marine drugs

Article
Bioactive Bromotyrosine-Derived Alkaloids from the
Polynesian Sponge Suberea ianthelliformis
Amr El-Demerdash 1,2 ID , Céline Moriou 1 , Jordan Toullec 3 , Marc Besson 4,5 ID ,
Stéphanie Soulet 6 , Nelly Schmitt 6 , Sylvain Petek 3 ID , David Lecchini 4 , Cécile Debitus 3, * and
Ali Al-Mourabit 1, *
1 Institut de Chimie des Substances Naturelles, CNRS UPR 2301, University Paris-Sud,
University of Paris-Saclay, 1, Avenue de la Terrasse, 91198 Gif-Sur-Yvette, France;
eldemerdash555@gmail.com (A.E.-D.); Celine.Moriou@cnrs.fr (C.M.)
2 Organic Chemistry Division, Chemistry Department, Faculty of Science, Mansoura University,
Mansoura 35516, Egypt
3 LEMAR, IRD, UBO, CNRS, IFREMER, IUEM, 29280 Plouzané, France;
Jordan.Toullec@univ-brest.fr (J.T.); sylvain.petek@ird.fr (S.P.)
4 CRIOBE, CNRS, EPHE, UPVD, PSL Research University, 98729 Moorea, French Polynesia;
bessonmarcluc@gmail.com (M.B.); david.lecchini@ephe.sorbonne.fr (D.L.)
5 Observatoire Océanologique de Banyuls-sur-Mer, Université Pierre et Marie Curie Paris,
66650 Banyuls-sur-Mer, France
6 EIO, UPF, ILM, IFREMER, IRD, Faa’a, 98702 Tahiti, French Polynesia;
stephanie.soulet@upf.pf (S.S.); nelly.schmitt@upf.pf (N.S.)
* Correspondance: cecile.debitus@ird.fr (C.D.); Ali.ALMOURABIT@cnrs.fr (A.A.-M.);
Tel.: +336-0448-1378 (C.D.); +336-1585-9203 (A.A.-M.)




Received: 29 March 2018; Accepted: 24 April 2018; Published: 27 April 2018


Abstract: Herein, we describe the isolation and spectroscopic identification of eight new tetrabrominated
tyrosine alkaloids 2–9 from the Polynesian sponge Suberea ianthelliformis, along with known major
compound psammaplysene D (1), N,N-dimethyldibromotyramine, 5-hydroxy xanthenuric acid,
and xanthenuric acid. Cytotoxicity and acetylcholinesterase inhibition activities were evaluated for
some of the isolated metabolites. They exhibited moderate antiproliferative activity against KB cancer
cell lines, but psammaplysene D (1) displayed substantial cytotoxicity as well as acetylcholinesterase
inhibition with IC50 values of 0.7 µM and 1.3 µM, respectively.

Keywords: brominated tyrosine alkaloids; Suberea ianthelliformis; cytotoxicity; acetylcholinesterase

inhibition

1. Introduction
Sponges of the marine genus Suberea (order: Verongiida) are known to produce a large array
of structurally diverse brominated tyrosine alkaloids, which are considered as chemotaxonomical
markers [1–3]. Nevertheless, bromotyrosine-derived metabolites have been isolated from sponges
belonging to distinct orders such as Agelasida [4] and others. In addition, an interesting recent study
showed that culture of the marine sponge-derived bacterium Pseudovibrio denitrificans Ab134 isolated
from the Arenosclera brasiliensis sponge (order Haplosclerida) can produce bromotyrosine-derived
alkaloids as well [5]. Although misidentifications and contaminations due to misplaced specimens
are possible, these reports compromise this family of compounds as a Verongiida marker and suggest
staying cautious about secondary metabolites markers for sponge phylogeny [6]. The bromotyrosine
derivatives’ architectures vary from the simple monomeric molecules such as subereaphenol K [7],
or the dimeric suberedamine A [8,9], to more complex derivatives like the well-known fistularin-3 [10].

Mar. Drugs 2018, 16, 146; doi:10.3390/md16050146 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2018, 16, 146 2 of 16

Many compounds of this class of bromotyrosine alkaloids display a wide scope of bioactivities
Mar. Drugs 2018, 16, x FOR PEER REVIEW 2 of 16
including cytotoxicity against murine leukemia L1201 cells and KB cells [8,9], DNA demethylating [11],
antiproliferative
wide scope [12], antimicrobial
of bioactivities including [13–16], and antiplasmodial
cytotoxicity activities
against murine leukemia L1201[17].
cells In
andcontinuation
KB cells of
our search
[8,9],for
DNA bioactive marine[11],
demethylating natural products [12],
antiproliferative fromantimicrobial
South Pacific sponges
[13–16], [18], in both health
and antiplasmodial
activities
and chemical [17]. In continuation
ecology of our search
fields, we report for bioactive
here the isolation, marine naturaldetermination,
structural products from South andPacific
cytotoxicity
sponges [18], in both health and chemical ecology fields, we report here
evaluation of eight new tetrabromotyrosine alkaloids (Figure 1), along with four known compounds the isolation, structural
determination, and cytotoxicity evaluation of eight new tetrabromotyrosine alkaloids (Figure 1),
from the sponge Suberea ianthelliformis. The known major compound psammaplysene D (1) was also
along with four known compounds from the sponge Suberea ianthelliformis. The known major
evaluated for its psammaplysene
compound acetylcholinesteraseD (1) was inhibition activity.
also evaluated for itsAlthough acetylcholinesterase
acetylcholinesterase inhibition
inhibition activity.
has been mostlyacetylcholinesterase
Although explored as treatment for has
inhibition Alzheimer’s
been mostly pathology,
explored asour attention
treatment has been brought
for Alzheimer’s
to the chemical
pathology, ecology
our attentionimportance of suchtoenzyme
has been brought inhibitors
the chemical ecologyinimportance
their environment
of such enzyme or against
inhibitors in their environment or against predation [19]. Only one publication
predation [19]. Only one publication relates fish toxicity on Gambusia affinis (western mosquitofish) relates fish toxicity
on Gambusia affinis (western
and butylcholinesterase from amosquitofish)
Latrunculiaand butylcholinesterase
magnifica toxin [20].from Wea Latrunculia
describe our magnifica toxin
observations of
[20]. We describe our observations of the inhibitory effect of psammaplysene D on
the inhibitory effect of psammaplysene D on acetylcholinesterase, using balneation experiments on
acetylcholinesterase, using balneation experiments on two fish species, Poecilia reticulata (guppy) and
species, Poecilia
two fishAcanthurus triostegusreticulata
(reef fish).(guppy) and Acanthurus triostegus (reef fish).

Figure 1. Structures of isolated compounds 1–12.

Figure 1. Structures of isolated compounds 1–12.
2. Results and Discussion
2. Results and Discussion
2.1. Isolation and Structure Elucidation
2.1. IsolationEach
and crude
Structure Elucidation
extract obtained by CH2Cl2 (10.5 g) and n-BuOH, (13 g) of Suberea ianthelliformis was
subjected to normal phase MPLC and eluted with gradients of CH 2Cl2/MeOH. The sub-fractions
Each crude extract obtained by CH2 Cl2 (10.5 g) and n-BuOH, (13 g) of Suberea ianthelliformis was
were purified by preparative, semi-preparative, and analytical reversed-phase HPLC and led to the
subjected to normal phase MPLC and eluted with gradients of CH2 Cl2 /MeOH. The sub-fractions
isolation of eight new compounds 2–9, along with the known derivatives psammaplysene D (1),
were purified by preparative, semi-preparative,
N,N-dimethyldibromotyramine and analytical
(10), 5-hydroxy xanthurenic reversed-phase
acid (11) HPLC
and xanthurenic and(12)
acid acid led to the
isolation of eight
(Figure new
1). The compounds
known 2–9, along
natural products with the Dknown
psammaplysene (1) [21],derivatives psammaplysene D (1),
N,N-dimethyldibromotyramine
(10) [22], 5-hydroxy xanthenuric
N,N-dimethyldibromotyramine acid (4,5,8-trihydroxyquinoline-2-carboxilic
(10), 5-hydroxy acid) (11) [23]
xanthurenic acid (11) and xanthurenic andacid (12)
acid
(Figure xanthurenic acid (12) [24] were identified by comparison of
Dtheir
1H NMR and MS data with those
1). The known natural products psammaplysene (1) [21], N,N-dimethyldibromotyramine
(10) [22], 5-hydroxy xanthenuric acid (4,5,8-trihydroxyquinoline-2-carboxilic acid) (11) [23] and
xanthurenic acid (12) [24] were identified by comparison of their 1 H NMR and MS data with those
reported in the literature. It is worth to point out that psammaplysene D (1) was isolated by normal
phase MPLC as the major compound (6.7 g) representing over 50% of the entire n-BuOH extract.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 16
Mar. Drugs 2018, 16, 146 3 of 16
reported in the literature. It is worth to point out that psammaplysene D (1) was isolated by normal
phase MPLC as the major compound (6.7 g) representing over 50% of the entire n-BuOH extract.
The molecular formula formula C C2323H 7979
H2727 BrBr
81
2812Br2Br N2O N32of
O3compound
of compound 2 was 2 was determined
determined by HRESIMS
by HRESIMS (m/z
(m/z 698.8675 [M + H] + ) indicating ten degrees of unsaturation. The isotopic pattern of the protonated
698.8675 [M + H] ) indicating ten degrees of unsaturation. The isotopic pattern of the protonated
+

