Next Article in Journal
Natural Product Potential of the Genus Nocardiopsis
Previous Article in Journal
Multiple Isoforms of Anti-Lipopolysaccharide Factors and Their Antimicrobial Functions in the Ridgetail Prawn Exopalaemon carinicauda
Article Menu
Issue 5 (May) cover image

Export Article

Mar. Drugs 2018, 16(5), 146; doi:10.3390/md16050146

Article
Bioactive Bromotyrosine-Derived Alkaloids from the Polynesian Sponge Suberea ianthelliformis
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
2
Organic Chemistry Division, Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
3
LEMAR, IRD, UBO, CNRS, IFREMER, IUEM, 29280 Plouzané, France
4
CRIOBE, CNRS, EPHE, UPVD, PSL Research University, 98729 Moorea, French Polynesia
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
*
Authors to whom correspondence should be addressed.
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 29 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 [1,2,3]. 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]. Many compounds of this class of bromotyrosine alkaloids display a wide scope of bioactivities including cytotoxicity against murine leukemia L1201 cells and KB cells [8,9], DNA demethylating [11], antiproliferative [12], antimicrobial [13,14,15,16], and antiplasmodial activities [17]. In continuation of our search for bioactive marine natural products from South Pacific sponges [18], in both health and chemical ecology fields, we report here the isolation, structural determination, and cytotoxicity evaluation of eight new tetrabromotyrosine alkaloids (Figure 1), along with four known compounds from the sponge Suberea ianthelliformis. The known major compound psammaplysene D (1) was also evaluated for its acetylcholinesterase inhibition activity. Although acetylcholinesterase inhibition has been mostly explored as treatment for Alzheimer’s pathology, our attention has been brought to the chemical ecology importance of such enzyme inhibitors in their environment or against predation [19]. Only one publication relates fish toxicity on Gambusia affinis (western mosquitofish) and butylcholinesterase from a Latrunculia magnifica toxin [20]. We describe our observations of the inhibitory effect of psammaplysene D on acetylcholinesterase, using balneation experiments on two fish species, Poecilia reticulata (guppy) and Acanthurus triostegus (reef fish).

2. Results and Discussion

2.1. Isolation and Structure Elucidation

Each crude 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 CH2Cl2/MeOH. The sub-fractions were purified by preparative, semi-preparative, and analytical reversed-phase HPLC and led to the isolation of eight new compounds 29, along with the known derivatives psammaplysene D (1), N,N-dimethyldibromotyramine (10), 5-hydroxy xanthurenic acid (11) and xanthurenic acid acid (12) (Figure 1). The known natural products psammaplysene D (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 1H 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.
The molecular formula C23H2779Br281Br2N2O3 of compound 2 was determined by HRESIMS (m/z 698.8675 [M + H]+) indicating ten degrees of unsaturation. The isotopic pattern of the protonated molecule confirmed the presence of four bromines. Analysis of the NMR data recorded for 2 revealed striking similarities with the NMR data recorded for the co-isolated psammaplysene D (1), except for the absence of the signal corresponding to the propylamine group, as confirmed by the HRESIMS spectrum. In general, the 1H NMR spectra of these families of compounds were complicated by the multiplicity of the signals due to the two rotational isomers trans/cis (6:4 ratio) along the amide bond [21]. The same behavior was already reported for tetrabromotyrosine derivatives psammaplysene A-D, isolated from the sponges Psammaplysilla and Psammoclemma [21,22,23,24,25]. The 1D and 2D NMR analysis revealed the existence of four aromatic protons comprising two singlets at δH 7.78 and 7.70, assigned to H-3/5, and another broad singlet at δH 7.57 integrating for 2 protons, attributed to H-15/17. An A-B system signal, corresponding to two olefinic protons at δH 7.39–7.36/7.38–7.35 (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 unsaturation. The HMBC data analysis showed correlations between H-7 and the two sp2 aromatic carbons C-3/5 (δC 133.09/133.01) and the sp2 non-protonated carbon C-4 (δC 130.98) confirming the connection of the trans-cinnamoyl motif to the 2,6-dibrominated phenolic ring (deduced with the chemical shifts of C-2/6 at δC 112.80 and C-1 at δC 154.10). The second olefinic proton H-8 exhibited an HMBC correlation with the non-protonated carbon C-9 at (δC 168.96/168.80) linking this end to an amidic carbonyl function. HSQC and COSY spectra analysis allowed the assignment of the signals at δH 4.11 and 4.05 (2H, 2t, J = 6.4), 3.89 and 3.74 (2H, 2t, J = 7.2 Hz), 2.22 and 2.16 (2H, 2m) to the three propylic carbon sequence N–CH2-10–CH2-11–CH2-12–O, while those at δH 3.30 (2H, m) and 3.00 (2H, m) were attributed to the ethylic chain CH2-19–CH2-20. The protons H-20 were correlated to N-Me22 and N-Me23 by HMBC analysis (Figure 2). The methylene group CH2-10 was connected to the amidic N-Me21 based on its chemical shift (δC 47.14/48.2) and the HMBC correlation to C-9 (δC 168.96/168.80) and C-21 (δC 34.74/36.55). The chemical shifts of CH2-12 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 carbon C-13 (δC 153.88/153.94) allowed the connection of this part of the molecule to the other 1,2,4,6-tetrasubstituted-phenyl ring. Furthermore, CH2-19 (δH 3.00) displayed HMBC correlations with the aromatic non-protonated carbon C-16 (δC 136.81/136.99) and CH-15/17 (134.63/134.56), confirming the substitution of this aromatic ring by the N,N-dimethylaminoethyl chain. Compound 2 was named psammaplysene F.
