Using UHPLC–MS profiling for the discovery of new sponge-derived metabolites and anthelmintic screening of the NatureBank bromotyrosine library

While the unique NatureBank HTS-compatible natural product screening libraries (>21K extracts and >105K fractions) [14] have been regenerated for biodiscovery collaborations with industry and academia over the past 5 years, the NatureBank open access compound library currently contains only ≈100 pure natural products. In an effort to expand this compound library, we have recently embarked on a program whereby a subset of the NatureBank marine and terrestrial extracts have been analysed by UHPLC–MS in order to determine those that contain abundant or potentially new natural products. One such example is the investigation into Verongid sponges; NatureBank holds 63 Verongida specimens that have been recently acquired from AIMS and of these, 39 have adequate material (≥10 g dry weight) for typical large-scale extraction and isolation work. Small-scale (300 mg) extraction of the abundant 39 sponge samples followed by UHPLC–MS and data analysis led to the prioritisation of several extracts for follow-up large-scale chemical investigations (see Figure S1 in Supporting Information File 1 for all UHPLC–MS chromatograms). One extract derived from the marine sponge Ianthella basta showed five UV active peaks (P1–5) at 254 nm in the UHPLC–MS chromatogram (Figure 1) with P1–3 and P5 displaying quasi-molecular ion clusters in the positive MS mode. Subsequent dereplication, literature and MarinLit database [1] mining tentatively identified P1 as a new monobrominated marine natural product with ions of equal relative intensity at m/z 380 and 382 [M + H]+. Complex ion clusters (abundance ratio: 1:4:6:4:1) at m/z 715/717/719/721/723 [M + H]+, 1014/1016/1018/1020/1022 [M + H]+ and 715/717/719/721/723 [M + H]+ associated with peaks two, three and five, respectively, were tentatively assigned as tetrabrominated metabolites. MS data of P4 was ambiguous and dereplication approaches were unable to successfully assign a tentative structure.

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Figure 1: UHPLC–UV chromatogram (254 nm) of the CH2Cl2/MeOH extract of Ianthella basta (NB6021519); retention times for major UV peaks are indicated.

Based on these data, large-scale extraction and mass-directed isolation studies were initiated. The freeze-dried and ground specimen of Ianthella basta (10 g) was sequentially extracted with n-hexane, CH2Cl2 and MeOH, and a portion of each extract was analysed by UHPLC–MS for the ions of interest. Subsequently, the CH2Cl2 and MeOH extracts were combined (which both contained targeted ion clusters) and subjected to reversed-phase C18 HPLC (MeOH/H2O/0.1%TFA) which led to the purification of the new metabolite, 5-debromopurealidin H (1) as its TFA salt along with the major previously reported metabolite, ianthesine E (2) (Figure 2). Comparison of the 1D NMR, MS and specific rotation data for compound 2 with literature values [15] identified this metabolite as ianthesine E, which had been previously identified from a Pseudoceratina sp. specimen collected from the Great Barrier Reef. 5-Debromopurealidin H and ianthesine E corresponded to peaks one and two in the UHPLC–MS trace, respectively, while metabolites associated with peaks 3–5 in the UHPLC–MS were unable to be obtained in sufficient purity or quantity for structure elucidation studies. Furthermore, insufficient quantities of the raw material of the I. basta specimen prevent further chemical investigation studies at this point in time.

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Figure 2: Chemical structures of 5-debromopurealidin H (1) and ianthesine E (2).

5-Debromopurealidin H (1) was isolated as a stable yellow film. The LRESIMS of 1 showed 1:1 ion cluster peaks at m/z 382/384 [M + H]+ and 380/382 [M − H]−, indicating the presence of one bromine atom [16]. Furthermore, a molecular formula of C14H17BrN5O3 was assigned following analysis of the 1D NMR data in conjunction with the HRESIMS ion at m/z 384.0487 [M + H]+. The 1H and COSY NMR spectra (Table 1) of 1 in DMSO-d6 revealed two distinct spin systems, which included a 1,3,4-trisubstituted aromatic ring [17,18], and an N-substituted ethylene system [19]. Remaining unassigned proton signals included a methylene singlet (δH 3.67), a downfield sp2 singlet (δH 6.55) and four exchangeable protons (δH 7.36, 8.07, 11.84, and 10.11). HMBC correlations from the methylene singlet (δH 3.67, δC 27.8) into the trisubstituted phenyl system and two downfield sp2 signals (δC 151.9 and 163.3), in conjunction with the HMBC data for both exchangeable signals at δH 7.36 and 11.84, and methylene moiety (δH 3.35, δC 39.4) to the sp2 signals (δC 151.9 and 163.3) allowed a N-oxime substructure to be assigned (see Figure 3).

Table 1: NMR data for the TFA salt of 5-debromopurealidin H (1) in DMSO-d6.a

position δC, type δH, mult (J in Hz) COSY HMBC ROESY 1 128.8, C         2 132.8, CH 7.27, d (2.1) 6 3, 4, 6, 7 7 3 108.9, C         4 152.4, C         4-OH   10.11, brs   3, 4b   5 116.2, CH 6.83, d (8.3) 6 1, 3, 4b 6 6 129.1, CH 6.99, dd (2.1, 8.3) 2, 5 2, 4, 7 5, 7 7 27.8, CH2 3.67, s   2, 6, 8, 9 2, 6 8 151.9, C         8-NOH   11.84, s   8   9 163.3, C         9-NH   8.07, t (6.0) 10 9b 10 10 37.2, CH2 3.35, dt (6.0, 6.9) 9-NH, 11 9, 12 9-NH, 11, 13 11 24.5, CH2 2.59, t (6.9) 10, 13 12, 13 10, 13 12 124.4, C         13 109.3, CH 6.55, s 11 12, 14 10, 11 14 146.8, C         14-NH2   7.36, brs      

a1H, 800 MHz; 13C, 200 MHz; bweak correlation.