molecule confirmed
confirmedthe thepresence
presence of four
of four bromines.
bromines. Analysis
Analysisof theofNMR the data
NMRrecordeddata recordedfor 2 revealed
for 2
striking
revealedsimilarities with the NMR
striking similarities with the dataNMR recorded data for the co-isolated
recorded psammaplysene
for the co-isolated D (1), except
psammaplysene for
D (1),
the absence of the signal corresponding to the propylamine
except for the absence of the signal corresponding to the propylamine group, as confirmed by the group, as confirmed by the HRESIMS
spectrum. 1 H NMR spectra
HRESIMS In general, In
spectrum. thegeneral, the 1H NMR of these families
spectra of compounds
of these families were complicated
of compounds wereby
the multiplicity of the signals due to the two rotational isomers
complicated by the multiplicity of the signals due to the two rotational isomers trans/cis (6:4 ratio) trans/cis (6:4 ratio) along the amide
bond
along[21].the Theamidesamebond behavior[21].was The already
same reported
behaviorforwas tetrabromotyrosine
already reported derivatives psammaplysene
for tetrabromotyrosine
A-D, isolated
derivatives from the sponges
psammaplysene A-D,Psammaplysilla
isolated from and Psammoclemma
the sponges [21–25].and
Psammaplysilla ThePsammoclemma
1D and 2D NMR [21–
analysis revealed the existence of four aromatic protons
25]. The 1D and 2D NMR analysis revealed the existence of four aromatic protons comprising comprising two singlets at δH 7.78 and 7.70,
two
assigned
singlets at toδH-3/5,
H 7.78 and and 7.70,
another broad singlet
assigned to H-3/5, at δand
H 7.57 integrating
another broadfor 2 protons,
singlet at δH attributed
7.57 integrating to H-15/17.
for 2
An A-B system
protons, attributedsignal, corresponding
to H-15/17. An A-B to system
two olefinicsignal, protons at δH 7.39–7.36/7.38–7.35
corresponding to two olefinic protons (J = 15.5atHz) δH
and δ 7.15–7.11/7.02–6.99
7.39–7.36/7.38–7.35
H (J = 15.5 Hz), were assigned to the E olefin
(J = 15.5 Hz) and δH 7.15–7.11/7.02–6.99 (J = 15.5 Hz), were assigned to the E olefin–CH-7=CH-8–, satisfying the
tenth degree of satisfying
–CH-7=CH-8–, unsaturation. the The tenth HMBC
degree data of analysis
unsaturation.showed The correlations
HMBC data betweenanalysis H-7 showed
and the
two sp 2 aromatic carbons C-3/5 (δ 133.09/133.01) and the sp 2 non-protonated carbon C-4 (δ 130.98)2
correlations between H-7 and theC two sp aromatic carbons C-3/5 (δC 133.09/133.01) andC the sp
2

confirming
non-protonated the connection
carbon C-4 of(δthe trans-cinnamoyl
C 130.98) confirming motif
the to the 2,6-dibrominated
connection phenolic ring
of the trans-cinnamoyl (deduced
motif to the
with the chemical phenolic
2,6-dibrominated shifts of C-2/6 at δC 112.80
ring (deduced withand the C-1 at δC 154.10).
chemical shifts of The C-2/6 second olefinic
at δC 112.80 proton
and C-1 at H-8δC
exhibited an HMBC correlation with the non-protonated
154.10). The second olefinic proton H-8 exhibited an HMBC correlation with carbon C-9 at (δ C 168.96/168.80)
the non-protonated linking
this endC-9
carbon to anat amidic carbonyl function.
(δC 168.96/168.80) linking this HSQC endand to anCOSY amidic spectra analysis
carbonyl allowed
function. HSQC the assignment
and COSY
of the signals
spectra analysis at allowed
δH 4.11 and 4.05 (2H, 2t,ofJ the
the assignment = 6.4), 3.89atand
signals δH 3.74
4.11 (2H,
and 4.052t, J (2H,
= 7.22t, Hz),
J = 2.22
6.4), and
3.89 2.16
and
(2H, 2m) to
3.74 (2H, 2t, the
J = three
7.2 Hz), propylic
2.22 and carbon
2.16 sequence
(2H, 2m) to N–CH 2 -10–CH
the three 2 -11–CH
propylic 2 -12–O,
carbon while those
sequence N–CHat δH
2-10–

3.30 (2H, m)2-12–O,

CH2-11–CH and 3.00 while(2H, m) were
those at δH attributed
3.30 (2H, m) to the
andethylic
3.00 (2H, chain m) CH were2 -19–CH 2 -20. to
attributed The
theprotons
ethylic
H-20
chain were correlated
CH2-19–CH 2-20.to TheN-Me 22 and
protons N-Me
H-20 were23 by HMBC analysis
correlated to N-Me(Figure
22 and N-Me 2). The 23 by methylene
HMBC analysis group
CH 2 -10 was
(Figure 2). Theconnected
methylene to the amidic
group CHN-Me
2-10 was 21 based on its chemical
connected to the amidicshift (δ C 47.14/48.2)
N-Me 21 based on anditsthe HMBC
chemical
correlation to C-9 (δ
shift (δC 47.14/48.2) C 168.96/168.80)
and the HMBC correlation and C-21to(δC-9 C 34.74/36.55).
(δC 168.96/168.80) The andchemical C-21 (δ shifts of CH2 -12The
C 34.74/36.55). at
(δ 4.11/4.05)
chemical
H and
shifts of CH δ (71.94/72.64) showed its link to the ether oxygen.
C 2-12 at (δH 4.11/4.05) and δC (71.94/72.64) showed its link to the ether oxygen. Furthermore, the correlation of
H-12 to the non-protonated
Furthermore, the correlationcarbon of H-12 C-13to (δ theC 153.88/153.94)
non-protonatedallowed carbon the C-13 connection of this part
(δC 153.88/153.94) of the
allowed
molecule
the connection to the other
of this 1,2,4,6-tetrasubstituted-phenyl
part of the molecule to the ring. Furthermore,
other CH2 -19 (δH 3.00) displayed
1,2,4,6-tetrasubstituted-phenyl ring.
HMBC correlations
Furthermore, CH2-19 with(δHthe aromatic
3.00) non-protonated
displayed HMBC correlationscarbon C-16 (δC the
with 136.81/136.99) and CH-15/17
aromatic non-protonated
(134.63/134.56),
carbon C-16 (δC confirming136.81/136.99) the andsubstitution
CH-15/17 of (134.63/134.56),
this aromatic ring by the N,N-dimethylaminoethyl
confirming the substitution of this
chain.
aromatic Compound
ring by the 2 was named psammaplysene
N,N-dimethylaminoethyl F. Compound 2 was named psammaplysene F.
chain.
Compound 3 displayed the same molecular formula than compound 2 obtained by HRESIMS
(m/z 698.8675 [M
(m/z 698.8675 [M + + H]++).).AApreliminary
preliminary1H 1 H NMR inspection (Table 1)
NMR inspection (Table 1) showed similarity between
compounds 11and and2,2,whichwhich was easily observed
was easily observed by superimposition by superimposition of their of spectra.
their spectra.
The A-B The A-B
system
system
of two of two downfield
downfield doublets doublets
of doublets of doublets at δH 6.49–6.52/6.56–6.58
at δH 6.49–6.52/6.56–6.58 (J = 12.5 (J =Hz,
12.5cis) Hz, andcis)δand
H 6.13–δH
6.13–6.16/6.09–6.12
6.16/6.09–6.12 (J = 12.5 (J = Hz)
12.5 were
Hz) were assigned
assigned to –CH-7=CH-8
to –CH-7=CH-8 of the of the cinnamoyl
cinnamoyl moietymoiety withwith a Za
Z configuration.
configuration. Furthermore,
Furthermore, the theCOSY, COSY, HSQC, HSQC, andand HMBC HMBC analysis
analysis data data
(Figure (Figure
2) showed2) showedthe same the
same correlations
correlations as previously
as previously found foundfor for 2. Thus,
2. Thus, thethe tetrabrominated
tetrabrominated tyrosine3 3was
tyrosine was assigned
assigned to
psammaplysene G.

Figure
Figure 2.
2. COSY
COSY and
and key
key HMBC
HMBC correlations for psammaplysenes
correlations for psammaplysenes F
F (2)
(2) and
and G
G (3).
(3).
Mar. Drugs 2018, 16, 146 4 of 16

Table 1. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data for Psammaplysenes F (2) and G (3) in
CD3 OD.

Position Psammaplysene F (2) Psammaplysene G (3)

No. δC , Type a δH mult, (J in Hz) a HMBC δC , Type a δH mult, (J in Hz) a HMBC
1 154.10, C - - 153.29/153.04, C - -
2 112.80, C - - 112.49, C - -
3 133.09/133.01, CH 7.78/7.70, s 1, 2, 5, 6, 7 133.70/133.42, CH 7.53/7.54, s 1, 2, 5, 6, 7
4 130.98, C - - 130.70/131.16, C - -
5 133.09/133.01, CH 7.78/7.70, s 1, 2, 3, 6, 7 133.70/133.42, CH 7.53/7.54, s 1, 2, 3, 6, 7
6 112.80, C - - 112.49, C - -
7.36–7.39/7.35–7.38, 6.49–6.52/6.56–6.58,
7 140.89/141.19, CH 3, 4, 5, 9 132.24/132.49, CH 3, 4, 5, 9
d (15.5) d (12.5)
7.11–7.15/6.99–7.02, 6.13–6.16/6.09–6.12,
8 118.90/118.72, CH 4, 7, 9 123.90/124.59, CH 4, 7, 9
d (15.5) d (12.5)
9 168.96/168.80, C - - 171.13/171.01, C - -
10 48.20/47.14, CH2 3.89/3.74, t (7.2) 9, 11, 12, 21 48.97/45.75, CH2 3.65/3.73, t (7.5) 9, 11, 12, 21
11 30.86/29.16, CH2 2.22/2.16, m 10, 12 29.32/28.68, CH2 2.02/2.18, m 10,12
12 71.94/72.64, CH2 4.11/4.05, t (6.4) 10, 11, 13 72.03/72.32, CH2 3.78/4.04, t (6.5) 10, 11, 13
13 153.88/153.94, C - - 153.55/153.86, C - -
14 119.57, C - - 119.36/119.59, C - -
15 134.63/134.56, CH 7.57, br s 13, 14, 17, 18, 19 134.51/134.57, CH 7.51/7.56, s 13, 14, 17, 18, 19
16 136.81/136.99, C - - 136.85, C - -
17 134.63/134.56, CH 7.57, br s 13, 14, 15, 18, 19 134.51/134.57, CH 7.51/7.56, s 13, 14, 15, 18, 19
18 119.57, C - - 119.36/119.59, C - -
19 30.45, CH2 3.00, m 15, 16, 17, 20, 22, 23 30.51/30.56, CH2 3.00, m 15, 16, 17, 20, 22, 23
20 59.24, CH2 3.30, m 16, 19, 22, 23 59.32/59.24, CH2 3.34, m 16, 19, 22, 23
21 34.74/36.55, CH3 3.09/3.28, s 9, 10 32.59/36.67, CH3 3.06/3.03, s 9, 10
22 43.62, CH3 2.91, s 20, 23 43.70/43.73, CH3 2.91, s 20, 23
23 43.62, CH3 2.91, s 20, 22 43.70/43.73, CH3 2.91, s 20, 22
a Chemical shifts are given for both rotamers when distinguishable signals were identified.