Compound 3 displayed the same molecular formula than compound 2 obtained by HRESIMS (m/z 698.8675 [M + H]+). A preliminary 1H NMR inspection (Table 1) showed similarity between compounds 1 and 2, which was easily observed by superimposition of their spectra. The A-B system of two downfield doublets of doublets at δH 6.49–6.52/6.56–6.58 (J = 12.5 Hz, cis) and δH 6.13–6.16/6.09–6.12 (J = 12.5 Hz) were assigned to –CH-7=CH-8 of the cinnamoyl moiety with a Z configuration. Furthermore, the COSY, HSQC, and HMBC analysis data (Figure 2) showed the same correlations as previously found for 2. Thus, the tetrabrominated tyrosine 3 was assigned to psammaplysene G.
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 C27H3679Br281Br2N3O3 (m/z 769.9521 [M + H]+) and C26H3479Br281Br2N3O3 (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 1H 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 CH2-3′, 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). 1H 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 (CH3-4′ and CH3-5′, 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-CH3-4′ and 5′ (δH 2.74–2.79, δC 44.7) to C-3′ (δC 57.2) along with the chemical shift of C-3′, showed the presence of two methyl groups on the nitrogen N-C-3′. The chemical shift of C-20 and C-22 were the same for compound 5, but the chemical shift of C-2′ (δC 27.9), C-3′ (δC 48.7), and CH3-4′ (δC 34.0), as well as the HMBC correlations between C-3′ and CH3-4′, suggested the presence of a NH-Me connected to the carbon C-3′. 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.
Compounds 6 and 7 exhibited the molecular formula were determined by HRESIMS as C25H3379Br281Br2N3O3 at m/z 743.9386 [M + H]+, and C24H3179Br281Br2N3O3 at m/z 729.9190 [M + H]+ respectively, indicating nine degrees of unsaturation with an isotopic pattern of four bromines. These NMR and mass data showed that compound 7 has one methyl group less than compound 6. Examination of the 1H NMR and HSQC spectra (Table 3) displayed signals with similar chemical shifts to those reported for the tetrabromotyrosine compounds anomoians A and B, previously isolated from Anomoianthella popeae [26] (order: Verongiida) and from an unknown species of the genus Hexadella (order: Verongiida) collected in Indonesia [27]. 2D NMR analysis including COSY, HSQC, and HMBC (Table 3, Figure 4), 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.
Compound 8 had a protonated molecular ion at m/z 815.0068 [M + H]+ corresponding to the molecular formula C29H4379Br281Br2N4O3, determined by HRESIMS. The mass spectrum indicated an isotopic matrix of a tetrabrominated compound. Quick examination of the 1H NMR spectrum (Table 4) showed signals with identical chemical shifts to those reported for anomoian C (6) and D (7). Furthermore, it revealed the presence of an additional dimethylaminopropylic chain, as observed for psammaplysene D (1). This was confirmed by the 2D NMR spectra analysis, including COSY, HSQC, and HMBC, enabling us to set the correlations shown in Figure 5. The new compound 8 was named anomoian E.
The HRESIMS data of 9 revealed the molecular formula C30H4579Br281Br2N4O3 (m/z 829.0259 [M + H]+). Its 1H NMR spectrum was very similar to anomoian E (8) spectrum, except for the two additional methyl groups at δH 2.51/2.54 integrating for six protons for 9. HSQC, COSY, and HMBC spectra analysis led to the same correlations as in compound 8 and enabled the assignment of the 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).
The absolute configuration of the stereocenter C-8 for anomoian C-F (69) can be proposed by comparison of the values and signs of their optical rotations in MeOH ([α] D 25 +9°, +11°, +7° and +6°, respectively), with the closely related congeners, anomoian A ([α]D +5.1°) [26], anomoian B ([α]D +10.5°) [27], iso-Anomoian A ([α] D 20 +4.5°) [28], suberedamines A ([α] D 25 +21°) and B ([α] D 25 +16°) [9], indicating that all are bearing l-tyrosine residue. The synthetic iso-Anomoian A ([α] D 20 +4.5°) and suberedamines A ([α] D 20 +19.5°) allowed the determination of their absolute configurations [28].

2.2. Biological Activities

The isolated compounds 13 and 611 were tested for their cytotoxic activity against human 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 (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 point of view, both motifs (N,N-dimethylaminopropyl and trans-cinnamoyl) seem to be together correlated to the activity of psammaplysene D (1).
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 Vmax 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).
Psammaplysene D (1) displayed clearly behavioral effects on both guppies and reef fish larvae: they showed a loss of appetite from the first day of experiment. Reef fish juveniles’ behavior did not seem to be impacted by psammaplysene D. It is interesting to note that only A. triostegus larvae were impacted by the pure psammaplysene D, suggesting a change of sensibility towards the compound during the metamorphosis. The toxicity test at a high dose (100 μg/mL of crude extract or purified psammaplysene D) on the guppies led to their death after one hour bathing with either the crude extract or the purified psammaplysene D fraction (acute toxicity experiment). Brains and gills of these guppies were extracted and the AChE activity measured on the extracts (Figure 7a,b) [29]. The results were clearer with the purified psammaplysene D, showing a mild but significant decrease of the activity of the enzyme in the gills, suggesting an action on the olfactory organs (peripheral nervous system) that could not be further investigated there. A great number of AChE inhibitors targeting Alzheimer’s disease have been isolated from natural sources [30]. Among them, 6 compounds with activities ranging from IC50 1 to 100 μM were isolated from Verongiida sponges (Table 6). It would be interesting also to further investigate those compounds as chemical anti-predatory defenses through fish antifeedant activity, since the fish model is easier to set up than related bioassays involving sponge predators, such as nudibranchs or crustaceans.
