Capoamycin (1, Figure 1) was originally isolated from the culture broth of soil Streptomyces capoamus strain No. 23–41 [1]. Its characteristic structure consists of a modified benz[a]anthraquinone chromophore, a deoxysugar unit and a long chain polyene acid. Capoamycin has been reported to inhibit the growth of gram-positive bacteria, yeasts and fungi, induce differentiation of mouse myeloid leukemia cells, and prolong the survival periods of mice bearing Ehrich ascites carcinoma [1,2]. The capoamycin-related antibiotics included dioxamycin (2) from soil Streptomyces xantholiticus strain MH406-SF1 [3] and MK844-mF10 (5), which was recently discovered from soil Streptomyces sp. MK844-mF10 [4]. MK844-mF10 had activities against multidrug-resistant Staphylococcus aureus and vancomycin-intermediated Staphylococcus aureus and inhibited the activities of Bacillus subtilis YycG histidine kinase, Streptococcus mutans Vick histidine kinase, and Erwinia carotovora PehS histidine kinase [4].

During the course of our ongoing program for the discovery of novel bioactive substances from marine microorganisms, a crude extract prepared from the pellet of the cultured Streptomyces fradiae strain PTZ00025 isolated from marine sediments was shown to be active against Staphylococcus aureus. Bioassay-guided isolation of this active crude resulted in the isolation and identification of the known antibiotics dioxamycin (2) and MK844-mF10 (5), new compounds of fradimycins A (3) and B (4) and fradic acids A (6) and B (7) (Figure 1). Compounds 3–5 showed antibacterial and antitumor activities. In this study, the isolation and structure elucidation of these new compounds and their antibacterial and antitumor properties are described.

The antibiotic-producing strain PTZ0025 was isolated from marine sediments. The taxonomic identity of the isolated strain was determined by 16S rDNA sequence analysis. The top sequence match as of August 2012, using Basic Local Alignment Search Tool (BLAST) (nucleotide sequence comparison) and the GenBank database was a 99.0% sequence similarity to Streptomyces fradiae strain CO-4 (GenBank accession number JF915304.1).

An acetone extract prepared from the pellet of the cultured broth of this isolated strain had activity against Staphylococcus aureus. This active crude was fractionated by Diaion HP-20 column chromatography, following by repeated high-performance liquid chromatography (HPLC) purification (supplementary Figure S43) to furnish Compounds 2–7.

Compound 2 was proven to be identical to dioxamycin by comparison of its physico-chemical properties and spectral data with the reported values [3]. Dioxamycin has reported to have activities in vitro against gram-positive bacteria and some tumor cells [3].

Compound 3 was obtained as an orange amorphous powder and had a molecular formula of C₃₉H₄₂O₁₅ deduced from its high resolution electron spray ionization mass spectroscopy (HRESIMS) m/z at 751.2673 [M + H]⁺ and 773.2463 [M + Na]⁺, 14 mass units higher than that of 2. Compound 3 exhibited similar UV absorption to 2. The ¹³C and ¹H nuclear magnetic resonance (NMR) data of 3 (Table 1 and Table 2) were also very close to those of 2 with the exception of additional signals at δ_H 3.76 (3H, s) and δ_C 53.1 for a methoxyl in 3. An upfield chemical shift (∆δ −1.3 ppm) for C-12″ between 3 and 2 suggested the methoxyl at C-12″. A key heteronuclear multiple bond correlation (HMBC) of δ_H 3.76 (OCH₃) to δ_C 172.6 (C-12″) further supported the above-mentioned conclusion. The trans-coupling constants of 15.1 Hz (H-2″ and H-3″), 14.8 Hz (H-4″ and H-5″), and 15.0 Hz (H-6″ and H-7″) determined the double bond geometries of 2″E, 4″E and 6″E in 3. In the rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectrum of 3, the key nuclear Overhauser effect (NOE) correlations (Figure 2) of H-1′ with H-5′, H-4′ with H-6′, H-7″ with H-9″, H-8″ with H-10″, and H-9″ with H-13″ suggested that 3 had the assigned relative stereochemistry at C-1′, C-4′, C-5′, C-8″, C-9″, and C-11″, which were the same as their counterparts in 2 [3]. After alkaline hydrolysis, both 3 and 2 furnished a same product 2a, confirmed by their UV, [α]_D, and co-HPLC analysis, suggesting that 3 and 2 also possessed the same stereochemistry at C-2, C-3, C-4a and C-12b. The ¹H and ¹³C NMR signals of 3 were assigned by a combination of ¹H, ¹³C, heteronuclear multiple quantum correlation (HMQC), HMBC, and ROESY NMR spectroscopic analysis, and by comparison with the NMR data of 2. The structure of 3 was thus established as fradimycin A, a new marine natural product.

