Lipoamicoumacin A (1) was obtained as a white amorphous solid. Based on HRESIMS (m/z 703.3921 [M + H]⁺) data we established its molecular formula as C₃₆H₅₄N₄O₁₀, indicating twelve degrees of unsaturation. The IR absorptions at 3432, 1667 and 1545 cm⁻¹ in association with the ¹³C NMR spectroscopic data suggested the presence of hydroxyl, carbonyl and aromatic groups. The ¹H NMR spectra exhibited three aromatic proton singlets for a tri-substituted aromatic unit at δ_H 7.47 (1H, dd, H-6), 6.85 (1H, d, H-7) and 6.81 (1H, d, H-5), three oxymethines at δ_H 4.79 (1H, t, H-9'), 4.68 (1H, dd, H-3) and 4.42 (1H, d, H-8'), three nitrogen-bond methines at δ_H 4.51 (1H, dt, H-10'), 4.65 (1H, dd, H-15') and 4.29 (1H, m, H-5'), a broad peak at δ_H 1.30 (10H, brs, H4''~8'') and four methyl resonances at δ_H 0.97 (3H, d, H-2'), 0.89 (3H, d, H-1') and 0.88 (6H, d, H-11'', H-12''). The ¹³C NMR spectra provided five ester/amide carbonyl resonances at δ_C (171.2–178.1), six aromatic carbons for a trisubstituted aromatic ring, three oxymethines and three nitrogen-bonded sp³ carbons. In addition, the UV spectrum was nearly identical to that reported for amicoumacins (λ_max 206, 247 and 314 nm) [8], suggesting that 1 possessed a similar dihydroisocoumarin chromophore. The gross structure of 1 was further established by analyses of the ¹H, ¹³C, ¹H–¹H COSY, HMQC and HMBC NMR spectral data (Figure 1 and Figure 2), indicating an amicoumacin unit was linked to a fatty acid chain. The NMR spectroscopic data of 1 were highly similar to those from the combination of amicoumacin C (9) and lipoamide B [8,11] with the exceptions of the Δδ = 1.6 ppm upfield shift of C-10' (δ_C 48.6) and Δδ = 0.23 ppm downfield shift of H-10' (δ_H 4.51). The differences were due to the linkage of the lipoamide group at C-10' in compound 1 when comparing it with 9. These assignments were also supported by HMBC correlations from H-10' to C-14' and ESI-MS fragmentations (Figure 3). The ESI-MS spectrum showed fragment ions corresponding to the loss of an acyl group from the iso C₁₂ fatty acid side strain (m/z 521) and sequential loss of asparagine (Asn) (m/z 407) and dihydroisocoumarin fragment ion (m/z 250). The relative configuration of 1 was determined by NOE correlations of H-3/H-4' and H-5'/H-8', which shares similar stereochemical configurations with amicoumacin C (9). To further confirm the absolute configuration of the dihydroisocoumarin unit, the CD spectrum of 1 and 9 was compared. The CD spectrum of 1 showed positive Cotton effects at 221 and 240 nm and negative Cotton effects at 260 and 310 nm, which were in good agreement with those for compound 8 and 9 [9,10]. Thus, the structure of 1 was determined as illustrated in Figure 1.

The molecular formula of lipoamicoumacin B (2) was established as C₃₇H₅₆N₄O₁₀, based on the HRESIMS (m/z 717.4062 [M + H]⁺, calcd. 717.4069), which was 14 amu higher than that of 1. The NMR spectroscopic data of 2 (Supporting Information, Table 1) indicated that its structure was closely related to that of 1. The only difference was the presence of an additional methyl group (δ_H 0.86, δ_C 11.9) in 2, located at C-11''. HMBC correlation between H-12'' and C-10'' and COSY correlations from H-12'' to H-11'', H-11'' to H-10'' and H-10'' to H-13'' confirmed that the structure of 2 was a C-11'' methylated analog of 1, seen also with the ESIMS fragmentation analysis.

Lipoamicoumacin C (3) had a molecular formula of C₃₇H₅₆N₄O₁₀, which was established by the HRESIMS data (m/z 717.4062 [M + H]⁺, calcd. 717.4069). The NMR data of 3 (Supporting Information, Table 1) were comparable to those of 1, with the exception of an additional methylene at δ_H 2.25 (2H, t, H-17') and the Δδ = 3.1 ppm downfield shift of C-18' (δ_C 177.8). Analysis of ESIMS fragmentation ions (Figure 3) revealed the presence of a glutamine group (∆m/z 535–407) in 3, instead of an asparagine group (∆m/z 521–407) in 1. The suggestion was further confirmed by HMBC correlations of H-10'/C-14', H-15'/C-1'' and ¹H–¹H COSY correlations of H-17'/H-16' and H-16'/H-15'.

