subtropical
regions, affecting 212 million people and causing 429 000
deaths in 2015. In Africa, P. falciparum is responsible for the
highest mortality rate of children under 5 years of age. Malaria
etiologic agents have developed resistance to several
antimalarial drugs, including emerging resistance to artemisinin
and artemisinin-based combination therapies that comprise
8
,9
da).
The compound displayed more potent antifouling
activity than its monosubstituted 4,5-dibromo-1H-pyrrole-2-
10
carboxylic acid spermidine analogues. In these previous
studies, it was found that both the substitution pattern of the
terminal rings and the length of the triamine chain of
pseudoceratidine derivatives affect the antifouling activity.
Furthermore, pseudoceratidine also displayed the most potent
antibacterial activity against Staphylococcus aureus, Listeria
monocytogenes, Pseudomonas aeruginosa, Escherichia coli, and
12
current first-line treatments. Thus, the discovery of new
antimalarial lead compounds is of utmost importance.
Leishmaniasis is a group of diseases caused by several species
of protozoan parasites belonging to the genus Leishmania
1
1
Serratia liquefaciens. In the present investigation, we report
the antiparasitic activity of pseudoceratidine and several new
synthetic pseudoceratidine derivatives against Plasmodium
B
DOI: 10.1021/acs.jnatprod.7b00876
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Data (δ, ppm) for Pseudoceratidines 2−8 in DMSO-d6
b
b
a
a
b
b
b
position
2
δC, type
3
δC, type
4
δC, type
5
δC, type
6
δC, type
7
δC, type
8
δC, type
2
3
4
5
6
8
9
104.6, C
97.9, C
112.5, CH
128.4, C
104.77/104.68, C
98.09/98.07, C
112.8/112.6, CH
128.53/128.47, C
159.2/159.15, C
38.5/38.3, CH2
26.8/26.4, CH2
25.8/24.5, CH2
46.4/40.1, CH2
44.2/41.0, CH2
28.2/27.4, CH2
36.6/36.0, CH2
159.2/159.13, C
128.50/128.41, C
112.8/112.6, CH
98.09/98.07, C
104.75/104.63, C
163.2/163.1, CH
102.7, C
104.7, C
98.0, C
112.9, CH
128.0, C
159.4, C
37.9, CH2
26.6, CH2
23.4, CH2
46.9, CH2
45.09, CH2
26.57, CH2
35.8, CH2
160.2, C
n.o.
n.o.
n.o.
n.o.
104.6, C
97.9, C
n.o.
n.o.
n.o.
n.o.
111.7, CH
111.0, CH
128.4, C
112.6, CH
128.3, C
159.1, C
38.0, CH2
26.5, CH2
23.3, CH2
46.7, CH2
44.7, CH2
26.5, CH2
35.3, CH2
162.0, C
n.o.
158.93/159.0, C
38.5/38.3, CH2
26.8/26.6, CH2
26.0/25.0, CH2
47.8/44.5, CH2
45.8/42.7, CH2
28.8/27.8, CH2
36.5/36.3, CH2
158.95/159.1, C
128.3, C
112.4, CH
97.9, C
104.5, C
169.6/169.3, C
21.4/21.3, CH3
159.9, C
n.o.
162.8, C
37.3, CH2
27.0, CH2
23.4, CH2
46.9, CH2
44.8, CH2
26.9, CH2
34.8, CH2
163.9, C
n.o.
38.0, CH2
26.52, CH2
23.3, CH2
46.8, CH2
45.07, CH2
26.4, CH2
36.0, CH2
159.1, C
38.3, CH2
26.37, CH2
23.31, CH2
46.80, CH2
44.9, CH2
26.2, CH2
36.0, CH2
159.4, C
128.3, C
112.8, CH
98.0, C
104.6, C
1
1
1
1
1
1
1
1
2
2
2
2
0
1
3
4
5
7
8
9
0
1
3
4
128.1, C
112.7, CH
98.1, C
128.3, C
111.1, CH
112.0, CH
102.4, C
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
104.9, C
a
b
1
00 MHz. 150 MHz; n.o.: not observed.
1
3
endemic in more than 80 countries. Transmitted by different
sandfly species, Leishmania parasites infect cells from the
mononuclear phagocytic system, leading to clinical manifes-
tations that vary from localized, disseminated, and diffuse skin
lesions (cutaneous leishmaniasis) to a systemic multiorgan
infection (visceral leishmaniasis). Visceral leishmaniasis is fatal
if left untreated. Treatment options are limited to parenteral
drugs, including pentavalent antimonials as the first-line drugs,
which very often present severe side effects. The utilization of
liposomal amphotericin B and oral miltefosine proved useful
for treating patients with visceral leishmaniasis. Nevertheless,
the high cost of liposomal amphotericin B and the miltefosine
teratogenic effect, in addition to its long half-life, hampers the
(4), 20-debromopseudoceratidine (5), 4-bromopseudocerati-
dine (6), 19-bromopseudoceratidine (7), and 4,19-dibromop-
seudoceratidine (8). Compounds 4 and 5, 6 and 7, 9 and 10,
as well as 11 and 12 have been isolated as pairs of inseparable
structural isomers differing in their sites of bromination or
oxidation. Since pseudoceratidine displayed good anti-P.
falciparum activity, we prepared a series of pseudoceratidine
derivatives with different polyamine and aromatic moieties,
aiming to assess their anti-P. falciparum, anti-Leishmania spp.,
and anti-T. cruzi activities and begin evaluation of their
structure−activity relationships.
RESULTS AND DISCUSSION
■
1
4,15
therapeutic success of such treatments.
Therefore, the
Isolation and Identification of Natural Pseudocer-
atidine Derivatives. Analysis of the HRMS and NMR data of
identification of new drug candidates for leishmaniasis
treatment remains imperative in the current therapeutic
scenario.
T. cruzi is the causative agent of Chagas disease, which may
lead to fatal disorders such as cardiomegaly and megacolon in
8,9
1
facilitated the identification of minor pseudoceratidine
congeners 4−12 (Tables 1−4) that were all isolated as their
formate salts. The inseparable mixture of 3-debromopseudo-
ceratidine (4) and 20-debromopseudoceratidine (5) displayed
a protonated molecule cluster at m/z 565.9409/567.9389/
16
approximately 30% of patients. The disease is an emerging
1
7−19
health issue in North America and Europe.
−10 million people in Latin American countries are infected
by T. cruzi, with an annual death rate of approximately 14 000
Benznidazole, toxic and of limited efficacy, is the
only available therapy in Brazil for Chagas disease. No effective
Approximately
5
1
69.9370/571.9354 with peaks showing relative intensities of
+
8
:3:3:1. The [M + H] ion at m/z 565.9409 corresponded to
7
9
the formula C H Br N O , with one fewer bromine atom
1
7
23
3
5
2
1
8,19
people.
and one more hydrogen atom than 1. Monobromo substitution
at one of the pyrrole moieties was verified in the COSY
spectrum of 4 and 5. Coupling between two hydrogens was
treatment exists for chronic phase patients affected by Chagas
2
0
disease. Thus, the discovery of new therapies for Chagas
disease is sorely needed. Furthermore, there are no approved
vaccines for the treatment of humans to prevent malaria,
leishmaniasis, or Chagas disease.
observed in one of the pyrrole rings, at δ 6.11/6.13 (dd, J =
H
2.5 and 3.7 Hz, H-4 or H-19) and at δ 6.71/6.72 (dd, J = 2.5
H
and 2.7 Hz, H-3 or H-20), both of which coupled with an NH
group at δ 12.13/12.18 (br s). A doublet at δ 6.89/6.90 (d, J
H
H
In our continuing search for antiparasitic marine metabo-
= 2.2 Hz, H-4 or H-19) was assigned to the hydrogen of a 2,3-
dibromo-substituted pyrrole group, which coupled only to the
2
1−24
lites,
we verified that the MeOH antiparasitic extract of T.
brasiliensis contained pseudoceratidine (1) as the most
abundant metabolite. Related minor constituents were
detected only by HPLC-UV-MS because of the high
abundance of 1 in the sponge extract. Several chromatographic
separations of this extract yielded a series of minor
pseudoceratidine derivatives. These include the new tedamides
A−D (9−12), along with the new 3-debromopseudoceratidine
NH group at δ 12.67/12.63 (d, J = 2.2 Hz). Analysis of the
H
COSY spectrum of 4 indicated that H-4 (δ 6.11, dd, J = 2.5
H
and 3.7 Hz) was vicinal to H-3 (δ 6.72, dd, J = 2.5 and 2.7
H
Hz). In the HMBC spectrum, H-4 coupled with C-2 (δC
102.7), C-5 (δ 128.4), and C-6 (δ 159.9), whereas NH-7
C
C
(δ 8.03, t, J = 5.7 Hz) coupled with C-6 and C-8 (δ 38.0).
