Brominated Bisindole Alkaloids from the Celtic Sea Sponge Spongosorites calcicola

As part of an ongoing program to identify new bioactive compounds from Irish marine bioresources, we selected the subtidal sponge Spongosorites calcicola for chemical study, as fractions of this species displayed interesting cytotoxic bioactivities and chemical profiles. The first chemical investigation of this marine species led to the discovery of two new bisindole alkaloids of the topsentin family, together with six other known indole alkaloids. Missing the usual central core featured by the representatives of these marine natural products, the new metabolites may represent key biosynthetic intermediates for other known bisindoles. These compounds were found to exhibit weak cytotoxic activity against HeLa tumour cells, suggesting a specificity towards previously screened carcinoma and leukaemia cells.


Introduction
Marine sponges are well-recognised as producers of a diverse array of bioactive metabolites for defence, competition, and communication [1]. For this reason, these invertebrates have been the subjects of extensive chemical study over the past half century. However, there still remain extensive regions of the world where sponges have been understudied, including much of the Irish coastal waters [2]. As part of an Irish marine biodiscovery initiative supported by the Marine Institute, invertebrates present in the shallow waters of Ireland have been inventoried and then chemically and biologically screened in the search for bioactive natural products. A priority list of species placed the sponge Spongosorites calcicola on the top of the list due to the amount of biomass available, good chemical profiles and interesting cytotoxic activity (Table S2). While this sponge species has never been studied chemically, other species of this genus have already led to interesting bioactive metabolites.
The sponge genus Spongosorites has been reported to produce many brominated bisindole alkaloids belonging to four classes that differ in the linkage between the indole moieties [3,4]. These bisindoles include topsentin and spongotine derivatives with imidazole or dihydroimidazole linkages [5,6], and also, dragmacidin and hamacanthin derivatives characterised by a pyrazine or piperazine
Compound 2 was isolated as a yellow amorphous solid that displayed an ion cluster in the (+)-HRESIMS for [M + H] + at m/z 425.0608/427.0590, indicative of a structural isomer of 1. The 1 H-NMR and 13 C-NMR data (Table 1) were similar to those of 1, again revealing resonances associated with one indole (δ H 12.26, 8.77, 8.22, 7.54, 7.27, 7.25) and one 6-bromoindole (δ H 11.45, 7.43, 7.71, 7.63, 7.23). The differences between 1 and 2 were therefore located in the linkage between the two indole moieties. The 13 C-NMR and IR data indicated the presence of an alpha-keto amide connected to the non-brominated indole. However, the spin-coupled system assigned through the COSY/HSQC spectra led us to identify the difference between 1 and 2 with the linkage from δ H 8.95 (NH-10) to δ H 3.83, 3.70 (H 2 -9) to δ H 4.83 (H-8) to δ H 8.33 (NH 3 -10 ). The absolute configuration of the C-8 stereocenter was again determined by ECD analysis. The theoretical ECD spectra of the enantiomers of 2 were calculated using the same methods and basis set/functional combination as used for 1, allowing the assignment of the same 8S configuration for calcicamide B ( Figure S15).   The differences between 1 and 2 were therefore located in the linkage between the two indole moieties. The 13 C-NMR and IR data indicated the presence of an alpha-keto amide connected to the non-brominated indole. However, the spin-coupled system assigned through the COSY/HSQC spectra led us to identify the difference between 1 and 2 with the linkage from δH 8.95 (NH-10) to δH 3.83, 3.70 (H2-9) to δH 4.83 (H-8) to δH 8.33 (NH3-10′). The absolute configuration of the C-8 stereocenter was again determined by ECD analysis. The theoretical ECD spectra of the enantiomers of 2 were calculated using the same methods and basis set/functional combination as used for 1, allowing the assignment of the same 8S configuration for calcicamide B ( Figure S15).
Calcicamides (1-2) differ from other bisindole compounds due to the lack of a central heterocyclic ring, which is instead opened in 1 and 2. As the tryptophan origin of bisindole alkaloids cannot be contested, we hypothesize that the nucleophilicity of one primary amine should enable the connection between two 8-ketotryptamine derivative units after a first condensation (Scheme 1) [3]. Hydration/oxidation of the intermediate would lead to compound 1. Oxidation of the 8ketotryptamine derivatives at C-9 prior to isomerisation would lead to the monoindole derivatives 7 and 8. The piperazine central core of hamacanthins and dragmacidins would subsequently be formed when the second condensation occurs at C-8′ of compound 1 or 2. To assess if calcicamides are natural products or hydrolysed artefacts produced during the isolation process, the extracts and fractions were analysed by LC-MS/MS prior to acidic HPLC isolation. We identified two peaks in the chromatogram with a molecular ion at m/z 425/427 (1:1) coeluting with the two isomers of calcicamides. This result came as a proof of their natural origin. Additionally, coscinamides isolated from the marine sponge Coscinoderma sp. are another family of bisindole alkaloids that lack a central ring and instead contain a linear chain with an alpha keto enamide [15]. We hypothesise that the coscinamides may also be biosynthetically related to calcicamide B (2) following a deamination at C-8.
Members of the bisindole alkaloid family have been previously reported with a large array of Calcicamides (1-2) differ from other bisindole compounds due to the lack of a central heterocyclic ring, which is instead opened in 1 and 2. As the tryptophan origin of bisindole alkaloids cannot be contested, we hypothesize that the nucleophilicity of one primary amine should enable the connection between two 8-ketotryptamine derivative units after a first condensation (Scheme 1) [3]. Hydration/oxidation of the intermediate would lead to compound 1. Oxidation of the 8-ketotryptamine derivatives at C-9 prior to isomerisation would lead to the monoindole derivatives 7 and 8. The piperazine central core of hamacanthins and dragmacidins would subsequently be formed when the second condensation occurs at C-8 of compound 1 or 2. To assess if calcicamides are natural products or hydrolysed artefacts produced during the isolation process, the extracts and fractions were analysed by LC-MS/MS prior to acidic HPLC isolation. We identified two peaks in the chromatogram with a molecular ion at m/z 425/427 (1:1) coeluting with the two isomers of calcicamides. This result came as a proof of their natural origin. Additionally, coscinamides isolated from the marine sponge Coscinoderma sp. are another family of bisindole alkaloids that lack a central ring and instead contain a linear chain with an alpha keto enamide [15]. We hypothesise that the coscinamides may also be biosynthetically related to calcicamide B (2) following a deamination at C-8.
Members of the bisindole alkaloid family have been previously reported with a large array of bioactivities including moderately potent antiviral, antitumoral, antibacterial, and antifungal activities [5,10,16]. The six bisindole alkaloids were screened for their antitumoral activity against a HeLa cell line. Compounds 2 and 4-6 exhibited weak activity against these cells with IC 50 s between 100 and 200 µM (Table S1). Compounds 1 and 3 did not induce detectable cell lysis at the highest tested concentrations (185 and 166 µM respectively). Most cell death occurred within the first 6 h of incubation. Cell lysis increased between 6 and 24 h incubation, corresponding to an increase in the cytotoxicity with longer exposure. Compounds 3-6 have previously been shown to have moderate cytotoxicity against adenocarcinoma (AGS) and lymphocytic leukaemia (L1210) cells. This previous study noted that the cytotoxicity of Spongosorites bisindoles is cell-line dependent with only weak activity reported against other cell lines screened [10]. The inability of these compounds to display comparable activity against HeLa cells again suggests that these compounds may be more specific against carcinoma and leukaemia cell lines. Therefore, based on these results we will continue a deeper evaluation of the cytotoxicity of Spongosorites bisindoles against carcinoma and leukaemia cell lines. study noted that the cytotoxicity of Spongosorites bisindoles is cell-line dependent with only weak activity reported against other cell lines screened [10]. The inability of these compounds to display comparable activity against HeLa cells again suggests that these compounds may be more specific against carcinoma and leukaemia cell lines. Therefore, based on these results we will continue a deeper evaluation of the cytotoxicity of Spongosorites bisindoles against carcinoma and leukaemia cell lines. Scheme 1. Biosynthetic hypothesis for the calcicamide, hamacanthin, coscinamides, and topsentin derivatives.

