The marine environment covers over two thirds of the earth’s surface and it encompasses a wide range of complex ecosystems that are highly variable in their physical attributes including pressure, salinity, temperature, and light availability. Both flora and fauna have evolved over billions of years to survive within this unique environment , which has led to the production and diversification of unique and novel metabolites with specialised biological functions . The richest diversity of marine metabolites are derived from invertebrates; most of which are sessile and lack the ability to physically defend themselves from both predators and competitors and thus rely on chemical mechanisms for their defense . Metabolites derived from marine sponges contribute more than half of all compounds identified from marine invertebrates , therefore it is no surprise that sponges are highly sought after for novel bioactive metabolites and have been a major focus of marine natural product drug discovery for over 70 years.

The identification of ianthelliformisamines A–C from the Australian marine sponge Suberea ianthelliformis, which displayed activity against both Pseudomonas aeruginosa and Staphylococcus aureus, has contributed to a surge in the interest of polyamines as new antibacterial leads . To date the total synthesis of ianthelliformisamines A–C (1–3) has been described and numerous synthetically related analogues have been published with their antibiotic assessment against numerous bacterial species reported . Our recent publication showed that of the naturally occurring metabolites, compound 3 inhibits the formation of P. aeruginosa PAO1 biofilms (MIC 53.1 µg/mL), whilst 1 and 2 enhanced the antibiotic effect of ciprofloxacin when used in combination . In efforts to further examine the chemistry of S. ianthelliformis and potentially discover new antibiotic leads, we undertook a scaled-up chemical investigation on the original specimen from which ianthelliformisamines A–C were isolated.

Herein, we describe the large-scale extraction, isolation, and structure elucidation of four new metabolites, ianthelliformisamines D–G (4–7). Additionally, we report the isolation of the known natural products, aplysterol (8) and ianthelliformisamines A–C (1–3). Owing to the novelty of ianthelliformisamine D (4) we undertook the first total synthesis of this natural product, which was successfully achieved in only three steps and respectable yield. All newly identified metabolites were subsequently assessed for their ability to inhibit the growth of planktonic P. aeruginosa PAO1 and formation of biofilms.

Figure 1: Chemical structures of ianthelliformisamines A–G (1–7) and aplysterol (8).

Table 1: NMR data of ianthelliformisamines D (4) and E (5) in DMSO-d6.a

aSpectra recorded at 25 °C (800 MHz for 1H NMR and 200 MHz for 13C NMR); bisolated as a TFA salt; cnot observed.

Figure 2: Key COSY (), HMBC () and ROESY () correlations for ianthelliformisamines D (4) and E (5).

Table 2: NMR data of ianthelliformisamines F (6) and G (7) in DMSO-d6.a

aSpectra recorded at 25 °C (800 MHz for 1H NMR and 200 MHz for 13C NMR); bisolated as a TFA salt.

Figure 3: Key COSY () and HMBC () correlations for ianthelliformisamines F (6) and G (7).

Compounds 6 and 7 are both commercially available but neither of these compounds has been fully characterised with only the total synthesis of 7 being reported in the literature . Our study reports the first identification of 6 and 7 from a natural origin and the first full characterisation of these molecules using NMR, UV, and MS data.

Scheme 1: Total synthesis of ianthelliformisamine D (4).

Due to our interest in the identification of potential new leads against Pseudomonas aeruginosa and supported by numerous reports of favourable activity for ianthelliformisamines A–C and their synthetic analogues , we investigated the planktonic and biofilm activity of the new natural products 4–7 in addition to the known metabolite, aplysterol (8) . Our biological assessment of compounds 4–8 showed no inhibition of planktonic growth or biofilm formation for P. aeruginosa when screened at 50 µM. Previously reported antibacterial assessment of ianthelliformisamines A–C (1–3) and their synthetic analogues has generated structure–activity relationship data, leading to speculation over the moieties responsible for their antibiotic effects. A reduction in the number of amines in the polymeric chain and the absence of a primary amine was noted to decrease bioactivity by Xu et al. . Research reported by Khan et al. in 2014 also supported the important role that the polyamine chain length plays in antibacterial activity. Of note, increasing research providing structure–activity relationship analyses shows that polyamines (including spermine) are bacterial membrane disruptors and are beneficial in enhancing activity of known antibacterial agents . The absence of polyamine chains in ianthelliformisamines D–G (4–7) could explain the loss of activity seen in our assessment against P. aeruginosa. The synthetic molecule of 7 has been tested by other researchers against Gram-negative bacteria including P. aeruginosa with reported MIC values of >200 µg/mL , which is consistent with the data we report in this paper.