H
C
These couplings indicated that in 4 the monobromopyrrole-2-
C
DOI: 10.1021/acs.jnatprod.7b00876
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H NMR Data (δ, ppm) for Tedamides A−D (9−
the isomeric compound 5 shows H-4 (δ 6.89, d, J = 2.2 Hz)
at the dibromopyrrolecarboxylic acid group coupling with C-2
H
1
2) in DMSO-d
6
(
(
δ 104.7), C-5 (δ 128.0), and C-6 (δ 159.4), whereas NH-7
a
a
b
b
C C C
position
9
(J in Hz)
10 (J in Hz) 11 (J in Hz) 12 (J in Hz)
δ 8.14, t, J = 5.7 Hz) coupled with C-6 at δ 159.4 and with
H
C
N−H
12.63, d (1.6) 9.07, d (1.6)
n.o.
9.07, br
C-8 (δ 37.9). At the other end of the spermidine chain of 5,
C
2
3
H-19 (δ 6.13, dd, J = 2.2 and 3.7 Hz) showed a coupling with
H
H-20 (δ 6.71, dd, J = 2.5 and 2.7 Hz) in the COSY spectrum
H
4
6.90, m
7.30, d (1.6)
7.29, s
and coupled with C-18 (δ 128.3) and with C-21 (δ 102.4) in
C
C
5
6
1
13
the HMBC spectrum. Unambiguous H and C assignments
were also based on the relative integration of the H signals of
1
N−H
8
9
8.14, t (5.7)
3.21, m
1.49, m
1.53, m
2.88, br
n.o.
8.28, m
3.08, m
1.46, m
1.57, m
2.88, m
n.o.
7.54, t (5.8) 8.27, t (5.9)
4
and 5, those of 4 being slightly higher than those of 5, with a
3.21, m
1.54, m
1.58, m
2.88, m
n.o.
3.10, m
1.48, m
1.54, m
2.88, m
n.o.
relative abundance of approximately 55/45, respectively. The
1
13
H and C signals for the methylene groups of the spermidine
1
1
0
1
chain were essentially the same for 4 and 5 (Tables 1 and 2)
and identical to those of 1. Thus, the structures of 3-
debromopseudoceratidine (4) and 20-debromopseudocerati-
dine (5) were established.
N−H
1
1
1
3
4
5
2.88, br
1.73, m
3.11, m
8.39, t (6.0)
2.88, m
1.78, m
3.26, m
8.25, m
2.85, m
1.74, m
3.15, m
2.85, m
1.77, m
3.26, m
A fraction presenting an inseparable mixture of the isomeric
-bromopseudoceratidine (6) and 19-bromopseudoceratidine
4
N−H
8.39, t (5.9) 7.71, t (6.0)
(
7) was obtained along with one distinct fraction providing
1
1
1
2
2
7
8
9
0
1
pure 7. Many attempts made to separate both 6 and 7 under
different HPLC conditions were unsuccessful. The HRMS
spectrum of the mixture of 6 and 7, as well as that of pure 7,
displayed a protonated molecule cluster at m/z 721.7632/
7.34, d (1.7)
9.09, d (1.5)
6.90, m
7.33, s
7
23.7587/ 725.7626/727.7581/ 729.7564/731.7520, with
N−H
O−CH3
12.67, d (1.4) 9.08, br
3.17, s 3.17, s
n.o.
3.16, s
peaks showing relative intensities of 1:5:10:10:5:1. The [M +
H] ion at m/z 721.7632 corresponded to the formula
C H Br N O , with one more bromine atom and one fewer
hydrogen atom than 1. The H NMR spectrum of 7 (Table 1)
3.18, s
+
a
b
4
00 MHz. 600 MHz; n.o.: not observed.
79
1
7
21
5
5
2
1
Table 4. 13C NMR Data (δ, ppm) for Tedamides A−D (9−
2) in DMSO-d6
1
presented a singlet at δ 6.89 integrating to one H (H-4),
H
1
indicating that one of the pyrrole moieties was substituted by
three bromines. The HMBC spectrum of 7 showed couplings
of H-4 with C-2 (δ 104.6), C-3 (δ 97.9, weak), C-5 (δ
C
a
a
b
b
position
9
δC, type
10 δC, type
11 δC, type
12 δC, type
C
C
2
3
4
5
6
8
9
1
1
1
1
1
1
1
1
2
2
2
104.6, C
97.9, C
167.1, C
92.1, C
n.o.
n.o.
n.o.
n.o.
167.3, C
92.29, C
144.73, CH
121.71, C
165.8, C
38.8, CH2
26.1, CH2
23.2, CH2
46.7, CH2
44.8, CH2
26.6, CH2
35.5, CH2
162.0, C
n.o.
1
28.3), and C-6 (δ 159.1), as well as between NH-7 (δ 8.14,
C
H
br t, J = 5.6 Hz) and C-6 and C-8 (δ 38.0), which enabled us
C
112.6, CH
128.3, C
159.0, C
38.0, CH2
26.4, CH2
23.2, CH2
46.7, CH2
44.7, CH2
25.8, CH2
36.5, CH2
166.0, C
121.6, C
144.5, CH
92.0, C
144.5, CH
121.5, C
165.6, C
38.7, CH2
26.1, CH2
23.1, CH2
46.6, CH2
44.9, CH2
26.3, CH2
35.9, CH2
159.3, C
128.1, C
112.8, CH
98.0, C
to establish to which of the two amides the dibrominated
pyrrole moiety was attached. Because the H -8 methylene
2
160.4, C
37.9, CH2
26.67, CH2
23.3, CH2
46.9, CH2
44.9, CH2
25.9, CH2
36.6, CH2
166.2, C
121.79, C
144.72, CH
92.21, C
167.4, C
51.3, CH3
protons (δ 3.21) showed HMBC couplings to C-9 (δ 26.5)
H
C
and to C-10 (δC 23.3), whereas H -11 (δH 2.89) showed
2
couplings to C-10, C-9, and C-13 (δ 44.7), it was possible to
C
0
1
3
4
5
7
8
9
0
1
3
determine the connectivity of the 2,3-dibromopyrrole-5-
carboxylic acid group to NH-7 in compound 7. Additional
HMBC couplings between NH-16 (δH 7.72) and C-17 (δC
1
62.0) and C-15 (δ 35.3) and between H -15 (δ 3.25) and
C 2 H
C-14 (δC 26.5) and C-13, along with couplings observed
between H -13 (δH 2.86) and C-11, C-14, and C-15,
2
confirmed the structure of 19-bromopseudoceratidine (7).
Finally, MS/MS analysis of 7 provided further support for its
structure (Figures S30 and S31, Supporting Information).
Fragment ions detected at m/z 395.0079, 397.0097, 399.0098
(1:2:1) and at m/z 320.9235, 322.9219, 324.9187 (1:2:1)
indicated that the dibromopyrrole-2-carboxylic acid group was
attached to the four-methylene amine moiety of 7, whereas
fragment ions observed at m/z 498.9012, 500.8963, 502.8913
(1:3:3:1), at m/z 472.9718, 474.9163, 476.9141, 478.9121
(1:3:3:1), and at m/z 384.8184, 386.8165, 388.8146, 390.8121
(1:3:3:1) indicated that the tribromopyrrole-2-carboxylic acid
group was attached to the three-methylene amine moiety of 7,
confirming its structure with no ambiguity.
n.o.
n.o.
n.o.
