Anti-acanthamoeba activity of brominated sesquiterpenes from laurencia johnstonii

Article abstract of DOI:10.3390/md16110443

Focused on our interest to develop novel antiparasistic agents, the present study was aimed to evaluate the biological activity of an extract of Laurencia johnstonii collected in Baja California Sur, Mexico, against an Acantamoeba castellanii Neff strain. Bioassay-guided fractionation allowed us to identify the amoebicidal diastereoisomers α-bromocuparane (4) and α-isobromocuparane (5). Furthermore, bromination of the inactive laurinterol (1) and isolaurinterol (2) yielded four halogenated derivatives, (6)-(9), which improved the activity of the natural sesquiterpenes. Among them, the most active compound was 3α-bromojohnstane (7), a sesquiterpene derivative which possesses a novel carbon skeleton johnstane.

Full text of DOI:10.3390/md16110443

marine drugs
Article
Anti-Acanthamoeba Activity of Brominated
Sesquiterpenes from Laurencia johnstonii
Sara García-Davis 1,2, Ines Sifaoui 3, María Reyes-Batlle 3, Ezequiel Viveros-Valdez 2,
José E. Piñero 3, Jacob Lorenzo-Morales 3, José J. Fernández 1,4,
*
and Ana R. Díaz-Marrero 1,
*
1
Instituto Universitario de Bio-Orgánica Antonio González (IUBO AG), Centro de Investigaciones
Biomédicas de Canarias (CIBICAN), Universidad de La Laguna (ULL), Avda. Astrofísico F. Sánchez, 2,
38206 La Laguna, Tenerife, Spain; sara.garciadv@uanl.edu.mx
2
3
Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León (UANL), Av. Pedro de Alba s/n,
66450 San Nicolás de los Garza, Nuevo León, Mexico; jose.viverosvld@uanl.edu.mx
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Islas Canarias,
Universidad de La Laguna, Av. Astrofísico Francisco Sánchez s/n, 38206 La Laguna, Tenerife, Spain;
ines.sifaoui@hotmail.com (I.S.); mreyesba@ull.edu.es (M.R.-B.); jpinero@ull.edu.es (J.E.P.);
jmlorenz@ull.edu.es (J.L.-M.)
4
Departamento de Química Orgánica, Universidad de La Laguna (ULL), Avda. Astrofísico F. Sánchez, 2,
38206 La Laguna, Tenerife, Spain
*
Correspondence: jjfercas@ull.edu.es (J.J.F.); adiazmar@ull.edu.es (A.R.D.-M.);
Tel.: +34-922-318-586 (J.J.F. & A.R.D.-M.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 October 2018; Accepted: 9 November 2018; Published: 11 November 2018
Abstract: Focused on our interest to develop novel antiparasistic agents, the present study was aimed
to evaluate the biological activity of an extract of Laurencia johnstonii collected in Baja California
Sur, Mexico, against an Acantamoeba castellanii Neff strain. Bioassay-guided fractionation allowed
us to identify the amoebicidal diastereoisomers
Furthermore, bromination of the inactive laurinterol (
halogenated derivatives, ( )–( ), which improved the activity of the natural sesquiterpenes. Among
them, the most active compound was 3 -bromojohnstane ( ), a sesquiterpene derivative which
possesses a novel carbon skeleton johnstane.
α-bromocuparane (4) and
α
-isobromocuparane (
5).
1) and isolaurinterol (
2) yielded four
6
9
α
7
Keywords: brominated sesquiterpene; marine natural products; Laurencia johnstonii; johnstane;
3-bromojohnstane; anti-amoeboid activity; Acanthamoeba
1. Introduction
Free-living amoeba (FLA) are widely distributed protozoa in the environment [
parasites present a life cycle with two different stages: the trophozoite and the resistant phase,
the cyst. Among FLA, Acanthamoeba genus [ ] has been isolated from air, soil, water, contact lenses, air
conditioning units, clinical samples, among others [ ]. These parasites are able to cause pathologies
in humans such as Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK) [
Regarding Acanthamoeba infections, an early diagnosis is crucial to achieve a successful treatment [
1–3]. These
4
5
1
–
3
3
,
,
5
6
].
].
Antimicrobial chemotherapy is the most widely used method for treating Acanthamoeba-caused
infections. Pentamidine, azoles, sulfonamides, and possibly flucytosine, are among the most frequently
used drugs in successfully treated cases of GAE, whereas topical chlorhexidine or polyhexamethylene
biguanide appear to be the most effective option in cases of AK [7]. However, the existing therapies
are not fully effective against these organisms mainly due to the existence of the cyst phase, and also
due to the presence of strains that are resistant to the currently used anti-amoebic drugs [2,3,8].
Mar. Drugs 2018, 16, 443; doi:10.3390/md16110443
www.mdpi.com/journal/marinedrugs

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In the past, natural products have been used for the treatment of different parasitic diseases.