Finally, and of note, our studies described here report the first isolation of aplysterol (8) from the genus Suberea. To date, there has been no evaluation of this compound’s antibiotic potential towards P. aeruginosa, including biofilm inhibition. Furthermore, this is the first report of any biological assessment of aplysterol (8) as a pure compound, since prior studies have only tested a mixture of 8 with 24,28-didehydroaplysterol, where it was found to inhibit DNA topoisomerase II-α (MIC = 50 µM) .

In summary, we report here the discovery and full characterisation of four new natural products, ianthelliformisamines D–G from the marine sponge Suberea ianthelliformis. Furthermore, we describe the first total synthesis of ianthelliformisamine D which contains a rare N-(3-aminopropyl)-2-pyrrolidone moiety. Whilst testing of these natural products against Pseudomonas aeruginosa showed that none of them inhibited planktonic growth or biofilm formation at 50 µM, synthetic efforts has generated sufficient quantities of the novel compound ianthelliformisamine D that will enable additional biological profiling.

Specific rotations were recorded using a JASCO P-1020 polarimeter. UV data was recorded on a JASCO V-650 UV–vis spectrophotometer. NMR spectra were recorded at 25 °C on a Bruker Avance III HD 800 MHz NMR spectrometer equipped with a cryoprobe. The 1H and 13C chemical shifts were referenced to solvent peaks for DMSO-d6 at δH 2.50 and δC 39.52, respectively. LRESIMS data was recorded on an Ultimate 3000 RS UHPLC coupled to a Thermo Fisher Scientific ISQEC single quadruple ESI mass spectrometer. HRESIMS data was acquired on a Bruker maXis II ETD ESI-qTOF. Alltech Davisil (30–40 µm, 60 Å) C8-bonded silica and Alltech Davisil (30–40 µm, 60 Å) diol-bonded silica were used for pre-adsorption work before RP- or NP-HPLC, respectively. The pre-adsorbed material was subsequently packed into an Alltech stainless steel guard cartridge (10 × 30 mm) then attached to a HPLC column prior to fractionation. A Waters 600 pump fitted with a Waters 996 photodiode array detector fitted with a Gilson 717-plus autosampler were used for RP-HPLC separations. A Thermo Fisher Scientific Dionex Ultimate 3000 UHPLC was used for NP-HPLC separations. Thermo Betasil phenyl-bonded silica (5 μm, 100 Å, 150 × 21.2 mm) and Phenomenex Luna C18 column (5 µm, 90–110 Å, 10 mm × 250 mm) were used for RP-HPLC separations. For NP-HPLC, a YMC diol-bonded silica (5 μm, 120 Å, 150 × 20 mm) column was used. The frozen marine sponge was dried using a Dynamic FD12 freeze dryer and ground using a Fritsch Universal Cutting Mill Pulverisette 19. The ground marine sponge was extracted at room temperature using an Edwards Instrument Company Bioline orbital shaker set to 200 rpm. Solvents were removed from extracts with a Büchi R-144 rotary evaporator and from HPLC fractions using a GeneVac XL4 centrifugal evaporator. All solvents used for chromatography, UV, MS, and [α]D were Honeywell Burdick & Jackson or Lab-Scan HPLC grade. H2O was filtered using a Sartorius Stedium Arium Pro VF ultrapure water system. Reagents used for the total synthesis of Ianthelliformisamine D were purchased from either Sigma or Aaron Chemicals.

The sponge sample was obtained from the NatureBank biota library housed at the Institute for Biomedicine and Glycomics, Griffith University, Australia. A voucher specimen of Suberea ianthelliformis (NB6014998; phylum Porifera, class Demospongiae, order Verongida, family Aplysinellidae) has been previously lodged (G322255) at the Queensland Museum, South Brisbane, Queensland, Australia .