51.3, CH3
167.2, C
51.1, CH3
104.8, C
51.1, CH3
a
b
1
00 MHz. 150 MHz; n.o.: not observed.
carboxylic acid group was attached to the NH amide
connected to the four-methylene moiety of spermidine,
because H -8 showed couplings to H -9 in the COSY
spectrum and to C-9 and C-10 in the HMBC spectrum.
Considering the NMR data of 4, the dibromopyrrole-2-
carboxylic acid group had to be attached to the three-
methylene moiety of spermidine. Indeed, H-19 (δ 6.90, d, J =
.2 Hz) coupled with C-18 (δ 128.1) and with C-21 (δ
04.9), whereas NH-16 (δ 8.24, t, J = 6.0 Hz) coupled with
C-15 (δ 36.0) and C-17 (δ 159.1). In the HMBC spectrum,
2
2
1
13
The assignments of H and C NMR signals of 6 were also
established by analysis of NMR data obtained for the 50/50
mixture of 6 and 7 (Tables 1 and 2). For compound 6, the
singlet at δH 6.90 (H-19) showed couplings in the HMBC
H
2
C C
1
H
C
C
D
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spectrum with C-17 (δ 159.4), C-18 (δ 128.3), and C-21
showed a strong NOE between H-4 and the methoxy group.
C
C
1
3
2
(
δ 104.6). The amide NH-16 (δ 8.24, t, J = 5.6 Hz) showed
The C signals of sp carbons of 9 and 10 were assigned to a
8
C
H
1
couplings with C-17 and C-15 (δ 36.0), whereas CH -15 (δ
2,3-dibromopyrrole moiety by comparison with data for 1. H
C
2
H
1
3
3
.29) showed couplings with C-17 (δ 159.4), C-14 (δ 26.2),
and C NMR assignments of the spermidine chain were based
C
C
1
3
and C-13 (δ 44.9) in the HMBC spectrum. In the COSY
on the higher intensity of the C NMR signals of 10, by
C
spectrum, H -15 was coupled with NH-16 and H -14 (δ
analysis of COSY and HMBC spectra as well as by comparison
2
2
H
1
3
1
.79), which was sequentially coupled with H -13 (δ 2.90).
with data for 1. Because C chemical shifts of the pyrrole
conjugated amide carbons lie below δ 160, for tedamide B
(10) C-6 was assigned at δ 165.6 and C-17 at δ
Couplings of NH-16 (δ 8.25) with C-17 and C-15 (δ
and of NH-7 (δ 8.28) with C-6 and C-8 (δ
2
H
The remaining four-methylene chain was shown to be attached
C
1
to the NH-7/C-6 amide group. Complete assignments of H
C
C
159.3.
35.9)
C
1
3
and C resonances of 6 and 7 (Tables 1 and 2) enabled the
identification of these pentabrominated alkaloids.
H
38.7) confirmed
H
C
The structure of 4,19-dibromopseudoceratidine (8) could be
established by HRMS analysis, which showed a protonated
molecule cluster at m/z 799.6352/801.6241/ 803.6213/
such assignments (Tables 3 and 4). Analogous reasoning
enabled the identification of tedamide A (9) as the minor
isomer of 10.
Tedamides C (11) and D (12) were also isolated as
inseparable isomers in a ratio of approximately 28:72,
presenting a protonated molecule cluster at m/z 689.8571/
8
05.6219/ 807.6194/809.6263/ 811.6205 with peaks showing
+
relative intensities of 1:6:15:20:15:6:1. The [M + H] ion at
m/z 799.6352 corresponded to the formula C H Br N O ,
79
20
17
6
5
2
6
91.8551/ 693.8535/695.8514/ 697.8499 with peaks showing
with two more bromine atoms and two fewer hydrogen atoms
+
1
relative intensities of 1:4:6:4:1. The [M + H] ion at m/z
6
than 1. Inspection of H NMR data obtained for 8 revealed
7
9
89.8571 corresponded to the formula C H Br N O , with
that it corresponded to the 4,19-dibrominated version of 1,
18 24 4 5 4
1
one more bromine atom than tedamides A and B. Analysis of
the NMR data of 11 and 12 indicated that the hydrogen at the
pyrrole moiety was missing, whereas the remaining structures
were essentially identical to those of compounds 9 and 10
because no pyrrole hydrogen was detected, whereas the H
signals of the spermidine chain remained very similar to the
1
corresponding H signals in compounds 1−7. The same
13
pattern was observed for the C NMR signals of 8 (Tables 1
and 2). Therefore, the structure of 8 was established as that of
(
Tables 3 and 4). Thus, the structures of tedamides 11 and 12
were assigned as those corresponding to the tribrominated
pyrrole derivatives of 9 and 10.
4
,19-dibromopseudoceratidine.
Isomeric tedamides A (9) and B (10) were isolated as an
α-Bromo-α-alkoxyamides are known stable chemical entities,
frequently utilized as functionalized substrates suitable for the
inseparable mixture and presented a protonated molecule
cluster at m/z 611.9452/613.9453/ 615.9435/617.9399, with
25−30
+
preparation of further derivatives in medicinal chemistry.
peaks showing relative intensities of 1:3:3:1. The [M + H] ion
However, we have been unable to find β,γ-unsaturated-α-
bromo-α-alkoxy lactams in the literature or any natural product
bearing such functionalities in the SciFinder, Dictionary of
Natural Products, or MarinLit natural products databases.
Thus, the 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-
at m/z 611.9452 corresponded to the formula
7
9
C H Br N O , with eight double-bond equivalents. A set
1
8
25
3
5
4
13
of doubled signals in the C NMR spectrum (Table 4)
indicated the presence of two closely related compounds.
Several attempts to separate them by HPLC using different
columns and/or solvent mixtures failed. Because the intensities
2
-carboxamide moieties of tedamides A and B are structurally
1
3
unprecedented. Although an artifactual origin could, in
principle, be considered for tedamides given that MeOH was
used as the extraction solvent, the mixture of compounds 9 and
of the C NMR signal pairs were not identical, we assumed a
mixture of a slightly major isomer (10) and a minor one (9), in
5
5:45 relative ratio. The assignments of the tedamide B isomer
1
0 displayed some optical activity ([α]D −7.8 (c 0.007,
(10) were established as follows.
MeOH)), while the mixture of 11 and 12 displayed significant
The β,γ-unsaturated lactam moiety in 10 was constructed by
optical activity ([α] +110 (c 0.0006, MeOH)). This provides
D
analysis of the HMBC spectrum. Correlations between NH-1
δ 9.07) and C-2 (δ 167.1), C-3 (δ 92.1), C-4 (δ 144.5),
evidence that these bromomethoxy acetals are not artifacts of
oxidation during the isolation process but are likely products of
an enzymatic oxidation.
(
H
C
C
C
and C-5 (δ 121.5), between H-4 (δ 7.30, d, J = 1.6 Hz) and
C
H
C-2, C-3, and C-5, and between the methoxy group at δ 3.17
H
The isolation of tri-, tetra-, penta-, and hexabrominated
pyrrole alkaloids from T. brasiliensis is noteworthy. Prior to the
present investigation, the 3,4,5-tribromo-1H-pyrrole-2-carbox-
ylic acid was the only alkaloid presenting a tribromo
and C-3 accounted for either one of the two hypothetical
lactam moieties, A or B (Figure 1). Fragment B was discarded
because the coupling constant observed for H-4 (J = 1.6 Hz)
1
1
agrees with a long-distance H− H coupling rather than a
31
acylpyrrole isolated from a marine sponge, Axinella sp.
1
1
vicinal H− H coupling between H-4 and NH-1. Moreover,
analysis of the 1D NOESY spectrum of the mixture of 9 and 10
Because several bromine-substituted pyrroles have been
32,33
isolated from cultures of marine bacteria,
it is possible
that sponge polybrominated pyrrole alkaloids may have a
bacterial origin. However, recent investigations have shown
that vanadium-dependent bromoperoxidases isolated from
marine algae promote bromination of a variety of pyrrole
34−36
derivatives,
including that of 1H-pyrrole-2-carboxamide
and 1H-pyrrole-2-carboxylate esters, which are commonly
found in Agelasida and other marine sponges. Therefore, the
distribution of brominated pyrroles in nature may be related
not only to the diversity of bacteria associated with
macroorganisms but possibly also to a widespread occurrence
of bromoperoxidases in phylogenetically distant taxa. A recent
Figure 1. NOE observed in the 1D NOESY spectrum of tedamides A
(9) and B (10) that supports the presence of fragment A instead of
fragment B in the structures of 9 and 10.