Artemisinin, quinine, amphotericin B, and ivermectin are examples of important antiparasitic
compounds isolated from plants and microorganisms [9,10]. From 1981 to 2014, 15 small molecules
were approved as antiparasitic drugs; among them, two were obtained from natural sources and
five are the result of semisynthetic derivatives of natural products [11], however, none of them
came from marine sources. Many other natural products of diverse molecular structures have
revealed antiparasitic potency in the laboratory and, thus, represent interesting lead structures for the
development of new and urgently needed antiparasitic agents.
The genus Laurencia is one of the richest sources of novel secondary metabolites among red
algae [12–15]. Although the chemistry of Laurencia species has been exhaustively investigated,
the biological activity of the isolated secondary metabolites has not been studied in a systematic
way. Furthermore, Laurencia metabolites have been revealed to possess antiparasitic properties against
a number of parasites and their vectors. However, no activity has been previously reported against
Acanthamoeba [10,16].
Focused on our interest to develop novel lead structures for the development of antiparasistic agents,
the present study was aimed to evaluate the biological activity of an extract of Laurencia johnstonii against
an Acanthamoeba castellanii Neff strain. To the best of our knowledge, johnstonol [17] and prepacifenol
epoxide [18] are the only sesquiterpenes isolated from specimens of Laurencia johnstonii, both with
a chamigrene carbon skeleton. In this paper, we report the isolation of the known brominated
metabolites laurinterol (
-bromocuparane ( ) and
backbones. (Figure 1). In addition, transformation of (
1
), isolaurinterol (
2
)
,
aplysin (
), with cyclolaurane, laurane and cuparane
) and ( ) has yielded four structural derivatives
3), and the antiparasitic diastereoisomers
α
4
α
-isobromocuparane (5
1
2
(6–9) which improved the antiparasitic activity with respect to the natural products.
Figure 1. Structures of natural sesquiterpenes (1)–(5) isolated from Laurencia jonhstonii.
2. Results and Discussion
2.1. Isolation and Identification of Natural Brominated Sesquiterpenes
Laurencia johnstonii was collected off the coast of the Gulf of California, Mexico. Clean and dry
specimens were extracted in ethanol to give a crude extract which showed moderate anti-Acanthamoeba
activity with an IC50 value of 125.14
extract by Sephadex LH-20 led us to an active fraction (SF3). This fraction was further chromatographed
±
4.5 µg/mL. The bioassay-guided fractionation of the ethanolic
over a silica gel column using a step-gradient from n-hexane to EtOAc to give seven subfractions.
The most active was SF3.1 (n-hexane fraction) with an IC50 value of 101.29
a separation on a silica gel open column eluted with mixtures of n-hexane:EtOAc, fraction SF3.1
yielded aplysin ( ) [19], and the active stereoisomers -bromocuparane ( ) and -isobromocuparane
) [20], which showed activity against Acanthamoeba castellanii Neff with IC50 values of 90.68 1.5
and 64.25 ± 1.5 µg/mL, respectively. Additionally, laurinterol ( ) [21], and isolaurinterol ( ) [22] were
isolated from the inactive fraction SF3.2. The NMR, mass spectrometry, and optical rotation data of
)–( ) were compared with those previously reported in the literature to confirm their structures
(Supplementary Materials).
±
0.2 µg/mL. After
3
α
4
α
(5
±
1
2
(1
5

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2.2. Derivatization of Laurinterol (1) and Isolaurinterol (2)
Halogenation is a common biosynthetic strategy found in marine organisms as is the case of
Laurencia species [23]. Halogen-containing natural products display a wide range of biological activities;
therefore, they are interesting structures for medicinal chemistry studies. The presence of halogen
substituents in many natural products also enhances their biological activities [24]. In order to evaluate
structure
) and (
−
activity relationships of laurinterol and its congeners in antiparasitic assays, bromination of
(
1
2
) were carried out to obtain their brominated derivatives. Hence, laurinterol ( ) was dissolved
1
in ethyl ether and treated with bromine. The reaction mixture was chromatographed on a silica gel
column to obtain the brominated sesquiterpene derivatives (6) and (7) (Figure 2).
Figure 2. Bromination reaction of natural laurinterol (1) and isolaurinterol (2).
Compound (6) was obtained as a colorless oil. Its molecular formula was established by HR-EI-MS
(High-Resolution Electron Impact Mass Spectrometry) analysis as C15H18Br2O by the presence of three
molecular ions [M]+ at m/z 371.9711/373.9702/375.9677 (calcd. 371.9724/373.9704/375.9683; ratio
50:100:48), indicative of two bromine atoms and six degrees of unsaturation. The structure of (
determined by comparison of its NMR data with those for aplysin (3) (Table 1). The main differences
observed in their 1H NMR spectra were the absence of one of the two aromatic singlets of (
), H-8
δH 6.59), and a deshielded aromatic methyl group H3-15 (δH 2.49) for ( ) compared with that of
6) was
3
(
6
3
H3-15 (δH 2.30). These values agree with the presence of a bromine atom at C-8 in the aromatic ring as
well as with the information provided by mass spectrometry.
1
Table 1. H and 13C NMR data for aplysin (3) and (6) (600 MHz, 150 MHz, CDCl3).
