Secondary Metabolites with Cytotoxic Activities from Streptomyces sp. BM-8 Isolated from the Feces of Equus quagga

A new aliphatic acid, compound 1, together with six known metabolites, including nonactic acid (2), homononactic acid (3), ethyl homononactate (4), homononactylhomononactate (5), valinomycin (6), and cyclo-(Pro-Leu) (7), was isolated from the culture broth of Streptomyces sp. BM-8, an actinobacterial strain isolated from the feces of Equus quagga. The structures of these compounds were established by analyses of spectroscopic data, including 1D and 2D nuclear magnetic resonance spectra (NMR), as well as by HR-ESI-MS spectrometry and chemical derivative analyses. Additionally, a serial analogue of nonactic acid and homononacticacid (8–21) was synthesized. The cytotoxicity of 1–21 wastested against a panel of cancer cell lines, such as HT-29, MCF-7, A375 and K562, with MTT assay. In addition, the cytotoxicity tests revealed that 1 was less cytotoxic toward a panel of cancerous cells, as compared with valinomycin (6).


Introduction
Thus far, a large number of drugs/leads were derived from microbial secondary metabolites that were isolated from microbes living in different environments all around the world [1]. Among them, the intestinal microbiota has proven to be a new source of bioactive molecules that are used for the treatment of different kinds of diseases [2]. The host-microbe mutualism is a special ecosystem associated with the interactions between symbiotic bacteria and the hosts of plants or animals. As we have known, the intestinal microbiota arecomposed of a large number of heterogeneous commensal bacterial species that have coevolved with the host for very long periods of time. Therefore, it can be inferred that the metabolites produced by these microbials residing in the intestine might be safe and provide additional protection for their hosts' homeostasis [3], and possess a strong potential for development as possible therapeutic agents [4], which has been supported by a lot of literature published in recent years. In addition, a large number of novel and bioactive metabolites associated with this special ecosystem have been identified [5][6][7].
In our previous study, a butanolide with potent anti-influenza A virus activity was isolated and identified from a bacterial strain of Streptomyces sp. SMU03 residing in an Asian elephant's intestine via the bioassay-guided approach [8]. In our continuous search for bioactive compounds from intestinal bacteria, we identified an actinobacterium of Streptomyces sp. BM-8 from the feces of Equus quagga. Notably, the ethyl acetate extracts

Structure Elucidation
Compound 1 was isolated as a colorless oil and was defined for the molecular formula of C11H20O5based on high-resolution mass spectrometry analysis (electrospray ionization, [ 1 H and 13 C NMR spectra (see Table 1). The 1 H NMR, 13 C NMR, and DEPT spectra (in CDOD3) of 1 showed three methine protons from 2.29 to 3.98 ppm, including an aliphatic methine proton at δH 2.29 (quint, J = 6.16, 1 H), and two oxygenated methine protons at δH 3.49-3.54(m, 1 H) and 3.92-3.98(m, 1 H). The 1 3 H). The 13 C NMR (see Table1), HMQC, and HMBC spectral data displayed one carbonyl carbon at δc 212.9 ppm, one carboxyl carbon at δc 184.5 ppm, two oxygenbearing carbons at δc 74.6 and 70.4 ppm, five aliphatic carbon signals at δc 50.92, 48.35, 30.35, 31.29, and 41.04 ppm, and two methyl carbon signals at δc 15.9 and 10.3 ppm. Considering the amount of the carbonyl carbon and the carboxyl carbon account for two unsaturation equivalents, it can be deduced that 1 possesses no ring system in the structure.

Structure Elucidation
Compound 1 was isolated as a colorless oil and was defined for the molecular formula of C 11 H 20 1 H and 13 C NMR spectra (see Table 1). The 1 H NMR, 13 C NMR, and DEPT spectra (in CDOD 3 ) of 1 showed three methine protons from 2.29 to 3.98 ppm, including an aliphatic methine proton at δ H 2.29 (quint, J = 6.16, 1 H), and two oxygenated methine protons at δ H 3 H). The 13 C NMR (see Table 1), HMQC, and HMBC spectral data displayed one carbonyl carbon at δ c 212.9 ppm, one carboxyl carbon at δ c 184.5 ppm, two oxygen-bearing carbons at δ c 74.6 and 70.4 ppm, five aliphatic carbon signals at δ c 50.92, 48.35, 30.35, 31.29, and 41.04 ppm, and two methyl carbon signals at δ c 15.9 and 10.3 ppm. Considering the amount of the carbonyl carbon and the carboxyl carbon account for two unsaturation equivalents, it can be deduced that 1 possesses no ring system in the structure.