Compounds 4 and 5 were obtained in a minute quantity (0.7 mg) as a 1:1 mixture that could
not be separated. Due to the minute quantity of the mixture, we decided to determine the structures
of the latter two metabolites from the mixture. HRESIMS analysis revealed the molecular formulas
C27 H36 79 Br2 81 Br2 N3 O3 (m/z 769.9521 [M + H]+ ) and C26 H34 79 Br2 81 Br2 N3 O3 (m/z 755.9309 [M + H]+ )
for 4 and 5, with similar isotopic patterns indicating the presence of four bromines, but 14 and 28 a.m.u.
(atomic mass unit) less than psammaplysene D (1), respectively. Direct comparison of the 1 H NMR
spectra of this mixture (Table 2) and the co-isolated major compound psammaplysene D (1) showed
good superimposition, including the rotameric forms. The main differences concerned the signals of
CH2 -30 , CH2 -19 and CH2 -20 (Figure 3) indicating modifications of the number of the N-methyl groups
linked to them. The 2D NMR experiments (COSY, HSQC, and HMBC) of both compounds showed
the same correlations as the co-isolated psammaplysene D (1) (Figure 2). 1 H NMR (Table 2) revealed
four signals of N-methyl groups at δH 3.10 and 3.29 (2s, 6H, s-trans-CH3 -21, s-cis-CH3 -21), δH 2.70
(s, CH3 -22, 3H) and δH 2.74–2.79 (CH3 -40 and CH3 -50 , 6H). The HMBC analysis showed correlation
from N-CH3 -22 (δH 2.70, δC 34.0) to C-20 (δC 51.1) for compound 4. The chemical shifts differences
observed for H-19 (δH 2.93), H-20 (δH 3.21) and CH3 -22 (δH 2.70) suggested a modification on this part
of the molecule. The chemical shifts of C-20 (δC 51.1) and CH3 -22 (δC 34.0) suggested the existence
of only one methyl group on this side of the chain. At the other end of the molecule, correlations of
N-CH3 -40 and 50 (δH 2.74–2.79, δC 44.7) to C-30 (δC 57.2) along with the chemical shift of C-30 , showed
the presence of two methyl groups on the nitrogen N-C-30 . The chemical shift of C-20 and C-22
were the same for compound 5, but the chemical shift of C-20 (δC 27.9), C-30 (δC 48.7), and CH3 -40
(δC 34.0), as well as the HMBC correlations between C-30 and CH3 -40 , suggested the presence of a
NH-Me connected to the carbon C-30 . Therefore, our mixture was composed of two psammaplysene D
derivatives lacking one and two N-methyls and named psammaplysene H (4) and psammaplysene
I (5), respectively.
Mar. Drugs 2018, 16, 146 5 of 16
Mar. Drugs 2018, 16, x FOR PEER REVIEW 5 of 16

Figure 3.
3. COSY and key HMBC correlations for
for psammaplysenes
psammaplysenes H
H (4)
(4) and
and II (5).
(5).

Table 2.2. 11H

Table H NMR
NMR (500
(500 MHz)
MHz)and CCNMR
and1313 NMR(125
(125MHz)
MHz)data
dataforfor
Psammaplysenes HH
Psammaplysenes (4)(4)
and I (5)
and in
I (5)
CDCD
in 3OD.
3 OD.
Position Psammaplysene H (4) Psammaplysene I (5)
Position Psammaplysene H (4) a Psammaplysene I (5) a
No δC, Type a δH mult, (J in Hz) HMBC δC, Type a δH mult, (J in Hz) HMBC
No1 Type a C
δC , 154.7, δH mult, (J in - Hz)
a HMBC- , Type C
δC154.7, a δH mult, (J- in Hz) a HMBC -
12 154.7, C C
119.6, - - - - 154.7,
119.6,CC -- --
23 119.6, C
133.5/133.4, CH -
7.92/7.85, d 1, -2, 5, 6, 7 119.6, C CH
133.5/133.4, -
7.92/7.85, d 1, 2,- 5, 6, 7
3 133.5/133.4, CH 7.92/7.85, d 1, 2, 5, 6, 7 133.5/133.4, CH 7.92/7.85, d 1, 2, 5, 6, 7
4
4 136.2,
136.2, C
C -
- -
- 136.2,
136.2, C
C -
- -
-
55 133.5/133.4,
133.5/133.4, CHCH 7.92/7.85,
7.92/7.85, d d 1, 3,
1, 2, 2,6,3,76, 7 133.5/133.4,CH
133.5/133.4, CH 7.92/7.85,dd
7.92/7.85, 1,1,2,2,3,3,6,6,7 7
66 119.6,
119.6, C C - - - - 119.6,CC
119.6, -- --
77 140.2/140.0,
140.2/140.0, CHCH 7.40–7.44,
7.40–7.44, m m 3, 4,
3,5,4,95, 9 140.2/140.0,
140.2/140.0,CH CH 7.40–7.44,
7.40–7.44,mm 3,3,4,4,5,5,9 9
8 121.6/121.7, CH 7.14–7.27, dd (15.0) 4, 7, 9 121.6/121.7, CH 7.14–7.27, dd (15.0) 4, 7, 9
8 121.6/121.7, CH 7.14–7.27, dd (15.0) 4, 7, 9 121.6/121.7, CH 7.14–7.27, dd (15.0) 4, 7, 9
9 168.5, C a - - 168.5, C a - -
109 168.5,CH
48.4/47.3, Ca
2 3.91/3.76, - (7.0)
t 9, 11, 12, -21 168.5, CH
48.4/47.3, Ca
2 3.91/3.76, - t (7.0) 9, 11, - 21
12,
1110 48.4/47.3,
30.6/29.1, CHCH2 2 3.91/3.76,
2.23/2.17, mt (7.0) 9,
10, 11,
12 12, 21 48.4/47.3,
30.6/29.1, CHCH 2 2 3.91/3.76,
2.23/2.17, tm(7.0) 9, 11,
10, 12, 21
12
1211 72.6/71.9,
30.6/29.1,CHCH2 2 4.12/4.07,
2.23/2.17, m m 10, 11, 10,1312 72.6/71.9,
30.6/29.1,CH CH 2 2 4.12/4.07,
2.23/2.17,mm 10,10,11, 12 13
1312 154.7, C CH2
72.6/71.9, -
4.12/4.07, m 10,- 11, 13 154.7, C CH2
72.6/71.9, -
4.12/4.07, m 10, -11, 13
14 119.6, C - - 119.6, C - -
1513 154.7,
134.5, CH C 7.56/7.53, -m 13, 14, 17, 18, - 19 154.7,
134.5, CHC - m
7.56/7.53, 13, 14, 17,- 18, 19
1614 119.6,
137.8, CbC - - - - 119.6,
137.8, C bC -- --
1715 134.5,
134.5, CHCH 7.56/7.53,
7.56/7.53, m m 13,13,
14,14,
15, 17,
18, 18,
19 19 134.5,CH
134.5, CH 7.56/7.53,mm
7.56/7.53, 13,14,
13, 14,15, 17,18,18,
1919
1816 119.6, CCb
137.8, - - - - 119.6,
137.8,CC b -- --
19 32.2, CH2 2.93, t (8.0) 16, 20, 22 32.2, CH2 2.93, t (8.0)
17 134.5, CH 7.56/7.53, m 13, 14, 15, 18, 19 134.5, CH 7.56/7.53, m 13,16,14,20, 15,2218, 19
20 51.1, CH2 3.21, m 16, 19, 22 51.1, CH2 3.21, m 16, 19, 22
21 18 119.6,
34.7/36.6, CH3 C -
3.10/3.29, s 9, 10 - 119.6, C
34.7/36.6, CH3 -
3.10/3.29, s 9, 10 -
2219 32.2,
34.0, CHCH3 2 2.93,
2.70, st (8.0) 16,
20 20, 22 32.2,CH
34.0, CH3 2 2.93,
2.70,t s(8.0) 16,2020, 22
1020 57.2, CHCH
51.1, 2 2 3.27, m m
3.21, 20 , 319,
1,16, 0
22 48.7,
51.1,CHCH2 2 3.35,
3.21,mm 1, 2019,
16, , 30 22
2021 27.6, CH2 CH3
34.7/36.6, 2.23, m
3.10/3.29, s 10 , 9,
30 10 30.9 CH2CH3
34.7/36.6, 2.23, m s
3.10/3.29,
0 , 30
19, 10
30 71.9, CH2 4.18, m 10 , 20 , 40 , 50 71.9, CH2 4.18, m 10 , 20 , 40
40
22 34.0, CH3
44.3, CH3
2.70, s
2.78/2.74, s 30 , 50
20 34.0, CH3
34.0, CH3
2.70, s
2.77/2.76, s 30
20
50 1′ 57.2,
44.3, CHCH3
2 3.27, m
2.79/2.74, s 301,
, 402′, 3′ 48.7,
- CH2 3.35,
- m 1, -2′, 3′
2′
a Chemical 27.6, CH2 2.23, m 1′, 3′ 30.9 CH2 2.23, m b 1′, 3′
shifts are given for both rotamers when distinguishable signals were identified; Detected by
3′ 71.9, CH2 4.18, m 1′, 2′, 4′, 5′ 71.9, CH2 4.18, m 1′, 2′, 4′
HMBC correlations.
4′ 44.3, CH3 2.78/2.74, s 3′, 5′ 34.0, CH3 2.77/2.76, s 3′
5′ 44.3, CH3 2.79/2.74, s 3′, 4′ - - -
a Chemical shifts are given for both rotamers when distinguishable signals were identified; b Detected
Compounds 6 and 7 exhibited the molecular formula were determined by HRESIMS as
C25 Hby 79HMBC 81 Br
correlations. + 79 81 +
33 Br 2 2 3 O3 at m/z 743.9386 [M + H] , and C24 H31 Br2 Br2 N3 O3 at m/z 729.9190 [M + H]
N
respectively, indicating nine degrees of unsaturation with an isotopic pattern of four bromines.
TheseCompounds
NMR and mass 6 and data7 exhibited
showed that thecompound
molecular 7formula
has one weremethyl determined
group less bythanHRESIMS
compound as
C25Examination
6. H33 Br2 Br2Nof3Othe
79 81 3 at1Hm/z
NMR743.9386 [M + spectra
and HSQC H] , and
+ C24H3)
(Table 31 Br2 Br2N3O3 at m/z 729.9190 [M + H]+
79 81
displayed signals with similar chemical
respectively,
shifts to those indicating
reported fornine degrees of unsaturation
the tetrabromotyrosine compoundswith anomoians
an isotopicApattern of four bromines.
and B, previously isolated
These NMR and mass data showed that compound 7 has one methyl
from Anomoianthella popeae [26] (order: Verongiida) and from an unknown species of the genus group less than compound
Hexadella6.
Examination
(order: of thecollected
Verongiida)
1 H NMRinand HSQC [27].
Indonesia spectra (Tableanalysis
2D NMR 3) displayed signals
including with
COSY, similar
HSQC, andchemical
HMBC
shifts to those reported for the tetrabromotyrosine compounds anomoians
(Table 3, Figure 4), allowed identification of the structures of 6 and 7 as the O-demethyl anomoian A and B, previouslyB
isolated
and from Anomoianthella
O-demethyl anomoian A,popeae [26] (order:
respectively. Thus,Verongiida) and fromwere
the new compounds an unknown species ofC the
named anomoian (6)
genus
and Hexadella
anomoian D (order:
(7). TheVerongiida)
1 H NMR signalscollected
of theinaromatic
Indonesia [27]. 2D
protons NMRand
CH-3/5 analysis including
CH-15/17 of theseCOSY,
new
HSQC, and HMBC (Table 3, Figure 4),
compounds indicated a 6:4 rotameric proportionality. allowed identification of the structures of 6 and 7 as the
O-demethyl anomoian B and O-demethyl anomoian A, respectively. Thus, the new compounds
were named anomoian C (6) and anomoian D (7). The 1H NMR signals of the aromatic protons
CH-3/5 and CH-15/17 of these new compounds indicated a 6:4 rotameric proportionality.
Mar. Drugs 2018, 16, 146 6 of 16
Mar. Drugs 2018, 16, x FOR PEER REVIEW 6 of 16

Figure 4. COSY and key HMBC correlations for anomoians C (6) and D (7).