A common side effect of AChE inhibition treatments is anorexia [34,35], which strengthens the importance of natural AChE inhibitors as potential deterrents of predators such as fishes. Furthermore, the olfactory dysfunction in Alzheimer’s disease has also been demonstrated [36]. Psammaplysene D is the major compound of the sponge (0.66% w/w). It has been shown that tyrosine derived compounds are located in sphaerus cells of the sponge, allowing their release in the aquifer channels and further in the close environment of the sponge [37]. It would be thus interesting to track and quantify the concentration of this compound in the close reef environmental water of the sponges, using for example, solid phase adsorption toxin tracking (SPATT) [38], to allow the on field deterrent or reef protective activity of this compound acting softly on olfactory sense of predators in the marine environment. The importance of AChE inhibitors 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 between ecology and evolution, secondary metabolites, behavior, and neurosciences [39].

3. Material and Methods

3.1. General Procedures

Optical rotations were measured using MCP-300 polarimeter (Anton Paar, Les Ulis, France). IR spectra 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, France). The chemical shifts are relative to the residual signal solvent (CD3OD: 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 (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°55′977′ S–140°01′17′ 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 CH2Cl2/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 CH2Cl2 and H2O. 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 CH2Cl2 extract was then subjected to normal phase silica-gel MPLC. A gradient composed of CH2Cl2/MeOH (1:0 to 0:1), followed by a mixture of EtOAc/Acetone/H2O/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, H2O + 0.1% formic acid/CH3CN + 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, H2O + 0.1% formic acid/CH3CN + 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; 1H and 13C NMR data, Supporting Materials; HRESIMS m/z 783.9673 [M + H]+ (calcd for C28H38N3O3Br4, 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; 1H and 13C NMR data, Table 1; HRESIMS m/z 698.8675 [M + H]+ (calcd for C23H27N2O379Br281Br2, 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; 1H and 13C NMR data, Table 1; HRESIMS m/z 698.8723 [M + H]+ (calcd for C23H27N2O379Br281Br2, 698.8714).
Psammaplysene H (4)/Psammaplysene I (5) mixture: pale yellow oil, (0.7 mg); 1H and 13C NMR data, Table 2; HRESIMS m/z 769.9521 [M + H]+ (calcd. for C27H3679Br2 81Br2N3O3, 769.9449)/HRESIMS m/z 755.9309 [M + H]+ (calcd. for C26H3479Br2 81Br2N3O3, 755.9293).
Anomoian C (6): colorless oil, (1.9 mg); [α] D 25 +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; 1H and 13C NMR data, Table 3; HRESIMS m/z 743.9386 [M + H]+ (calcd for C25H34N3O379Br281Br2, 743.9293).
Anomoian D (7): pale yellow oil, (1.2 mg); [α] D 25 +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; 1H and 13C NMR data, Table 3; HRESIMS m/z 729.9190 [M + H]+ (calcd for C24H32N3O379Br281Br2, 729.9136).
Anomoian E (8): colorless oil, (3.8 mg); [α] D 25 +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; 1H and 13C NMR data, Table 4; HRESIMS m/z 815.0068 [M + H]+ (calcd for C29H43N4O379Br281Br2, 815.0028).
Anomoian F (9): yellow oil, (3.8 mg); [α] D 25 +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; 1H and 13C NMR data, Table 4; HRESIMS m/z 829.0259 [M + H]+ (calcd for C30H45N4O379Br281Br2, 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; 1H and 13C NMR data, Supplementary Materials; HRESIMS m/z 323.9388 [M + H]+ (calcd for C10H14NO79Br81Br, 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; 1H and 13C NMR data, Supplementary Materials; HRESIMS m/z 222.0441 [M + H]+ (calcd for C10H8NO5, 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; 1H and 13C NMR data, Supplementary Materials; HRESIMS m/z 206.0511 [M + H]+ (calcd for C10H8NO4, 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 H2O 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. 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, 1H NMR, 13C NMR, COSY, HSQC and HMBC) for compounds 112.

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 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.

