Aurantoside L, a New Tetramic Acid Glycoside with Anti-Leishmanial Activity Isolated from the Marine Sponge Siliquariaspongia japonica

A new tetramic acid glycoside, aurantoside L (1), was isolated from the sponge Siliquariaspongia japonica collected at Tsushima Is., Nagasaki Prefecture, Japan. The structure of aurantoside L (1) composed of a tetramic acid bearing a chlorinated polyene system and a trisaccharide part was elucidated using spectral analysis. Aurantoside L (1) showed anti-parasitic activity against L. amazonensis with an IC50 value of 0.74 µM.


Introduction
Leishmaniases are vector-borne parasitic diseases caused by several different species of Leishmania [1].It is estimated that there are 12 million patients suffering from leishmaniasis with around 1 million new cases annually {www.who.int(accessed on 13 July 2023)}.Visceral leishmaniasis, caused by L. donovani in Asia and Africa and L. infantum in the Mediterranean Basin, Middle East, Central Asia, South America, and Central America, is characterized by clinical symptoms such as fever, anemia, splenomegaly, hepatomegaly, and weight loss and is fatal unless treated appropriately [2].Cutaneous leishmaniasis is a skin manifestation that sometimes heals naturally over 3-18 months, but the ulcer can lead to serious permanent scarring, disfigurement, and stigmatization [3].Mucocutaneous leishmaniasis leads to the partial or total destruction of the mucous membranes of the nose, mouth, and throat, and the disease can be found mostly in South America (www.who.int(accessed on 13 July 2023)).Because no effective vaccines to prevent the disease for humans are commercially available yet, the control of the disease mostly relies on chemotherapy.These leishmaniases are treated by, for example, pentavalent antimonials, liposomal amphotericin B, and milterfosine.However, drug side effects, high costs, potential teratogenicity, and the emergence of drug-resistant strains pose a serious potential threat to endemic countries where leishmaniases are prevalent [4,5].
Consequently, there have been efforts to discover new candidate compounds for chemotherapeutic use against leishmaniasis.Although target-based screening is becoming Mar.Drugs 2024, 22, 171 2 of 9 a more popular way for drug discovery, the phenotypic screening of natural compounds is still a vital choice [6].In fact, marine bioproducts are attractive sources of anti-parasitic agents for various diseases including malaria and leishmaniasis [7].Gracilioethers A-C were isolated from the marine sponge Agelas gracilis as anti-protozoan natural compounds, which had anti-malarial activity [8].A xenicane diterpenoid, cristaxenicin A, was found in the deep-sea gorgonian Acanthoprimnoa cristata, showing anti-leishmanial and anti-trypanosomal activities (against Leishmania amazonensis and Trypanosoma congolense, respectively) [9].
To discover potential drug leads against leishmaniasis, we focused on marine invertebrates, whose extracts are a rich source of various bioactive compounds [10].Marine organism extracts (1565 samples) have been tested against the recombinant L. amazonensis doped with a green fluorescent protein (La/egfp).In this screening approach, a marine sponge Siliquariaspongia japonica extract showed strong anti-parasitic activity against La/egfp.From the Lithistida order of sponges, to which S. japonica belongs, a wide variety of compounds that are thought to be produced by the symbiotic bacteria [11] have been isolated.The marine sponge Siliquariaspongia sp. has been also reported several times as the source of unique and bioactive metabolites, for example, aurantosides D-E [12], rubrosides A-H [13], motualevic acids A-F [14], mirabamides A-D [15], celebesides A-C, and theopapuamides B-D [16].Based on the result of the screening and the abundant discoveries reported so far, we considered the marine sponge S. japonica to be a suitable candidate for searching for substances with anti-leishmanial activity.The bioassay-guided fractionation of this sponge extract provided a new anti-leishmanial tetramic acid glycoside, aurantoside L (1) (Figure 1).In this paper, the isolation, structure elucidation, and biological activities of this compound are discussed.Consequently, there have been efforts to discover new candidate compounds for chemotherapeutic use against leishmaniasis.Although target-based screening is becoming a more popular way for drug discovery, the phenotypic screening of natural compounds is still a vital choice [6].In fact, marine bioproducts are attractive sources of antiparasitic agents for various diseases including malaria and leishmaniasis [7].Gracilioethers A-C were isolated from the marine sponge Agelas gracilis as anti-protozoan natural compounds, which had anti-malarial activity [8].A xenicane diterpenoid, cristaxenicin A, was found in the deep-sea gorgonian Acanthoprimnoa cristata, showing anti-leishmanial and anti-trypanosomal activities (against Leishmania amazonensis and Trypanosoma congolense, respectively) [9].
To discover potential drug leads against leishmaniasis, we focused on marine invertebrates, whose extracts are a rich source of various bioactive compounds [10].Marine organism extracts (1565 samples) have been tested against the recombinant L. amazonensis doped with a green fluorescent protein (La/egfp).In this screening approach, a marine sponge Siliquariaspongia japonica extract showed strong anti-parasitic activity against La/egfp.From the Lithistida order of sponges, to which S. japonica belongs, a wide variety of compounds that are thought to be produced by the symbiotic bacteria [11] have been isolated.The marine sponge Siliquariaspongia sp. has been also reported several times as the source of unique and bioactive metabolites, for example, aurantosides D-E [12], rubrosides A-H [13], motualevic acids A-F [14], mirabamides A-D [15], celebesides A-C, and theopapuamides B-D [16].Based on the result of the screening and the abundant discoveries reported so far, we considered the marine sponge S. japonica to be a suitable candidate for searching for substances with anti-leishmanial activity.The bioassay-guided fractionation of this sponge extract provided a new anti-leishmanial tetramic acid glycoside, aurantoside L (1) (Figure 1).In this paper, the isolation, structure elucidation, and biological activities of this compound are discussed.