Table 3. 11H NMR (500 MHz) and 13

Table 3. 13C NMR (125 MHz) data for Anomoians
C NMR (125 (7) in CD
C (6) and D (7) CD33OD.
Position Anomoian C (6) Anomoian D (7)
Position Anomoian C (6) Anomoian D (7)
No δC, Type a δH mult, (J in Hz) a HMBC δC, Type a δH mult, (J in Hz) a HMBC
1No δC , Type a C
δH mult, (J in- Hz) a
151.65/151.47, HMBC- δC ,152.7,
Type Ca δH mult, (J in Hz)
-
a HMBC -
21 151.65/151.47,
112.36, C C - - - - 152.7,
112.69,CC - - - -
32 112.36, C CH
134.47/134.41, -
7.34/7.32, s - 4, 5, 6, 7
1, 2, 112.69,
134.40,CCH -7.37/7.35, s 1,- 2, 4, 5, 6, 7
3 134.47/134.41, CH 7.34/7.32, s 1, 2, 4, 5, 6, 7 134.40, CH 7.37/7.35, s 1, 2, 4, 5, 6, 7
44 132.77/133.49,CC
132.77/133.49, - - - - 130.53/129.90,CC
130.53/129.90, - - - -
55 134.47/134.41,CH
134.47/134.41, CH 7.34/7.32,
7.34/7.32, s s 1, 2, 3,
1, 2, 3, 4, 6, 74, 6, 7 134.40,
134.40, CH CH 7.37/7.35,
7.37/7.35, s s 1,4,2,6,3,74, 6, 7
1, 2, 3,
66 112.36,CC
112.36, - - - - 112.69,
112.69, CC - - - -
77 33.14/32.14,
33.14/32.14,CH
2.90/2.83–2.99,
CH22 m m
2.90/2.83–2.99, 3, 4,3,5,4,8,5,98, 9 37.40/38.02,
37.40/38.02,CHCH2 2 2.83–3.04/2.92, m m
2.83–3.04/2.92, 3, 4, 5,3,8,4,95, 8, 9
8 66.86, CH 3.82, m 7, 9, 24, 25 60.79/61.29, CH 4.26/4.17, m 7, 9, 24
8 66.86, CH 3.82, m 7, 9, 24, 25 60.79/61.29, CH 4.26/4.17, m 7, 9, 24
9 172.36/172.63, C - - 171.40/172.00, C - -
9 172.36/172.63, C - - 171.40/172.00, C 3.48–3.80/3.26–3.51,
- -
10 47.14/48.24, CH2 3.40–3.72, m 9, 11, 21 47.67/47.78, CH2
10 47.14/48.24, CH2 3.40–3.72, m 9, 11, 21 47.67/47.78, CH2 3.48–3.80/3.26–3.51,
m m 9, 11,9,2111, 21
11 1.92–2.00/1.73–1.92,
29.09/30.45,CH
CH2
1.92–2.00/1.73–1.92, m
11 29.09/30.45, 2 10, 10,
12 12 29.15/30.50,CH
29.15/30.50, CH2
2
1.95–2.05/1.95,
1.95–2.05/1.95, m m 10, 1210, 12
m
12 72.31/71.72, CH2 3.85–3.93/3.88, m 11, 13 72.35/71.61, CH2 3.97/3.99, m 11, 13
12 72.31/71.72, CH2
3.85–3.93/3.88, m 11, 13 72.35/71.61, CH2 3.97/3.99, m 11, 13
1313 153.61/153.30,CC
153.61/153.30, - - - - 153.72,
153.72, CC - - - -
1414 119.43,CC
119.43, - - - - 119.47,
119.47, CC - - - -
15 134.47/134.50, CH 7.52/7.53, s 13, 14,13,
16,14,17, 16,
18, 17,
19 134.51/134.53, CH 7.54/7.55, s 13, 14, 16,
13,17,14,18,16,1917,
15
16 134.47/134.50, CH
137.97/138.24, C 7.52/7.53,
- s -18, 19 134.51/134.53,
137.72, C CH -7.54/7.55, s - 18, 19
17 134.47/134.50, CH 7.52/7.53, s 13, 14, 15, 16, 18, 19 134.51/134.53, CH 7.54/7.55, s 13, 14, 15, 16, 18, 19
16 137.97/138.24, C - - 137.72, C - -
18 119.43, C - - 119.47, C - -
19 31.40/31.46, CH 2 2.92, m 15,13,
16, 14,
17, 15,
20 16, 31.22, CH 2 CH 2.93, m 13,17,
15, 16, 14,2015, 16,
17 134.47/134.50, CH 7.52/7.53, s 134.51/134.53, 7.54/7.55, s
20 60.12/60.14, CH2 3.08, m 16, 19, 18,
22, 1923 59.96, CH2 3.21, m 16, 19, 22, 18,2319
21
18 36.64/34.26, CH3
119.43, C 2.92/2.93,- s 9, 10 - 36.25/34.29,
119.47,CHC 3 2.50/2.47, s- 9, 10 -
22 44.30/44.34, CH3 2.72/2.71, s 20, 23 44.18/44.21, CH3 2.74/2.74, s 20, 23
19 31.40/31.46, CH2 2.92, m 15, 16, 17, 20 31.22, CH2 2.93, m 15, 16, 17, 20
23 44.30/44.34, CH3 2.72/2.71, s 20, 22 44.18/44.21, CH3 2.74/2.74, s 20, 22
20
24 60.12/60.14, CH
42.31/41.19, CH3 2 3.08,
2.45/2.42, sm 16, 19,
8, 25 22, 23 59.96, CH
33.26/33.68, CH32 3.21,
2.82/3.00, s m 16,
8 19, 22, 23
21
25 36.64/34.26,CH
42.31/41.19, CH33 2.92/2.93,
2.45/2.42, s s 8, 24 9, 10 36.25/34.29,
- CH3 -2.50/2.47, s - 9, 10
22 44.30/44.34,
a ChemicalCH3 2.72/2.71, s 20, 23 44.18/44.21, CH3 2.74/2.74, s 20, 23
shifts are given for both rotamers when distinguishable signals were identified.
23 44.30/44.34, CH3 2.72/2.71, s 20, 22 44.18/44.21, CH3 2.74/2.74, s 20, 22
24 42.31/41.19, CH3 2.45/2.42, s 8, 25 33.26/33.68, CH3 2.82/3.00, s 8
25 42.31/41.19,
Compound 8 hadCH3 a protonated 2.45/2.42, s molecular8, ion 24 at m/z 815.0068- [M + H]+ -corresponding- to the
molecularChemical
formula shifts
C29 Hare 79given
81for both rotamers when distinguishable signals were identified.
a
43 Br2 Br2 N4 O3 , determined by HRESIMS. The mass spectrum indicated
an isotopic matrix of a tetrabrominated compound. Quick examination of the 1 H NMR spectrum
Compound 8 had a protonated molecular ion at m/z 815.0068 [M + H]+ corresponding to the
(Table 4) showed signals with identical chemical shifts to those reported for anomoian C (6) and D (7).
molecular formula C29H4379Br281Br2N4O3, determined by HRESIMS. The mass spectrum indicated an
Furthermore, it revealed the presence of an additional dimethylaminopropylic chain, as observed for
isotopic matrix of a tetrabrominated compound. Quick examination of the 1H NMR spectrum (Table
psammaplysene D (1). This was confirmed by the 2D NMR spectra analysis, including COSY, HSQC,
4) showed signals with identical chemical shifts to those reported for anomoian C (6) and D (7).
and HMBC, enabling us to set the correlations shown in Figure 5. The new compound 8 was named
Furthermore, it revealed the presence of an additional dimethylaminopropylic chain, as observed for
anomoian E.
psammaplysene D (1). This was confirmed by the 2D NMR spectra analysis, including COSY,
The HRESIMS data of 9 revealed the molecular formula C30 H45 79 Br2 81 Br2 N4 O3 (m/z 829.0259
HSQC, and HMBC, enabling us to set the correlations shown in Figure 5. The new compound 8 was
[M + H]+ ). Its 1 H NMR spectrum was very similar to anomoian E (8) spectrum, except for the
named anomoian E.
two additional methyl groups at δH 2.51/2.54 integrating for six protons for 9. HSQC, COSY,
The HRESIMS data of 9 revealed the molecular formula C30H4579Br281Br2N4O3 (m/z 829.0259 [M +
and HMBC spectra analysis led to the same correlations as in compound 8 and enabled the assignment
H]+). Its 1H NMR spectrum was very similar to anomoian E (8) spectrum, except for the two
of the N-8-methyl groups through an additional correlation from the N–CH3 -24/25 (δH 2.51/2.54, 6H)
additional methyl groups at δH 2.51/2.54 integrating for six protons for 9. HSQC, COSY, and HMBC
to the methine group CH-8 (δH 3.97/3.89, m; δC 66.79/66.58). Therefore, structure 9 was determined
spectra analysis led to the same correlations as in compound 8 and enabled the assignment of the
as anomoian F, existing as 6:4 rotamers (Table 4, Figure 5).
N-8-methyl groups through an additional correlation from the N–CH3-24/25 (δH 2.51/2.54, 6H) to the
methine group CH-8 (δH 3.97/3.89, m; δC 66.79/66.58). Therefore, structure 9 was determined as
anomoian F, existing as 6:4 rotamers (Table 4, Figure 5).
Mar. Drugs 2018, 16, 146 7 of 16
Mar. Drugs 2018, 16, x FOR PEER REVIEW 7 of 16