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Figure 3: Key COSY, HMBC and ROESY correlations for 5-debromopurealidin H (1).

MarinLit database mining using this fragment readily identified that compound 1 belonged to the purealidin structure class, the majority of which all contain a terminal imidazole system. HMBC data from the one of the methylene protons (δH 2.59) that constituted the -NHCH2CH2- spin system to the imidazole carbons at δC 124.4 and 109.3 linked these fragments, while a strong HMBC correlation from the imidazole proton at δH 6.55 to the downfield carbon at δC 146.8 indicated a 2-amino substituted histamine moiety [19]. Finally, the unassigned exchangeable protons at δH 10.11 and 7.36, were assigned to phenol (4-OH) and amino groups (14-NH2), respectively, following comparison of NMR data with related previously reported purealidins [19]. While no HMBC correlations were identified for these exchangeable proton signals, the presence of a phenol was further supported by a bathochromic shift that was seen in the UV spectrum of 1 upon addition of base [20]. The bromine atom and phenol hydroxy group were positioned at C-3 and C-4 of the aromatic ring, respectively, based on NMR chemical shift data comparison with related marine natural products [19]. The E configuration for the oxime in 1 was assigned by the diagnostic carbon chemical shifts of the benzylic methylene (C-7, δC 27.8) [21]. Thus, the chemical structure 1 was assigned as 5-debromopurealidin H.

Due to our interest in discovering new anthelmintics from marine sources [22-26], we decided to test these compounds for their nematocidal activity against Haemonchus contortus, a highly pathogenic parasitic nematode of ruminants [27]. A structure-based search of the ≈100 NatureBank pure compound library identified seven previously reported bromotyrosines that were available in quantities that would enable in vitro anthelmintic evaluations. These compounds (Figure 4) included psammaplysins F (3) and H (4), bastadins 4 (5), 8 (6) and 13 (7), aerothionin (8) and hexadellin A (9). The extraction and isolation of these known compounds has been previously reported elsewhere [11,28-30]

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Figure 4: Chemical structures of the NatureBank bromotyrosine derivatives: psammaplysins F (3) and H (4), bastadins 4 (5), 8 (6) and 13 (7), aerothionin (8) and hexadellin A (9).

Compounds 19 were assessed for their activities and potencies in a dose-response assay by measuring the inhibition level on larval motility after 72 h of exposure (Figure 5). Compound 6 was the most potent compound at inhibiting exsheathed third-stage larvae (xL3) motility after 72 h, with an IC50 value of 1.6 ± 0.4 µM (65.2 ± 4.7% inhibition at 100 µM). Two other compounds showed relatively high potency: compounds 9 (IC50 = 10.0 ± 1.9 µM; 47.7 ± 9.5% inhibition at 100 µM) and 5 (IC50 = 33.3 ± 4.7 µM; 49.0 ± 10.2% inhibition at 100 µM). Although not as potent, compounds 1 and 3 were active against xL3s and reduced motility at 100 µM (30.4 ± 16.4% and 35.5 ± 7.1% inhibitions, respectively). Compounds 4, 7 and 8 had no effects on xL3 after 72 h of exposure.

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Figure 5: Dose-response assessment of in vitro activity of compounds 19 against exsheathed third-stage larvae (xL3) of Haemonchus contortus. The potency of compounds 19 on the larval motility was assessed after 72 h of exposure by establishing potency (IC50). Two positive controls, monepantel and moxidectin, were included as active compound references. Data points represent three independent experiments conducted in triplicate: the mean ± standard error of the mean (SEM).

None of the bromotyrosines tested in these studies had previously been tested for anthelmintic activity, however, several of them have been shown to display a variety of biological activities. For example, some ianthesines have been shown to display Na,K-ATPase inhibitory activity [31] and antimicrobial activity [32]; ianthesine E has rarely been tested for bioactivity, however, some weak binding has been identified for this molecule against human adenosine A1 receptor 19, along with some weak cytotoxicity towards HeLa cells [15]. Limited testing has also been conducted on the purealidin bromotyrosine structure class, however, quorum sensing inhibition and antifouling activities against several strains of bacteria and microalgae appears in the literature [33,34], while HIV-1 replication inhibition and anti-Leishmania and Plasmodium activity has also been recorded [35,36]. The psammaplysin structure class has also had antimalarial [28], cytotoxicity [37] and antimicrobial data reported, albeit with low to moderate potencies [7]. More recently psammaplysin F and several semi-synthetic analogues have been shown to cause loss of mitochondrial membrane potential, fragmentation of the mitochondrial tubular network, chromosome misalignment, and cell cycle arrest in mitosis in LNCaP prostate cancer cells [38]. The bastadin structure class is well-documented within the literature for their cytotoxic activity [37,39-41], with both bastadins 4 and 8 exhibiting in vitro cytotoxicity against leukemia cell line L-1210 (ED50 5 µg/mL) [39]. Aerothionin has been previously shown to exhibit antimycobacterial activity against monoresistant variants of Mycobacterium tuberculosis H37Rv, leading to further reported activity in various M. tuberculosis clinical isolates as well as non-tuberculosis mycobacteria [42]; while antitumour [43] and antifouling [44] activities have also been published.

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