A Collection of Bioactive Nitrogen-Containing Molecules from the Marine Sponge Acanthostrongylophora ingens

Thirteen nitrogen-containing molecules (1a/1b and 2–12) were isolated from the Indonesian sponge Acanthostrongylophora ingens, highlighting the richness of this organism as a source of alkaloids. Their structures were elucidated using one- and two-dimensional NMR spectroscopy and HR-ESI-MS, while the stereochemistry of the diketopiperazines was established using Marfey’s method. All compounds were screened in our standard bioactivity assays, including antibacterial, antikinases, and amyloid β-42 assays. The most interesting bioactivity result was obtained with the known acanthocyclamine A (3), which revealed for the first time a specific Escherichia coli antimicrobial activity and an inhibitory effect on amyloid β-42 production induced by aftin-5 and no cytotoxicity at the dose of 26 µM. These results highlight the potentiality of a bipiperidine scaffold as a promising skeleton for preventing or reducing the production of amyloid β-42, a key player in the initiation of Alzheimer’s disease.


Introduction
Over the last thirty years, research programs focusing on the chemical study of the metagenomic content of marine sponges have led to a plethora of novel molecules, often showing innovative skeletons, and being used as lead compounds in the search for novel therapeutic approaches [1][2][3][4].
In the framework of our research program, named BlueGenics (https://cordis.europa.eu/result/rcn/ 193365_en.html), devoting to the sustainable exploitation of bioactive marine compounds, the chemical composition of the dichloromethane extract of the marine sponge Acanthostrongylophora ingens, collected off the coast of South Sulawesi, Indonesia, was analyzed, and chloromethylhalicyclamine B (5), a novel selective CK1δ/ε kinase inhibitor with an IC 50 value of 6 µM, was described [5]. An in-depth re-examination of the organic extracts of A. ingens revealed, in addition to halicyclamine B (4) and chloromethylhalicyclamine B (5), small amounts of tetradehydrohalicyclamine B (1b) and its epimer, a new dehydrohalicyclamine derivative named epi-tetradehydrohalicyclamine B (1a) as well as its chloromethyl derivative (2), acanthocyclamine A (3), and seven diketopiperazines (DKPs) (6)(7)(8)(9)(10)(11)(12). Tetradehydrohalicyclamine B (1b) was recently reported from the same Indonesian A. ingens by a ported from the same Indonesian A. ingens by a Japanese group [6]. They investigated the proteasome inhibitor activity of tetradehydrohalicyclamine B (1b) and halicyclamine B (4), showing that both have proteasome inhibitor activity at micromolar concentration, 4 being more potent than 1b, suggesting their possible role as anticancer lead compounds [7].
Our screening program includes antibacterial, antikinases, and amyloid β-42 assays. The obtained data are reported here, together with structural elucidation of the new alkaloid epi-tetradehydrohalicyclamine B (1a) found in the sponge extract.

Epi-Tetradehydrohalicyclamine B (1a) and Tetradehydrohalicyclamine B (1b)
Compounds 1a and 1b could not be obtained in pure form, but only as mixtures enriched in either 1a or 1b. These mixtures, allowing a clear distinction of the 1 H and 13 C signals of either stereoisomer, could be successfully used for structure elucidation. Compound 1a showed the same molecular formula C26H39N2 + as the recently reported [6] tetradehydrohalicyclamine B (1b), accounting for 9 degrees of unsaturation, as deduced by the [