E
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Scheme 1. Syntheses of Pseudoceratidine Derivatives with Variations in the Tether
1
metagenomic analysis of T. brasiliensis and other sponges
presenting bromopyrrole alkaloids indicates that the biosyn-
thesis of these compounds is related to the less abundant
Compounds 2 and 3 were fully characterized by HRMS, H,
1
3
C, HSQC, and HMBC spectra (Supporting Information).
In order to prepare additional pseudoceratidine derivatives
in sufficient amounts for testing in several antiparasitic assays,
the natural product was first synthesized based on a previously
Commercially available 2,2,2-tri-
chloro-1-(1H-pyrrol-2-yl)ethenone (13) was brominated with
37
microbes in these sponges.
Derivatization of Pseudoceratidine and Synthesis of
Pseudoceratidine Derivatives. Aiming to evaluate struc-
ture−activity relationships of pseudoceratidine and congeners
in antiparasitic assays, a series of 23 pseudoceratidine
derivatives were synthesized. Natural pseudoceratidine was
10,11
reported procedure.
38
Br to give the known dibrominated pyrrole 14 in 87% yield.
2
1
2
Coupling of 14 with spermidine (15) in tetrahydrofuran
provided synthetic pseudoceratidine in 72% yield (Scheme 1).
N-Methylpseudoceratidine (16) was then prepared by
reductive amination with formaldehyde in 70% yield to
probe the importance of the basic secondary amine as a
proton donor or acceptor, or both. Toward a derivative (17)
amenable to further coupling with a fluorophore via
acetylated with Ac O in pyridine to obtain its N -acetyl
2
derivative (2). After HPLC purification of the acetylated
12
product, we obtained both N -acetylpseudoceratidine (2) and
1
2
N -formylpseudoceratidine (3). Compound 3 is likely derived
from reaction of pseudoceratidine with acetic formic anhydride
resulting from the reaction of Ac O and residual formic acid
2
from the HPLC solvent used to purify pseudoceratidine.
F
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Scheme 2. Syntheses of Pseudoceratidine Derivatives with Variations in the Pyrrole (Aromatic) Moiety
Sharpless−Hu
̈
isgen cycloaddition, pseudoceratidine (1) was
(Scheme 1). Compounds 20 and 21 were prepared using the
same conditions for the preparation of 1 employing 1,5-
diaminopentane or 1,8-diaminooctane, in 50% and 74% yield
from 14, respectively. Pseudoceratidine derivatives 23 and 25
were prepared using similar conditions with spermine (22) and
coupled to 2-azidoacetic acid in 72% yield (Scheme 1).
A series of derivatives with different chains between the two
pyrrole moieties were also synthesized to probe the importance
of the chain length and the requirement of basic amines
G
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H
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Figure 2. Structures and antiplasmodial activities of pseudoceratidine derivatives against Plasmodium falciparum.
1
N -(6-aminohexyl)hexane-1,6-diamine (24), in 60% and 68%
yield, respectively. An additional variation introduced into the
polyamine chain was introduction of a sulfur atom into the
chain through synthesis of dialkyl sulfide 27 from 2-(2-
aminoethylthio)ethanamine (26) and ketone 14 in a micro-
wave reactor in 38% yield.
48 and 49 with spermidine enabled us to obtain bis-furans 50
and 51 in moderate yields.
Antiparasitic Activity and SAR Investigation of
Pseudoceratidine and Derivatives. Pseudoceratidine (1)
was assayed against four protozoan parasite species: P.
falciparum, L. (L.) amazonensis (etiological agent of localized
and diffuse cutaneous leishmaniasis in South America), L. (L.)
infantum (causative agent of visceral leishmaniasis in the
Mediterranean and in Latin America), and T. cruzi. Although 1
was essentially inactive against L. (L.) amazonensis, L. (L.)
infantum, and T. cruzi, it showed very good antiplasmodial
activity (1.1 ± 0.1 μM) against P. falciparum (Table 5). The
inseparable mixture of structural isomers 3-debromopseudo-
ceratidine (4) and 20-debromopseudoceratidine (5) showed 5-
fold decreased antiparasitic activity on P. falciparum (5.8 ± 0.5
μM), while N-acetylpseudoceratidine (2) did not display any
antiparasitic activity. The isomeric pair of tedamides A (9) and
B (10) was also inactive as antiparasitic agent. The preparation
of pseudoceratidine derivatives 2, 16, 17, 20, 21, 23, 25, 27−
We also investigated changes in substituents on the pyrrole
moieties while keeping the spermidine linker unchanged, with
the exception of a nonbrominated derivative of 20, namely, 28,
prepared in 60% yield by coupling of pyrrole ketone 13 and
1
,5-diaminopentane (18). The spermidine-derived nonbromi-
nated variant 29 was obtained in a similar manner in 70% yield.
Monobromination of 13 at C-4 to provide 30 in 44% yield was
a c h i e v e d w i t h 1 - c h l o r o m e t h y l - 4 - fl u o r - 1 , 4 -
diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (Select-
fluor, or F-TEDA-BF ) in the presence of KBr. Reaction of
0 with spermidine (15) under the same conditions for the
synthesis of pseudoceratidine gave 2,21-debromo-
pseudoceratidine (31) in 65% yield. Chlorination of 13 with
sulfuryl chloride provided 2,2,2-trichloro-1-(4,5-dichloro-1H-
pyrrol-2-yl)ethenone (32) in 15% yield. Coupling of 32 with
spermidine led to the chlorinated version of pseudoceratidine
33) in 38% yield. Monofluorination at C-5 of 13 was achieved
with Selectfluor in a microwave reactor at 70 °C to provide 34
39
4
3
2
9, 31, 33, 35, 38, 39, 42, 43, 46, 47, 50, and 51 in adequate
amounts enabled us to explore the impact on bioactivity
through variation of the pseudoceratidine polyamine chain and
aromatic end groups. N-Methylpseudoceratidine (16) showed
good antiplasmodial activity against P. falciparum (4 ± 1 μM).
Compound 23, with a larger polyamine chain bearing two
basic nitrogens, showed antiplasmodial activity similar to that
of the natural product (3 ± 1 μM), while compound 25, with a
polyamine chain of similar length but with only one basic
nitrogen, displayed a 2-fold decrease in antiplasmodial activity
(
4
0
in 20% yield. Coupling of 34 with spermidine under basic
conditions gave 35 in 65% yield.
More substantial changes in the aromatic moieties of
pseudoceratidine were also introduced. Coupling of indole-2-
carboxylic acid (36) and 1H-benzo[d]imidazole-2-carboxylic
acid (37) with spermidine gave derivatives 38 and 39 in
moderate yields. Coupling with 5-bromothiophene-2-carbox-
ylic acid (40) and 5-methylthiophene-2-carboxylic acid (41)
gave 42 and 43 in low yield. Reaction of 6-chloropyridine-2-
carboxylic acid (44) or 6-methylpyridine-2-carboxylic acid
(
2 ± 1 μM). The 2,21-debromopseudoceratidine (31)
derivative showed a decrease in antiplasmodial activity (7 ±
μM) relative to pseudoceratidine (1). Finally, the furan
1
derivative 50, bearing four bromine atoms, also showed good
antiplasmodial activity against P. falciparum (3 ± 1 μM).
Although pseudoceratidine (1) was inactive against L. (L.)
infantum promastigotes, derivatives 20, 23, 27, 42, and 50
showed enhanced, but yet moderate and similar, antileishma-
(
4
45) with spermidine provided the pyridine derivatives 46 and
7 also in low yield. Finally, the coupling of furan derivatives
I
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Figure 3. Structures and antileishmanial activities of pseudoceratidine derivatives against Leishmania (L.) infantum.
nial activity (EC s ∼20 μM). The same derivatives were
atidine. Therefore, a basic linker in the form of the polyamine
chain appears important for antiplasmodial activity.