Aplysin (3)
6
Position
δC, Type
δ
(J in Hz)
δC, Type
δ (J in Hz)
H
H
1
2
3
54.3, C
99.8, C
46.0, CH
56.0, C
100.5, C
46.4, CH
1.75, ddd (12.8, 6.9, 6.5)
1.62, m
1.79, ddd (13.3, 6.5, 6.5)
1.65, m
31.1, CH2
31.3, CH2
4
1.15, m
1.15, m
1.85, dd (6.4, 12.1)
1.58, d (6.2)
1.86, dd (12.1, 6.2)
1.64, ddd (12.1, 6.2, 6.2)
42.5, CH2
42.7, CH2
5
6
7
8
136.2, C
158.1, C
110.8, CH
136.9, C
136.2, C
156.0 1, C
105.1, C
6.59, s
9
136.0, C
10
11
12
13
14
15
113.9, C
114.1, C
126.5, CH
13.0, CH3
23.1, CH3
23.3, CH3
29.9, CH3
7.14, s
1.10, d (6.8)
1.30, s
125.4, CH
13.3, CH3
20.4, CH3
23.2, CH3
23.5, CH3
7.11, s
1.14, d (6.8)
1.34, s
1.29, s
2.30, s
1.32, s
2.49, s
1
Chemical shift deduced from the HMBC experiment.

Mar. Drugs 2018, 16, 443
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Aplysin (
3
) ([
) was easily obtained after treatment of laurinterol (
1
) with HBr (Figure 2). The specific
25
25
rotations for (
1
α
]
+17) and
3
([
3
α
]
−
49) are consistent with those reported in the literature for
D
D
α
25
both compounds (
by X-ray crystallography [21
coupling constants of H3-12, H3-13, H3-14 and H-3 between
configurations of are 1S, 2S, and 3S. Caccamese and Rinehart identified
by GC-MS [26]. Later, in 2010, a patented methodology for the extraction of antiobesity sesquiterpenes
from Laurencia species [27] refers as 8-bromoaplysin, however no complete NMR data has been
reported for this brominated sesquiterpene.
1): [
α
]
25 +13 and
: [
25]. According to this, and supported by consistent chemical shifts and
and aplysin ( ) (Table 1), the absolute
as bromoaplysine in 1978
]
−
86, for which the absolute configuration was stablished
D
D
,
6
3
6
6
6
Compound
7 was obtained as a colorless oil. The HREIMS analysis provided four molecular ions
[M]+ at m/z 449.8840/451.8800/453.8798/455.8758 (calcd. 449.8829/451.8809/453.8789/455.8768; ratio
26:78:67:26) suggesting a molecular formula C15H17OBr3 and six degrees of unsaturation. The structure
of 7 was established based on the analysis of its spectroscopic data and comparison with those of 6.
1D and 2D NMR spectra evidenced three methyl, three methylenes, and two methine groups (one on
sp2 carbon and one bearing heteroatom), in addition to five sp2 and two sp3 quaternary carbons.
Similar to 6, the pentasubstituted phenyl ring was confirmed by the presence of a singlet at δH 7.14
(H-12) and the methyl group at δH 2.53 (H3-15) in the 1H NMR spectrum (Table 2), and the HMBC
correlations from H3-13 (δH 2.54, s) and H3-14 (δH 1.04, s) to a quaternary carbon at δC 92.3 (C-2),
and from H3-14 to C-7 (δC 136.3) confirming the presence of the same dihydrofuran ring found
in 6. The main difference between both compounds was a COSY spin system from the diasterotopic
protons H2-3 (δH 2.42/1.99), sequentially coupled with the bromomethine H-4 (δH 3.84), methylene
H2-5 (δH 2.18/1.76), and methylene H2-6 (δH 2.18/1.54). HMBC correlations from H3-14 (δH 1.04) to
C-6 (δC 32.8) and H3-13 (δH 2.54) to C-3 (δC 47.6) led to the connection of this substructure within
the molecule and thus established the planar structure of the rearranged sesquiterpene
in Figure 3.
7 as shown
Table 2. NMR data for 3α-bromojohnstane (7) (600 MHz, 150 MHz, CDCl3).
3α-Bromojohnstane (7)
Position
δC, Type
δ (J in Hz)
H
1
2
48.1, C
92.3, C
β: 2.42 dd (3.6, 12.9)
α: 1.99 dd (12.6, 12.9)
47.6, CH2
45.8, CH
34.2, CH2
3
4
3.84 dddd (3.6, 3.6, 12.3, 12.6)
β: 2.18 dddd (3.2, 3.5, 3.6, 12.9)
α: 1.76 dddd (3.5, 12.3, 12.3, 12.6)
β: 1.54 ddd (3.5, 12.3, 12.9)
α: 2.18 ddd (3.2, 3.5, 12.9)
5
32.8, CH2
6
7
8
136.3, C
155.3, C
9
107.6, C
10
11
12
13
14
15
136.6, C
115.5, C
124.6, CH
19.7, CH3
26.7, CH3
23.3, CH3
7.14 s
1.54 s
1.04 s
2.53 s

Mar. Drugs 2018, 16, 443
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(c)
) Selected COSY and HMBC correlations, (b) key-NOE correlations and (c) 1D-NOE
Figure 3.