Epi-Tetradehydrohalicyclamine B (1a) and Tetradehydrohalicyclamine B (1b)
Compounds 1a and 1b could not be obtained in pure form, but only as mixtures enriched in either 1a or 1b. These mixtures, allowing a clear distinction of the 1 H and 13 C signals of either stereoisomer, could be successfully used for structure elucidation. Compound 1a showed the same molecular formula C 26 H 39 N 2 + as the recently reported [6] tetradehydrohalicyclamine B (1b), accounting for 9 degrees of unsaturation, as deduced by the [M] + peak at m/z 379.3132 and the doubly charged [M + H] 2+ peak at m/z 190.1596 in the HR-ESIMS. Analysis of the one-and two-dimensional (COSY, TOCSY, ROESY, HSQC, and HMBC) NMR spectra of 1a allowed the full assignment of all the 1 H and 13 C signals of both compounds 1a and 1b, and showed for 1a the same planar structure as 1b ( Figure 2). Comparison of 1 H NMR chemical shifts and coupling constants of compound 1a (Table 1) with those of compound 1b (Table S1) highlighted some remarkable differences, clearly implying a stereochemical difference between 1a and 1b. This could be linked not only to a different configuration at C-14 or at C-15, but also to atropisomerism (conformers separated by a high-energy barrier) of the same configurational stereoisomer, which is reasonable in the strained tetracyclic structure of 1a/1b, and has been observed in other natural products [9]. The most notable feature of the 1  conformational rigidity of the C-6/C-13 carbon chain (containing the shielded protons H-8a and H-13a), which showed the same conformation in all the 16 lowest-energy conformers, accounting for over 96% of population. In this conformation, H-8a and H-13a (marked in yellow in Figure 3) are located in the shielding cone of the pyridine ring and in the shielding cone of the double bond at position 10, thus accounting for their unusually shielded chemical shift. In contrast, the C-19/C-26 carbon chain showed a much higher degree of flexibility. Finally, the piperidine ring showed an equilibrium between chair and twist-boat conformations, with the twist-boat largely dominating and accounting for over 78% of population ( Figure 3). This conformational change involves pyramidal inversion at the nitrogen atom, and the minor chair conformation, with the lone pair towards the outside of the molecule, is important in the reaction with dichloromethane leading to compound 2 (see below). Because compound 1a could not be obtained in pure form, it was not possible to measure its optical rotation nor its electronic circular dichroism (ECD) spectrum. Therefore, its absolute configuration could not be established experimentally, but was assumed to be (14R,15R) by analogy with absolute configuration of acanthocyclamine A (3) [8].  To discriminate between configurational stereoisomerism and atropisomerism, the conformational behavior of 1b was studied. A conformational search was performed by molecular dynamics (MD), because MD-based methods are more suitable than Monte Carlo methods for flexible polycyclic molecules [10]. A series of 10-ns MD simulations was performed using the CFF91 force field, and geometries were extracted every 50 ps, resulting in 200 conformations from each MD simulations. Because we were looking for conformational changes much slower than the ns timescale at room temperature, simulation was performed at increasing temperatures, from 600 K to 3000 K. Overall, 108 conformers were obtained for tetradehydrohalicyclamine B (1b), considering only low-energy conformers in a range of 5 kcal/mol, and the sets of conformers obtained from the different simulations were very similar. These results suggested that no slow conformational equilibrium occurs for 1b, thus excluding the hypothesis of atropisomerism.
Therefore, it was assumed that 1a is a diastereomer of 1b, i.e., the stereoisomer with cis substituents at C-14 and C-15. Unfortunately, the relative configuration of C-14 and C-15 could not be confirmed on the basis of the coupling constants of H-14 and H-15. In fact, even though C-14 and C-15 are part of a six-membered piperidine ring, the ring is in equilibrium between a chair and a twist-boat conformation (see below); in addition, the almost coincident chemical shifts of H-14 and H-16b, and of H-28a and H-28b produced a non-first-order spin system. Therefore, structure 1a was validated using quantum-mechanical prediction of 1 H and 13 C chemical shift and 1 H-1 H coupling constants (see Table S2). The conformational space of compound 1a was explored using again an MD-based conformational search, performed at 300 K. The search generated 42 conformers within 4 kcal/mol from the lowest-energy conformer. The geometry of each conformer was optimized quantum mechanically at the B3LYP/6-31G(d) level of theory and the continuum-solvent (PCM) model for MeOH, and 1 H-1 H coupling constants were calculated for each conformer at the B3LYP/6-31G(d,p) level of theory, according to the suggestion by Bally and Rablen [11]. Boltzmann-averaged coupling constants were then calculated, and compared with the respective experimental values. An excellent agreement was found between calculated and experimental coupling constants (Table 2), which strongly supported both the relative stereochemistry of structure 1a and the quality of the conformational search.
Examination of the conformers produced by the conformational search revealed an unexpected conformational rigidity of the C-6/C-13 carbon chain (containing the shielded protons H-8a and H-13a), which showed the same conformation in all the 16 lowest-energy conformers, accounting for over 96% of population. In this conformation, H-8a and H-13a (marked in yellow in Figure 3) are located in the shielding cone of the pyridine ring and in the shielding cone of the double bond at position 10, thus accounting for their unusually shielded chemical shift. In contrast, the C-19/C-26 carbon chain showed a much higher degree of flexibility. Finally, the piperidine ring showed an equilibrium between chair and twist-boat conformations, with the twist-boat largely dominating and accounting for over 78% of population ( Figure 3). This conformational change involves pyramidal inversion at the nitrogen atom, and the minor chair conformation, with the lone pair towards the outside of the molecule, is important in the reaction with dichloromethane leading to compound 2 (see below).    Because compound 1a could not be obtained in pure form, it was not possible to measure its optical rotation nor its electronic circular dichroism (ECD) spectrum. Therefore, its absolute configuration could not be established experimentally, but was assumed to be (14R,15R) by analogy with absolute configuration of acanthocyclamine A (3) [8].