Synthesis of Serial Compounds Based on 2 and 3
Since the main components of the culture broth of Streptomyces sp. BM8 were found to be known natural products nonactic acid (2) and homononactic acid (3), which showed different cytotoxicity against a panel of cancer cell lines, as indicated in Table 4, synthesis was employed for serial compounds 8-21, as shown in Figure 4. Nonactic acid thatwas isolated from BM8 was reacted with appropriate derivatives, which led to the generation of compounds 8, 11, and 17, while the synthesis of homononactic acid resulted in the preparation of the analogues 9-10, 12-16, and 18-

Cytotoxicity
Compounds 1-21 were tested for their cytotoxicity against a panel of cell lines, including human colon cancer cell line HT-29, human breast cancer cell line MCF-7, human malignant melanoma cell line A375 and human chronic myeloid leukemia cell line K562. In addition, the human normal colon epithelial cell line NCM460 was also tested for comparison. As a result, 6 showed the most potent cytotoxicities toward all cell lines with the IC50 values ranging from 0.04 ± 0.01 µM to 1.66 ± 0.19 µM, while others were less active under the conditions tested ( Table 4).

Cytotoxicity
Compounds 1-21 were tested for their cytotoxicity against a panel of cell lines, including human colon cancer cell line HT-29, human breast cancer cell line MCF-7, human malignant melanoma cell line A375 and human chronic myeloid leukemia cell line K562. In addition, the human normal colon epithelial cell line NCM460 was also tested for comparison. As a result, 6 showed the most potent cytotoxicities toward all cell lines with the IC 50 values ranging from 0.04 ± 0.01 µM to 1.66 ± 0.19 µM, while others were less active under the conditions tested ( Table 4).
The synthesized analogues of nonactic acid or homononactic acid (8-21) were also evaluated for their in vitro cytotoxicity against these cell lines by the MTT assay with paclitaxel as apositive control. As shown in Table 4, the saturated fatty alcoholicesters of nonactic acid or homononactic acid, 8-13, exhibited moderate cytotoxic activities with IC 50 values in the micromolar range. Although 11 showed a slightly higher inhibitory effect toward the tested cells with IC 50 ranging from 24.29 ± 0.03 µM to 44.21 ± 0.64 µM, structure-activity relationship could not be clearly observed since the cytotoxicities of 8-13 did not increase as the side chains of these compounds becoming longer. Replacing the fatty alcohol moiety with benzyl (14) led to average IC 50 values ranging from 21.05 ± 3.66 µM to 89.92 ± 1.09 µM, suggesting that phenylesters of homononactic acid may have higher cytotoxicities. However, when the carboxyl group of the homononactic acid reacted with vanillin (15), 4-biphenylmethanol (16), pyridine (17), O-phenylenediamine (18), 2-Benzothiazolamine (19), the reaction products (15-19) showed different cytotoxic activities without an obvious structure-activity relationship, as did silylether (20) and the benzimidazole derivative (21). Furthermore, higher toxicity of most of the synthesized compounds towards the nontumorigenic cell line (NCM460) was observed in the MTT assay, as shown in Table 4, indicating that these compounds, including 9, 14, 18, 20, and 21, may possess broad toxicity toward the cell lines tested. means 50% inhibitory concentration, and expressed as the means ± standard deviation (SD), each of which was repeated at least three times independently; b HT-29: human colon cancer cell line; c MCF-7: human breast cancer cell line; d A375: human malignant melanoma cell line; e K562: human chronic myeloid leukemia cell line, and it was cultured in RPMI1640 containing FBS while the other cell lines were all cultured in medium without the addition of FBS; f NCM460: human normal colon epithelial cell line; g Paclitaxel was used as a positive control; h NA: not active; i NT: not tested.

Collection and Phylogenetic Analysis of Strain BM-8
The animal-derived bacterial strain BM8 was isolated from a feces sample excreted by adult Equus quagga collected in Guangzhou Zoo, Guangdong Province, China. Based on its 16S rRNA sequence (GenBank accession number MT912570) and morphology characteristics, the strain BM-8 was determined to be Streptomyces sp. by Guangzhou IGE biotechnology LID. A voucher specimen is preserved and available from the collection (No. BM-8) at Group of peptides and natural products research, School of Pharmaceutical Sciences, Southern Medical University in Guangzhou (510515), China.