References

  1. Rahboek, L.; Christophersen, C. The Alkaloids: Chemistry and Biology; Academic Press: Cambridge, MA, USA, 2001; Volume 57, p. 185. [Google Scholar]
  2. Nuñez, C.V.; Almeida, E.V.R.; Granato, A.C.; Marques, S.O.; Santos, K.O.; Pereira, F.R.; Macedo, M.L.; Ferreira, A.G.; Hajdu, E.; Pinheiro, U.S.; et al. Chemical variability within the marine sponge Aplysina fulva. Biochem. Syst. Ecol. 2008, 36, 283–296. [Google Scholar] [CrossRef]
  3. Puyana, M.; Pawlik, J.; Blum, J.; Fenical, W. Metabolite variability in Caribbean sponges of the genus Aplysina. Rev. Bras. Farmacogn. 2015, 25, 592–599. [Google Scholar] [CrossRef]
  4. König, G.M.; Wright, A.D. Agelorins A and B, and 11-Epi-fistularin-3, Three New Antibacterial Fistularin-3 Derivatives from the Tropical Marine Sponge Agelas oroides. Heterocycles 1993, 36, 1351–1358. [Google Scholar] [CrossRef]
  5. Nicacio, K.J.; Ióca, L.P.; Fróes, A.M.; Leomil, L.; Appolinario, L.R.; Thompson, C.C.; Thompson, F.L.; Ferreira, A.G.; Williams, D.E.; Andersen, R.J.; et al. Cultures of the Marine Bacterium Pseudovibrio denitrificans Ab134 Produce Bromotyrosine-Derived Alkaloids Previously Only Isolated from Marine Sponges. J. Nat. Prod. 2017, 80, 235–240. [Google Scholar] [CrossRef] [PubMed]
  6. Erpenbeck, D.; Van Soest, R.W.M. Status and Perspective of Sponge Chemosystematics. Mar. Biotechnol. 2007, 9, 2–19. [Google Scholar] [CrossRef] [PubMed]
  7. Shaker, K.H.; Zinecker, H.; Ghani, M.A.; Imhoff, J.F.; Schneider, B. Bioactive metabolites from the sponge Suberea sp. Chem. Biodivers. 2010, 7, 2880–2887. [Google Scholar] [CrossRef] [PubMed]
  8. Hirano, K.; Kubota, T.; Tsuda, M.; Watanabe, K.; Fromont, J.; Kobayashi, J. Ma’edamines A and B, cytotoxic bromotyrosine alkaloids with a unique 2 (1H) pyrazinone ring from Sponge Suberea sp. Tetrahedron 2002, 56, 8107–8110. [Google Scholar] [CrossRef]
  9. Tsuda, M.; Sakuma, Y.; Kobayashi, J. Suberedamines A and B, new bromotyrosine alkaloids from a sponge Suberea species. J. Nat. Prod. 2001, 64, 950–982. [Google Scholar] [CrossRef]
  10. Kijjoa, A.; Watanadilok, R.; Sonchaeng, P.; Puchakarn, S.; Sawangwong, P.; Herz, W. Bromotyrosine derivatives from the marine sponge Suberea aff. praetensa. Boll. Mus. Ist. Biol. Univ. Genova 2004, 68, 391–397. [Google Scholar]
  11. Florean, C.; Schnekenburger, M.; Lee, J.; Kim, K.R.; Mazumder, A.; Song, S.; Kim, J.; Grandjenette, C.; Kim, J.; Yoon, A.; et al. Discovery and characterization of Isofistularin-3, a marine brominated alkaloid, as a new DNA demethylating agent inducing cell cycle arrest and sensitization to TRAIL in cancer cells. Oncotarget 2016, 7, 24027–24049. [Google Scholar] [CrossRef] [PubMed]
  12. Shaala, L.A.; Youssef, D.T.A.; Badr, J.M.; Sulaiman, M.; Kherd, A. Bioactive secondary metabolites from the Red Sea marine Verongid sponge Suberea species. Mar. Drugs 2015, 13, 1621–1631. [Google Scholar] [CrossRef] [PubMed]
  13. Shaala, L.A.; Khalifa, S.I.; Mesbah, M.K.; van Soest, R.W.M.; Youssef, D.T.A. Subereaphenol A, a new cytotoxic and antimicrobial dibrominated phenol from the Red Sea sponge Suberea mollis. Nat. Prod. Commun. 2008, 3, 219–222. [Google Scholar] [CrossRef]
  14. Abou-Ashour, M.I.; Shaala, L.A.; Youssef, D.T.A.; Bader, J.M.; Habib, A.M. Bioactive brominated metabolites from the Red Sea sponge Suberea mollis. J. Nat. Prod. 2008, 71, 1464–1467. [Google Scholar] [CrossRef] [PubMed]
  15. Shaala, L.A.; Bamane, F.H.; Badr, J.M.; Youssef, D.T.A. Brominated arginine-derived alkaloids from the Red Sea sponge Suberea mollis. J. Nat. Prod. 2001, 74, 1517–1520. [Google Scholar] [CrossRef] [PubMed]
  16. Xu, M.; Davis, R.A.; Feng, Y.; Sykes, M.L.; Shelper, T.; Avery, V.M.; Camp, D.; Quinn, R.J. Ianthelliformisamines A-C, antibacterial bromotyrosine-derived metabolites from the marine sponge Suberea ianthelliformis. J. Nat. Prod. 2012, 75, 1001–1005. [Google Scholar] [CrossRef] [PubMed]
  17. Mani, L.; Jullian, V.; Mourkazel, B.; Valentin, A.; Dubois, J.; Cresteil, T.; Folcher, E.; Hooper, J.N.A.; Erpenbeck, D.; Aalbersberg, W.; et al. New antiplasmodial bromotyrosine derivatives from Suberea ianthelliformis (Lendenfeld, 1888). Chem. Biodivers. 2012, 9, 1436–1451. [Google Scholar] [CrossRef] [PubMed]
  18. El-Demerdash, A.; Moriou, C.; Martin, M.T.; Rodrigues-Stien, A.; Petek, S.; Demoy-Schnider, M.; Hall, K.; Hooper, J.N.A.; Debitus, C.; Al-Mourabit, A. Cytotoxic guanidine alkaloids from a French Polynesian Monanchora n. sp. sponge. J. Nat. Prod. 2016, 79, 1929–1937. [Google Scholar] [CrossRef] [PubMed]
  19. Kashman, Y.; Groweiss, A.; Shmueli, U. Latrunculin, A new 2-thiazolidinone macrolide from the marine Sponge Latrunculia magnifica. Tetrahedron Lett. 1980, 21, 3629–3632. [Google Scholar] [CrossRef]
  20. Neeman, I.; Fishelson, L.; Kashman, Y. Isolation of a new toxin from the sponge Latrunculia magnifica in the Gulf of Aquaba (Red Sea). Mar. Biol. 1975, 30, 293–296. [Google Scholar] [CrossRef]
  21. Buchanan, M.S.; Carroll, A.R.; Addepalli, R.; Avery, V.M.; Hooper, J.N.A.; Quinn, R.J. Psammaplysenes C and D, Cytotoxic Alkaloids from Psammoclemma sp. J. Nat. Prod. 2007, 70, 1827–1829. [Google Scholar] [CrossRef] [PubMed]
  22. Yin, L.W.; Feng, Y.; Shimizu, Y.; Pfeifer, T.; Wellington, C.; Hooper, J.N.A.; Quinn, R. Aplysinellamides A–C, Bromotyrosine-Derived Metabolites from an Australian Aplysinella sp. Marine Sponge. J. Nat. Prod. 2014, 77, 1210–1214. [Google Scholar] [CrossRef]
  23. Molinski, T.F.; Faulkner, D.J. An antibacterial pigment from the sponge Dendrilla membranosa. Tetrahedron Lett. 1988, 29, 2137–2138. [Google Scholar] [CrossRef]
  24. Spectral Data for Xanthurenic Acid Are Available on Line at the Spectral Database for Organic Compounds. Available online: http://sdbs.db.aist.go.jp/sdbs/cgi-bin/cre_index.cgi (accessed on 26 April 2018).