5
0
active, but considerably less potent, against L. (L.)
amazonensis. Only compound 20 showed good antiparasitic
activity against T. cruzi epimastigotes, while compound 27
showed weak activity in the same bioassay.
Derivatives with an N-acyl group (2 and 17) are inactive
against P. falciparum, suggesting the importance of the basic
nitrogens in the polyamine chain. These compounds were also
inactive against Leishmania species and T. cruzi tested in our
assays, which prevents simple acylation of pseudoceratidine to
access cellular probes. However, alkylation of pseudoceratidine
derivatives was tolerated since they maintain the basic nitrogen
atom. The selectivity index of the antiplasmodial activity of
compounds 1, 4, 5, 16, 23, 25, 31, and 50 was determined by
measuring cytotoxicity on the human liver cancer HepG2 cell
line (Table 5). Pseudoceratidine (1) was the most cytotoxic
compound (16 ± 1 μM), but with a selectivity index of 15.
Compounds 16, 4, 5, 23, 25, 31, and 50 displayed overall weak
cytotoxicity, with selectivity indices between 35 and 125, which
are considered excellent, as this shows dramatically reduced
toxicity to healthy cells.
Leishmania and Trypanosoma species are Euglenozoa
kinetoplastida parasites, while P. falciparum belongs to
41
Hemosporida within the phylum Apicomplexa. Constituting
two groups of phylogenetically distant parasites, not surpris-
ingly compounds affecting these two groups of organisms may
show distinct effects. Our results indicated a selective, more
potent activity against P. falciparum for compounds 1, 4 and 5,
1
6, 23, 25, 31, and 50 (Table 5 and Figure 2). It is evident the
importance of bromine substituents in the aromatic extremities
of pseudoceratidine derivatives for the antiplasmodial activity.
Fully debrominated pseudoceratidine (29) is completely
inactive, as well as the chlorinated derivative 33 and fluorinated
3
5. Although methyl groups represent a structural variation
42
similar to bromine groups in terms of van der Waals radii, the
bis-methylated bis-furan pseudoceratidine derivative 51 is
completely inactive as well. Thus, bromination in both pyrrole
and furan pseudoceratidine derivatives is essential for the
expression of antiplasmodial activity against P. falciparum,
which seems to be related to the electronic effect of bromine
on aromatic rings rather than to its van der Waals radius or to
an enhanced lipophilic character promoted by the bromine
substituents. The nature of the polyamine chain is an
additional structural characteristic that determines the
antiplasmodial activity of pseudoceratidine derivatives. Com-
pounds 20 and 21, which are devoid of a basic nitrogen, but
have similar chain length (e.g., 21) when compared to 1, are
inactive as antiplasmodial agents. The sulfur-bearing derivative
As for the anti-Leishmanial activity, compounds 20, 23, 27,
42, and 50 are moderate to weakly active (EC50 in the range
between 19 and 24 μM; Figure 3). However, a clear picture of
the structural requirements for anti-Leishmanial activity of
these pseudoceratidine derivatives did not emerge. Com-
pounds 20 and 27 have shorter polyamine chains devoid of a
basic nitrogen, but the opposite is true for compounds 23, 42,
and 50. Bromination seems to be important, but the nature of
the aromatic ring is apparently less relevant for the
antileishmanial activity. Therefore, further variations on this
series of antileishmanial compounds need to be explored in
order to clarify structure−activity relationships aiming to
improve the effectiveness of toxicity toward L. (L.) infantum
promastigotes.
4
3
2
7 is inactive as well. On the other hand, compounds 23 and
5, with longer polyamine chains, are as active as
In vitro infections were performed aiming to evaluate the
activity of compounds 23, 42, and 50 against Leishmania
intracellular amastigotes. Because amastigotes must reside
inside macrophages, we first determined the cytotoxicity of
2
pseudoceratidine (1). The N-methyl derivative 16 is still
active, but approximately 4-fold less active than pseudocer-
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Figure 4. In vitro activity of 23, 42, and 50 against intracellular Leishmania (L.) amazonensis amastigotes. Macrophages derived from BALB/c mice
bone marrow were infected with L. (L.) amazonensis stationary promastigotes for 1 h (MOI = 10). After 24 h, infected cells were incubated with
6
.125, 25, 50, or 100 μM of each compound for 24 h. MeOH-fixed cells were stained, and infection was determined by counting 300 cells/coverslip.
Experiments were performed in triplicate. The results shown are representative of two independent experiments. (A) Bars indicate the number of
intracellular amastigotes. Numbers above each bar indicate the percentage of reduction over control untreated infected macrophages. (B)
Photomicrograph examples showing untreated infected macrophages (a) and infected macrophages incubated with compound 42 at 100 μM (b).
Arrows point to intracellular amastigotes. Bar = 10 μm.
active pseudoceratidine derivatives against bone-marrow-
derived macrophages from BALB/c mice. Compounds 23
and 42 showed low toxicity (CC50 > 100 μM). The half-
maximal cytotoxic concentration for compound 50 was 83 ± 4
μM. Compounds 23, 42, and 50 were selected for further
investigation of their activities on intracellular amastigotes.
Bone-marrow-derived macrophages were infected with L. (L.)
amazonensis stationary phase promastigotes (MOI = 10) for 24
h and then incubated with increasing concentrations of
compounds 23, 42, and 50 for subsequent counting of
intracellular amastigotes. Intracellular parasitism was reduced
in a dose-dependent manner after 24 h for 23, 42, and 50
Figure 5. HepG2 cell morphology before (left) and after (right)
treatment with pseudoceratidine (1) at 10 μM.
(
Figure 4), leading to approximately 80% reduction of the
Table 6. Antiplasmodial Activities of Pseudoceratidine (1)
and Standard Antimalarials against Sensitive (3D7 Strain)
and Resistant Plasmodium falciparum (K1 Strain)
amastigote number over the control group at higher
concentrations. The intracellular effect against Leishmania
amastigotes was almost equivalent when comparing com-
pounds 23, 42, and 50. Further studies should evaluate the
effects of compounds 23, 42, and 50 in experimental
leishmaniasis.
In order to better characterize the antiplasmodial activity of
pseudoceratidine (1), the most active compound against P.
falciparum, its impact on the morphology of HepG2 cells was
evaluated after 24 h of parasite treatment at a concentration
P. falciparum IC50 (μM)
compound
3d7 strain
K1 strain
pseudoceratidine (1)
chloroquine
pyrimethamine
cycloguanil
1.1 ± 0.1
0.013 ± 0.002
0.03 ± 0.01
0.010 ± 0.002
0.004 ± 0.001
1.1 ± 0.1
0.167 ± 0.002
3.9 ± 0.1
0.54 ± 0.02
0.003 ± 0.001
artesunate
1
0-fold higher than its IC50 value on the parasites.
Pseudoceratidine (1) was not toxic to HepG2 cells at this
high concentration, as HepG2 cells showed microscopic
morphology similar to untreated cells. Pseudoceratidine (1)
was then tested against multiresistant P. falciparum strain (K1
strain) and still presented very good potency against this K1
resistant strain, with an IC50 value of 1.1 ± 0.1 μM (Figure 5
and Table 6).
Cell morphological changes were observed by microscopy at 0,
8, 16, and 32 h postsynchronization (Figure 6). Pseudocer-
atidine (1) showed inhibitory activity in the early ring stages,
inducing alterations in P. falciparum morphology between 8
and 16 h after incubation. These data suggest a fast-acting
mechanism in which young forms of P. falciparum in the
intraerythrocytic cycle are highly susceptible to the antipar-
asitic activity of 1. The potential drug interactions of
pseudoceratidine (1) with sodium artesunate against P.
falciparum were also assayed, in order to elucidate potential
Aiming to assess the stage-specific inhibitory activity of
pseudoceratidine (1), it was incubated at a concentration 10-
fold higher than IC values with highly synchronized parasites.