Compound 4 was obtained as an orange amorphous powder and had a molecular formula of C₃₈H₃₈O₁₄ as determined by its HRESIMS ions at m/z 719.2408 [M + H]⁺ and 741.2247 [M + Na]⁺. Its UV spectrum displayed maxima absorption at 294 and 427 nm. The ¹³C NMR data (Table 1) of 4 showed 38 signals, of which six were assigned to the 2,3-dideoxysugar moiety (ring E), 13 to the polyene acid unit (part F), and the remaining 19 to the anthraquinone chromophore (rings A–D). Detailed analysis of the NMR data of 4 and 2 concluded that both compounds shared identical substructures of ring E and part F.

The anthraquinone chromophore in 4 was constructed by NMR spectroscopic analysis and by comparison of its NMR data with those of its counterpart in 2. In the ¹H NMR spectrum (Table 2, in DMSO-d₆) of the anthraquinone part in 4, three aromatic proton signals at δ 7.66 (1H, s), δ 7.85 (1H, d, 7.8 Hz), and δ 7.54 (1H, d, 7.8 Hz) were attributed to H-6, H-10, and H-11, respectively. The three singlet signals at δ 4.02 (1H, s), 2.88 (2H, s), and 1.20 (3H, s) were assigned to H-2, H-4, and H-13, respectively. In addition, two singlets at δ 11.4 (1H, s) for 5-OH and δ 12.6 (1H, s) for 8-OH were also observed in the ¹H NMR spectrum of 4. The ¹³C NMR spectrum of 4 (in DMSO-d₆) displayed signals at δ 127.5 for C-4a and δ 136.1 for C-12b, instead of two signals at δ 76.1 and δ 76.8 for the two oxyquaternary carbons (C-4a and C-12b) as in 2. Above evidence suggested the existence of a double bond between C-4a and C-12b in 4, instead of the two hydroxyl groups at C-4a and C-4b as in 2. The double bond geometries for the polyene acid in 4 were assigned as 2″E, 4″E and 6″E based on the trans-coupling constants (Table 2). Unfortunately, ROESY experiment for 4 did not provide useful NOE information due to its limited amount for the assignment of the relative stereochemistry at C-2 and C-3 in 4. However, the relative stereochemistry at C-1′, C-4′, C-5′, C-8″, C-9″, and C-11″ in 4 was proposed to be the same as those in 2 because the NMR values for ring E and part F between 4 and 2 were almost the same (Table 1 and Table 2). After alkaline hydrolysis, both 4 and 2 produced the same compound 6 as confirmed by UV, [α]_D, and co-HPLC analysis, further supporting the assigned stereochemistry at C-8″, C-9″, and C-11″ in 4. The complete assignments of ¹H and ¹³C NMR data of 4 were made by HMQC and HMBC correlations (Figure 2). The structure of 4 was determined as fradimycin B, a new compound.

The molecular formula of C₃₈H₃₈O₁₃ for 5 was determined by HRESIMS and indicated that it had one less oxygen atom when compared with 4. Its UV spectrum and [α]_D value were very similar to those of 4. Examination of the NMR data of 5 and 4 showed that 5 differed from 4 only in the substituent at C-5 of ring B. In the NMR spectra, the signals at δ_C 159.5 (C, C-5), δ_C 112.0 (CH, C-6), and δ_H 7.66 (1H, s, H-6) in 4 were replaced by the signals at δ_C 134.1 (CH, C-5), δ_C 128.7 (CH, C-6), δ_H 7.71 (1H, d, J = 8.0 Hz, H-5), and δ_H 8.23 (1H, d, J = 8.0 Hz, H-6) in 5. The above information demonstrated that 5 was related to 4 without the hydroxyl group at C-5. Accordingly, the structure of 5 was assigned as MK844-mF10 that was recently isolated from Streptomyces sp. MK844-mF10 [4].