(
a
experiments of 3α-bromojohnstane (7).
NOE correlations observed from H3-13 to H3-14 and H-4, and from H-4 to the diastereotopic
proton H-3 located all these protons on the same face of the molecule. Since the absolute configuration
of laurinterol ( ) has been confirmed and configuration at C-1 is conserved in , it is possible to establish
the absolute configuration of its chiral centers as 1S, 2S, and 4S.
In 1972, Yamada et al. reported the synthesis of 2,3-dimethyl-3-(5-bromo-2-methoxy-4-methylphenyl)
-cyclohexane from an anisole derivative as a key intermediate to obtain aplysin ( ) and other related
β
1
7
3
sesquiterpenes [28]. Also, synthetic efforts to obtain the sesquiterpene cuparene through radical
cyclization exclusively yielded 1-(1,2-dimethylcyclohexyl)-4-methylbenzene [29].
It is noteworthy that, even though chemical investigations on the prolific genus Laurencia have
yielded over 500 sesquiterpene metabolites belonging to more than 50 carbon frameworks [16], so far
none has been reported to possess that of
7. Thus, for this carbon backbone we propose the name
johnstane and the name 3α-bromojohnstane for 7 (Figure 4).
Figure 4. Johnstane carbon skeleton and numbering system.
In the same way, isolaurinterol (
afford , and the known sesquiterpenes 8,10-dibromoisoaplysin (
derivative 9, also obtained after bromination of debromoaplysinol [31].
2) was dissolved in ethyl ether and treated with bromine to
6
8) [30] as well as the 13-hydroxy

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2.3. Antiparasitic Effect Against Acanthamoeba Castellanii Neff Strain
As well as in the case of natural sesquiterpenes
1–5, the antiamoeboid activity of the brominated
sesquiterpene derivatives was tested against Acanthamoeba castellanii Neff (Inhibitory Concentrations
6–9
(IC50) shown in Table 3). Moreover, their toxicity (Cytotoxic Concentrations (CC50) at 24 h, Table 3) against
murine macrophages J774.A1 (ATCC # TIB-67) at 24 h was evaluated as previously described [32,33].
Table 3. Effect of Laurencia johnstonii ethanolic extract and
1–9 against Acanthamoeba castellanii Neff
(IC50) and murine macrophage J774.A1 (CC50). * Reference compounds.
Sample
IC50 (µg/mL)
IC50 (µM)
CC50 (µg/mL)
Crude extract
125.14 ± 4.5
n/d
1
2
3
4
5
6
7
8
>100
23.65 ± 2.3
>100
>100
7.25 ± 0.7
323.69 ± 12.0
33.04 ± 4.2
90.674 ± 1.529
64.251 ± 3.492
24.559 ± 1.105
18.804 ± 0.198
22.818 ± 1.896
29.937 ± 2.918
1.526 ± 0.45
0.33 ± 0.1
322.41 ± 5.44
228.46 ± 12.42
65.64 ± 2.95
41.51 ± 0.44
50.37 ± 4.19
76.74 ± 7.48
3.02 ± 0.89
85.34 ± 10.9
32.880 ± 3.125
62.341 ± 2.589
70.365 ± 3.245
74.931 ± 2.769
6.64 ± 0.35
9
Chlorhexidine *
Voriconazole *
0.94 ± 0.29
2.64 ± 0.27
As shown in Table 3, all brominated derivatives (
sesquiterpenes ( ). Monobrominated compounds in the aromatic ring,
suggesting that dibromination of the aromatic moiety is relevant to obtain antiamoeboid activity.
On the other hand, natural diastereoisomers and , which only differ in the configuration of the
bromine atom attached to C-3 of cyclopentane ring, showed a differentiated activity. It is interesting
to note that an -oriented bromine substitution at C-3 in increases the activity against A. castellanii
while decreases the toxicity, a structural feature also found in compound . 3 -bromojohnstane (
was the most active molecule against A. castellanii with an IC50 value of 41.51 M, and one of those
6
–
9) improved the activity of the natural
1–
5
1–3, were inactive, thus
4
5
α
5
7
α
7)
µ
with lower toxicity values against murine macrophages among the assayed compounds.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were measured in CH2Cl2 on a PerkinElmer 241 polarimeter (Waltham, MA,
USA), by using a sodium lamp operating at 589 nm. NMR spectra were recorded on a Bruker AVANCE
500 MHz or 600 MHz, as required. NMR spectra were obtained dissolving samples in CDCl3 (99.9%)
and chemical shifts are reported relative to solvent (δH 7.26 and δC 77.0 ppm) and TMS as internal
pattern. Bruker AVANCE 600 MHz instrument is equipped with a 5 mm TCI inverse detection
cryoprobe. Standard Bruker NMR pulse sequences were utilized. HR-ESI-MS (High-Resolution
ElectroSpray Ionization Mass Spectrometry) and HR-EI-MS (High-Resolution Electron Impact Mass
Spectrometry) data were obtained on a Waters LCT Premier XE Micromass (Manchester, UK) and
VG-AutoSpec Micromass (Manchester, UK) spectrometers, respectively. IR spectra were recorded
on a Bruker IFS66/S (Ettlingen, Germany) equipped with an ATR accessory using CH2Cl2 solutions.