Results
The frozen specimen of S. japonica (170 g, wet weight) was extracted with MeOH and CHCl3/CH3OH (1:1) repeatedly.The extracts were combined, and the concentrated extract was partitioned between H2O and CHCl3.The water-soluble layer was further extracted with n-C4H9OH, and the n-C4H9OH layer was combined with the former CHCl3 layer.The combined organic layer was fractionated using the Kupchan procedure [17] to yield nhexane, CHCl3, and 60% CH3OH layers.The anti-leishmanial aqueous CH3OH layer was subjected to octadecylsilyl (ODS) column flash chromatography to give six fractions (fr.A-F).Among them, active fr.E, eluting with 100% CH3OH, was subjected to reversedphase high-performance liquid chromatography (RP-HPLC) to afford compound 1 as the active substance (12.5 mg, 7.4 × 10 -3 % yield based on the wet weight).
Compound 1 was obtained as a red amorphous solid.The electrospray ionization mass spectrum (ESIMS) (positive mode) showed clusters of ions at m/z 865, 867, and 869 [M + Na] + in the ratio of 9:6:1, indicating the presence of two chlorine atoms in 1 since the natural abundance of chlorine atoms is 75% with integer mass 35 and 25% with 37. The molecular formula was established as C38H48Cl2N2O15 by high-resolution electrospray
Aurantoside L (1) exhibited anti-leishmanial activity against La/egfp with an I value of 0.74 µM, while it showed modest cytotoxicity against HeLa cells and P388 c with IC50 values of 2.4 and 1.1 µM, respectively.In contrast, aurantoside L (1) was inact at 3.0 µM against Trypanosoma congolense, indicating selective anti-parasitic activity wit the same family of Trypanosomatidae.According to the literature, aurantosides A-F and rubrosides A-H are all derived from L-aspartic acid (detected via the GC analysis of the acid hydrolysate of the Lemieux oxidation product) and carry D-saccharides [12,13,[21][22][23][24][25].This structural information indicates that a common biosynthetic pathway produces these polyene tetramic acid glycosides.Based on biogenetic reasoning and comparing spectroscopic data with those of analogs, the absolute configurations at C-4 and each saccharide were presumed to be identical to that of analogs.Therefore, the absolute configuration of aurantoside L (1) was tentatively assigned as 4S and the saccharides as D-forms.
Aurantoside L (1) exhibited anti-leishmanial activity against La/egfp with an IC 50 value of 0.74 µM, while it showed modest cytotoxicity against HeLa cells and P388 cells with IC 50 values of 2.4 and 1.1 µM, respectively.In contrast, aurantoside L (1) was inactive at 3.0 µM against Trypanosoma congolense, indicating selective anti-parasitic activity within the same family of Trypanosomatidae.

General Experimental Procedures
NMR spectra were recorded on an Avance (400 MHz) spectrometer (Bruker Corporation, Billerica, MA, USA). 1 H and 13 C NMR chemical shifts were referenced to the CD 3 OD solvent peaks δ H 3.31 and δ C 49.15 (Wako, Osaka, Japan).HRESI-MS spectra were measured on an Exactive Plus (Thermo Fisher Scientific Inc., Waltham, MA, USA).ESIMS/MS spectra were measured on a TripleTOF 4600 (AB Sciex Pte. Ltd., Tokyo, Japan) in the positive mode.Optical rotation was determined on a P-2200 polarimeter (JASCO Corporation, Tokyo, Japan) in CH 3 OH.UV spectra were recorded using a V-630 spectrophotometer (JASCO).IR spectra were measured on a Nicolet6700 spectrometer (Thermo Fisher Scientific Inc.).