5
0
K
DOI: 10.1021/acs.jnatprod.7b00876
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Journal of Natural Products
Article
CONCLUSION
■
In the present investigation, we report the isolation of eight
new pseudoceratidine derivatives from the sponge T.
brasiliensis, of which the tedamides encompass a new 4-
bromo-4-methoxy-5-oxo-4,5-dihydro-1H-pyrrole-2-carboxa-
mide moiety. Pseudoceratidine displayed very good antima-
larial activity and justified the preparation of 23 of its
derivatives, aiming to establish initial structure−activity
relationships. Pseudoceratidine and seven synthetic derivatives
indicated that the length of the polyamine chain bearing a basic
nitrogen and the presence of bromine atoms on pyrrole or
furan terminal moieties represent essential structural features
for the expression of antiplasmodial activity. Pseudoceratidine
Figure 6. Microscopy of synchronized parasites continuously treated
with pseudoceratidine (1) at a concentration 10-fold the IC50 value
(
top line) and DMSO (control, bottom line). Images are
(
1) showed antiplasmodial activity against both sensitive (3D7
representative of three independent experiments.
strain) and resistant (K1 strain) P. falciparum strains and also
an additive interaction effect in combination with artesunate,
indicating that derivatives of 1 may be used in combination
with artemisinin for the treatment of malaria. The results
described demonstrate that the pseudoceratidine scaffold,
which is easily obtained by total synthesis, constitutes a useful
lead to be developed as potential antiplasmodial agents.
benefits and limitations of candidate molecules in combination
44
with antimalarial drugs. The isobologram analysis and FIC
index (1.0 ± 0.2) indicate an additive interaction effect of
pseudoceratidine (1) in combination with artesunate, thereby
suggesting that pseudoceratidine derivatives may be used in
artemisinin-based combination therapies (Figure 7).
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV spectra were recorded
on a Shimadzu UV-3600 spectrophotometer. IR spectra were
obtained on a Shimadzu IRAffinity-1 Fourier transform infrared
spectrophotometer on a silica plate. NMR spectra were obtained at 25
°
C, with tetramethylsilane as an internal standard, using a Bruker ARX
1
9
.4 T spectrometer operating at either 400.35 MHz ( H) or 100.10
1
3
MHz ( C) and a Bruker AV-600 spectrometer operating at either
6
1
13
1
00 MHz ( H) or 150 MHz ( C) with a 2.5 mm cryoprobe. The H
chemical shifts are referenced to the residual DMSO-d (δ 2.49),
6
1
3
whereas C chemical shifts are referenced to the DMSO-d solvent
6
peaks (δ 39.5). HRMS and direct insertion MS/MS analyses were
performed on a Waters Xevo QTOF MS/MS instrument using the
following conditions: capillary voltage, 1.20 kV; desolvation temper-
ature, 450 °C; voltage cone, 30 V; electrospray, positive mode;
detection range, 100−1000 Da with total ion count extracting
−
1
acquisition. Cone and desolvation gas flows were set to 700 L h ,
respectively, with a nitrogen source. HPLC semipreparative and
preparative separations were performed with a Waters instrument
(
0
600 quaternary pump and 2487 double-beam UV detector), with
.1% formic acid in all eluents utilized. HPLC-UV-ELSD-MS analyses
were performed using a Waters Alliance 2695 instrument coupled
online with a Waters 2996 photodiode array detector and a Waters
Figure 7. Isobologram plot for drug interaction analysis of
pseudoceratidine (1) and sodium artesunate.
2424 evaporative light-scattering detector, followed by a Micromass
ZQ 2000 detector with an electrospray interface. The mass
spectrometer detector was optimized using the following conditions:
capillary voltage, 3.00 kV; source block temperature, 100 °C;
desolvation temperature, 350 °C; voltage cone, 25 V; electrospray,
positive mode; detection range, 200−900 Da with total ion count
extracting acquisition. Cone and desolvation gas flows were set to 50
Bromopyrrole alkaloids related to oroidin have been assayed
against a series of parasites. Dispacamide B, spongiacidin B,
and dibromopalau’amine displayed antiplasmodial activity
against P. falciparum comparable to that of pseudoceratidine
45
(
1.34, 1.09, and 1.48 mg/mL, respectively). Dibromopalau’-
−1
and 350 L h , respectively, with a nitrogen source.
amine was also very active against T. brucei rhodesiense (0.46
μg/mL) and against L. donovani (1.48 μg/mL). Longamide B
also showed good antiparasitic activity against T. brucei
rhodesiense (1.53 μg/mL) and against L. donovani (3.85 μg/
Animal Material. The sponge Tedania brasiliensis was collected at
Cabo Frio (Rio de Janeiro state, in April 2011) and identified by one
of the authors (E.H.). A voucher of the collected sponge has been
deposited at the Museu Nacional do Rio de Janeiro, Universidade
Federal do Rio de Janeiro (MNRJ 16876).
Extraction and Isolation. A 136.2 g freeze-dried sample of
T. brasiliensis was homogenized and exhaustively extracted with 5 L of
MeOH. The solvent was evaporated to 500 mL. The resulting MeOH
4
5
mL). Oroidin showed good antiparasitic activity against P.
falciparum (3.9 μg/mL) and moderate activity against T. brucei
rhodesiense (17.3 μg/mL), being inactive against L. donovani
4
6
and T. cruzi. Simpler derivatives 4,5-dibromo-1H-pyrrole-2-
extract was diluted with 50 mL of H O and partitioned with hexane
2
carboxylic acid and the respective methyl ester showed better
(
3 × 500 mL). The MeOH/H O fraction was evaporated to dryness
46
2
antiplasmodial activity (5.8 and 7.9 μg/mL, respectively). It
is clear that a detailed investigation of bromopyrrole alkaloids
as antiparasitic agents, particularly as antiplasmodial com-
pounds, is a worthy area for further research.
and dissolved in 1 L of 1:1 EtOAc/H O. Partitioning was performed
2
two additional times. The EtOAc fraction was evaporated to dryness
to yield 5.37 g. The hexane extract was also evaporated to dryness to
yield 1.79 g of an apolar fraction. The aqueous fraction of the EtOAc/
L
DOI: 10.1021/acs.jnatprod.7b00876
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Journal of Natural Products
Article
H O partition was extracted with a 1:1 mixture of XAD-4 and XAD-7,
3-Debromopseudoceratidine (4) and 20-debromopseudocerati-
dine (5): colorless, glassy solid; UV (MeOH) λmax (log ε) 215 (4.2),
235 (4.2), 274 (4.6) nm; IR (film) νmax 3186, 2945, 1677, 1629, 1133,
2
after which the resins were desorbed with MeOH, then with 1:1
MeOH/acetone, and the organic solvents were evaporated to dryness.
Antiparasitic assays performed with the hexanes, EtOAc, and resin-
−
1
1
13
740, and 615 cm ; H and C NMR data, Tables 1 and 2;
+
79
extracted H O fractions indicated bioactivity exclusively in the EtOAc
HRESIMS m/z 565.9409 [M + H] (calcd for C17
H
23 Br
3
N
O
5
,
2
2
fraction. HPLC-UV-MS analysis of these three fractions indicated
pseudoceratidine (1) and its minor derivatives only in the EtOAc
fraction as well. Therefore, neither the hexanes nor resin-extracted
565.9402).
4-Bromopseudoceratidine (6) and 19-bromopseudoceratidine
(
(
7): colorless, glassy solid; UV (MeOH) λ (log ε) 215 (4.2), 235
max
1
13
4.2), 277 (4.6) nm; H and C NMR data, Tables 1 and 2;
H O fractions were investigated.
2
+
79
HRESIMS m/z 721.7632 [M + H] (calcd for C H Br N O ,
The EtOAc fraction (5.37 g) was subjected to solid-phase
extraction on a C18 reversed-phase cartridge (Waters) eluted with a
17 21
5
5
2
7
21.7612).
4
,19-Dibromopseudoceratidine (8): glassy solid; UV (MeOH)
gradient of MeOH in H O. After their evaporation and TLC analysis,
2
1
13
the fractions obtained were pooled into eight fractions, named TBA1
λ
max (log ε) 222 (4.2), 235 (4.2), 274 (4.6) nm; H and C NMR
+
(
(
608 mg), TBA2 (567 mg), TBA3 (347 mg), TBA4 (377 mg), TBA5
251 mg), TBA6 (383 mg), TBA7 (338 mg), and TBA8 (399 mg).
data, Tables 1 and 2; HRESIMS m/z 799.6749 [M + H] (calcd for
C
7
9
H
17
20 Br
N
5
O
2
, 799.6717).