Chloromethyltetradehydrohalicyclamine B (2)
Compound 2 was isolated as a colorless solid. Analysis of the HR-ESIMS showed a doubly charged [M] 2+ ion peak at m/z 214.1473, with an M+2 isotope peak whose intensity (35% compared to M) suggested the presence of a chlorine atom in the molecule. The molecular formula was deduced as C 27 H 41 ClN 2 2+ , indicating nine degrees of unsaturation in the molecule no single-charge ion was observed in the MS spectrum, suggesting the presence of two quaternary ammonium nitrogen atoms in the molecule. Detailed examination of NMR spectra revealed that compound 2 had the same atom connectivity as in compounds 1a and 1b (Figure 1), except for an additional methylene signal (δ H 5.38, δ C 69.9). This was indicative of a chloromethyl group linked to N-18, as observed in chloromethylhalicyclamine B (5) [5]. Consequently, this product resulted is an artifact produced during the extraction process, but its structure was not studied in depth because it did not reveal any activity in our panel of assays.

Diketopiperazines 6-12
In addition, seven diketopiperazines were isolated ( Figure 4). Their planar structures were easily identified by their spectroscopic data in comparison with those found in the literature, while the absolute configurations of their stereogenic carbons were determined using Marfey's analyses. Therefore, after hydrolysis and derivatization with the L-enantiomer of Marfey's reagent, the obtained derivatives were analysed by HR-ESIMS-HPLC. On the basis of the retention times of their respective Marfey's derivatives, the absolute configuration of the stereogenic carbons of the seven diketopiperazines were deduced as depicted in Figure 4. As for the cyclo (Pro-Ser) (10), only the proline residue was determined as L-Pro.
during the extraction process, but its structure was not studied in depth because it did not reveal any activity in our panel of assays.

Diketopiperazines 6-12
In addition, seven diketopiperazines were isolated (Figure 4). Their planar structures were easily identified by their spectroscopic data in comparison with those found in the literature, while the absolute configurations of their stereogenic carbons were determined using Marfey's analyses. Therefore, after hydrolysis and derivatization with the L-enantiomer of Marfey's reagent, the obtained derivatives were analysed by HR-ESIMS-HPLC. On the basis of the retention times of their respective Marfey's derivatives, the absolute configuration of the stereogenic carbons of the seven diketopiperazines were deduced as depicted in Figure 4. As for the cyclo (Pro-Ser) (10), only the proline residue was determined as L-Pro.