Figure 5. COSY and key HMBC correlations for anomoians E (8) and F (9).
Figure 5. COSY and key HMBC correlations for anomoians E (8) and F (9).
Table 4. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Anomoians E (8) and F (9) in CD3OD.
Table 4. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data for Anomoians E (8) and F (9) in CD3 OD.
Position Anomoian E (8) Anomoian F (9)
No δC, Type a δH mult, (J in Hz) a HMBC δC, Type a δH mult, (J in Hz) a HMBC
Position 1 153.29/153.15, C Anomoian E - (8) - 152.76/152.57, C Anomoian- F (9) -
No 2 δC ,119.08.119.14,
Type a C δ mult, (J in- Hz) a HMBC - 118.92, C a
δC , Type δH mult, - (J in Hz) a -HMBC
H
3 135.35/135.19, CH 7.51/7.48, s 1, 2, 5, 6, 7 135.32/135.22, CH 7.51/7.48, s 1, 2, 5, 6, 7
1 153.29/153.15, C - - 152.76/152.57, C - -
4 137.14/138.15, C - - 138.66/137.72, C - -
2 119.08.119.14, C - - 118.92, C - -
5 135.35/135.19, CH 7.51/7.48, s 1, 2, 3, 6, 7 135.32/135.22, CH 7.51/7.48, s 1, 2, 3, 6, 7
3 135.35/135.19, CH 7.51/7.48, s 1, 2, 5, 6, 7 135.32/135.22, CH 7.51/7.48, s 1, 2, 5, 6, 7
6 119.08.119.14, C - - 118.92, C - -
4 137.14/138.15, C - - 138.66/137.72, C - -
7 38.03/38.65, CH2 2.84–3.03/2.93, m 3, 4, 5, 8, 9 33.05/31.91, CH2 3.00/2.92–3.07, m 3, 4, 5, 8, 9
5 135.35/135.19, CH 7.51/7.48, s 1, 2, 3, 6, 7 135.32/135.22, CH 7.51/7.48, s 1, 2, 3, 6, 7
8 60.72/61.23, CH 4.15/4.06, m 4, 7, 9, 24 66.58/66.79, CH 3.97/3.89, m 4, 7, 9, 24, 25
6 119.08.119.14, C - - 118.92, C - -
7 9 38.03/38.65,
172.73/173.46,
CH2 C 2.84–3.03/2.93, - m 3, 4, 5, -8, 9 172.10/171.65,
33.05/31.91, C CH2 -
3.00/2.92–3.07, m 3,- 4, 5, 8, 9
8 10 47.57/47.82,
60.72/61.23, CH CH 2 3.51–3.76/3.26–3.60,
4.15/4.06, m m 4, 7, 9, 12,
9, 11, 24 21 48.26/47.36, CH
66.58/66.79, CH 2 3.41–3.74/3.51–3.64,
3.97/3.89, m m 9, 11, 12, 21
4, 7, 9, 24, 25
9 11 172.73/173.46,
29.16/30.64,C CH2 1.95–2.03/1.95,
- m 10,
- 12 30.71/29.08, CH2 C 1.69–1.98/1.90–1.98,
172.10/171.65, - m 10, 12 -
12 72.36/71.71, CH2 3.51–3.76/3.26–3.60,
3.99, m 10, 11, 13 71.82/72.40, CH2 3.94/3.95, m
3.41–3.74/3.51–3.64, 10, 11, 13
10 13 47.57/47.82, CH2C
153.54, 9, 11, 12, - 21 48.26/47.36, C CH2 9,- 11, 12, 21
m - 153.77/153.51, - m
14 119.42, C - - 119.52, C -
1.69–1.98/1.90–1.98, -
11 15 29.16/30.64, CH2 CH 1.95–2.03/1.95,
134.54/134.52, 7.55/7.56,ms 13,10,
14, 1217, 18, 19 30.71/29.08,CH
134.57/134.60, CH2 7.57/7.58, 13, 14, 17,10,18,12
ms 19
12 16 72.36/71.71,
137.74/138.52,
CH2 C 3.99, m - 10, 11,-13 137.28, C CH2
71.82/72.40, -
3.94/3.95, m - 11, 13
10,
13 17 134.54/134.52,
153.54, C CH 7.55/7.56,
- s 13, 14,- 15, 18, 19 134.57/134.60,
153.77/153.51,CH C 7.57/7.58,- s 13, 14, 15, 18, - 19
14 18 119.42, C C
119.42, - - - - 119.52,
119.52, CC - - - -
15 19 134.54/134.52, CHCH2
31.31/31.41, 7.55/7.56,
2.93,sm 13, 14,
15, 17,
16, 18,
17, 19
20 134.57/134.60,
30.67/30.59, CH2CH 7.57/7.58,
2.99, m s 15,13,
16,14,17,17,
20 18, 19
16 20 137.74/138.52,
60.01/60.08,C CH2 - 3.09, m - 22, 23
16, 19, 137.28,
59.43, CH2C 3.26, m- 16, 19, 22, -23
17 21 134.54/134.52, CHCH3
36.24/34.33, 7.55/7.56, s s
2.88/2.99, 13, 14, 15, 18, 19
9, 10 134.57/134.60,
34.31/36.64, CH3CH 7.57/7.58,
2.92/2.94, s s 13,9, 14,
10 15, 18, 19
18 22 119.42, C
44.19/44.24, CH3 -
2.73/2.71, s - 23
20, 119.52,
43.75, CH3C 2.85/2.85,- s 20, 23 -
19 23 31.31/31.41, CH2 CH3
44.19/44.24, 2.93, m
2.73/2.71, s 15, 16,20, 17,2220 30.67/30.59,
43.75, CH3 CH2 2.99, sm
2.85/2.85, 15,22
20, 16, 17, 20
20 24 60.01/60.08, CH2 CH3
33.71/34.11, 3.09, m
2.42/2.39, s 16, 19, 4, 22,8 23 59.43, CH
42.23/42.07, CH23 3.26, sm
2.51/2.45, 4,16,
8, 2519, 22, 23
21 25 36.24/34.33, CH - 3 2.88/2.99,- s 9, 10- 34.31/36.64,
42.23/42.07, CHCH3 3 2.92/2.94,
2.51/2.45, s s 4, 8, 24 9, 10
22 1′ 44.19/44.24, CH3 CH2
71.66/71.62, 2.73/2.71,
4.09,sm 20,1, 23
2′, 3′ 43.75,
71.44, CHCH2 3
2.85/2.85,
4.01, t (5.5) s 1, 2′, 20,
3′ 23
23 2′
44.19/44.24, CH3 2
26.95, CH
2.73/2.71, s
2.25, m
20,1′, 223′ 43.75, CH
26.62, CH2 3
2.85/2.85, s
2.27, m 1′, 3′
20, 22
24 33.71/34.11, CH3 2.42/2.39, s 4, 8 42.23/42.07, CH3 2.51/2.45, s 4, 8, 25
3′ 57.15, CH2 3.36, m 1′, 2′, 4′, 5′ 57.07, CH2 3.45, m 1′, 2′, 4′, 5′
25 - - - 42.23/42.07, CH3 2.51/2.45, s 4, 8, 24
0 4′ 44.05, CH3 2.85, s 3′, 5′ 43.79, CH3 2.92/2.91, s 3′, 5′ 0 0
1 71.66/71.62, CH2 4.09, m 1, 20 , 30 71.44, CH 4.01, t (5.5) 1, 2 , 3
0 5′ 44.05, CH3 2.85, s 0 3′,0 4′ 43.79, CH3 2 2.92/2.91, s 3′, 4′ 0 0
2 26.95, CH2 2.25, m 1,3 26.62, CH2 2.27, m 1,3
30 a Chemical
57.15, CH2 shifts are given
3.36, m 10 , 20 , 40 , 50when distinguishable
for both rotamers 57.07, CH2 signals were identified.
3.45, m 10 , 20 , 40 , 50
40 44.05, CH3 2.85, s 30 , 50 43.79, CH3 2.92/2.91, s 30 , 50
50 44.05, CH3 2.85, s 30 , 40 43.79, CH3 2.92/2.91, s 30 , 40
The absolute configuration of the stereocenter C-8 for anomoian C-F (6–9) can be proposed by
a Chemical shifts are given for both rotamers when distinguishable signals were identified.
25
comparison of the values and signs of their optical rotations in MeOH ([α] D +9°, +11°, +7° and +6°,
respectively), with the closely related congeners, anomoian A ([α]D +5.1°) [26], anomoian B ([α]D
The+10.5°)
absolute
[27], configuration
iso-Anomoian A of ([α]the
20
stereocenter
D +4.5°) C-8 for anomoian
[28], suberedamines
25
C-Fand
A ([α] D +21°) (6–9) can25D be
B ([α] +16°)proposed
[9], by
indicating that all are bearing -tyrosine residue. The synthetic
comparison of the values and signs of their optical rotations in MeOH ([α]D +9 , +11
L iso-Anomoian 25 A ◦
([α]
20 ◦ , +7 and +6◦ ,
+4.5°) ◦
and
D
20
suberedamines A ([α]
respectively), with the closely D +19.5°) allowed the determination of their absolute ◦
configurations
related congeners, anomoian A ([α] +5.1 ) [26], anomoian B ([α] [28].
D D
+10.5◦ ) [27], iso-Anomoian A ([α]20 ◦ 25 ◦ 25
D +4.5 ) [28], suberedamines A ([α]D +21 ) and B ([α]D +16 ) [9],
◦
2.2. Biological Activities 20 ◦
indicating that all are bearing L-tyrosine residue. The synthetic iso-Anomoian A ([α]D +4.5 ) and
The isolated20compounds 1–3 and 6–11 were tested for their cytotoxic activity against human
suberedamines A ([α]D +19.5◦ ) allowed the determination of their absolute configurations [28].
epidermoid carcinoma cells (KB) cancer cell lines (Table 5) and acetylcholinesterase. The results
showed of the compounds exhibited moderate cytotoxicity. The major compound psammaplysene D
2.2. Biological Activities
(1) showed the most interesting cytotoxicity with an IC50 = 0.7 μM. Anomoian E (8) and F (9),
Thecontaining
isolatedthe N,N-dimethylaminopropyl
compounds 1–3 and 6–11 side
were as tested
psammaplysene
for theirD,cytotoxic
but lacking the double
activity bond human
against
–CH-7=CH-8,
epidermoid carcinomadisplayed a lower
cells (KB) activity.
cancer Moreover,
cell lines (Tablepsammaplysene F (2), which bear
5) and acetylcholinesterase. Thea results
showed of the compounds exhibited moderate cytotoxicity. The major compound psammaplysene
D (1) showed the most interesting cytotoxicity with an IC50 = 0.7 µM. Anomoian E (8) and F (9),
containing the N,N-dimethylaminopropyl side as psammaplysene D, but lacking the double bond
–CH-7=CH-8, displayed a lower activity. Moreover, psammaplysene F (2), which bear a trans-cinnamoyl
part, was found to exhibit a lower activity than psammaplysene D (1). Therefore, from a structural
Mar. Drugs 2018, 16, 146 8 of 16

point of view, both motifs (N,N-dimethylaminopropyl and trans-cinnamoyl) seem to be together

correlated to the activity of psammaplysene D (1).