EnSpire® Multimode Reader (Perkin Elmer, Walt, MA, USA) using absorbance values of Alamar Blue®
reagent. TLC (Thin layer chromatography) (Merck, Darmstadt, Germany) was visualized by UV light
(254 nm) and spraying with cobalt chloride reagent (2% in sulfuric acid, 10%) and heating.

Mar. Drugs 2018, 16, 443
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3.2. Biological Material
Laurencia johnstonii was collected by hand during June–July 2015 off the coast of Baja California
Sur, Mexico (24◦21010.800 N, 110◦16058.800 W). A voucher specimen (code 13-003) was deposited at the
Herbarium of the Laboratory of Marine Algae of the CICIMAR (Centro Interdisciplinario de Ciencias
Marinas, Mexico). This seaweed was identified to genus and species level by Dr. Rafael Riosmena
Rodríguez (Universidad Autónoma de Baja California Sur) using taxonomic keys for future reference.
3.3. Extraction and Isolation
Specimens of Laurencia johnstonii were cleaned of sand and epiphytes, and dried. Dried alga
was crushed using a blender and extracted with EtOH for three days at 25 ◦C under gentle agitation.
The EtOH was replaced (3
no. 4 filter paper. Solvent was removed using a rotatory vacuum evaporator. 10.0 g of the resulting
extract were chromatographed in Sephadex LH-20 (Sigma, St. Louis, MO, USA) (500 70 mm, CH3OH,
100%) to obtain five fractions. The active fraction SF3 was separated in flash Silicagel 0.2–0.5 mm
(Sigma-Aldrich, St. Louis, MO, USA) (130 70 mm) using a stepwise gradient of n-hexane: ethyl
acetate to obtain 7 fractions. Fractions SF3.1 (100% n-hexane) and SF3.2 (95% n-hexane) were separated
on a normal phase open column (Silicagel, 0.2–0.5 mm, 300 50 mm, using a stepwise gradient of
n-hexane: ethyl acetate) to yield pure compounds (10.5 mg), (7.2 mg) and (45.9 mg) from SF3.1 and
compounds (10.2 mg), (8.1 mg) and (55.7 mg) from SF3.2. (Supplementary Materials, Scheme S1)
×
1200 mL), and the combined extracts were filtered through a Whatman
×
×
×
5
4
3
3
2
1
3.3.1. Laurinterol (1)
White crystal; [
293.0541), 295.0518 [M
α
]
25 +17 (c 0.15, CH2Cl2); HRESIMS m/z 293.0531 [M
−
−
H]− (calc. C15H18O79Br,
0.55 (1H, dd,
D
−
H] (calc. C15H18O81Br, 295.0521) 1H NMR (500 MHz, CDCl3)
δ
J = 7.9, 4.8 Hz, H-12), 0.58 (1H, t, J = 4.6 Hz, H-12), 1.15 (1H, dt, J = 8.1, 4.3 Hz, H-3), 1.28 (1H, m, H-5),
1.32 (3H, s, H-13), 1.41 (3H, s, H-14), 1.66 (1H, dd, J = 12.3, 8.0 Hz, H-4), 1.95 (1H, tdd, J = 12.3, 8.1, 4.4,
H-4), 2.09 (1H, dd, J = 13.2, 8.1 Hz, H-5), 2.29 (3H, s, H-15), 5.26 (1H, br, s, 7-OH), 6,61 (1H, s, H-8),
7.61 (1H, s, H-11); 13C NMR (125 MHz, CDCl3)
δ 16.2 (C-12), 18.6 (C-13), 22.2 (C-14), 23.5 (C-15), 24.4
(C-3), 25.3 (C-4), 29.6 (C-2), 35.9 (C-5), 114.9 (C-10), 118.8 (C-8), 132.3 (C-11), 134.0 (C-6), 135.9 (C-9),
153.3 (C-7).
3.3.2. Isolaurinterol (2)
25
Colorless amorphous solid; [
α
]
−
46 (c 0.14, CH2Cl2); HRESIMS m/z 293.0536 [M
−
H]− (calc.