Evaluation of Biological Activities in the Antibacterial, Antikinases, and Amyloid β-42 Assays
All the isolated compounds were tested against the bacteria Staphylococcus aureus and Escherichia coli as well as against eight different protein kinases relevant to cell proliferation, cancer, diabetes, and neurodegenerative disorders (CDK1, CDK2, CDK5, CDK9, CK1, CLK1, DYRK1A, and GSK3) and in both amyloid β-42 assays (amyloid β-42 induction assay and inhibition of amyloid β-42 production induced by aftin-5 assay). The main significant results are presented in Table 3. Only acanthocyclamine A (3) and halicyclamine B (4) showed a selective antimicrobial activity against E. coli and S. aureus, respectively (diameter inhibition of 12 and 10 mm at 100 µ g/disk, respectively). Furthermore, while chloromethylhalicyclamine B (5) showed a selective inhibitory activity against the protein kinase CK1δ/ε with an IC50 value of 6 µ M [5], the diketopiperazine cyclo(D-Pro-L-Phe) (6) displayed a selective kinase inhibitory activity against CDK2/cyclin A with an IC50 value of 1 µ M. The amyloid β-42 assays were performed preliminary on both CH2Cl2 and BuOH extracts, showing an inhibition of the amyloid β-42 production induced by aftin-5, without reducing survival of the N2a-APP695 cell line. Their activity was confirmed in dose-response (0.1, 1.0 and 10 µ g/mL). Therefore, 1-12 were tested in both amyloid β-42 assays at the dose of 10 µ g/mL. Among the compounds tested, acanthocyclamine A (3), showed an inhibition of amyloid β-42 production induced by aftin-5 at 26 µ M, without cytotoxicity at this dose.

Evaluation of Biological Activities in the Antibacterial, Antikinases, and Amyloid β-42 Assays
All the isolated compounds were tested against the bacteria Staphylococcus aureus and Escherichia coli as well as against eight different protein kinases relevant to cell proliferation, cancer, diabetes, and neurodegenerative disorders (CDK1, CDK2, CDK5, CDK9, CK1, CLK1, DYRK1A, and GSK3) and in both amyloid β-42 assays (amyloid β-42 induction assay and inhibition of amyloid β-42 production induced by aftin-5 assay). The main significant results are presented in Table 3. Only acanthocyclamine A (3) and halicyclamine B (4) showed a selective antimicrobial activity against E. coli and S. aureus, respectively (diameter inhibition of 12 and 10 mm at 100 µg/disk, respectively). Furthermore, while chloromethylhalicyclamine B (5) showed a selective inhibitory activity against the protein kinase CK1δ/ε with an IC 50 value of 6 µM [5], the diketopiperazine cyclo(d-Pro-l-Phe) (6) displayed a selective kinase inhibitory activity against CDK2/cyclin A with an IC 50 value of 1 µM. The amyloid β-42 assays were performed preliminary on both CH 2 Cl 2 and BuOH extracts, showing an inhibition of the amyloid β-42 production induced by aftin-5, without reducing survival of the N2a-APP695 cell line. Their activity was confirmed in dose-response (0.1, 1.0 and 10 µg/mL). Therefore, 1-12 were tested in both amyloid β-42 assays at the dose of 10 µg/mL. Among the compounds tested, acanthocyclamine A (3), showed an inhibition of amyloid β-42 production induced by aftin-5 at 26 µM, without cytotoxicity at this dose.