Table 5. Cytotoxic activities in vitro (KB Cell Line) for compounds 1–3, 6–11.

Compounds 10 µM a 1 µM a
Psammaplysene D (1) 100 ± 0.2 95 ± 0.5
Psammaplysene F (2) 73 ± 2 20 ± 0.2
Psammaplysene G (3) 75 ± 0.4 17 ± 1
Anomoian C (6) 28 ± 5 15 ± 3
Anomoian D (7) 29 ± 2 17 ± 2
Anomoian E (8) 82 ± 1 6±2
Anomoian F (9) 100 ± 0.5 20 ± 0.6
N,N-dimethyldibromotyramine (10) 100 ± 1 89 ± 1
5-hydroxy xanthenuric acid (11) 25 ± 1 6 ± 0.5
a Cell proliferation was measured with Celltiter 96 Aqueous One solution reagent (Promega), and results are
expressed as the percentage of inhibition of cellular proliferation of KB cells treated for 72 h with compounds
compared to cells treated with DMSO only (mean ± SE of triplicate).

The screening of our French Polynesian sponges extract library was performed on
acetylcholinesterase inhibitor (AChE) in the aim to investigate a possible chemical defense of sessile
organisms on the reef against fishes, their main predators. The chemical study of the bioactive extract of
Suberea ianthelliformis using AChE led to the identification of psammaplysene D (1) as its unique in vitro
AChE inhibitor with an IC50 = 1.3 µM. None of the other closely related compounds displayed any
activity on this target enhancing here again the importance of the phenoxy-1-N,N-dimethyl propane
chain and the trans-cinnamoyl motifs (Figure 1). Further kinetics investigation of the mode of AChE
inhibition of psammaplysene D (convergence of the lines on the Lineweaver Burk plot, decreasing
V max values (from 0.0036 to 0.0007 µmol·min−1 ) and increasing KM values (from 0.14 to 0.23 µM))
suggested a mixed competitive/non-competitive inhibition suggesting that psammaplysene D (1) not
only binds to the free enzyme, but also to the enzyme-substrate complex. In order to try to evaluate
the effect of psammaplysene D in the environment, it was further tested on fresh water fish (guppy,
Poecilia reticulata) and reef fish (Acanthurus triostegus) feeding experiment using bathing with or without
the bioactive compound. Guppies are fed with commercial flakes, but the wild reef fish feed on natural
coral heads, and their appetite evaluated by counting the bites on the corals. Two experiments were
run on guppies using Suberea ianthelliformis crude extract and purified psammaplysene D (1): an acute
toxicity bioassay (100 µg/mL of crude extract or purified psammaplysene D (1), 1 h) and a chronic
toxicity (5 µg/mL of crude extract or 1 µg/mL of purified psammalysene D (1), 72 h).
During the acute exposure, fish were totally confused and had uncontrolled mobility, displaying
sudden jumps and balance control loss, as soon as 20 min after the addition of psammaplysene D in
the tank. This result can be compared to the observation reported by Nèeman et al. with Latrunculia
species toxins [20]. Exposure of guppies during 24 h of at a sub-lethal dose (5 µg/mL of crude extract
or 1 µg/mL of purified psammalysene D (1) of the compound caused passivity of the fish towards the
food flakes offered daily, whereas those of the controls ate normally. These observations indicate a
clear link between the loss of appetite and psammaplysene D (1) toxicity. Similar activity was observed
on reef fish recruits (fish larvae undergoing metamorphosis) of Acanthurus triostegus: the presence
psammaplysene D (1) (1 µg/mL) in tanks significantly and readily decreased the number of bites of
recruits, suggesting a rapid saturation of the environment perception sensors of the fish larvae during
the 72 h of experiment (Figure 6).
Mar. Drugs Drugs16,
Mar.2018, 14616, x FOR PEER REVIEW
2018, 9 of 16
9 of 16

Mar. Drugs 2018, 16, x FOR PEER REVIEW 9 of 16

C = Control
S = Solvent
P = Psammaplysene D
C = Control
S = Solvent
P = Psammaplysene D
Bites. Fish-1. h-1 -1 -1
Bites. Fish . h

C S P C S P C S P

Figure 6. Number ofC bites S on coral

P pieces per C A. triostegus
S recruit
P and perC hour. Error
S bars
P represent
Figure 6. Number of bites on coral pieces per A. triostegus recruit and per hour. Error bars represent
standard deviation of the mean (N = 6). ** p < 0.01.
standard deviation of the mean (N = 6). ** p < 0.01.
Figure 6. Number of bites on coral pieces per A. triostegus recruit and per hour. Error bars represent
Psammaplysene
standard
Psammaplysene deviation
D (1)Dthe
of (1) mean
displayed
displayed clearly
(N =clearly
6). behavioral effects
** p <behavioral
0.01. effects on
onboth
bothguppies
guppies and reefreef
and fishfish
larvae:
larvae:
they showed
they showed a lossa of loss of appetite
appetite from fromthethe first
first daydayofofexperiment.
experiment. Reef Reeffish fishjuveniles’
juveniles’ behavior
behavior diddidnot not
seemPsammaplysene
to be impacted D by(1) displayed clearly
psammaplysene D. Itbehavioral effects
is interesting on both
to note thatguppies and reef fish
only A. triostegus larvae:
larvae were
seemthey
to beshowed
impacted byofpsammaplysene from the D. Itday
is interesting to note that only A. triostegus larvae
did notwere
impacted byathe loss pureappetite
psammaplysene first
D, suggesting of experiment.
a change Reef fish juveniles’
of sensibility towardsbehavior
the compound
impacted
seem by the
to be pure psammaplysene D, suggesting a change of sensibility towards the compound
during theimpacted by psammaplysene
metamorphosis. The toxicityD.test It isatinteresting
a high dose to (100
note μg/mL
that only of A. triostegus
crude extractlarvae were
or purified
during the
impactedmetamorphosis.
by the pure The
psammaplysenetoxicity test
D, at a
suggesting high a dose
change (100
of
psammaplysene D) on the guppies led to their death after one hour bathing with either the crude µg/mL
sensibility of crude
towards extract
the or
compound purified
psammaplysene
extract or the purified psammaplysene D fraction (acute toxicity experiment). Brains and gills crude
during the D) on
metamorphosis.the guppies
The led
toxicity to their
test at death
a high after
dose one
(100 hour
μg/mL bathing
of crude with
extracteither
or the
purified of
psammaplysene
extractthese
or the purified
guppies D) on
were the guppies
psammaplysene
extracted and the ledAChE
Dto fraction
their death
activity after toxicity
(acute
measured one onhour the bathing
extractswith
experiment). either
Brains
(Figure theand
7a,b) crude
[29]. gills of
The
theseextract
resultsor
guppies werethe clearer
were purified
extracted psammaplysene
with and
the the AChE
purified D fraction
activity (acute
psammaplysene measured toxicity
D, showing on experiment).
the extracts
a mild Brains
(Figureand
but significant 7a,b)gills
decrease ofofThe
[29].
these
resultsthe
wereguppies
activity
clearer were
of with extracted
the enzyme and the AChE
in the psammaplysene
the purified activity
gills, suggesting an measured
D,action on
showing the
on the extracts (Figure
olfactory
a mild 7a,b) [29].
organs (peripheral
but significant The
decrease of
results
nervouswere clearerthat
system) with the purified
could not be psammaplysene
further investigated D, showing
there. A agreat
mildnumber
but significant
of AChE decrease
inhibitorsof
the activity of the enzyme in the gills, suggesting an action on the olfactory organs (peripheral nervous
the activity
targeting of the
Alzheimer’s enzyme in the gills,
disease investigated suggesting
have been isolated an action
from on
natural the olfactory
sources organs (peripheral
[30]. inhibitors
Among them, 6
system) that could not be further there. A great number of AChE targeting
nervous
compoundssystem) withthat could not
activities be further
ranging from IC investigated
50 1 to 100 μM there.
wereA great
isolatednumber
from of AChE inhibitors
Verongiida sponges
Alzheimer’s
targeting disease have been isolated from isolated
natural sources [30]. Among them,Among
6 compounds 6 with
(Table 6).Alzheimer’s
It would be disease have been
interesting also to further from natural
investigate sources [30].
those compounds as them,
chemical
activities rangingwith
compounds
anti-predatory
from IC50 1through
activities
defenses
to 100 fish
ranging µM were
from IC50isolated
antifeedant 100from
1 toactivity, Verongiida
μMsince
were isolated
the
sponges (Table 6).sponges
from isVerongiida
fish model easier to set
Itupwould
than
be
interesting
(Table also
6). to
It further
would investigate
be interesting those
also compounds
to further
related bioassays involving sponge predators, such as nudibranchs or crustaceans. as chemical
investigate anti-predatory
those compounds defenses
as chemicalthrough
fish antifeedant
anti-predatory activity,
defensessince the fish
through fishmodel is easier
antifeedant to setsince
activity, up than
the fishrelated
model bioassays involving
is easier to set up than sponge
relatedsuch
predators, bioassays involving sponge
as nudibranchs predators,
or crustaceans.
Brain
such as nudibranchs or crustaceans.
Gill
0.12 0.12
Brain Gill
0.10 0.10
0.12 0.12
U. mg-1

0.08 0.08
0.10 0.10
0.06 0.06
U. mg-1

0.08 0.08
0.04 0.04
0.06 0.06
0.02 0.02
0.04 0.04
0 0
0.02 0.02
Control 100 µg. mL-1 Control 100 µg. mL-1
0 0

Control 100 µg. mL-1(a) Control 100 µg. mL-1

(a)

Figure 7. Cont.
Mar. Drugs 2018, 16, 146 10 of 16
Mar. Drugs 2018, 16, x FOR PEER REVIEW 10 of 16

