Marine sponges are a rich source of structurally novel chemical leads [1] that are interesting for drug discovery. In recent years, advances in chromatographic and spectroscopic techniques have facilitated the identification of structurally complex natural products [2] from sponges with >30% of all marine natural product discovered to date [3, 4]. Members of the genus Hyrtios (Demospongiae class, Dictyoceratida order, Thorectidae family) are known to produce alkaloids, sesterterpene, sesquiterpene, sterols, and macrolides [5,6,7,8,9], of which diverse activities are attributed such as cytotoxicity against murine P388 lymphocytic leukemia cells and various human tumor cell lines, antimalarial, antifungal, and antitrypanosomal activities [10, 11].

Dereplication of the secondary metabolites [12] from the antitrypanosomally active (90% growth inhibition) crude extract marine sponge Hyrtios sp. was achieved by high resolution Fourier transform mass spectrometry. Compounds identified and dereplicated from high-resolution mass spectral data sets of the marine sponge Hyrtios sp. by utilizing macros and algorithms that coupled MZmine with both in-house and commercial database MarinLit. The dereplicated compounds were showed in Fig. 1a, The crude extract of the Red Sea sponge Hyrtios sp. was then subjected to vacuum-liquid chromatography, followed by chromatography on silica gel and Sephadex LH-20, and finally, purification on semi-preparative high-pressure liquid chromatography (HPLC) to afford compounds 1–5 (Fig. 1b).

Fig. 1
figure 1

a Compounds identified and dereplicated from high-resolution mass spectral data sets of the Hyrtios sp. b Structures of the isolated compounds 1–5. c Significant HMBC and COSY correlations of compound 1

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Compound 1 (2.5 mg) was isolated as a white powder. The molecular formula was determined to be C12H15N3O2Na, from its high-resolution electrospray ionization mass spectrometry, m/z calcd. 256.1064 for C12H15N3O2Na (HRESI-MS). The 1H-NMR spectrum (Table 1) revealed the presence of four proton resonances for the aromatic ABCD system, at δH 6.95 (1H, td, J = 7.0, 1.0 Hz; H-5), δH 7.03 (1H,td, J = 8.1, 1.1 Hz; H-6), δH 7.37 (1H, br d, J = 7.2 Hz; H-4), δH 7.45 (1H, br d, J = 8.0 Hz; H-7). In addition to two methylene protons at δH 2.82 (1H, dt, J = 4.8, 15.6; H-8a), δH 2.92 (1H, dt, J = 6.6, 15.6; H-8b), δH 3.27 (1H, m, H-9a), and δH 3.42 (1H, m; H-9b) overlapped with the water peak, in addition to a downfield broad singlet proton at δH 4.68 (1H, br s; H-10). Finally, a singlet peak at δH 10.68 (1H, s; NH-1) was inspected. On the other hand, the 13C NMR, DEPT spectra revealed the presence of 12 signals classified into five methine carbon signals at δC 117.9 (CH-4), δC 118.9 (CH-5), δC 121.4 (CH-6), δC 112.2 (CH-7), and δC 55.3 (CH-10); and five quaternary carbon signals at δC 125.8 (C-2), δC 104.2 (C-3), δC 128.6 (C-3a), δC 136.0 (C-7a), and δC 165.5 (C-11), in addition to two methylene carbons at δC 18.1 (CH2-8) and δC 41.2 (CH2-9). Investigation of the heteronuclear multiple bond correlation (HMBC) spectrum showed strong correlations between the δH 10.68 assignable to NH-1 and δC 104.2 (C-3); δC 128.6 (C-3a) confirmed the position of NH-1; and finally, the significant correlations between δH 2.82 for H-8a with δC 128.6 (C-3a), and between δH 2.92 corresponding to H-8b with δC 128.6 (C-3a),125.8 (C-2), confirmed the position of the aliphatic side chain at C-3. The HMBC spectrum showed the correlation between H-10 (δH 4.68) and the carboxylic carbon (C-11). The complete assignment of compound 1 was confirmed by investigation of heteronuclear single quantum coherence (HSQC), correlation spectroscopy (COSY), and HMBC as showed in Fig. 1c (Table 1). From the previous data, compound 1 (2-amino-4 (2-amino-1H-indole-3-yl) butanoic acid) was confirmed to be a new alkaloid, which we named Hyrtiodoline A.