Lipoamicoumacin D (4) had a molecular formula of C₃₈H₅₈N₄O₁₀, which was established from the HRESIMS (m/z 731.4215 [M + H]⁺, calcd. 731.4222). The molecular weight of 4 was also 14 amu higher than that of 3. The NMR and MS spectroscopic data of 4 indicated that its structure is nearly identical to that of 3, with the exception of an additional methyl group located at C-11'' in 4.

Bacilosarcin C (5) had a molecular formula of C₂₄H₃₄N₂O₉, which was established from the HRESIMS (m/z 495.2344 [M + H]⁺, calcd. 495.2337). The ¹H NMR spectrum exhibited three aromatic proton singlets for one aromatic spin system at δ_H 7.46 (1H, dd, H-6), 6.85 (1H, d, H-7) and 6.82 (1H, d, H-5), three oxymethines at δ_H 4.68 (1H, dd, H-9'), 4.66 (1H, dd, H-3) and 4.14 (1H, d, H-8'), two nitrogen-bonded methines at δ_H 4.34 (1H, dt, H-5'), 3.91 (1H, m, H-10') and 3.43 (1H, q, H-14'), two methylenes at δ_H 3.10 (1H, dd, H-4) and 2.96 (1H, dd, H-4), δ_H 3.06 (1H, dd, H-11') and 2.92 (1H, dd, H-11'), and four methyl resonances at δ_H 1.28 (3H, d, H-13'), 1.26 (3H, s, H-16'), 0.98 (3H, d, H-2') and 0.94 (3H, d, H-1'). The UV absorptions at λ_max 206, 247 and 314 nm in association with the NMR data suggested that compound 5 also possesses an amicoumacin unit. The NMR data of 5 (Supporting Information, Table 2) were mostly the same as bacilosarcin B (6) [10], except for the Δδ = 0.20 ppm downfield shift of H-11' (δ_H 3.06). The difference was due to the linkage of a hydroxyl group (5) as opposed to an amide group (6) at C-12'. These assignments were also supported by molecular composition and a HRESI-MS fragment ion (m/z: 477.2238 [M − H₂O + H]⁺) corresponding to the intermolecular dehydration between C-9' and C-12'. The relative configurations of 5 were determined by the NOE correlations of H-4'/H-9', H-12'/H-8' and H-12'/14', which were in good agreement with those for compound 6. The stereochemistry of 5 was assigned to be the same as that of 6 based on their similar Cotton effects observed in the CD spectrum and similar levo specific rotation.

Based on spectroscopic analyses and the comparison of their spectroscopic data with those reported in the literature, six known compounds were identified as bacilosarcin B (6) [10], amicoumacin A–C (7–9) [6,9], 6-[[1-(3,4-dihydro-8-methoxy-l-oxo-1H-2-benzopyran-3-yl)-3-methylbutyl]amino]-4,5-dihydroxy-6-oxo-3-ammoniohexanoate (10) and AI-77-F (11) [8].

All isolated compounds were evaluated for their cytotoxicity against human cervical carcinoma epithelioid cell line HeLa using MTT methods, and for their antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Loktanella hongkongensis, Peseudomonas aeruginosa and Chromobacterium violaceum by MIC assay .Only compounds amicoumacin A (7) and bacilosarcin B (6), which have an amide functional group at C-12', exhibited cytotoxicity against the HeLa cells with IC₅₀ values of 33.60 and 4.32 μM respectively, indicating that the C-12' amide group of amicoumcin plays a critical role in cytotoxicity. This was further supported by comparision of cytotoxicity between compounds 7 and 8 and between compounds 5 and 6. Compound 7, with an IC₅₀ value of 4.32 μM showed 30-fold higher cytotoxicity than 8. Previous research also reported that C-12 amide groups of amicoumacin played a critical role in antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) [12], which was based on the comparison of amicoumacin A (7) and amicoumacin B (8). Herein, we studied the antibacterial structure-activity relationships based on 11 amicoumacins against three target bacterial strains. Our results indicate that only those compounds with C-12' amide groups exhibited antibacterial activities against B. subtilis, S. aureus and L. hongkongensis (Supporting Information, Table 3), which strongly supported that the C-12' amide group of amicoumcin acts as a pharmacophore in antibacterial activities. This conclusion was further supported by comparing antibacterial activities between compounds 7 and 8 and between compound 5 and 6. Compound 7 exhibited antibacterial activities against B. subtilis, S. aureus and L. hongkongensis, which were about six-fold higher than those of compound 8.

While microbial secondary metabolites have been extensively used as antimicrobial or antitumor therapeutic agents, the biological functions of the metabolites in microbes that produce them have only been studied in a few cases. For instance, xenocoumacin-1, the major antimicrobial compound produced by Xenorhabdus nematophila, was shown to suppress their competitors of the host and subsequently convert xenocoumacin-2, which has a much weaker antimicrobial activity, to avoid self-toxicity [13]. In Bacillus genus, surfactins were produced to serve as signaling molecules to activate the pathway related to biofilm information [14], while sporulenes was shown to serve as a chemical barrier against oxidative stress [15]. However, the biological role of amicoumacins in the life cycle of B. subtillis is far from being understood. It will therefore be intriguing to investigate the chemical ecology of amicoumacins in the future.