Anti-Leishmanial Assay
La/egfp promastigotes (1 × 10 5 cells) were cultured for 72 h in 199 medium (NISSUI Pharmaceutical, Tokyo, Japan) in 96-well plates with various concentrations of marine invertebrate extracts, as previously reported [26].Fluorescence was measured with excitation at 485 nm and emission at 538 nm.

Anti-Trypanosomal Assay
The procyclic form of the parasite (2 × 10 5 cells per well) Trypanosoma congolense IL 3000 was cultured for 48 h in TVM-1 medium [27] in 96-well plates with various concentrations of aurantoside L (1).Ten microliters of TetraColor ONE (Seikagaku Biobusiness, Tokyo, Japan) was added to each well.After 4 h, the absorbance of the samples was read at 450 nm using a microplate reader.

Cytotoxic Test
HeLa human cervical cancer cells in DMEM (cell concentration, 10,000 cells/mL, 200 µL) were added to each well of the microplates (96-well microplates, Costar, Washington, DC, USA) and kept in the incubator at 37 • C under an atmosphere of 5% CO 2 for 24 h.The sample solution (2 µL in MeOH or DMSO) at 1 mg/mL was added to each well with the medium.As the positive control, 2 µL of 1 mg/mL adriamycin was added to a well of each microplate.One-fourth of this medium (ca.50 µL) with a sample was transferred to a second well with medium (200 µL) to give a 1/5 dilution of the sample concentration.Two or six additional dilution steps gave four or eight sample concentrations.The prepared sample solutions (200 µL) were transferred to wells seeded with HeLa cells and then cultured at 37 • C under an atmosphere of CO 2 for 72 h.Cytotoxic tests against P388 murine leukemia cells were carried out in the same manner except for the medium (RPM1 medium, as described in Cell Culture).After 72 h of cultivation, 50 µL of 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) saline solution (1 mg/mL) was added to each well and the sample further incubated at 37 • C under an atmosphere of 5% CO 2 .After 4 h, the medium was removed via aspiration, and 150 µL of CH 3 COCH 3 was added to each well to lyse the cells.The concentration of the reduced MTT was quantified by measuring the absorbance at 650 nm to estimate IC 50 values.

Conclusions
Bioassay-guided isolation for anti-leishmanial activity afforded a new tetramic acid glycoside, aurantoside L (1), from the marine sponge S. japonica.The structure was elucidated using NMR and MS analyses.By combining high-temperature measurements and 2D NMR in different deuterated NMR solvents, the broadened 1 H signals and the unobserved 13 C signals that are measured by 1D NMR in CD 3 OD at room temperature were successfully assigned.Since the MS/MS experiment gives remarkably characteristic fragment ions for tetramic acid glycosides with polyene side chains, it was found to be useful for the structural analysis of compounds containing a tetramic acid moiety such as aurantoside analogs.Cytotoxicity against leukemia cells and antifungal activity have been reported for aurantoside analogs so far.There is a report that the number and structure of saccharide moiety are related to the strength and selectivity of bioactivity [21].Notably,

Compound 1
was obtained as a red amorphous solid.The electrospray ionization mass spectrum (ESIMS) (positive mode) showed clusters of ions at m/z 865, 867, and 869 [M + Na] + in the ratio of 9:6:1, indicating the presence of two chlorine atoms in 1 since the natural abundance of chlorine atoms is 75% with integer mass 35 and 25% with 37. The molecular formula was established as C 38 H 48 Cl 2 N 2 O 15 by high-resolution electrospray ionization mass spectrum (HRESIMS) (positive mode) (m/z 865.2310 [M + Na] + , calcd for C 38 H 48 35 Cl 2 N 2 O 15 Na, 865.2324, ∆ −1.6 mmu).

Figure 3 .
Figure 3. Substructure c deduced from COSY and key HMBC and NOESY correlations.

