Sponges (Porifera) remain the most important phylum in the field of marine drugs discovery, since they produce a great number of novel natural products with a variety of potent pharmacological activities. Nevertheless, the transformation of marine sponge-derived active compounds into drugs has been hindered by supply limitation. It is well known that sponges are hosts for a large amount of microorganisms, demonstrating interactions such as epibiotic, symbiotic and parasitic relationships, etc. Symbiotic functions that have been attributed to marine sponge microbial associates include nutrient acquisition and secondary metabolite production [1]. The various secondary metabolites synthesized by microbial associates inhabiting marine sponges also possess good bioactivities in many studies [2–6]. In recent years, microbial diversity in the marine environment, particularly marine sponge-associated microorganisms, have become a significant source of new lead compounds for marine drugs with vast biotechnological potential.

During the course of our investigation for bioactive natural products from marine sponge-associated microorganisms, we have isolated some known metabolites from marine sponge-derived fungi. In this paper, we report the isolation and characterization of two new compounds of kijanimicin derivatives from the fermentation broth of a variant strain of Streptomyces carnosus associated with marine sponges Hymeniacidon sp. The in vitro cytotoxic activities were detected against two human cancer cell lines. This is the first report of bioactive metabolites derived from Streptomyces associated with marine sponges of the genus Hymeniacidon according to the current review of Thomas [7].

Compound 1 (lobophorin C) was obtained as a white amorphous powder, soluble in dimethyl sulphoxide (DMSO) and methanol. It gave the [M + Na]⁺ ion at m/z 1209 in the ESI-MS, indicating a molecular weight of 1186. HR-ESI-MS analysis displayed the [M + Na]⁺ signal at m/z 1209.5958 (calculated 1209.5934). Element analysis revealed the compound consists of N (2.019%), C (54.11%), H (7.219%), O (36.65%). Thus, the molecular formula of AS7-2 was established to be C₆₁H₉₀N₂O₂₁ based on HR-ESIMS, ¹H and ¹³C NMR data (Table 1) and element analysis, with 18 sites of unsaturation. The IR spectrum of 1 showed bands characteristic of hydroxyl (3432 cm⁻¹), ester carbonyl (1721 cm⁻¹) groups and olefinic bonds (1631 cm⁻¹). UV spectrum exhibited the maximum absorption bands at 267 and 241 nm. The ¹H-NMR spectrum demonstrated the presence of eleven methyl protons, two methoxyl protons (δ 3.59, s; 3.26, s), one anomeric proton and a NH proton signal (δ 7.40). The ¹³C-NMR spectrum of 1 (Table 1) revealed signals for 61 carbons. ¹³C-NMR and various DEPT spectra allowed the assignments of the 61 carbon signals to eleven methyl, two methoxyl (δ 55.7, 51.9), eight methylene and 29 methine and eleven quaternary carbon atoms. Among them, four anomeric carbons (δ 99.08, 97.63, 97.34, 91.20) suggested the presence of four sugar residues. The correlation of the adjoining proton resonances was investigated by ¹H-¹H COSY, showing the HH connectivities as: H₁₅-H₁₆-H₁₇, H₁₉-H₂₀-H₂₁, H₂₄-H₂₃-H₃₃, H₁₃-H₁₂-H₁₁-H₁₀-H₉-H₈-H₂₉, H₈-H₇-H₆ and H₁₀-H₅-H₆-H₂₈ (Figure 1). The correlation of neighboring protons in A-ring sugar moiety was found to be H_1A-H_2A-H_3A-H_4A-H_5A-H_6A, similar proton connectivities in B-ring and C-ring sugar moieties can be derived from the cross peaks of the ¹H-¹H COSY spectrum. In the HMBC experiment, the following key correlations were observed in the aglycone part of compound 1: H-5 to C-7, C-6, C-4, C-28; H-7 to C-29, C-28; H-8 to C-29; H-13 to C-27, C-14, C-12, C-11, C-4; H-21 to C-19, C-20, C-23, C-32; H-24 to C-33, C-25, C-22, C-20; H-27 to C-5, C-4, C-3; H-28 to C-7, C-6, C-5; H-29 to C-9, C-8, C-7; H-30 to C-15, C-14, C-13; H-32 to C-23, C-22, C-21; H-33 to C-24, C-23, C-22 (Table 1 and Figure 1). HMBC correlations were detected within the sugar units as: H-6_D with C-5_D, C-4_D; H-5_C with C-6_C, C-4_C; H-4_C with C-5_C, C-3_C, C-6_C; H-2_B with C-3_B; H-3_B with C-1_B; H-5_A with C-6_A, C-1_A; H-4_A with C-6_A; H-1_A with C-3_A, C-5_A (Table 1 and Figure 1). The points of attachment for the sugar moieties of ring A and ring D to the C-9 and C-17 sites of the aglycone, respectively, were determined via the long-range correlations of H-1_A to C-9, H-1_D to C-17, as well as H-17 to C-1_D.