Cultivation and Extraction
Bacterium BM-8 was cultivated in a 250 mL Erlenmeyer flask containing 50 mL of seed medium and shaken on a rotary shaker at 200 rpm at 28 • C for 2 days. Then, the seed medium was transferred to a 2 L Erlenmeyer flask containing 0.5 L of culture medium and cultivated for another 7 days at 200 rpm at 28 • C. The seed medium was composed of glucose 10 g/L, beef extract 3 g/L, peptone 3 g/L, soluble starch 20 g/L, yeast extract 5 g/L, and CaCO 3 3 g/L, adjusted to pH 7.0 by 1 M NaOH solution. The culture medium contained mannitol 30 g/L, glucose 10 g/L, yeast extract 5 g/L, ammonium succinate 1 g/L, K 2 HPO 4 0.5 g/L, MgSO 4 ·7H 2 O 0.5 g/L, and 1 mL/L of trace element concentrate, and adjusted to pH 7.5 by 1 M NaOH solution. The trace element concentrate was prepared with FeSO 4 ·7H 2 O 0.2 g, MnCl 2 ·2H 2 O 0.1 g, and ZnSO 4 ·7H 2 O 0.1 g dissolved in 100 mL of deionized water. The seed medium and culture medium were autoclaved before use.
After seven days of cultivation, the culture broth was transferred to a 50 mL test tube and centrifuged at 8000 rpm for 6 min. A total culture broth of 22.5 L was collected. The supernatant was subjected to a D-101 resin column, washed with deionized water until the effluent became colorless, and then eluted with ethanol to generate the crude extract (20 g) after being concentrated under reduced pressure. Then the crude extract was partitioned with CH 2 Cl 2 (2.4 g) and EtOAc (1.6 g). The mycelium was extracted with ethanol three times to give 6.6 g of brown crude extract, which was partitioned with CH 2 Cl 2 (1.2 g) and EtOAc (1.4 g).

General Procedures for Preparation of Compounds 8-21
General procedure for compounds 8-16: A solution of nonactic/homononactic acid (4.3 mg) and vanillin (6.5 mg) in DCM/DMF (100 µL) was treated with 4-DMAP (1 mg, 0.008 mmol), and the mixture was cooled at 0 • C in an ice bath. Dicyclohexylcarbodiimide (20 mg) dissolved in 100 µL DCM was added dropwise to the above solution. The reaction mixture was stirred at room temperature overnight. After the end of the reaction (monitoring by thin-layer chromatography), the resulting solution was quenched with water (2 mL) and extracted with ethyl acetate (2 mL×3). The combined organic layers were dried under vacuum and purified by column chromatography on silica gel.  at room temperature, the optical density value was measured spectrophotometrically at 570 nm using the microplate reader. The IC 50 value (50% inhibitory concentration) was determined to evaluate the cell viability of these cell lines. In this experiment, Graph Pad Prism 5software (San Diego, CA, USA) was used to calculate the half inhibitory concentration (IC 50 ) values. Each experiment was independently repeated at least three times and represented as the means ± standard deviation (SD).

Conclusions
In summary, a new compound (1) and six known compounds, including nonactic acid (2), homononactic acid (3), ethyl homononactate (4), homononactylhomononactate (5), valinomycin (6), and cyclo-(Pro-Leu) (7), were isolated from the culture broth of Streptomyces sp. BM-8 residing in the feces of Equus quagga. Subsequently, 13 derivatives of nonactic/homononactic acid were synthesized. In this study, 6 showed significant cytotoxic activities against all tested cell lines, while 1 exhibited less activity toward these cells. Some synthesized compounds showed higher toxicity towards the nontumorigenic line and the absence of a structure-activity relationship, indicating that these compounds may have broad cellular toxicity toward the cell lines tested. The synthesized compounds did not show attractive selectivity in the cytotoxic assay, suggesting that more synthesis strategies should be proposed. Since the dimeric nonactic acid show less antibacterial and antifungal activity than that of dimeric dinactin [14], it might be worth synthesizing such kinds of cyclic analogues with nonactic acid or homononactic acid in further research. Nevertheless, these results suggested that microbes inhabiting the animal feces may represent an important source for new and bioactive natural compounds.