Brain Gill

0.10 0.10

0.08 0.08

U. mg-1
0.06 0.06

0.04 0.04

0.02 0.02

0 0

Control 5 µg. mL-1 Control 5 µg. mL-1

(b)
Figure 7. (a) AChE activity, U·mg−1, after 1 h exposure to 100 µ g/mL of Psammalysene D (1). Boxplot
Figure 7. (a) AChE activity, U·mg−1 , after 1 h exposure to 100 µg/mL of Psammalysene D (1). Boxplot
distribution was made with 5 fish (N = 5). * p < 0.05; (b) AChE activity, U·mg−1, after 72 h exposure to
distribution was made with 5 fish (N = 5). * p < 0.05; (b) AChE activity, U·mg−1 , after 72 h exposure to
5 µ g/mL of crude extract. Boxplot distribution was made with 5 fish (N = 5).
5 µg/mL of crude extract. Boxplot distribution was made with 5 fish (N = 5).
Table 6. AChE inhibition of bromotyrosine compounds isolated from Verongiida sponges.
Table 6. AChE inhibition of bromotyrosine compounds isolated from Verongiida sponges.
Sponge/Compounds AChE Origin Activities Ref.
Pseudoceratina purpurea Eel
Sponge/Compounds
Purealidine Q AChE Origin Activities
IC50 = 1.2 µ M (NCI a) Reference
Olatunji et al. 2014 [31]
Isoanomoian
Pseudoceratina A
purpurea Eel IC50 = 70 µ M (NCI)
Purealidine Q A
Aplysanzine IC50 =a )104 µ M (NCI)
IC50 = 1.2 µM (NCI
Olatunji et al. 2014 [31]
Isoanomoian A
Acanthodendrilla sp IC50 = 70 µM (NCI)
Human recombinant AChE
Aplysanzine A
Homoaerothionin IC50 = 104 µM (NCI)
IC50 = 4.5 µ M (CI b) Sirimangkalakitti et al. 2015 [32]
Acanthodendrilla
Fistularin 1 sp. Human recombinant
IC50 = AchE
47.5 µ M (ND c)
Homoaerothionin IC50 = 4.5 µM (CI b
Eel ) Insect recombinant Sirimangkalakitti et al. 2015 [32]
Unidentified
Fistularin 1 Verongida Aplysamine-4
IC50 = 47.5 µM (ND c Sepcic et al. 2001 [33]
KI = 16) µ M (NCI) KI = 2 µ M (NCI)
Unidentified Verongida Eel
a NCI: non competitive Insect recombinant
inhibition; b CI: competitive inhibition; c ND: nonetdescribed.
Sepcic al. 2001 [33]
Aplysamine-4 KI = 16 µM (NCI) KI = 2 µM (NCI)
a
NCI:side
noneffect
competitive inhibition; b
CI: competitive c
A common of AChE inhibition treatments inhibition;
is anorexiaND: non described.
[34,35], which strengthens the
importance of natural AChE inhibitors as potential deterrents of predators such as fishes.
AFurthermore,
common side the effect of AChE
olfactory inhibition
dysfunction treatmentsdisease
in Alzheimer’s is anorexia [34,35],
has also been which strengthens
demonstrated [36]. the
Psammaplysene
importance of naturalDAChEis theinhibitors
major compound
as potentialof the sponge of
deterrents (0.66% w/w). such
predators It hasasbeen shown
fishes. that
Furthermore,
tyrosine derived compounds are located in sphaerus cells of the sponge, allowing
the olfactory dysfunction in Alzheimer’s disease has also been demonstrated [36]. Psammaplysene their release in the D is
aquifer channels and further in the close environment of the sponge [37]. It would
the major compound of the sponge (0.66% w/w). It has been shown that tyrosine derived compounds be thus interesting
to track and quantify the concentration of this compound in the close reef environmental water of
are located in sphaerus cells of the sponge, allowing their release in the aquifer channels and further
the sponges, using for example, solid phase adsorption toxin tracking (SPATT) [38], to allow the on
in thefield
closedeterrent
environment of the sponge [37]. It would be thus interesting to track and quantify the
or reef protective activity of this compound acting softly on olfactory sense of
concentration of this compound in the close
predators in the marine environment. Thereef environmental
importance of AChEwater of the
inhibitors insponges,
the marine using for example,
environment
solid phase
can be adsorption toxin tracking
included in neuroecology of (SPATT) [38], to allow
chemical defenses, the on
a merging field
field of deterrent or reef protective
chemical ecology, which
activity of thisthe
displays compound
importance acting softly
of the linksonbetween
olfactory sense and
ecology of predators
evolution,insecondary
the marine environment.
metabolites,
The importance
behavior, and ofneurosciences
AChE inhibitors [39]. in the marine environment can be included in neuroecology of
chemical defenses, a merging field of chemical ecology, which displays the importance of the links
3. Material and Methods
between ecology and evolution, secondary metabolites, behavior, and neurosciences [39].
3.1. General Procedures
3. Material and Methods
Optical rotations were measured using MCP-300 polarimeter (Anton Paar, Les Ulis, France). IR
3.1. General
spectraProcedures
were recorded on a BX FT-IR spectrometer (Perkin Elmer, Courtaboeuf, France). 1D and 2D
NMR spectra were recorded on a Avance 500 and 600 MHz (CNRS-ICSN, Bruker, Wissembourg,
Optical rotations were measured using MCP-300 polarimeter (Anton Paar, Les Ulis, France).
France). The chemical shifts are relative to the residual signal solvent (CD 3OD: δH 3.31; δC 49.20;
IR spectra were recorded on a BX FT-IR spectrometer (Perkin Elmer, Courtaboeuf, France). 1D and
DMF-d7: δH 8.03, 2.92, 2.75; δC 163.2, 34.9, 29.8). High-resolution mass spectra were obtained on a LCT
2D NMR spectra
Premier were recorded
XE spectrometer on a Avance
(Waters, 500 and
Guyancourt, 600 in
France) MHz (CNRS-ICSN,
electrospray Bruker,
ionization modeWissembourg,
by direct
France). The chemical shifts are relative to the residual signal solvent (CD3 OD: δH 3.31; δC 49.20;
DMF-d7 : δH 8.03, 2.92, 2.75; δC 163.2, 34.9, 29.8). High-resolution mass spectra were obtained on
a LCT Premier XE spectrometer (Waters, Guyancourt, France) in electrospray ionization mode by
direct infusion of the purified compounds. Preparative HPLC was performed using Auto Prep system
Mar. Drugs 2018, 16, 146 11 of 16

(Waters 600 controller and Waters 600 pump, equipped with a 996 Photodiode Array Detector, Waters,
Guyancourt, France). A Varioskan® Flash microplate reader (Thermo Scientific, Villebon-sur-Yvette,
France) was used for the acetylcholinesterase experiments.

3.2. Animal Material

The sponge was collected off the coast of Nuku Hiva (8◦ 550 9770 S–140◦ 010 170 W) between 5 and
42 m deep using SCUBA on the 28 August 2009 [40]. It was identified as Suberea ianthelliformis and a
reference specimen is deposited at the Queensland Museum (Brisbane, Australia) under the accessing
number G331075.

3.3. Isolation of Bioactive COMPOUNDS

The freeze-dried sponge sample (960 g) of Suberea ianthelliformis was extracted three times at
room temperature with a 1:1 mixture of CH2 Cl2 /MeOH (1.5 L). The extracts were combined and
dried under reduced pressure to afford a brown residue (50 g). Forty-five grams of this residue were
partitioned between CH2 Cl2 and H2 O. The resulting organic layer was collected and dried under
reduced pressure to afford a brown material (10.5 g). The aqueous layer was further extracted with
n-BuOH to afford the butanolic extract (13 g). The remaining aqueous fraction was dried to afford
21.2 g material. The CH2 Cl2 extract was then subjected to normal phase silica-gel MPLC. A gradient
composed of CH2 Cl2 /MeOH (1:0 to 0:1), followed by a mixture of EtOAc/Acetone/H2 O/Formic Acid
(5/3/0.5/0.5) were used to afford 8 fractions F1 to F8. The n-BuOH extract was also subjected to normal
phase silica gel MPLC, using the same gradient as before to afford 6 fractions F1 to F6. Promising
fractions were selected based on the LC-MS analytical profile, and then each fraction was submitted
for purification by preparative reversed phase HPLC (column: Waters Sunfire C18 19 mm × 150 mm,
5 µ, H2 O + 0.1% formic acid/CH3 CN + 0.1% formic acid). Some sub-fractions were further purified
using semi-preparative reversed phase column (Waters Sunfire C18 , 10 mm × 150 mm, 5 µ, H2 O + 0.1%
formic acid/CH3 CN + 0.1% formic acid).
Psammaplysene D (1): brown oil, (2.6 g); UV (MeOH) λmax (log ε) 241 (1.30), 279 (2.90) nm; IR (neat)
νmax 2943, 1649, 1598, 1455, 1379, 738 cm−1 ; 1 H and 13 C NMR data, Supporting Materials; HRESIMS
m/z 783.9673 [M + H]+ (calcd for C28 H38 N3 O3 Br4 , 783.9606).
Psammaplysene F (2): pale yellow oil, (18.4 mg); UV (MeOH) λmax (log ε) 208 (0.84), 270 (0.20) nm;
IR (neat) νmax 3381, 2941, 1636, 1596, 1471, 1456, 1399, 1257, 738 cm−1 ; 1 H and 13 C NMR data, Table 1;
HRESIMS m/z 698.8675 [M + H]+ (calcd for C23 H27 N2 O3 79 Br2 81 Br2 , 698.8714).
Psammaplysene G (3): white powder, (8.1 mg); UV (MeOH) λmax (log ε) 208 (0.24), 270 (0.08) nm; IR (neat)
νmax 2938, 1600, 1457, 1398, 1258, 738 cm−1 ; 1 H and 13 C NMR data, Table 1; HRESIMS m/z 698.8723
[M + H]+ (calcd for C23 H27 N2 O3 79 Br2 81 Br2 , 698.8714).
Psammaplysene H (4)/Psammaplysene I (5) mixture: pale yellow oil, (0.7 mg); 1 H and 13 C NMR data,
Table 2; HRESIMS m/z 769.9521 [M + H]+ (calcd. for C27 H36 79 Br2 81 Br2 N3 O3 , 769.9449)/HRESIMS
m/z 755.9309 [M + H]+ (calcd. for C26 H34 79 Br2 81 Br2 N3 O3 , 755.9293).
Anomoian C (6): colorless oil, (1.9 mg); [α]25
D +9 (c 0.19, MeOH, as formate salt); UV (MeOH) λmax (log ε)
211 (0.80), 283 (0.10) nm; IR (neat) νmax 3396, 2938, 1635, 1458, 1258, 1039, 737 cm−1 ; 1 H and 13 C NMR
data, Table 3; HRESIMS m/z 743.9386 [M + H]+ (calcd for C25 H34 N3 O3 79 Br2 81 Br2 , 743.9293).
Anomoian D (7): pale yellow oil, (1.2 mg); [α]25
D +11 (c 0.12, MeOH, as formate salt); UV (MeOH) λmax
(log ε) 227 (2.00), 283 (0.40) nm; IR (neat) νmax 3356, 2934, 1600, 1598, 1458, 1259, 1041, 738 cm−1 ;
1 H and 13 C NMR data, Table 3; HRESIMS m/z 729.9190 [M + H]+ (calcd for C H N O 79 Br 81 Br ,
24 32 3 3 2 2
729.9136).
Mar. Drugs 2018, 16, 146 12 of 16