  25. Schroeder, F.C.; Kau, T.R.; Silver, P.A.; Clardy, J. The Psammaplysenes, Specific Inhibitors of FOXO1a Nuclear Export. J. Nat. Prod. 2005, 68, 574–576. [Google Scholar] [CrossRef] [PubMed]
  26. Kernan, M.R.; Cambie, R.C.; Bergquist, P.R. Chemistry of Sponges, VIII. Anomoian A, a Bromotyrosine Derivative from Anomoiantbella popeae. J. Nat. Prod. 1990, 53, 720–723. [Google Scholar] [CrossRef]
  27. Tarazone, G.; Santamaria, G.; Cruz, P.G.; Ferandez, R.; Pérez, M.; Martinez-Leal, J.F.; Rodriguez, J.; Jiménez, C.; Cuevas, C. Cytotoxic Anomoian B and Aplyzanzine B, New Bromotyrosine Alkaloids from Indonesian Sponges. ACS Omega 2017, 2, 3494–3501. [Google Scholar] [CrossRef]
  28. Kottakota, S.K.; Evangelopoulos, D.; Alnimr, A.; Bhakta, S.; McHugh, T.D.; Gray, G.; Groundwater, P.W.; Marrs, E.C.L.; Perry, J.D.; Spilling, C.D.; et al. Synthesis and Biological Evaluation of Purpurealidin E-Derived Marine Sponge Metabolites: Aplysamine-2, Aplyzanzine A, and Suberedamines A and B. J. Nat. Prod. 2012, 75, 1090–1101. [Google Scholar] [CrossRef] [PubMed]
  29. Bocquené, G.; Galgani, F. Biological Effects of Contaminants: Cholinesterase Inhibition by Organophosphate and Carbamate Compounds; ICES Techniques in Marine Environmental Sciences, No. 22; International Council for the Exploration of the Sea: Copenhagen, Denmark, 1998. [Google Scholar]
  30. Silva, T.; Reis, J.; Teixeira, J.; Borges, F. Alzheimer’s disease, enzyme targets and drug discovery struggles: From natural products to drug prototypes. Ageing Res. Rev. 2014, 15, 116–145. [Google Scholar] [CrossRef] [PubMed]
  31. Olatunji, O.J.; Ogundajo, A.L.; Oladosu, I.A.; Changwichit, K.; Ingkanina, K.; Yuenyongsawad, S.; Plubrukarn, A. Non-competitive inhibition of acetylcholinesterase by bromotyrosine alkaloids. Nat. Prod. Commun. 2014, 9, 1559–1561. [Google Scholar] [PubMed]
  32. Sirimangkalakitti, N.; Olatunji, O.J.; Changwichit, K.; Saesong, T.; Chamni, S.; Chanvorachote, P.; Ingkaninan, K.; Plubrukarn, A.; Suwanborirux, K. Bromotyrosine Alkaloids with Acetylcholinesterase activity from the Thai sponge Aconthodendrilla sp. Nat. Prod. Commun. 2015, 10, 1945–1949. [Google Scholar] [PubMed]
  33. Sepcic, K.; Mancini, I.; Vidic, I.; Jovan, U.; Frassanito, R.; Pietra, F.; Macek, P.; Turk, T. Antibacterial and anticholinesterase activities of aplysamine-4, a bromotyrosine-derived metabolite of a Red Sea marine sponge. J. Nat. Toxins 2001, 10, 181–191. [Google Scholar] [PubMed]
  34. Schneider, S.L. A critical review of cholinesterase inhibitors as a treatment modality in Alzheimer’s disease. Dialogues Clin. Neurosci. 2000, 2, 111–128. [Google Scholar]
  35. Aagaard, L. Chapter 1-Central nervous system stimulants and drugs that suppress appetite. Side Effects Drugs Annu. 2014, 35, 1–25. [Google Scholar]
  36. Zou, Y.; Lu, D.; Liu, L.; Zhang, H.; Zhou, Y. Olfactory dysfunction in Alzheimer’s disease. Neuropsychiatr. Dis. Treat. 2016, 12, 869–875. [Google Scholar] [CrossRef] [PubMed]
  37. Thompson, J.E.; Barrow, K.D.; Faulkner, D.J. Localization of Two Brominated Metabolites, Aerothionin and Homoaerothinin, in Spherulous cells of the Marine Sponge Aplysina fistularis (=Verongia thiona). Acta Zool. 1983, 64, 199–210. [Google Scholar] [CrossRef]
  38. Lane, J.Q.; Roddam, C.M.; Langlois, G.W.; Kudela, R.M. Application of Solid Phase Adsorption Toxin Tracking (SPATT) for field detection of the hydrophilic phycotoxins domoic acid and saxitoxin in coastal California. Limnol. Oceanogr. Methods 2010, 8, 645–660. [Google Scholar] [CrossRef]
  39. Derby, C.D.; Aggio, J.F. The Neuroecology of Chemical Defenses. Integr. Comp. Biol. 2011, 51, 771–780. [Google Scholar] [CrossRef] [PubMed]
  40. Debitus, C. BSMPF-1 Cruise, RV Alis, 2009. Available online: http://dx.doi.org/10.17600/9100030 (accessed on 26 April 2018).
