Isostreptazolin and Sannaphenol, Two New Metabolites from Streptomyces sannanensis

Two new compounds, isostreptazolin (1) and sannaphenol (2), were isolated from the culture broth of Streptomyces sannanensis and their structures elucidated on the basis of 1D and 2D NMR as well as MS, IR and UV spectroscopic data analysis. The cytotoxic activity of 1 and 2 were evaluated. Both compounds were inactive against H460 and HeLa cell lines at 100 μM.


Introduction
In the course of screening for new bioactive compounds from microbial sources, two new constituents, isostreptazolin (1) and sannaphenol (2), were isolated from the fermentation broth of Streptomyces sannanensis, from which a new cytotoxic alkaloid, sannanine, had been isolated by the authors in earlier work [1]. The structures of 1 and 2 were determined by detailed spectroscopic investigation and the results of biological testing for the cytotoxic activity of 1 and 2 are presented. The remarkable structural feature of 1 is a unique tricyclic skeleton with a urethane moiety of natural OPEN ACCESS origin. The unusual ring system is rarely seen in Nature and only found in streptazolin [2,3] and its analogues [4,5]. The biosynthesis of streptazolin had been discussed by M. Mayer in 1993 [6]. As streptazolin and 1 bear analogous structural elements, both of compounds possibl originate from a similar biosynthetic pathway or derive from the same biosynthetic polyketide precursor.
The 13 C-NMR spectrum of 1 (Table 1)  The key HMBC correlations between three olefinic protons at  = 5.44, 6.21 and 6.80 in fragment D, an oxygenated methine proton at  = 5.50 in fragment D, one methylene proton at  = 2.05 in fragment C, and another oxygenated methine proton at  =4.11 in fragment C and quaternary carbon at  = 79.0 suggested both ends of fragment D and one end of fragment C were connected with the same quaternary carbon  = 79.0. Therefore, a fragment E was built up as shown in Figure 1. The same molecular formula and similar NMR data suggested 1 was related to streptazolin, which possesses a unique tricyclic ring system embodying an internal urethane unit and an exocyclic ethylidene side chain [7] and had been found from several Stretomyces sp. [2,4,8]. According to the molecular formula C 11 H 13 NO 3 of 1 and the similar NMR data with those of streptazolin, the quaternary carbon at  = 160.1 was considered belonging to a carbamoyl ester moiety just like that in streptazolin.
Significant HMBC correlations between oxygenated methine proton at  = 5.50, and methylene protons at  = 3.58 and 3.11 and the acyl carbon at  = 160.1 indicated the oxygen atom of carbamoyl ester unit was connected with the terminal carbon at  = 82.6 in fragment E, and the nitrogen atom of the carbamoyl ester unit was connected with the terminal carbon at  = 42.9 in fragment E. Thus, another terminal carbon at  = 79.0 in fragment E was inevitably linked with the nitrogen atom of the carbamoyl ester moiety, which was confirmed by HMBC correlations between one methylene proton at  = 3.58 and the quaternary carbon at  = 79.0. Therefore, the planar structure of 1 was elucidated as shown in Figure 1 and named isostreptazolin. NOE correlations between H-6 ( = 6.80) and H-13 ( = 1.75); H-2 ( = 5.44) and H-10a ( = 2.48) suggested the double bond at  7,12 has a Z conformation. NOE correlations between H-4 ( = 5.50) and H-9 ( = 4.11) indicated the cis configuration of H-4/H-9. Just like streptazolin, 1 possesses an unusual ring system, leading the authors to postulate that 1 is related to streptazolin, and the two compounds originate from a similar biosynthetic pathway or derive from the same biosynthetic polyketide precursor [6].  (Table 1) showed three aromatic proton signals at  = 6.14 (1H, brs), 6.09 (1H, brs), and 5.98 (1H, brt, J =1.8), indicating a meta trisubstituted benzene ring moiety exists in 2. Two substituents could be clearly determined as a phenolic hydroxyl by a proton signal at  = 8.97 (1H, brs), and a dimethylamino group according to proton signal at  = 2.83 (6H, s) and the corresponding carbon signal at  = 40.0 in the 1 H-and 13 C-NMR of 2. Complete assignments of the 1 H-and 13 C-NMR spectra of 2 were achieved with 1D and 2D NMR experiments (Table 1). Partial structures were assembled by interpretation of COSY data and connected by analysis of the HMBC NMR data. The key correlations are shown in Figure 2. As not enough sample was available, the authors could not determine the stereochemistry of C-7 and C-8 by chemical methods. Therefore, the planar structure of

Biological Activity
Streptazolin showed no inhibition against several human cancer cell lines, while its precursors [9] and derivatives [4,10] indicated significant cytotoxic activity. In this paper, the cytotoxic effects of 1 and 2 were tested against human large-cell lung carcinoma cell line (H460) and human cervix carcinoma cell line (HeLa). Both compounds were inactive against these two cell lines at 100 M (inhibition ratio lower than 50%).

Producing Organism
The organism Streptomyces sannanensis and fermentation conditions were identical to those in reference [1].

Extraction and Isolation
The completed fermentation broth (80 L) was separated into filtrate and mycelium by centrifugation. The culture filtrate was absorbed onto the polymeric resin Amberlite XAD-16. The salt and high molecular materials were washed out with water, and then, other absorbed organic material was eluted with MeOH to yield 55 g of dried extract after removing the solvent under vacuum. The dried extract was extracted with petroleum, CHCl 3 and EtOAC respectively, the CHCl 3 extract fraction (2 g) was subjected to gel chromatography on Sephadex LH-20 (MeOH) to produce three fractions. Fraction 2.4.6 was purified by silica gel column chromatography (hexane-EtOAc = 2:1) to give compound 2 (13 mg). Fraction 2.4.7 was separated by ODS column chromatography, eluting with water-methanol (4:6) yielding compound 1 (9.8 mg).

Cytotoxic Assay
The human large-cell lung carcinoma cell line (H460) and human cervix carcinoma cell line (HeLa) were used to evaluate cytotoxic effects of 1 and 2 with an MTT method [11]. H460 was grown in RPM1-1640 medium plus 10% heat-inactived fetal bovine serum and HeLa was grown in DMEM medium plus 10% heat-inactived fetal bovine serum. The assays were performed in 96-well microtiter plates. Compounds 1 and 2 was dissolved in DMSO and diluted to six different concentations (10, 3.3, 1.0, 0.33, 0.10, and 0.033 mM), and each solution was ten-fold diluted to six different concentations using culture medium (1.0, 0.33, 0.10, 0.033, 0.010, and 0.0033 mM), then each solution (10 µL) was added to culture medium wells (90 µL, about 5000 cells). After incubation at 37 °C for 72 hours, MTT (10 µL, 5 mg/mL) was added to each well and incubated for four hours, and then liquid in the wells was removed. DMSO (150 µL) was added to each well. The absorbance was recorded on a microplate reader at a wavelength of 570 nm.

Conclusions
Two new metabolites have been isolated from Streptomyces sannanensis. The new compounds were spectroscopically identified and given the trivial names isostreptazolin and sannaphenol. Both the new compounds were inactive against the H460 and HeLa cell lines at 100 M.