In a manner similar to that reported by Xu et al. , the freeze-dried and ground specimen of S. ianthelliformis (10 g) was extracted sequentially with n-hexane (250 mL, 2 h), CH2Cl2 (250 mL, 2 h), and MeOH (250 mL, 2 h; 250 mL, 16 h). The n-hexane extract was discarded (as it contained only highly lipophilic material) and the CH2Cl2 and MeOH extracts were combined to produce a brown gum (173.3 mg) that was pre-adsorbed to C8-bonded silica (≈1 g), packed into a stainless-steel guard cartridge, and subjected to phenyl semipreparative RP-HPLC separation. Isocratic conditions of 90% H2O (0.1% TFA)/10% MeOH (0.1% TFA) were initially employed for the first 10 min, then a linear gradient to 100% MeOH (0.1% TFA) was run over 40 min, followed by isocratic conditions of 100% MeOH (0.1% TFA) for a further 10 min, all at a flowrate of 9 mL/min; 60 fractions (60 × 1 min) were collected. This first HPLC fractionation afforded ianthelliformisamine B (2, 10.4 mg, tR 36–37 min, 0.104% dry wt) and several other UV-active fractions that contained mixtures of polyamine-type alkaloids, which were combined for further isolation work. The following describes the purification of these fractions. Fractions 34–35 (5.0 mg) were purified by semipreparative phenyl RP-HPLC using isocratic conditions of 70% H2O (0.1% TFA)/30% MeOH (0.1% TFA) for the first 10 min, then a linear gradient to 10% H2O (0.1% TFA)/90% MeOH (0.1% TFA) was run over 50 min at a flowrate of 9 mL/min; 60 fractions (60 × 1 min) were collected and resulted in the purification of ianthelliformisamine A (1, 1.0 mg, tR 16 min, 0.010% dry wt). Fractions 38–40 were combined (45.0 mg) and purified by semipreparative phenyl RP-HPLC using the same solvent gradient as above and afforded the new natural products, ianthelliformisamines G (7, 1.2 mg, tR 19 min, 0.012% dry wt) and E (5, 1.5 mg, tR 23 min, 0.015% dry wt). Fractions 41–42 were combined (22.4 mg) and purified by semipreparative RP-HPLC using the same solvent gradient and afforded the new natural products, ianthelliformisamines F (6, 1.2 mg, tR 19 min, 0.012% dry wt) and D (4, 1.3 mg, tR 41 min, 0.013% dry wt). Fractions 43–45 were combined (28.7 mg) and purified by semipreparative RP-HPLC using the same solvent gradient and afforded the known metabolite, ianthelliformisamine C (3, 1.8 mg, tR 38 min, 0.018% dry wt). Fractions 46–60 (20.1 mg) was purified by semipreparative NP-HPLC. Isocratic conditions of 100% n-hexane were initially employed for the first 10 min, then a linear gradient to 80% n-hexane/20% iPrOH was run over 50 min at a flow rate of 9 mL/min; 60 fractions (60 × 1 min) were collected and resulted in the purification of the known sterol, aplysterol (8, 0.9 mg, tR 12–13 min, 0.009% dry wt). The extraction and isolation process described above was repeated twice more (identical scale) to obtain sufficient quantities of the minor natural products for characterisation and biological assessment with similar recoveries obtained.

3,5-Dibromo-4-hydroxybenzaldehyde (279.0 mg, 1.0 mmol) was dissolved in CH2Cl2/MeOH 1:1 (1 mL) then a solution of TMSCHN2 in hexanes (2.0 M, 1.5 mL, 11 mmol) was slowly added and the mixture stirred at room temperature for 6 h. The reaction crude was pre-adsorbed to C8-bonded silica, packed into a stainless steel guard cartridge then purified by Luna C18 semipreparative RP-HPLC using isocratic conditions of 50% H2O (0.1% TFA)/50% MeOH (0.1% TFA) for the first 5 min, then a linear gradient to 100% MeOH (0.1% TFA) was run over 20 min and held at 100% MeOH (0.1% TFA) for a further 5 min at a flowrate of 4 mL/min; 30 fractions (30 × 1 min) were collected and resulted in the purification of 3,5-dibromo-4-methoxybenzaldehyde (9, 50.1 mg, tR 18–22 min, 17% yield) .

3,5-Dibromo-4-methoxybenzaldehyde (9): White powder; 1H NMR (DMSO-d6, 800 MHz) δH 9.89 (s, 1H, H-7), 8.17 (s, 2H, H-2, H-6), 3.88 (s, 3H, 4-OCH3); 13C NMR (DMSO-d6, 200 MHz) δC 190.1 (C-7), 158.1 (C-4), 134.5 (C-1), 133.8 (2C, C-2, C-6), 118.6 (2C, C-3, C-5), 60.8 (4-OCH3); LRESIMS (m/z): 293/295/297 [M + H]+.