D
C15H18O79Br, 293.0541), 295.0528 [M
−
H] (calc. C15H18O81Br, 295.0521) 1H NMR (500 MHz, CDCl3)
−
δ
1.21 (3H, d, J = 7.0 Hz, H-12), 1.42 (1H, ddd, J = 12.8, 8.2, 6.6 Hz, H-5), 1.46 (3H, s, H-14), 1.59 (1H, dt,
J = 12.9, 7.1, 7.1 Hz, H-4), 2.05 (1H, ddt J = 12.8, 8.5, 7.0, 7.0 Hz, H-3), 2.20 (1H, ddd, J = 13.0, 8.1 6.7 Hz,
H-4), 2.31 (3H, s, H-15), 2.85 (1H, ddt, J = 9.1, 6.9, 6.9, 2.3, 2.3 Hz, H-5), 4.94 (1H, d, J = 2.2 Hz, H-13),
5.11 (1H, d, J = 2.2 Hz, H-13), 5.56 (1H, br, s, 7-OH), 6.73 (1H, s, H-8), 7.45 (1H, s, H-11); 13C NMR
(125 MHz, CDCl3)
δ 21.2 (C-14), 22.2 (C-15), 27.8 (C-12), 31.2 (C-4), 37.6 (C-3), 39.1 (C-5), 49.8 (C-1),
106.9 (C-13), 115.5 (C-10), 120.4 (C-8), 131.2 (C-11), 132.7 (C-6), 137.2 (C-9), 153.0 (C-7), 165.4 (C-2).
3.3.3. Aplysin (3)
25
D
−
H] (calc. C15H18O79Br,
Colorless needles; [
α
]
−
49 (c 0.19, CH2Cl2); HRESIMS m/z 293.0551 [M
−
293.0541), 295.0529 [M − H]− (calc. C15H18O81Br, 295.0524). 1H and 13C NMR data (Table 1).
3.3.4. α-Bromocuparane (4)
25
White amorphous solid; [
α
]
+21 (c 0.19, CH2Cl2); HREIMS [M
−
H]+ m/z 279.0744 (calc. for
Br]+ m/z 201.1640 (calc. for
0.62 (3H, s, H-13), 1.09 (3H, s, H-12), 1.43 (3H, s, H-14),
1.96 (1H, m, H-5), 2.24 (1H, m, H-4), 2.27 (1H, m, H-5), 2.32 (3H, s, H-15), 2.52 (1H, ddt, J = 14.3, 9.3, 5.2,
D
C15H2079Br, 279.0748), 282.0723 (calc. for C15H2081Br, 282.0728), [M
C15H21, 201.1643); 1H NMR (500 MHz, CDCl3)
−
δ

Mar. Drugs 2018, 16, 443
8 of 12
H-4), 4.07 (1H, t, J = 9.4 Hz, H-3), 7.09 (4H, m, H-7, H-8, H-10, H-11); 13C NMR (125 MHz, CDCl3)
δ
20.7 (C-15), 20.9 (C-13), 22.4 (C-12), 25.0 (C-14), 33.1 (C-4), 36.4 (C-5), 47.4 (C-2), 48.4 (C-1), 62.0 (C-3),
127.3 (C-7, C-11), 128.1 (C-8, C-10), 135.4 (C-9), 143.9 (C-6).
3.3.5. α-Isobromocuparane (5)
25
Colorless oil; [
α
]
+37 (c 0.14, CH2Cl2); HREIMS [M]+ m/z 280.0826 (calc. for C15H2179Br,
D
280.0827), 282.0801 (calc. for C15H2181Br, 282.0806), [M
1H NMR (500 MHz, CDCl3)
−
Br]+ m/z 201.1649 (calc. for C15H21, 201.1643);
δ
0.65 (3H, s, H-13), 1.10 (3H, s, H-12), 1.28 (3H, s, H-14), 1.61 (1H, m, H-5),
2.20 (1H, m, H-4), 2.33 (3H, s, H-15), 2.51 (1H, dtd, J = 14.2, 9.5, 6.5 Hz, H-4), 2.71 (1H, td, J = 12.6,
6.4 Hz, H-5), 4.46 (1H, t, J = 9.2 Hz, H-3), 7.12 (1H, d, J = 8.1 Hz, H-10), 7.13 (1H, d, J = 8.1 Hz, H8),
7.27 (2H, d, J = 8.1, H-7, H-11); 13C NMR (125 MHz, CDCl3)
δ 20.6 (C-15), 20.8 (C-13), 22.2 (C-12), 25.3
(C-14), 31.2 (C-4), 33.5 (C-5), 47.7 (C-2), 48.4 (C-1), 63.6 (C-3), 126.5 (C-7, C-11), 128.5 (C-8, C-10), 135.4
(C-9), 143.0 (C-6).
3.4. Transformaction of Natural Sesquiterpenes 1 and 2
Laurinterol (1, 8.0 mg, 0.027 mmol) was dissolved in ethyl ether (5 mL) and bromine (100 µL,
1.95 mmol) was added. The reaction was left under magnetic stirring for 15 min at room temperature,
after which the solvent was evaporated in vacuo. The reaction mixture was purified on a silica gel
0.2–0.5 mm (Sigma-Aldrich, St. Louis, MO, USA) column (10 Ø
of n-Hex/EtOAc (100:0–95:5) to yield 8-bromoaplysin ( , 1.49 mg, 14.8%) and
Similarly, isolaurinterol ( , 5.0 mg, 0.017 mmol) was treated with bromine (100 L, 1.95 mmol) in the
same experimental conditions. After evaporation of the solvent, the reaction mixture was purified on a
×
50 mm) using a step gradient
6
7
(3.45 mg, 29.6%).