Discussion and Conclusions
Alkaloids are pharmacologically well characterized and are used in therapy, ranging from chemotherapeutics to analgesics.
This chemical study of the marine sponge A. ingens allowed the isolation of 13 alkaloids, of which, one, 1a is the epimer of the tetradehydrohalicyclamine B (1b), just recently published. Compounds 1-12 were analyzed for their biological activity using our standard panel assays that include antibacterial, antikinases, and amyloid β-42 assays. Acanthocyclamine A (3) showed a selective antimicrobial activity against E. coli and an inhibition of the amyloid β-42 production induced by aftin-5 at 26 µM, without cytotoxicity at this dose. These results highlight the potentiality of a bipiperidine scaffold as a promising skeleton to develop products able to prevent or reduce the production of amyloid β-42, a key player in the initiation of Alzheimer's disease. A previous study revealed that chloromethylhalicyclamine B (5) appeared to be a selective CK1δ/ε inhibitor at low micromolar concentrations, while halicyclamine B (4) was inactive. Docking studies showed that chloromethylhalicyclamine B (5) can efficiently interact with the ATP-binding site of CK1δ in spite of its globular structure, very different from the planar structure of known inhibitors of CK1δ [5]. Because no CK1δ inhibitory activity was observed for the chloromethyltetradehydrohalicyclamine B (2), the presence of a tetrahydropyridine appears to be essential for the inhibitory activity towards CK1δ.
Moreover, the diketopiperazine cyclo (d-Pro-l-Phe) (6) revealed a selective antikinase activity against CDK2/cyclin A with an IC 50 value of 1 µM. In comparison with the inactive diketopiperazine cyclo (L-Pro-L-Tyr) (12), we can hypothesize that hydroxylation of the phenyl group leads to a loss of CDK2/cyclin A kinase inhibitory activity.
The marine sponge A. ingens is showed to be a rich source of a number of bioactive alkaloids, some of them having an unusual skeleton, and sometimes being halogenated.
This study completes the previous works on A. ingens and points out the growing interest in studying this sponge family with unique and diverse chemical structures. These data highlight the potentiality of these molecules as lead compounds.
The Marfey's experiments were performed using a Thermo LTQ Orbitrap XL mass spectrometer coupled to a Thermo Ultimate 3000 RS system (Thermo Fisher Scientific Spa, Rodano, Italy), which included solvent reservoir, in-line degasser, ternary pump, column thermostat, and refrigerated autosampler. LC-MS data were recorded and analyzed using the software Thermo Xcalibur 2.07 (Thermo Fisher Scientific Spa). The samples (5 µL) were applied on to an analytical reversed-phase column (Phenomenex Kinetex C18, 100 × 2.1 mm, particle size 5 µm), which was eluted at 200 µL/min. The elution procedure consisted of an isocratic profile of acetonitrile-water (5:95, v/v) for 3 min, followed by a linear gradient from 5% to 60% ACN/H 2 O over 20 min, a linear gradient from 60% to 90% ACN/H 2 O over 1 min, and an isocratic profile over 5 min.
The samples were treated with 6 N HCl and heated in a flame-sealed glass tube at 180 • C for 2 h. The residual HCl fumes were removed in vacuo. The hydrolysate of the diketopiperazines were dissolved in triethylamine/ACN (

Quantum Mechanical Prediction of 1 H-1 H Coupling Constants of epi-Tetradehydrohalicyclamine B (1a)
Initial conformational search was performed using molecular dynamics simulations at different temperatures, performed in vacuo in the CFF91 force field using the Insight II/Discover package (BIOVIA: San Diego, CA, USA). The resulting conformers (42 conformers within 4 kcal/mol from the lowest-energy conformer) were used as starting structure for quantum mechanical calculations with the Gaussian 09 program [13]. Geometries were optimized at the B3LYP/6-31G(d) level of theory and the continuum-solvent (PCM) model for MeOH, and 1 H-1 H coupling constants were calculated for each conformer at the B3LYP/6-31G(d,p) level of theory according to the suggestion by Bally and Rablen [11], i.e., considering only the Fermi contact contribution to J and scaling the calculated value In DKP cyclo(Pro-Gly) (7), L configuration was found for proline residue ( Figure S1). In DKP cyclo(Pro-Ala) (8), both alanine and proline residues were found to have L configuration ( Figure S2).
In DKP cyclo(Pro-Val) (9), the valine residue was found to have L configuration while the proline residue was found to have D configuration ( Figure S3).
In DKP cyclo(Pro-Ser) (10), L configuration was found for proline residue while it was not possible to establish the configuration for the serine residue, probably because of the low amount of diketopiperazine ( Figure S4).
In DKP cyclo(Pro-Ile) (11), the isoleucine residue was found to have L configuration while the proline residue was found to have the D configuration ( Figure S5).
In DKP cyclo(Pro-Tyr) (12), the tyrosine and the proline residues were found to have the L configuration ( Figure S6).

Quantum Mechanical Prediction of 1 H-1 H Coupling Constants of epi-Tetradehydrohalicyclamine B (1a)
Initial conformational search was performed using molecular dynamics simulations at different temperatures, performed in vacuo in the CFF91 force field using the Insight II/Discover package (BIOVIA: San Diego, CA, USA). The resulting conformers (42 conformers within 4 kcal/mol from the lowest-energy conformer) were used as starting structure for quantum mechanical calculations with the Gaussian 09 program [13]. Geometries were optimized at the B3LYP/6-31G(d) level of theory and the continuum-solvent (PCM) model for MeOH, and 1 H-1 H coupling constants were calculated for each conformer at the B3LYP/6-31G(d,p) level of theory according to the suggestion by Bally and Rablen [11], i.e., considering only the Fermi contact contribution to J and scaling the calculated value by 0.9117. The averaged coupling constants shown in Table 2 were obtained using populations calculated from the CFF91 energies, and including in the calculations only the conformers populated by more than 1%. It is interesting to note that using quantum mechanical calculations to evaluate energies and populations of conformers produced a remarkably worse fit with the experimental values. The inaccuracy of relative energies of conformers is a known problem of DFT calculations [10], which in this particular study could not be avoided even when a higher level of theory (e.g., B3LYP-D3/TZVP) was used.