Anomoian E (8): colorless oil, (3.8 mg); [α]25

D +7 (c 0.23, MeOH, as formate salt); UV (MeOH) λmax (log ε)
222 (1.60), 276 (0.20) nm; IR (neat) νmax 3382, 2937, 2782, 1635, 1457, 1257, 1040, 738 cm−1 ; 1 H and 13 C
NMR data, Table 4; HRESIMS m/z 815.0068 [M + H]+ (calcd for C29 H43 N4 O3 79 Br2 81 Br2 , 815.0028).
Anomoian F (9): yellow oil, (3.8 mg); [α]25
D +6 (c 0.38, MeOH, as formate salt); UV (MeOH) λmax (log ε)
220 (1.30), 277 (0.20) nm; IR (neat) νmax 3402, 2940, 2780, 1641, 1460, 1258, 1041, 737 cm−1 ; 1 H and 13 C
NMR data, Table 4; HRESIMS m/z 829.0259 [M + H]+ (calcd for C30 H45 N4 O3 79 Br2 81 Br2 , 829.0184).
N, N-Dimethyldibromotyramine (10): yellow oil, (10.5 mg); UV (MeOH) λmax (log ε) 222 (1.50), 291 (0.60)
nm; IR (neat) νmax 3255, 2957, 1636, 1596, 1283, 737 cm−1 ; 1 H and 13 C NMR data, Supplementary
Materials; HRESIMS m/z 323.9388 [M + H]+ (calcd for C10 H14 NO79 Br81 Br, 323.9422).
5-Hydroxy xanthenuric acid (11): reddish brown amorphous material, (3.2 mg); UV (MeOH) λmax (log ε)
210 (0.55), 236 (0.8), 336 (0.20) nm; IR (neat) νmax 3360, 3250, 2980, 1726, 1644, 1590, 1447, 1246, 1051,
757, 683 cm−1 ; 1 H and 13 C NMR data, Supplementary Materials; HRESIMS m/z 222.0441 [M + H]+
(calcd for C10 H8 NO5 , 222.0402).
Xanthurenic acid (12): pale green oil, (1.5 mg); UV (MeOH) λmax (log ε) 220 (0.35), 248 (0.60), 345 (0.15)
nm; 1 H and 13 C NMR data, Supplementary Materials; HRESIMS m/z 206.0511 [M + H]+ (calcd for
C10 H8 NO4 , 206.0453).

3.4. Cell Culture and Cell Proliferation Assay

The human cell line KB originated from the National Cancer Institute (NCI) and was grown in
Dulbecco’s Modified Eagle Medium (D-MEM) medium supplemented with 10% foetal calf serum,
in the presence of penicillin, streptomycin, and fungizone in a 75 mL flask under 5% CO2 . A total of
600 cells were plated in 96-well tissue culture plates in 200 L of medium and treated 24 h later with
2 µL of stock solution of compounds dissolved in DMSO using a Biomek 3000 (Beckman-Coulter,
Brea, CA, USA). Controls received the same volume of DMSO (1% final volume). After 72 h
exposure, cell titer 96 Aqueous One solution (Promega, Madison, WI, USA) was added and incubated
for 3 h at 37 ◦ C: the absorbance was monitored at 490 nm, and results were expressed as the
inhibition of cell proliferation calculated as the ratio [(1 − (OD490 treated/OD490 control)) × 100] in
triplicate experiments.

3.5. Acetylcholinesterase Inhibition Assay

The crude extract of the sponge and the purified psammaplysene D (1) were dissolved in DMSO at
10 mg/mL, stored at −20 ◦ C and diluted subsequently 100 times in H2 O prior testing. Determination
of AChE-inhibitory activity was measured according the Ellman’s colorimetric method [41], in 96-well
tissue culture plates. All reagents, buffers and salts were purchased from Sigma-Aldrich (Saint-Louis,
MO, USA). Galantamine was used as a positive control for each analysis. Solvent controls received
the same volume of water. After a 30 min incubation at 25 ◦ C in the dark, absorbance was monitored
at λ = 405 nm. Results were expressed as the percentage of enzyme inhibition calculated as the
ratio: 100 × (ODcontrol − ODtreated )/(ODcontrol − ODblank ), where the blanks are the wells without
AChE. Serial dilutions of the compound or extract were prepared (100, 50, 25, 12.5, 6.25, 3.12,
1.56, and 0.78 µg/mL) and tested according to the same procedure using 10 µL of each solution.
IC50 values were graphically determined by plotting the percentage of inhibition against the compound
concentrations. Each assay was performed in triplicate and results are expressed as mean values ± S.D.

3.5.1. Kinetic Study

The inhibition mode of psammaplysene D on AChE was determined while measuring the enzyme
activity in presence of an increasing concentration of ATCI (0.024, 0.049, 0.098, 0.19, 0.39, 0.78, 1.57,
and 3.15 µM), in absence or presence of 2 µg/mL final concentration of purified psammaplysene D.
Mar. Drugs 2018, 16, 146 13 of 16

The inhibition mode, Vmax , and KM values were determined by double-reciprocal Lineweaver and
Burk plot analysis (1934) of the data obtained.

3.5.2. Guppy Bioassay

5 to 8 cm long guppies (Poecilia reticulata) were collected at night in a private fresh water pool at
Papara (Tahiti, French Polynesia) and immediately acclimated at the laboratory temperature (25 ◦ C).
After acclimatization, fishes were parted in two 2 L tanks: one for the control group treated with
solvent (N = 5). Another experiment was performed using a 1 h treatment with 100 µg/mL of crude
extract of the sponge or purified psammaplysene D (1) to check their acute toxicity.

3.5.3. Reef Fish Feeding Behavior Experiment

Manini (Acanthurus triostegus) were caught during new moon nights on the foreshore puddles at
Tema’e public beach [42]. Recruits (fish larvae undergoing metamorphosis) and juveniles (larvae after
the recruits stage) were collected and immediately used for the experiments. Natural lagoon sea-water
was used and UV-sterilized prior use. Three conditions were tested on three groups: control, solvent,
1 µg/mL sponge extract treatment, with 4 or 5 fishes in each 3 L tank. The fishes were exposed under
semi-static system for 72 h, the experimental solutions were renewed every 24 h to maintain clean
experimental conditions. The experiments were performed with fish recruits and juveniles. Rubbles
were disposed in each aquarium 1 h/day during 3 days. Feeding behavior experiment consists in
counting the overall number of bites on those rubbles in each aquarium. Six videos sequences (N = 6)
of 5 or 10 min per aquarium and per day were analyzed. Results are expressed in number of bites per
fish and per hour.

3.5.4. Statistical Analysis

All the values of AChE activity are represented in a boxplot distribution, because of non-normality
of variable distribution (N = 4 or 5). The significant differences between controls and treatments
(p-value p < 0.05) were analyzed with non-parametric test of Wilcoxon Mann Whitney. In feeding
deterrent assay, pairs significant differences between different conditions (p < 0.05) were tested with
non-parametric Kruskal Wallis test. All statistical assays were executed using R software (R-3.2.0,
The R Foundation for Statistical Computing, Vienna, Austria).

4. Conclusions
This paper describes the isolation of eight new bromotyrosine secondary metabolites from the
Polynesian Sponge Suberea ianthelliformis. Their structures are close to those of psammaplysenes and
anomoians but all displaying methylated amidic nitrogen and rotameric mixtures. Since flexible
syntheses were developed for psammaplysene A [43], and iso-anomoian A [28] and other analogues,
our molecules present new derivatives for further optimization of biologically interesting molecules.
They all exhibited moderate antiproliferative activity against KB cancer cell lines, but psammaplysene
D (1) displayed substantial cytotoxicity as well as acetylcholinesterase inhibition with IC50 values of
0.7 µM and 1.3 µM, respectively. It would be interesting to further investigate the ecological role of
the major psammaplysene D as chemical anti-predatory defenses through fish antifeedant activity.
The available synthetic strategies would allow the preparation of our metabolites and a rage of their
analogues for the screening of the inhibition of various cancer cell lines and SAR studies.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/5/146/s1,

Spectroscopic Data (HRMS, 1 H NMR, 13 C NMR, COSY, HSQC and HMBC) for compounds 1–12.
Author Contributions: Ali Al-Mourabit and Cécile Debitus designed the project and the whole experiment and
prepared the manuscript. Amr El-Demerdash and Céline Moriou did the chemical experimental part (isolation
and structural identification of the compounds) and wrote the first draft of manuscript. The biological assays
were designed and performed by Jordan Toullec, Marc Besson and David Lecchini on fish, by Stéphanie Soulet
on acetylcholinesterase, by Nelly Schmitt on KB cells. Cécile Debitus and Sylvain Petek organized the field
Mar. Drugs 2018, 16, 146 14 of 16

work and sponge collection, the conditioning and the realization of the library of extracts in preliminary to this
thorough study.
Acknowledgments: We thank the French Polynesian authorities, as well as the communities, for allowing us to
collect in Marquesas Islands. Financial support from CNRS-ICSN, IRD for the collecting trip aboard R/V Alis,
French and French Polynesian governments for the Marquesas and Biopolyval projects, ANR (POMARE project,
2011-EBIM-006-01), as well as project TASCMAR which is funded by the European Union under grant agreement
number 634674. Labex MER and Labex CORAIL, are gratefully acknowledged. Amr El-Demerdash’s PhD was
granted and financed by the Egyptian Government (Egyptian Cultural Bureau in Paris, Ministry of Higher
Education), they are gratefully acknowledged. We thank IRD’s diving team (SEOH IRD Noumea, New Caledonia)
and the R/V crew for their on-field help, O. Thoison for HPLC assistance J.-F. Gallard and K. Hammad for
technical assistance and NMR measurements, J. Bignon for cytotoxicity evaluations. We gratefully acknowledge
Laurent Calcul for the improvements to this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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