Compounds 6 and 7 had molecular formulae of C₁₃H₁₆O₆ and C₁₄H₁₈O₆, respectively, deduced from their HRESIMS and ¹³C NMR data. Both compounds contained characteristic NMR signals of two methyls, two oxymethines, one oxyquaternary carbon, six olefinic carbons, and two carbonyls. The ¹³C and ¹H chemical shifts of 6 (Table 3) were very close to those of the polyene acid unit (part F) in 2 (Table 1 and Table 2, in DMSO-d₆). The ¹³C chemical shift differences for C-1 (∆δ +2.0 ppm), C-2 (∆δ +1.6 ppm), and C-3 (∆δ −1.0 ppm) between 6 and the part F of 2 accounted for the existence of a free carboxylic acid at C-1 in 6. The assigned structure of 6 was also confirmed by its [α]_D and co-HPLC analysis with an authentic sample, the polyene acid obtained from a product of alkaline hydrolysis of 2. The ¹H and ¹³C NMR signals of 6 were assigned using HMQC and HMBC correlations (Table 3). Therefore, the structure of 6 was assigned as fradic acid A.

The NMR data of 7 were almost superimposable on those of 6 with the exception of additional signals at δ_C 52.3 and δ_H 3.70 (3H, s) for a methoxyl. A chemical shift difference (Δδ −2.1 ppm) for C-1 (δ 165.5) of 7 when compared to C-1 (δ 167.6) of 6 indicated that the methoxyl was linked to C-1 position. Both 7 and 6 had very similar UV spectra and [α]_D values. Therefore, the structure of 7, a new compound, was established as fradic acid B.

It was possible that the isolates 3, 6, and 7 were artifacts during the work-up isolation. In order to exclude this possibility, an ethanol extract prepared from the pellet of the isolate strain PTZ0025 was analyzed by co-HPLC with authentic samples using acetonitrile/water without acid as flow phase. The results indicated that compounds 3, 6, and 7 as new natural products were detected in the original extract (supplementary Figure S43).

Compounds 3–7 were assayed in vitro for their antimicrobial activity against Staphylococcus aureus. The results showed that compounds 3–5 were active against Staphylococcus aureus with a MIC of 6.0, 2.0, and 4.0 μg/mL, respectively, whereas 6 and 7 were inactive.

Compounds 3–5 were also tested in vitro for their cell-growth inhibitory activity against human colon cancer HCT-15 and SW620 cells as well as rat glioma C6 cells. All compounds showed dose-dependence activities of inhibiting growth of the tested tumor cells with IC₅₀ values ranging from 0.13 ± 0.04 to 6.46 ± 1.44 μM (Figure 3 and Table 4).

Cell cycle arrest and induction of apoptosis by fradimycin B (4) in HCT-15 cells was further investigated. To determine if an alteration of the cell cycle occurred following the treatment of fradimycin B (4), the DNA content was measured by flow cytometric analysis. The percentages of each phase in the cell cycle are represented in Figure 4 and Table 5. The proportion of cells in the G₀/G₁ phase of the cell cycle increased 4.34%, 5.89%, 10.75%, and 21.91% after 3 h, 6 h, 12 h, and 24 h exposure to 1.25 μM fradimycin B (4). The alteration occurring in the cell cycle suggested that fradimycin B (4) blocked HCT-15 cells at the G₀/G₁ phase.

After 72 h of treatment, fradimycin B (4, 0.625 and 1.25 μM) induced apoptosis and necrosis in HCT-15, SW620 and C6 cells as detected by Hoechst 33342 and propidium iodide (PI) double staining (Figure 5 and Supplementary Figure S45 and Figure S46). In the cytometric analysis with annexin V-FITC/PI double staining, treatment with fradimycin B (4, 1.25 μM) in HCT-15 cells caused an increase of apoptotic and necrotic cells from 16.44% (control) to 28.46% (after 24 h treatment) and 43.04% (after 72 h treatment) (Figure 6).

Four new compounds of fradimycins A (3) and B (4) and fradic acids A (6) and B (7) were isolated and identified from marine Streptomyces fradiae PTZ0025. Fradimycins A (3) and B (4) showed potent antibacterial activity against Staphylococcus aureus and significantly inhibited cell-growth of human colon cancer HCT-15 and SW620 cells as well as rat glioma C6 cells. Fradimycin B (4) induced apoptosis and necrosis of HCT-15, SW620, C6 cells and blocked HCT-15 cells at the G₀/G₁ phase. Taken together, the results from this study demonstrated that the new marine natural products fradimycins A (3) and B (4), particularly fradimycin B (4) from marine Streptomyces fradiae strain PTZ0025 had potent antimicrobial and antitumor activities.