Table 1 NMR-spectroscopic data of compound 1 in DMSO-d6 (1H: 600 MHz; 13C: 150 MHz)
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Compounds 2–5 were identified based on HSQC, HMBC, COSY spectra, HRESI-MS and in comparison with the literature [9, 13,14,15,16] as 4-hydroxy-1H-indole-6-carboxylic acid methyl ester, synthetic known dimer of indole-3-carbaldehyde, 3-methylene hydroxy β-carboline alkaloid and 2,3-dibromo-4-hydroxybenzaldehyde, respectively. The five compounds were tested against Trypanosoma brucei brucei, and for cytotoxicity against J774.1 macrophages. Antitrypanosomal activity after 48 and 72 h was detected for compound 1 with IC50 values of 15.26 and 7.48 μM, respectively, with no cytotoxicity against J774.1 macrophages (IC50 of >200 μM). However, the other compounds showed no activity against T. brucei.

Material and methods

General experimental procedures

1H, 13C, COSY, HSQC, and HMBC NMR spectra were recorded on a Bruker Avance III HD 600 (Bruker Daltonics, Bremen, Germany) instrument. Accurate electrospray ionization mass spectra (ESI) were obtained by a micrOTOF focus (Bruker Daltonics, Bremen, Germany. TLC was performed on TLC plates precoated with silica gel F254 (Merck, Darmstadt, Germany). HPLC separation and purification were performed on a Agilent Technologies Series 1100 (ALS, UV Detect00719, USA) on a semi-preparative RP-C18 column (5 µm, 10 × 250 mm, Waters XBridge, city, Germany). MR 700 Microplate Reader (optical density measurement) (Dynatech Engineering Ltd., Willenhall, UK). The NMR spectral data of compound 1 were acquired using a 600 MHz instrument: 1H, 13C, 13C-DEPT135, 1H-1H COSY, HSQC, and HMBC (optimized to J = 8.3 Hz) in DMSO-d6 (Table 1).

Sponge material

The sponge material used in this work was collected by Safwat Ahmed (Suez Canal University) from the Egyptian coasts of the Red Sea at Sharm el-Sheikh, using scuba diving at a depth of 9.14 m. The collected material was immediately frozen and kept at −20 °C until investigation. The sponge biomass was identified by van Soest (Institute of Systematic Population Biology, Amsterdam University, The Netherlands). A voucher specimen was kept in the collections of the Zoological Museum of the University of Amsterdam, under registration number ZMAPOR19762. Another voucher sample was deposited under the number SAA-61 at the Pharmacognosy Department, Faculty of Pharmacy, Suez Canal University, Egypt.

Extraction and isolation

The freeze-dried sponge was extracted exhaustively with methanol–methylene chloride 1:1. The resulting crude extract was first partitioned between H2O and EtOAc, followed by evaporation of the EtoAc layer and repartitioning of the aqueous fraction with butanol. The EtOAc-soluble material of Hyrtios (6 g) was subjected to vacuum-liquid ochrmatography on silica gel, using gradient elution with Pet-Ether:EtoAc then EtoAc 100% followed by methanol. The methanol fraction was subjected to silica gel column chromatography and eluted with a dichloromethane methanol (DCM–MeOH) gradient elution, followed by sephadex LH-20 (Merck, Bremen, Germany), and finally purified on semi-preparative HPLC using an acetonitrile (MeCN) and water solvent mixture complemented by 0.05% trifluoroacetic acid (10% MeCN/H2O to 100% MeCN over 30 min at a flow rate of 5 mL/min), to yield compound 3 (Rt = 17.877 min) and compound 5 (Rt = 16.8 min); while the butanol fraction was subjected to sephadex LH-20 CC using a MeOH–H2O gradient elution (10:100%), followed by final purification on semi-preparative HPLC using an acetonitrile (MeCN) and water solvent mixture complemented by 0.05% trifluoroacetic acid (10% MeCN/H2O to 100% MeCN over 30 min at a flow rate of 5 mL/min), to afford the three compounds 1 (Rt = 18.1 min), 2 (Rt = 16.4 min), and 4 (Rt = 17.1 min).

Antitrypanosomal assay

Antitrypanosomal activity [17, 18] was tested, following the protocol of Huber and Koella [19, 20].