Antimicrobial Assays
Assays were performed using the disk diffusion assay method. Pure compounds (100 µg) were solubilized in DMSO and deposited on a 6 mm paper disk and put on agar plated seeded with reference strains Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739). Antimicrobial activity was determined by measuring the diameter of the inhibition zone after 24 h of incubation at 37 • C. Cefotaxime (30 µg) and amoxicillin (25 µg) were used as positive controls against S. aureus and E. coli, giving 30 and 21 mm of inhibition zones, respectively.

Amyloid β42 Induction Assay
This assay, described in detail in reference [15] allows the detection of molecules able to induce the production of extracellular amyloid β-42 peptide.
Amyloid β-42 levels were measured in a double antibody sandwich ELISA using a combination of monoclonal antibody (mAb) 6E10 (SIG-39320, Covance, Eurogentec, Seraing, Belgium) and biotinylated polyclonal amyloid β-42 antibody (provided by Dr. P.D. Mehta, Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA). Briefly, wells of microtiter plates (Maxisorp, Nunc, ThermoFisher Scientific, Illkirch, France) were coated 100 µL mAb 6E10 diluted in carbonate-bicarbonate buffer (buffer (0.015 M Na 2 CO 3 + 0.035 M NaHCO 3 ) pH 9.6) at a 1.5 µg/mL final concentration, and plates were incubated overnight at 4 • C. The plates were then washed with PBST (PBS containing 0.05% Tween-20) and blocked for 1 h with 1% BSA in PBST to avoid non-specific binding. Following a washing step, 100 µL of cell supernatant was added and incubated for 2 h at room temperature (RT) on a shaking device. Plates were then washed with PBST and 100 µL of biotinylated antibodies (diluted to 1 µL/mL in PBST containing 0.5% BSA) were added and incubation was carried out for 75 min at RT under constant shaking. After a washing step, streptavidin-Poly-HRP (horseradish peroxidase) conjugate (Pierce, ThermoFisher Scientific, Illkirch, France), diluted in PBS + 1% BSA, was added and incubation was carried out for 45 min at RT under continuous shaking. After washing, 100 µL of OPD (o-Phenylenediamine dihydrochloride, Pierce, ThermoFisher Scientific, Illkirch, France) in pH 5.0 citrate buffer (0.049M citric acid monohydrate + 0.1M Na 2 HPO 4 ·2H 2 O + 1 mL H 2 O 2 30%/L) were added as a substrate and after 15 min incubation at room temperature, the reaction was stopped by addition of 100 µL 1 N sulfuric acid. Optical density (OD) was measured at 490 nm using a plate reader (BioTek Instruments, El 800, Gen 5 software, Winooski, VT, USA).
Amyloidβ-42 quantification was calculated using standard curves, which were prepared with synthetic Aβ-42 HFIP treated (JPT Peptide Technologies, Berlin, Germany) and Aβ-42 specific polyclonal antibody. Curve fitting was performed using a 4 parameters sigmoid equation (SigmaPlot, Systat, Sigma). Results are expressed as fold change ± s.d. All experiments were performed in triplicate.

Inhibition of Amyloid β-42 Production Induced by Aftin-5 Assay
This assay allows the detection of molecules able to inhibit the production of extracellular Amyloid β peptides induced by a pre-treatment with 100 µM of aftin-5. Aftin-5 is available from Adipogen International, San Diego, CA, USA.

Cytotoxic Assay: Effects on N2a-APP695 Viability (MTS Survival Assay)
This assay allows evaluating the survival rate of cultured mammalian cells exposed to extracts or pure compounds. It allows the detection of cell death-inducing molecules.