Lobophorin C and D, New Kijanimicin Derivatives from a Marine Sponge-Associated Actinomycetal Strain AZS17

Marine sponge Hymeniacidon sp. was collected from coastal waters of the East China Sea to isolate symbiotic microorganisms. The resulting sponge-associated actinomycete, Streptomyces carnosus strain AZS17, was cultivated in a 20 L volume of medium for production of bioactive secondary metabolites. Bioassay-guided isolation and purification by varied chromatographic methods yielded two new compounds of kijanimicin derivatives, AS7-2 and AS9-12. Their structures were elucidated by spectroscopy and comparison with literatures. Results showed these two compounds were structurally similar to the previously reported compounds lobophorin A and B, yet differed in specific bond forms, stereochemistry and optical activities. The two novel compounds were named lobophorin C and D. In vitro cytotoxicity investigation by MTT assay indicated their selective activities. Lobophorin C displayed potent cytotoxic activity against the human liver cancer cell line 7402, while lobophorin D showed significant inhibitory effect on human breast cancer cells MDA-MB 435.


Introduction
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][3][4][5][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].
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 CH 3 group in site 28 was identical with the CH 3 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 CH 3 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.  1 H and 13 C NMR spectra and element constitution analysis. 13 C NMR and DEPT spectra displayed 11 CH 3 carbon signals, 2 -OCH 3 carbons；8 CH 2 carbons and 29 CH carbons. HSQC spectrum revealed 11 quaternary carbons without protons attachment. Compound 2 resembled lobophorin C (1) in signals assignments by 1 H-1 HCOSY coupled with HMBC spectra. Both showed great structural similarities except that the substitute group in C-3 of sugar ring D was -NH 2 in compound 2 instead of -NO 2 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 IC 50 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 IC 50 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.

General Procedures
Optical rotations were measured on a Horiba High Sensitivity Polarimeter SEPA-300. UV and IR spectra were measured on a Shimadzu UV-2501PC spectrophotometer and a Bruker VECTOR 22 FT-IR spectrophotometer, respectively. NMR spectroscopic data were recorded on a Varian INOVA 500NB NMR spectrometer. ESIMS and HR-ESIMS spectra were measured on a Thremo LCQ DECA XP LC