6
Fraction TBA2 was separated by HPLC using an Inertsil ODS-2
Tedamide A (9) and tedamide B (10): colorless, glassy solid; [α]
D
column (250 × 9.4 mm, 5 μm), with a H O/MeCN ratio of 75:25, a
−7.75 (c 0.007, MeOH); UV (MeOH) λmax (log ε) 218 (4.2), 275
(4.2) nm; IR (film) νmax 3103, 2938, 2842, 1718, 1677, 1526, 1202,
2
flow rate of 1.5 mL/min, and detection performed at λmax 254 nm. Six
fractions were obtained, among which fraction TBA2B (42.1 mg) was
further investigated and fraction TBA2F (28.1 mg) was identified as
−
1
1
13
1139, and 719 cm ; H and C NMR data, Tables 3 and 4;
+
79
HRESIMS m/z 611.9452 [M + H] (calcd for C18
H
25 Br
N
O
5
,
4
3
4
,5-dibromo-1H-pyrrole-2-carboxylic acid. Fraction TBA2B was
611.9456).
separated by HPLC using an Inertsil ODS-2 column (250 × 9.4
mm, 5 μm), with a H O/MeCN ratio of 67:33, a flow rate of 1.5 mL/
Tedamide C (11) and tedamide D (12): colorless, glassy solid;
[α] +107.33 (c 0.0006, MeOH); UV (MeOH) λmax (log ε) 218
2
D
1
13
min, and detection performed at λmax 254 nm. Seven fractions were
obtained, among which fraction TBA2B3 (9.4 mg) was identified as a
mixture of tedamides A (9) and B (10), fraction TBA2B5 (1.3 mg)
was identified as 3-debromopseudoceratidine (4) and 20-debromop-
seudoceratidine (5), and fraction TBA2B7 (10.2 mg) was identified as
(4.2), 275 (4.2) nm; H and C NMR data, Tables 3 and 4;
HRESIMS m/z 689.8571 [M + H] (calcd for C18
+
79
H
24 Br
N
O
5
,
4
4
689.8562).
12
12
Preparation of N -Acetylpseudoceratidine (2) and N -
Formylpseudoceratidine (3). Pseudoceratidine (1, 5.0 mg, 7.7
mM) was dissolved in freshly distilled pyridine (1 mL), and acetic
anhydride (1 mL, 10.6 mM) was added. The reaction was left under
8
pseudoceratidine (1).
HPLC separation of fraction TBA3 using an Inertsil ODS-2 column
magnetic stirring for 60 h, after which the pyridine/Ac O mixture was
(
250 × 9.4 mm, 5 μm), with a gradient of MeOH in H O from 40%
2
2
evaporated in vacuo. The reaction mixture was purified by HPLC
using an Inertsil ODS-3 column (250 × 4.6 mm, 5 μm), with 65%
MeOH to 100% MeOH over 45 min (flow rate of 1.5 mL/min and
detection at λmax 254 nm), yielded six fractions, among which fraction
TBA3-4 (71.8 mg) was identified as pure pseudoceratidine (1) and
fractions TBA3-3 (17.0 mg), TBA3-5 (13.3 mg), and TBA3-6 (4.3
mg) were further investigated. Fraction TBA3-3 was separated using
an Inertsil ODS-3 column (250 × 4.6 mm, 5 μm), with a gradient of
MeOH, a flow rate of 1.0 mL/min, and detection performed at λ
max
1
2
12
2
54 nm. N -Acetylpseudoceratidine (2) (2.7 mg) and N -
formylpseudoceratidine (3) (1.4 mg) were obtained in 50.7% and
26.9% yields, respectively.
1
2
MeOH in H O from 38% MeOH to 60% MeOH over 38 min (flow
N -Acetylpseudoceratidine (2): colorless, glassy solid; UV
2
rate of 1.0 mL/min and detection at λmax 254 nm), to yield six
fractions, among which TBA3-3C (2.3 mg) was identified as a mixture
of tedamides C (11) and D (12). Fraction TBA3-5 (13.3 mg) was
separated by HPLC using an Inertsil ODS-3 column (250 × 4.6 mm,
(MeOH) λmax (log ε) 222 (4.2), 235 (4.2), 275 (4.6) nm; IR
−
1
1
13
(film) νmax 2924, 2842, 1608, 1560, 1526, and 664 cm ; H and
C
−
NMR data, Tables 1 and 2; HRESIMS m/z 683.8433 [M − H]
7
9
(calcd for C19H22 Br N O , 683.8456).
4 5 3
1
2
5
μm), with a gradient of MeOH in H O from 10% MeOH to 100%
N -Formylpseudoceratidine (3): colorless, glassy solid; UV
2
MeOH over 35 min (flow rate of 1.0 mL/min and detection at λmax
54 nm), to yield four fractions, among which fraction TBA3-5B (2.7
(MeOH) λmax (log ε) 222 (4.2), 235 (4.2), 275 (4.6) nm; IR
−
1
1
13
2
(film) νmax 2924, 2842, 1587, 1360, and 650 cm ; H and C NMR
−
mg) was further purified using an Inertsil ODS-3 column (250 × 4.6
data, Tables 1 and 2; HRESIMS m/z 669.8283 [M − H] (calcd. for
7
9
mm, 5 μm), with a gradient of MeOH in H O from 55% MeOH to
C H22 Br N O , 671.8299).
18 4 5 3
2
6
0% MeOH over 30 min (flow rate of 1.0 mL/min and detection at
Bioassay Procedures. Chemical Compounds. Alamar blue
λmax 254 nm), to yield pure 19-bromopseudoceratidine (7) (1.0 mg).
Fraction TBA3-6 (4.3 mg) was purified by HPLC using an Inertsil
ODS-3 column (250 × 4.6 mm, 5 μm), with a gradient of MeOH in
(resazurin), DMSO, MeOH, M-199 medium, RPMI-1640, phos-
phate-buffered saline (PBS), sodium azide (99.5% purity), bacterial
lipopolysaccharide (99% purity), TritonX-100 (99% purity), and
benznidazole (97% purity) were purchased from commercial
suppliers.
H O from 10% MeOH to 100% MeOH over 35 min (flow rate of 1.0
2
mL/min and detection at λmax 254 nm), to yield 0.5 mg of 4,19-
dibromopseudoceratidine (8).
Anti-Leishmanial and Anti-Trypanosoma cruzi in Vitro Assays.
L. (L.) amazonensis (MHOM/BR/1973/M2269) promastigotes were
maintained at 26 °C in Medium 199 (Sigma-Aldrich) supplemented
with 5% penicillin/streptomycin, 0.1% hemin (25 mg/mL in 50%
triethanolamine), 20% heat-inactivated fetal bovine serum, 10 mM
Separation of fraction TBA-5 (250.5 mg) by HPLC using an
Inertsil ODS-2 column (250 × 9.4 mm, 5 μm) and a gradient of 1:1
MeOH/MeCN in H O, from 44% to 100% of the organic mixture
2
over 30 min, yielded seven fractions, among which TBA-5D (80.7
mg) was identified as pure pseudoceratidine (1).