Optical rotations were recorded on an Autopol III Rudolph research automatic polarimeter, whereas IR spectra were obtained on a Thermo Nicolet Nexus 470 FT-IR spectrometer. NMR spectra were measured on a Bruker Avance-500/700 FT 500/700 MHz NMR spectrometer using trimethylsilane (TMS) as an internal standard. CD spectra were recorded on J-810-150s spectropolarimeter (Jasco, Germany). HRESIMS spectra were recorded on a Bruker micrOTOF II ESI-TOF-MS spectrometer. Sephadex LH-20 (18–110 μm) was purchased from Pharmacia Co (Japan), whereas octadecylsilane (ODS) (50 μm) was purchased from YMC Co. (Japan). HPLC was performed on a Waters 600 apparatus using a semi-preparative C-18 Phenomenex Luna 5 μm (10 mm × 250 mm) column and monitored by a UV detector (Waters 2475).

The Bacillus subtilis strain B1779 was isolated from a marine sediment sample which was collected by a multicore sampler at a depth of 1000 m in the Red Sea, in April 2010. This strain was isolated by a standard dilution-plating technique. Bacteria in the sediment were released by vigorous shaking in autoclaved filtered seawater and inoculated in a serial dilution on 1.5% agar composed of 10 g starch and 2 g yeast extract dissolved in 1 L filtered seawater. According to its 16S rRNA gene sequence (accession number: HM585050.1), this strain belongs to Bacillus subitlis group. A voucher strain of this Bacillus subtilis strain has been preserved at the Costal Marine Lab, Hong Kong University of Science and Technology. Genomic DNA was extracted from the cells using the TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, China). The 16S rRNA gene of B1779 was amplified by a pair of primers of 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1525R (5'-AAGGAGTGWTCCARCC-3') with Vent DNA polymerase (NEB) and sequenced using Applied Biosystems 3100 automated DNA sequencer, as previously described [16,17]. The resulting 16S rRNA gene sequence was compared with sequences obtained from the NCBI nucleotide database using BLAST search [18] to locate approximate phylogenetic affiliation.

The bacterium was cultured in multiple 3 L flasks containing SYT culture medium (1% of starch, 0.4% of yeast extract and 0.2% tryptone) in seawater at 25 °C with agitation (160 rpm) for 4 days until the stationary phase was reached. In total, 20 L of fermented broth were obtained. The bacterial culture broth was extracted with an equal volume of ethyl acetate (EtOAc) three times and gave a total of 11 g of crude extract.

The crude EtOAc extract (11.0 g) was fractionated by ODS open column chromatography eluting with a step gradient from 40% methanol in water to 100% methanol to give six fractions (F1–F6). UPLC-MS analysis and cytotoxicity bio-assay showed that bioactive isocoumacins were mainly concentrated in fractions 3 and 4 (named as F3 and F4). F3 (0.39 g) was separated by semi-preparative HPLC with a gradient mobile phase (MeCN-H₂O, from 30 % to 50 %) to obtain 5 (3.6 mg), 6 (4.7 mg), 7 (25.0 mg), 8 (11.5 mg), 9 (18.0 mg), 10 (22 mg) and 11(6.2 mg), respectively. F4 (0.42 g) was subjected to Sephadex LH-20 and subsequently separated with semi-preparative HPLC eluting with a gradient mobile phase (MeCN-H₂O, from 60% to 80%) to yield the compounds 1 (1.9 mg), 2 (1.6 mg), 3 (3.5 mg) and 4 (4.8 mg), respectively.

Human HeLa cells were used in the assay; 80 μL of 1 × 10⁵/mL cells were planted into 96-microwell plates. Twelve hours later the cells were treated with testing samples at various concentrations and then incubated at 37 °C for 48 hours. Afterward, the supernatant was removed and 20 μL of MTT (2.5 mg mL⁻¹) were added to each well. After incubation at 37 °C for 4 hours, 100 μL of dimethyl sulfoxide (DMSO) were added to each well and incubated for an additional 20 min. The absorbance of each well was then measured at 570 nm by a Thermo scientific Multiskan FC multiplate photometer (Waltham, MA, USA).

The antibacterial activities of compound 1–11 were evaluated by MIC assay against Staphylococcus aureus, Bacillus subtilis, Loktanella hongkongensis, Peseudomonas aeruginosa and Chromobacterium violaceum. Briefly, the bacterial strains were inoculated in YP broth (0.2% yeast extract, 0.1% peptone and 1.7% sea salts) and were incubated at 28 °C for 12 h. The stock solution of samples were prepared at 50 mg/mL in DMSO and further diluted to varying concentrations in 96 well plates that contained the incubated microbial strains. After incubation at 28 °C for 24 hours, the absorbance of each well was then measured at 600 nm by a Thermo scientific Multiskan FC multiplate photometer.