Figure 4 .
Figure 4. Substructure b deduced from COSY and key HMBC and NOESY correlations.The whole planar structure of 1 was constructed using HMBC and MS/MS data analysis.HMBC correlations for H-8/C-7 (δH 7.22, d, J = 15.1 Hz/δC 175.2) and H-9/C-7 (δH 7.60, dd, J = 15.1, 11.5 Hz/δC 175.2) observed in CD3OD at 320 K indicated that partial structures a and b were connected between C-8 and C-7.MS/MS (positive ion mode) fragmentation analysis resolved the connection of partial structures a, b, and c and the sequence of the trisaccharide.The ion giving m/z 865.2193 (composed of C38H48Cl2N2O15Na) was chosen as the precursor ion for the experiment.The intensity of the sodium-cationized ion peak at m/z 599.1586 (calcd for C23H32N2O15Na, 599.1700) was the strongest among the fragment ion peaks observed, suggesting the conjugated system in the tetramic acid moiety was formed by the desorption of substructure a and stabilized in MS/MS fragmentation.The second strongest fragment ion peak appeared as m/z 337.0563 (calcd for C12H14N2O8Na, 337.0648), which was thought to be composed of the tetramic acid moiety (m/z 205.0161) and the xylopyranose (sugar-I).Besides these peaks, fragment ion peaks at m/z 603.1158 (intermediate ion peaks from m/z 865.2193 to 337.0563) and m/z 417.1274 to 285.0872

Figure 3 .
Figure 3. Substructure c deduced from COSY and key HMBC and NOESY correlations.

Figure 3 .
Figure 3. Substructure c deduced from COSY and key HMBC and NOESY correlations.

Figure 4 .
Figure 4. Substructure b deduced from COSY and key HMBC and NOESY correlations.The whole planar structure of 1 was constructed using HMBC and MS/MS data analysis.HMBC correlations for H-8/C-7 (δH 7.22, d, J = 15.1 Hz/δC 175.2) and H-9/C-7 (δH 7.60, dd, J = 15.1, 11.5 Hz/δC 175.2) observed in CD3OD at 320 K indicated that partial structures a and b were connected between C-8 and C-7.MS/MS (positive ion mode) fragmentation analysis resolved the connection of partial structures a, b, and c and the sequence of the trisaccharide.The ion giving m/z 865.2193 (composed of C38H48Cl2N2O15Na) was chosen as the precursor ion for the experiment.The intensity of the sodium-cationized ion peak at m/z 599.1586 (calcd for C23H32N2O15Na, 599.1700) was the strongest among the fragment ion peaks observed, suggesting the conjugated system in the tetramic acid moiety was formed by the desorption of substructure a and stabilized in MS/MS fragmentation.The second strongest fragment ion peak appeared as m/z 337.0563 (calcd for C12H14N2O8Na, 337.0648), which was thought to be composed of the tetramic acid moiety (m/z 205.0161) and the xylopyranose (sugar-I).Besides these peaks, fragment ion peaks at m/z 603.1158 (intermediate ion peaks from m/z 865.2193 to 337.0563) and m/z 417.1274 to 285.0872

Figure 4 .
Figure 4. Substructure b deduced from COSY and key HMBC and NOESY correlations.The whole planar structure of 1 was constructed using HMBC and MS/MS data analysis.HMBC correlations for H-8/C-7 (δ H 7.22, d, J = 15.1 Hz/δ C 175.2) and H-9/C-7 (δ H 7.60, dd, J = 15.1, 11.5 Hz/δ C 175.2) observed in CD 3 OD at 320 K indicated that partial structures a and b were connected between C-8 and C-7.MS/MS (positive ion mode) fragmentation analysis resolved the connection of partial structures a, b, and c and the sequence of the trisaccharide.The ion giving m/z 865.2193 (composed of C 38 H 48 Cl 2 N 2 O 15 Na) was chosen as the precursor ion for the experiment.The intensity of the sodium-cationized ion peak at m/z 599.1586 (calcd for C 23 H 32 N 2 O 15 Na, 599.1700) was the strongest among the fragment ion peaks observed, suggesting the conjugated system in the tetramic acid moiety was formed by the desorption of substructure a and stabilized in MS/MS fragmentation.The second strongest fragment ion peak appeared as m/z 337.0563 (calcd for C 12 H 14 N 2 O 8 Na, 337.0648), which was thought to be composed of the tetramic acid moiety (m/z 205.0161) and the xylopyranose (sugar-I).Besides these peaks, fragment ion peaks at m/z 603.1158 (intermediate ion peaks from m/z 865.2193 to 337.0563) and m/z 417.1274 to 285.0872 corresponding to the sugar sequence also supported the structure deduced by NMR experiments (see Figure5).These experiments confirmed the planar structure of compound 1 as a new tetramic acid glycoside, aurantoside L (1) (Figure5).