2
µ
silica gel column (10 Ø
(1.32 mg, 23.5%), 8 (0,38 mg, 6%) and 9 (1.81 mg, 27.3%).
Laurinterol ( , 10.0 mg, 0.034 mmol) was dissolved in ethyl ether (5 mL) and HBr (10
×
50 mm; step gradient of n-Hex/EtOAc (100:0–95:5)) to give compound
6
1
µL, 48%)
was added. The reaction was left under magnetic stirring for 15 min at room temperature, after which
the solvent was evaporated in vacuo. Quantitative conversion was observed to aplysin (3) which was
purified by crystallization in n-hexane.
3.4.1. 8-Bromoaplysin (6)
20
Colorless oil; [
α
]
−
30 (c 0.12, CHCl3); IR υ
max 3264, 1652, 1315, 1103, 1022, 904 cm−1; HREIMS
D
[M]+ m/z 371.9711 (calc. for C15H18O79Br2, 371.9724), 373.9702 (calc. for C15H18O79Br81Br, 373.9704),
375.9677 (calc. for C15H18O81Br2, 375.9683); 1H and 13 C NMR data (Table 1).
3.4.2. 3α-Bromojohnstane (7)
20
Colorless oil; [
α
]
−
35 (c 0.19, CHCl3); IR
υ
3259, 2362, 2158, 2031, 1975, 906, 800 cm−1
;
max
D
HREIMS [M]+ m/z 449.8840 (calc. for C15H17O79Br3, 449.8829), 451.8800 (calc. for C15H17O79Br281Br,
451.8809), 453.8798 (calc. for C15H17O79Br81Br2, 453.8789), 455.8758 (calc. for C15H17O81Br3, 455.8768);
1H and 13 C NMR data (Table 2).
3.4.3. 8,10-Dibromoisoaplysin (8)
20
Colorless oil; [
α
]
−
17 (c 0.04, CHCl3); HREIMS [M]+ m/z 449.8836 (calc. for C15H17O79Br3,
D
449.8829), 451.8815 (calc. for C15H17O79Br281Br, 451.8809), 453.8783 (calc. for C15H17O79Br81Br2,
453.8789), 455.8764 (calc. for C15H17O81Br3, 455.8768).
3.4.4. 8,10-Dibromoaplysinol (9)
20
Colorless oil; [
α
]
−
6 (c 0.08, CHCl3); HREIMS [M]+ m/z 387.9680 (calc. for C15H18O279Br2,
D
387.9674), 389.9667 (calc. for C15H18O279Br81Br, 389.9653), 391.9639 (calc. for C15H18O281Br2, 391.9633).

Mar. Drugs 2018, 16, 443
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3.5. Cell Culture
The amoeba strain used in this study was the type strain: Acanthamoeba castellanii Neff (ATCC
30010) which was axenically grown in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast
extract and 1.5% (w/v) glucose) containing 40 µg/mL of gentamicin (Biochrom AG, Cultek, Granollers,
Barcelona, Spain).
The murine macrophages J774A.1 (ATCC# TIB-◦67) cell line was cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum at 37 C and 5% CO2 atmosphere, was used for the
cytotoxicity assays.
3.6. Anti-Acanthamoeba Activity
To evaluate the biological activity of the fractions and molecules, the anti-Acanthamoeba assays
were determined using the alamarBlue® method as previously described [15
,
16]. Firstly, Acanthamoeba
L from a stock solution of
104 cells/mL. Amoebae were left to adhere to the bottom of the well for 15 min, which was checked
using a Leika DMIL inverted microscope (Leika, Wetzlar, Germany). After that, 50 L of serial
strain was seeded in triplicate on a 96-well microtiter plates with 50
µ
µ
dilutions of the molecules were added to the wells (1% DMSO was used to dissolve the highest dose of
the compounds with no effects on the parasites). As a control, we use chlorhexidine (chlorhexidine
digluconate; Alfa Aesar), which is a standard antiseptic belonging to the biguanide family of antiseptics
that is commonly used in contact lens maintenance solutions; and voriconazole (Sigma) is an inhibitor
of ergosterol synthesis that has been proven previously to be highly effective against clinical strains of
Acanthamoeba [26,
27]. Finally, the alamarBlue Reagent® (Life Technologies, Madrid, Spain) was placed
into each well at an amount equal to 10% of the final volume. Then, plates containing alamarBlue®
were incubated for 96 h at 26 ◦C with a slight agitation.
3.7. Cytotoxicity Test
The cytotoxicity assay was carried out using the alamarBlue® method as previously described [15
Firstly, the macrophages were seeded in triplicate on a 96-well microtiter plates with 50 L from a stock
solution of 2
105 cells/mL. Macrophages were left to adhere to the bottom of the well for 15 min,
which was checked using a Leika DMIL inverted microscope (Leika, Wetzlar, Germany). After that,
50
L of serial dilutions of the molecules were added to the wells. Finally, the alamarBlue Reagent®
,16].