General Experimental Procedures
Optical rotations were recorded on a Unipol L1000 polarimeter at the sodium D-line (589.3 nm) with a 5 cm cell at 20 °C (Schmidt+Haensch, Berlin, Germany). UV and ECD were recorded on a Chirascan V100 with a 1.0 cm quartz cuvette (Applied Photophysics, Leatherhead, UK). IR data were recorded on a PerkinElmer spectrum 100 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). NMR experiments were performed on a 500 MHz Varian Inova spectrometer (Agilent, Santa Clara, CA, USA). Chemical shifts (δ in ppm) were referenced to the carbon (δC 39.52) and proton (δH 2.50) signals of DMSO-d6. HRESIMS were obtained using an Agilent 6540 Q-Tof mass spectrometer equipped with an Agilent 1290 UPLC and autosampler (Agilent). Preparative and semipreparative HPLC was carried out on a Jasco LC-2000 series equipped with a coupled UV detector. Analytical HPLC was carried out on an Agilent 1260 HPLC system equipped with a DAD detector coupled with Scheme 1. Biosynthetic hypothesis for the calcicamide, hamacanthin, coscinamides, and topsentin derivatives.

General Experimental Procedures
Optical rotations were recorded on a Unipol L1000 polarimeter at the sodium D-line (589.3 nm) with a 5 cm cell at 20 • C (Schmidt+Haensch, Berlin, Germany). UV and ECD were recorded on a Chirascan V100 with a 1.0 cm quartz cuvette (Applied Photophysics, Leatherhead, UK). IR data were recorded on a PerkinElmer spectrum 100 FT-IR spectrometer (PerkinElmer, Waltham, MA, USA). NMR experiments were performed on a 500 MHz Varian Inova spectrometer (Agilent, Santa Clara, CA, USA). Chemical shifts (δ in ppm) were referenced to the carbon (δ C 39.52) and proton (δ H 2.50) signals of DMSO-d 6 . HRESIMS were obtained using an Agilent 6540 Q-Tof mass spectrometer equipped with an Agilent 1290 UPLC and autosampler (Agilent). Preparative and semipreparative HPLC was carried out on a Jasco LC-2000 series equipped with a coupled UV detector. Analytical HPLC was carried out on an Agilent 1260 HPLC system equipped with a DAD detector coupled with an Agilent 385-ELSD. All solvents used for extraction and separation were HPLC grade, and H 2 O was Milli-Q (Millipore Ireland B.V., Carrigtwohill, County Cork, Ireland) filtered.

Biological Material
Spongosorites calcicola was collected at Rathlin Island (Co. Antrim, Northern Ireland) at 18 m depth by SCUBA. The sponge was taxonomically identified by Christine Morrow through morphological and spicule analysis. A voucher specimen of this sample "BDV10015" is kept at the National Marine Biodiscovery Laboratory (Marine Institute, Oranmore, Co. Galway, Ireland).