  41. Ellman, G.L.; Courtney, D.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [Google Scholar] [CrossRef]
  42. Lecchini, D.; Dufour, V.; Carleton, J.; Strand, S.; Galzin, R. Estimating the Patch size of larval fishes during colonization on coral reefs. J. Fish Biol. 2004, 65, 1142–1146. [Google Scholar] [CrossRef]
  43. Georgiades, S.N.; Clardy, J. Total Synthesis of Psammaplysenes A and B, Naturally Occurring Inhibitors of FOXO1a Nuclear Export. Org. Lett. 2005, 7, 4091–4094. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Structures of isolated compounds 112.
Figure 1. Structures of isolated compounds 112.
Marinedrugs 16 00146 g001
Figure 2. COSY and key HMBC correlations for psammaplysenes F (2) and G (3).
Figure 2. COSY and key HMBC correlations for psammaplysenes F (2) and G (3).
Marinedrugs 16 00146 g002
Figure 3. COSY and key HMBC correlations for psammaplysenes H (4) and I (5).
Figure 3. COSY and key HMBC correlations for psammaplysenes H (4) and I (5).
Marinedrugs 16 00146 g003
Figure 4. COSY and key HMBC correlations for anomoians C (6) and D (7).
Figure 4. COSY and key HMBC correlations for anomoians C (6) and D (7).
Marinedrugs 16 00146 g004
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).
Marinedrugs 16 00146 g005
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.
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.
Marinedrugs 16 00146 g006
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 5 µg/mL of crude extract. Boxplot distribution was made with 5 fish (N = 5).
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 5 µg/mL of crude extract. Boxplot distribution was made with 5 fish (N = 5).
Marinedrugs 16 00146 g007aMarinedrugs 16 00146 g007b
Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Psammaplysenes F (2) and G (3) in CD3OD.
Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Psammaplysenes F (2) and G (3) in CD3OD.
PositionPsammaplysene F (2)Psammaplysene G (3)
No.δC, Type aδH mult, (J in Hz) aHMBCδC, Type aδH mult, (J in Hz) aHMBC
1154.10, C--153.29/153.04, C--
2112.80, C--112.49, C--
3133.09/133.01, CH7.78/7.70, s1, 2, 5, 6, 7133.70/133.42, CH7.53/7.54, s1, 2, 5, 6, 7
4130.98, C--130.70/131.16, C--
5133.09/133.01, CH7.78/7.70, s1, 2, 3, 6, 7133.70/133.42, CH7.53/7.54, s1, 2, 3, 6, 7
6112.80, C--112.49, C--
7140.89/141.19, CH7.36–7.39/7.35–7.38, d (15.5)3, 4, 5, 9132.24/132.49, CH6.49–6.52/6.56–6.58, d (12.5)3, 4, 5, 9
8118.90/118.72, CH7.11–7.15/6.99–7.02, d (15.5)4, 7, 9123.90/124.59, CH6.13–6.16/6.09–6.12, d (12.5)4, 7, 9
9168.96/168.80, C--171.13/171.01, C--
1048.20/47.14, CH23.89/3.74, t (7.2)9, 11, 12, 2148.97/45.75, CH23.65/3.73, t (7.5)9, 11, 12, 21
1130.86/29.16, CH22.22/2.16, m10, 1229.32/28.68, CH22.02/2.18, m10,12
1271.94/72.64, CH24.11/4.05, t (6.4)10, 11, 1372.03/72.32, CH23.78/4.04, t (6.5)10, 11, 13
13153.88/153.94, C--153.55/153.86, C--
14119.57, C--119.36/119.59, C--
15134.63/134.56, CH7.57, br s13, 14, 17, 18, 19134.51/134.57, CH7.51/7.56, s13, 14, 17, 18, 19
16136.81/136.99, C--136.85, C--
17134.63/134.56, CH7.57, br s13, 14, 15, 18, 19134.51/134.57, CH7.51/7.56, s13, 14, 15, 18, 19
18119.57, C--119.36/119.59, C--
1930.45, CH23.00, m15, 16, 17, 20, 22, 2330.51/30.56, CH23.00, m15, 16, 17, 20, 22, 23
2059.24, CH23.30, m16, 19, 22, 2359.32/59.24, CH23.34, m16, 19, 22, 23
2134.74/36.55, CH33.09/3.28, s9, 10 32.59/36.67, CH33.06/3.03, s9, 10
2243.62, CH32.91, s20, 2343.70/43.73, CH32.91, s20, 23
2343.62, CH32.91, s20, 2243.70/43.73, CH32.91, s20, 22
a Chemical shifts are given for both rotamers when distinguishable signals were identified.
Table 2. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Psammaplysenes H (4) and I (5) in CD3OD.
Table 2. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Psammaplysenes H (4) and I (5) in CD3OD.