µ
×
µ
(Life Technologies, Madrid, Spain) was placed into each well at an amount equal to 10% of the final
volume. Then, plates containing alamarBlue® were incubated for 24 h at 37 ◦C in presence of CO2 at 5%.
3.8. Statical Analysis
Briefly, the test plates were analyzed, during an interval of time between 24 and 96 h, on an
EnSpire® Multimode Plate Reader (Perkin Elmer, Madrid, Spain) using fluorescence, a test wavelength
of 570 nm and a reference wavelength of 630 nm. The percentage of the growth inhibition, 50%
inhibitory concentration (IC50 or CC50) was calculated by linear regression analysis with 95%
confidence limits using Sigma Plot 12.0 statistical analysis software (Systat Software). All experiments
were performed three times, and the mean values were also calculated. A paired two-tailed t-test was
used for analysis of the data. Values of p < 0.05 were considered significant.
4. Conclusions
To the best of our knowledge, red algae have never been evaluated as a potential source
of amoebicidal agents. Furthermore, this is the first time that the anti-Acantamoeba activity of
the brominated sesquiterpenes
Laurencia johnstonii, is reported. On the other hand, after chemical modification of the inactive
metabolites laurinterol ( ) and isolaurinterol ( ), a set of brominated derivatives, , that substantially
improve the activity of the natural sesquiterpenes have been obtained. The addition of a second
α-bromocuparane (4) and α-isobromocuparane (5), isolated from
1
2
6–9

Mar. Drugs 2018, 16, 443
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bromine atom in the aromatic ring of
6–
9
seems to be relevant to increase the antiamoeboid activity.
Whereas all obtained derivatives possess a laurane-type framework, the most active compound,
-bromojohnstane ( ), possesses a different rearranged carbon skeleton, johnstane, never found
3
α
7
among natural sesquiterpene metabolites isolated from Laurencia species so far. Our results suggest
that Laurencia-based brominated sesquiterpenes could be a potential source of novel therapeutic agents
against Acanthamoeba in the near future.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1660-3397/16/11/
443/s1. Scheme S1: Isolation process of sesquiterpenes 1–5 from Laurencia johnstonii; Figure S1:1H-NMR spectrum
1
1
for laurinterol (1); Figure S2: H-NMR spectrum for isolaurinterol (2); Figure S3: H-NMR spectrum for aplysin (3);
1
1
Figure S4: H-NMR spectrum for
α
-bromocuparane (4); Figure S5: H-NMR spectrum for
α
-isobromocuparane
1
(5); Figure S6: H-NMR spectrum of 8-bromoaplysin (6); Figure S7: COSY spectrum of 8-bromoaplysin (6); Figure
S8: HSQC-ed spectrum of 8-bromoaplysin (6); Figure S9: HMBC spectrum of 8-bromoaplysin (6); Figure S10:
13C NMR spectrum of 8-bromoaplysin (6); Figure S11: HREIMS spectrum of 8-bromoaplysin (6); Figure S12: H
1
NMR spectrum of 3
HSQC-ed spectrum of 3
13C NMR spectrum of 3
α
-bromojohnstane (7); Figure S13: COSY spectrum of 3
α
α
-bromojohnstane (7); Figure S14:
-bromojohnstane (7); Figure S16:
-bromojohnstane (7); Figure
α
-bromojohnstane (7); Figure S15: HMBC spectrum of 3
α
-bromojohnstane (7); Figure S17: 1D-NOE experiments of 3
α
1
S18: HREIMS spectrum of 3
α
-bromojohnstane (7); Figure S19: H NMR spectrum of 8,10-dibromoisoaplysin 8;
1
Figure S20: H NMR spectrum of 8,10-dibromoaplysinol 9.
Author Contributions: J.J.F., A.R.D.-M., and J.L.-M. conceived and designed the experiments; S.G.-D. and E.V.-V.
collected the algae and prepared extracts; S.G.-D. performed isolation, purification and chemical experiments;
J.L.-M., M.R.-B. and I.S. designed and performed the anti-Acanthamoeba activity assays and analyzed the activity
data; J.E.P contributed to activity analysis tools and discussed activity data; J.J.F. and A.R.D.-M. analyzed the
chemical data and wrote the paper. All authors contributed to the final version of the manuscript.
Funding: This research was funded by Ministerio de Economía y Competitividad: grant number CTQ2014-
55888-C03-01/R.
Acknowledgments: This research has been supported by Red de Investigation Colaborativa en Enfermedades
Tropicales RICET (project no. RD16/0027/0001 of the Program of Redes Temáticas de Investigación Cooperativa).
Authors thank funds from: CONACYT fellowship 504923/290666, (S.G.D.), Plan Propio ULL 2018, Agustín de
Betancourt Program and Cabildo de Tenerife (A.R.D.M., I.S.), the use of General Research Support Services of
University of La Laguna (SEGAI-ULL), and Rafael Riosmena Rodríguez (UABCS) for taxonomic classification of
specimens of Laurencia johnstonii.
Conflicts of Interest: The authors declare no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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