The NOESY spectrum provided information regarding the relative configuration of the compound 1. Key NOE interactions were recorded between H-5 (δ 2.00) and H-9 (δ 3.29), H-5 and H-28 (δ 0.55) as shown in Figure 2, implying H-9, H-5 and the methyl group in site 28 (H-28) were close to each other with same orientation. Also, NOE interaction can be observed between H-28 and H-29 (δ 1.01), indicating the orientation of the CH₃ group in site 28 was identical with the CH₃ group in site 29. Accordingly, they should be in the same side of the aglycone plane.

The structure of compound 1 was very similar to the compound lobophorin B, derived from an unidentified actinomycete strain CNC-837 isolated from the surface of the Caribbean brown alga Lobophora variegata [8]. Both of them were derivatives of the terrestrial antibiotic kijanimicin [9], with one less sugar moiety than kijanimicin and contained one 2,6-dideoxy-4-O-methyl-l-ribo-hexopyranose and two 2,6-dideoxy-l-ribo-hexopyranose in their sugar units. By comparison of their spectral data, compound 1 is distinctly different from the known lobophorin B in these aspects: Firstly, a –OH group and a C=O group linked to the sites of C-3 and C-26 in compound 1 respectively, while a C=O and a –OH attached to these positions in lobophorin B, respectively. Consequently, the unsaturated double bond between C-3 and C-2 and the saturated single bond between C-2 and C-26 in compound 1 were substituted by a saturated bond and an unsaturated double bond in lobophorin B, respectively, exhibiting transformations between enol and ketone forms. These changes can be confirmed by chemical shifts in the corresponding positions (Table 1). Secondly, the two CH₃ groups in C-28 and C-29, which were in opposite orientation in lobophorin B, were in the same side in compound 1 by NOE analysis. Besides, the optical rotation of compound 1 was −150.9°, in contrast with +104° of lobophorin B when measured in same condition. To sum up, the structure of compound 1, which derived from a marine sponge symbiotic actinomycete, was not identical to the previously reported compound lobophorin B due to mutual enol and ketone transformations in local groups, relative stereochemistry and optical activities. Therefore, compound 1 was named lobophorin C. Its structure is shown in Figure 3.

Compound 2 (lobophorin D) was obtained as a white amorphous powder like lobophorin C (1). The molecular weight was deduced as 1156 from the pseudomolecular ions [M + H]⁺ at m/z 1157 in positive ESI-MS and [M − H]⁻ at m/z 1155 in negative ESI-MS. Element analysis showed the compound consist of N (2.049%), C (50.10%), H (7.103%), O (40.75%). The molecular formula of compound 2 was determined as C₆₁H₉₂N₂O₁₉ based on ESI-MS, ¹H and ¹³C NMR spectra and element constitution analysis. ¹³C NMR and DEPT spectra displayed 11 CH₃ carbon signals, 2 –OCH₃ carbons; 8 CH₂ carbons and 29 CH carbons. HSQC spectrum revealed 11 quaternary carbons without protons attachment. Compound 2 resembled lobophorin C (1) in signals assignments by ¹H-¹HCOSY coupled with HMBC spectra. Both showed great structural similarities except that the substitute group in C-3 of sugar ring D was –NH₂ in compound 2 instead of –NO₂ in lobophorin C (1). Compound 2 exhibited almost identical structure with the published compound lobophorin A, except that a hydroxyl group and a carbonyl group linked to the sites of C-3 and C-26 in the aglycone part of compound 2, respectively, deduced by its spectral NMR data. In addition, both the methyl groups of C-29 and C-28 in compound 2 were in the same spatial orientation, and compound 2 displayed an optical rotation of −138.4° while −175° was observed for lobophorin A. Hence, compound 2 was named lobophorin D (Figure 3).

The cytotoxicity of compounds 1 and 2 were tested in vitro against a human liver cancer cell line 7402 and a human breast cancer cell line MDA-MB 435. The results showed compound 1 demonstrated a strong cytotoxic activity while compound 2 was inactive against the cell proliferation of 7402 hepatoma cells, with IC₅₀ values of 0.6 μg/mL and 723.1 μg/mL, respectively. On the contrary, compound 2 displayed a good inhibitory effect on the growth of human breast cancer cells MDA-MB 435 with an IC₅₀ value of 7.5 μM, in contrast to the negligible activity of 61.8 μM for compound 1. Apoptosis of the breast adenocarcinoma cells can be observed with increasing concentrations of compound 2 (Figure 4). Compounds 1 and 2 seemed to have selective cytotoxicity for these cancer cell lines. Though as derivatives of kijanimicin with similar structures, compounds 1 and 2, in contrast with lobophorin A and B demonstrated different bioactivities. The previously reported lobophorins A and B did not exhibit significant antibiotic properties, whereas they showed potent anti-inflammatory activities. It is suggested that the distinction of bioactivities was likely attributed to the differences of stereochemistry of the two groups of compounds and their optical activities, as well as the slight modification of specific bonds. The mechanism of cytotoxic selectivity of compounds 1 and 2 to various cancer cell lines needs to be further investigated.