PositionPsammaplysene H (4)Psammaplysene I (5)
NoδC, Type aδH mult, (J in Hz) aHMBCδC, Type aδH mult, (J in Hz) aHMBC
1154.7, C--154.7, C--
2119.6, C--119.6, C--
3133.5/133.4, CH7.92/7.85, d1, 2, 5, 6, 7133.5/133.4, CH7.92/7.85, d1, 2, 5, 6, 7
4136.2, C--136.2, C--
5133.5/133.4, CH7.92/7.85, d1, 2, 3, 6, 7133.5/133.4, CH7.92/7.85, d1, 2, 3, 6, 7
6119.6, C--119.6, C--
7140.2/140.0, CH7.40–7.44, m3, 4, 5, 9140.2/140.0, CH7.40–7.44, m3, 4, 5, 9
8121.6/121.7, CH7.14–7.27, dd (15.0)4, 7, 9121.6/121.7, CH7.14–7.27, dd (15.0)4, 7, 9
9168.5, C a--168.5, C a--
1048.4/47.3, CH23.91/3.76, t (7.0)9, 11, 12, 2148.4/47.3, CH23.91/3.76, t (7.0)9, 11, 12, 21
1130.6/29.1, CH22.23/2.17, m10, 1230.6/29.1, CH22.23/2.17, m10, 12
1272.6/71.9, CH24.12/4.07, m10, 11, 1372.6/71.9, CH24.12/4.07, m10, 11, 13
13154.7, C--154.7, C--
14119.6, C--119.6, C--
15134.5, CH7.56/7.53, m13, 14, 17, 18, 19134.5, CH7.56/7.53, m13, 14, 17, 18, 19
16137.8, C b--137.8, C b--
17134.5, CH7.56/7.53, m13, 14, 15, 18, 19134.5, CH7.56/7.53, m13, 14, 15, 18, 19
18119.6, C--119.6, C--
1932.2, CH22.93, t (8.0)16, 20, 2232.2, CH22.93, t (8.0)16, 20, 22
2051.1, CH23.21, m16, 19, 2251.1, CH23.21, m16, 19, 22
2134.7/36.6, CH33.10/3.29, s9, 1034.7/36.6, CH33.10/3.29, s9, 10
2234.0, CH32.70, s2034.0, CH32.70, s20
1′57.2, CH23.27, m1, 2′, 3′48.7, CH23.35, m1, 2′, 3′
2′27.6, CH22.23, m1′, 3′30.9 CH22.23, m1′, 3′
3′71.9, CH24.18, m1′, 2′, 4′, 5′71.9, CH24.18, m1′, 2′, 4′
4′44.3, CH32.78/2.74, s3′, 5′34.0, CH32.77/2.76, s3′
5′44.3, CH32.79/2.74, s3′, 4′---
a Chemical shifts are given for both rotamers when distinguishable signals were identified; b Detected by HMBC correlations.
Table 3. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Anomoians C (6) and D (7) in CD3OD.
Table 3. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Anomoians C (6) and D (7) in CD3OD.
PositionAnomoian C (6)Anomoian D (7)
NoδC, Type aδH mult, (J in Hz) aHMBCδC, Type aδH mult, (J in Hz) aHMBC
1151.65/151.47, C--152.7, C--
2112.36, C--112.69, C--
3134.47/134.41, CH7.34/7.32, s1, 2, 4, 5, 6, 7134.40, CH7.37/7.35, s1, 2, 4, 5, 6, 7
4132.77/133.49, C--130.53/129.90, C--
5134.47/134.41, CH7.34/7.32, s1, 2, 3, 4, 6, 7134.40, CH7.37/7.35, s1, 2, 3, 4, 6, 7
6112.36, C--112.69, C--
733.14/32.14, CH22.90/2.83–2.99, m3, 4, 5, 8, 937.40/38.02, CH22.83–3.04/2.92, m3, 4, 5, 8, 9
866.86, CH3.82, m7, 9, 24, 2560.79/61.29, CH4.26/4.17, m7, 9, 24
9172.36/172.63, C--171.40/172.00, C--
1047.14/48.24, CH23.40–3.72, m9, 11, 2147.67/47.78, CH23.48–3.80/3.26–3.51, m9, 11, 21
1129.09/30.45, CH21.92–2.00/1.73–1.92, m10, 1229.15/30.50, CH21.95–2.05/1.95, m10, 12
1272.31/71.72, CH23.85–3.93/3.88, m11, 1372.35/71.61, CH23.97/3.99, m11, 13
13153.61/153.30, C--153.72, C--
14119.43, C--119.47, C--
15134.47/134.50, CH7.52/7.53, s13, 14, 16, 17, 18, 19134.51/134.53, CH7.54/7.55, s13, 14, 16, 17, 18, 19
16137.97/138.24, C--137.72, C--
17134.47/134.50, CH7.52/7.53, s13, 14, 15, 16, 18, 19134.51/134.53, CH7.54/7.55, s13, 14, 15, 16, 18, 19
18119.43, C--119.47, C--
1931.40/31.46, CH22.92, m15, 16, 17, 2031.22, CH22.93, m15, 16, 17, 20
2060.12/60.14, CH23.08, m16, 19, 22, 2359.96, CH23.21, m16, 19, 22, 23
2136.64/34.26, CH32.92/2.93, s9, 1036.25/34.29, CH32.50/2.47, s9, 10
2244.30/44.34, CH32.72/2.71, s20, 2344.18/44.21, CH32.74/2.74, s20, 23
2344.30/44.34, CH32.72/2.71, s20, 2244.18/44.21, CH32.74/2.74, s20, 22
2442.31/41.19, CH32.45/2.42, s8, 2533.26/33.68, CH32.82/3.00, s8
2542.31/41.19, CH32.45/2.42, s8, 24---
aChemical shifts are given for both rotamers when distinguishable signals were identified.