47
adenine (pH 7.5), and 5 mM L-glutamine. L. (L.) infantum
(MHOM/BR/1972/LD) was cultured as described above, with the
exception that 5% human sterile urine was added to the culture
medium. T. cruzi epimastigotes (Y strain) were grown in liver infusion
tryptose medium supplemented with 20 mg/L hemin and 10% fetal
Separation of the fraction TBA-7 (338 mg) by HPLC using an
Inertsil ODS-2 column (250 × 9.4 mm, 5 μm), with a gradient of
MeOH/MeCN (1:1) in H O from 44% MeOH to 100% MeOH/
2
MeCN (1:1) over 35 min (flow rate of 1.5 mL/min and detection at
λmax 254 nm), yielded fraction TBA-7A (98.2 mg). This fraction was
further separated by HPLC using an Inertsil ODS-3 column (250 ×
48
calf serum at 28 °C as previously described. The antiparasitic
activity of pseudoceratidine derivatives was assessed against
49
4
3
.6 mm, 5 μm), with a gradient of MeOH/MeCN (1:1) in H O from
0% to 100% MeOH/MeCN (1:1) over 30 min (flow rate of 1.0 mL/
trypanosomatids, using an MTT viability assay after 24 h of
incubation with the series of pseudoceratidine derivatives. The half-
2
min and detection at λmax 254 nm), to yield 10 fractions, among which
TBA-7A8 (5.0 mg) was identified as a mixture of 4-bromopseudocer-
atidine (6) and 19-debromopseudoceratidine (7).
maximal effective concentration (EC ) values for the Leishmania spp.
and T. cruzi populations were calculated from sigmoidal regression of
the concentration−response curves using Scientific Graphing and
50
M
DOI: 10.1021/acs.jnatprod.7b00876
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Analysis Software Origin 5.0. Each experiment was performed in
triplicate and repeated two or three times. Cytotoxic experiments
were conducted using bone marrow macrophages from BALB/c
female mice. Differentiated cells were cultured in 96-well plates at 37
drug using complete media, such that the final concentration
approximates IC50 following three or four 2-fold dilutions. Using
these stock solutions, volume−volume (v/v) mixtures of artesunate
and pseudoceratidine (1) were prepared in 0:5, 1:4, 2:3, 1:4, and 5:0
ratios. These mixtures were 2-fold serially diluted to generate a range
of seven concentrations in each case. The ∑FIC50 were calculated
using the following equation: FIC50 artesunate (IC50 of artesunate
when combined with pseudoceratidine/artesunate IC50) + FIC50
pseudoceratidine (IC50 of pseudoceratidine when combined with
artesunate/pseudoceratidine IC50). Isobologram curves were con-
structed by plotting FIC50 pseudoceratidine vs FIC50 artesunate. A
straight diagonal line (∑FIC50 = 1) indicates an additive effect
between artesunate and pseudoceratidine, a concave curve below the
diagonal (FICindex < 1) indicates a synergistic effect, and a convex
°
C for posterior incubation with increasing concentrations of
pseudoceratidine derivatives for 24 h. The MTT method was also
49
performed, and CC50 values were calculated accordingly.
Intracellular infections were obtained by infecting bone-marrow-
derived macrophages with stationary phase L. (L.) amazonensis
promastigotes (10 parasites: 1 macrophage; MOI = 10). After 1 h of
incubation with the parasites, cultures were washed three times with
PBS 1× and maintained at 33 °C, 5% CO , for 24 h. After this period,
2
infections were established and different concentrations of pseudocer-
atidine compounds were added to the cultures, which remained at 33
54
°C for 24 h. Cells were then washed three times with warm PBS 1×
curve above the diagonal (FICindex > 1) indicates antagonism.
and fixed with MeOH for subsequent staining using the Instant Prov
kit (Newprov). The number of intracellular amastigotes was obtained
by counting 300 cells in triplicate coverslips. Photomicrographs of
infections were recorded using the Leica LAS Core microscope
system. Experiments using BALB/c mice were approved by the
Ethical Committee for Animal Experimentation of the Biology
Institute of the State University of Campinas−UNICAMP (4535-1/
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00876.
■
*
S
2
017).
Isolation procedures, HRMS, IR, and 1H and 13C NMR
Antiplasmodial in Vitro Assays against P. falciparum Blood
Parasites. P. falciparum blood parasites [3D7, sensitive strain; K1,
spectra of compounds 1−12, synthesis procedures and
1
13
IR, HRMS, and H and C NMR spectra of compounds
chloroquine-, cycloguanil-, and pyrimethamine-resistant strain] were
50
cultured as previously described. Freshly sorbitol synchronized ring
16, 17, 20, 21, 23, 25, 27−29, 31, 33, 35, 38, 39, 42, 43,
51
stages were incubated with the test samples at various concen-
trations, previously solubilized in 0.05% DMSO (v/v). Each assay was
performed in triplicate. Results were compared with control cultures
in complete medium with no assay samples. Pyrimethamine,
chloroquine, cycloguanil, and sodium artesunate were used in each
experiment as antimalarial controls. The activity of test samples was
4
6, 47, 50, and 51 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +55-16-33739954. Fax: +55-16-33739952. E-mail:
rgsberlinck@iqsc.usp.br.
ORCID
Daniel Romo: 0000-0003-3805-092X
Roberto G. S. Berlinck: 0000-0003-0118-2523
■
52
measured using the SYBR green assay. Briefly, the plates were
centrifuged at 700g for 5 min at room temperature to remove the
medium, washed with 1× PBS, and incubated for 30 min with lysis
buffer solution [2.4228 g of TRIS, ultrapure (for 20 mM solution),
pH 7.5; 1.8612 g of EDTA 5 mM ultrapure (for 5 mM solution); 80
μg of saponin (0.008% w/v); 800 μL of Triton X-100 (0.08% v/v);
Present Address
Departamento de Quimica, Universidad Santiago de Cali,
H O Type I] and SYBR green I DNA stain (1:20 000). The
‡
2
́
fluorescence of uninfected erythrocytes was considered as a
background. Fluorescence was measured on a SpectraMax340PC384
fluorimeter at 485/535 nm. The half-maximal compound inhibitory
concentration (IC ) was estimated by curve fitting using software
Calle 5#62-00, Santiago de Cali, Valle del Cauca, Colombia.
Author Contributions
L. L. L. Parra and A. F. Bertonha contributed equally.
Notes
#
50
from the OriginLab Corporation and comparing to the parasite
growth in test-sample-free medium.
Cytotoxicity Tests Using Immortalized Cells. The cytotoxicity of
The authors declare no competing financial interest.
test compounds was evaluated in a human hepatoma cell line
2
ACKNOWLEDGMENTS
(
HepG2) using cells cultured in 75 cm sterile flasks containing
■
RPMI-1640 medium (supplemented with 10% heat-inactivated fetal
The authors thank Dr. H. Xue (Baylor University) for help in
the characterization of some of the synthetic derivatives and
Dr. D. E. Williams (Department of Earth, Ocean and
Atmospheric Sciences, University of British Columbia), for
his assistance in some of the HRMS measurements. Financial
support was provided by a FAPESP BIOTA/BIOprospecTA
grant (2013/50228-8) awarded to R.G.S.B., CEPID-CIBFar
grant (2013/07600-3) to G.O., CNPq (grants 471509/2012-4
and 405330/2016-2) to R.V.C.G., FAPESP Young Inves-
tigators Grant (2014/21129-4) to D.C.M., and a FAPESP
Regular Research Grant (2015/24595-9) to F.R.G. L.L.L.P.
and A.F.B. thanks CAPES for a Ph.D. scholarship and also for a
Science without Borders scholarship at Baylor University for
A.F.B.
bovine serum and 40 mg/L gentamicin) under a 5% CO atmosphere
2
at 37 °C. When confluent, the cell monolayer was washed with culture
medium, trypsinized, distributed in a flat-bottomed 96-well plate (5 ×
3
53
1
0 cells/well), and incubated for 18 h at 37 °C for cell adherence.
The compounds (in 20 μL of solution) at various concentrations
(
1000−1 μg/mL) were placed in 96-well plates and incubated with
the cultured cells for 24 h under a 5% CO atmosphere at 37 °C.
Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
2
(
MTT) solution (5 mg/mL; 20 μL/well for 3 h) was used to evaluate
the mitochondrial viability. The supernatants were carefully removed,
and 100 μL of DMSO was added to each well and mixed to solubilize
the formazan crystals. The optical density was determined at 570 and
6
30 nm. The cell viability was expressed as the percentage of the
control absorbance in the untreated cells after subtracting the
appropriate background.
In Vitro Association with Artesunate. The isobologram was built
to analyze the effects of drug combination aiming to determine the
additive, synergic, or antagonistic effect. Briefly, the fractional half-
maximum inhibitory concentration (FIC ) was calculated for each
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5
0
drug pair combination. A stock drug solution was prepared for each
N
DOI: 10.1021/acs.jnatprod.7b00876
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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DOI: 10.1021/acs.jnatprod.7b00876
J. Nat. Prod. XXXX, XXX, XXX−XXX