Table 4. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Anomoians E (8) and F (9) in CD3OD.
Table 4. 1H NMR (500 MHz) and 13C NMR (125 MHz) data for Anomoians E (8) and F (9) in CD3OD.
PositionAnomoian E (8)Anomoian F (9)
NoδC, Type aδH mult, (J in Hz) aHMBCδC, Type aδH mult, (J in Hz) aHMBC
1153.29/153.15, C--152.76/152.57, C--
2119.08.119.14, C--118.92, C--
3135.35/135.19, CH7.51/7.48, s1, 2, 5, 6, 7135.32/135.22, CH7.51/7.48, s1, 2, 5, 6, 7
4137.14/138.15, C--138.66/137.72, C--
5135.35/135.19, CH7.51/7.48, s1, 2, 3, 6, 7135.32/135.22, CH7.51/7.48, s1, 2, 3, 6, 7
6119.08.119.14, C--118.92, C--
738.03/38.65, CH22.84–3.03/2.93, m3, 4, 5, 8, 933.05/31.91, CH23.00/2.92–3.07, m3, 4, 5, 8, 9
860.72/61.23, CH4.15/4.06, m4, 7, 9, 2466.58/66.79, CH3.97/3.89, m4, 7, 9, 24, 25
9172.73/173.46, C--172.10/171.65, C--
1047.57/47.82, CH23.51–3.76/3.26–3.60, m9, 11, 12, 2148.26/47.36, CH23.41–3.74/3.51–3.64, m9, 11, 12, 21
1129.16/30.64, CH21.95–2.03/1.95, m10, 1230.71/29.08, CH21.69–1.98/1.90–1.98, m10, 12
1272.36/71.71, CH23.99, m10, 11, 1371.82/72.40, CH23.94/3.95, m10, 11, 13
13153.54, C--153.77/153.51, C--
14119.42, C--119.52, C--
15134.54/134.52, CH7.55/7.56, s13, 14, 17, 18, 19134.57/134.60, CH7.57/7.58, s13, 14, 17, 18, 19
16137.74/138.52, C--137.28, C--
17134.54/134.52, CH7.55/7.56, s13, 14, 15, 18, 19134.57/134.60, CH7.57/7.58, s13, 14, 15, 18, 19
18119.42, C--119.52, C--
1931.31/31.41, CH22.93, m15, 16, 17, 2030.67/30.59, CH22.99, m15, 16, 17, 20
2060.01/60.08, CH23.09, m16, 19, 22, 2359.43, CH23.26, m16, 19, 22, 23
2136.24/34.33, CH32.88/2.99, s9, 1034.31/36.64, CH32.92/2.94, s9, 10
2244.19/44.24, CH32.73/2.71, s20, 2343.75, CH32.85/2.85, s20, 23
2344.19/44.24, CH32.73/2.71, s20, 2243.75, CH32.85/2.85, s20, 22
2433.71/34.11, CH32.42/2.39, s4, 842.23/42.07, CH32.51/2.45, s4, 8, 25
25---42.23/42.07, CH32.51/2.45, s4, 8, 24
1′71.66/71.62, CH24.09, m1, 2′, 3′ 71.44, CH24.01, t (5.5)1, 2′, 3′
2′26.95, CH22.25, m1′, 3′26.62, CH22.27, m1′, 3′
3′57.15, CH23.36, m1′, 2′, 4′, 5′57.07, CH23.45, m1′, 2′, 4′, 5′
4′44.05, CH32.85, s3′, 5′43.79, CH32.92/2.91, s3′, 5′
5′44.05, CH32.85, s3′, 4′43.79, CH32.92/2.91, s3′, 4′
a Chemical shifts are given for both rotamers when distinguishable signals were identified.
Table 5. Cytotoxic activities in vitro (KB Cell Line) for compounds 13, 611.
Table 5. Cytotoxic activities in vitro (KB Cell Line) for compounds 13, 611.
Compounds10 μM a1 μM a
Psammaplysene D (1)100 ± 0.295 ± 0.5
Psammaplysene F (2)73 ± 220 ± 0.2
Psammaplysene G (3)75 ± 0.417 ± 1
Anomoian C (6)28 ± 515 ± 3
Anomoian D (7)29 ± 217 ± 2
Anomoian E (8)82 ± 16 ± 2
Anomoian F (9)100 ± 0.520 ± 0.6
N,N-dimethyldibromotyramine (10)100 ± 189 ± 1
5-hydroxy xanthenuric acid (11)25 ± 16 ± 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).
Table 6. AChE inhibition of bromotyrosine compounds isolated from Verongiida sponges.
Table 6. AChE inhibition of bromotyrosine compounds isolated from Verongiida sponges.
Sponge/CompoundsAChE Origin ActivitiesRef.
Pseudoceratina purpurea
Purealidine Q
Isoanomoian A
Aplysanzine A
Eel
IC50 = 1.2 µM (NCI a)
IC50 = 70 µM (NCI)
IC50 = 104 µM (NCI)
Olatunji et al. 2014 [31]
Acanthodendrilla sp.
Homoaerothionin
Fistularin 1
Human recombinant AchE
IC50 = 4.5 µM (CI b)
IC50 = 47.5 µM (ND c)
Sirimangkalakitti et al. 2015 [32]
Unidentified Verongida Aplysamine-4Eel
KI = 16 µM (NCI)
Insect recombinant
KI = 2 µM (NCI)
Sepcic et al. 2001 [33]
a NCI: non competitive inhibition; b CI: competitive inhibition; c ND: non described.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Mar. Drugs EISSN 1660-3397 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top