The bacteria belonging to the genus Chromobacterium produce a wide variety of antibiotics, such as aerocyanidin, arphamenine A and violacein . Chromobacterium violaceum No. 968 is one of the most studied bacterium because of the production of romidepsin , which was approved by FDA for the treatment of Recurrent T Lymphocytic Carcinoma in 2009  and Peripheral T Lymphocytic Carcinoma in 2011  respectively. Romidepsin is a promising antitumor drug and may also be used for the treatment of HIV . Inspired by these discoveries, a series of romidepsin derivatives, such as thailandepsins , spiruchostatins  and chromopeptide A , have been isolated and characterized.
A chemical investigation of the extract from a large-scale fermentation of the Chromobacterium violaceum No. 968 led to the isolation of two new romidepsin derivatives, romipeptides A and B (1 and 2), together with three known compounds chromopeptide A (3) and romidepsin (4) and valine-leucine dipeptide (5) (Fig. 1). Their isolation, structural elucidation and cytotoxicity are reported herein.
Compound 1 was obtained as a colorless crystal, with the molecular formula of C48H72O12N8S4 based on HR-ESI-MS, 1H NMR and 13C NMR data. Compared with the molecular formula (C24H36O6N4S2) of compound 4, compound 1 might be the dimer of compound 4. The 13C NMR and DEPT data of compound 1 (Table 1) revealed the presence of 24 carbon signals, including one quaternary carbon, five carbonyl carbons, five methyl carbons, four methylene carbons and nine methine carbons, which were identical to those of compound 4. Based on above analysis, the structure of compound 1 might be symmetrical. The 13C NMR signals of compound 1 (Table 1) were similar to those of compound 4, except the signals for C-20 (C-20'), C-16 (C-16'), C-11 (C-11'), C-10 (C-10') and C-9 (C-9'), which related to disulfide bonds. By comparison of the 13C NMR data of compounds 1 and 4, chemical shift values for C-11 (C-11') and C-16 (C-16') were very different with δC 52.3 and δC 41.4 presented in compound 1, while δC 56.6 and δC 35.2 presented in compound 4, respectively. The carbon C-16 was directedly connected at the disulfide bond and the carbon C-11 was connected at C-16 in compound 4. Based on the above analysis, the disulfide bonds of compound 1 might be formed between two molecules of compound 4. The structure of compound 1 was further deduced by comprehensive interpretation of its 1H-1H COSY and HMBC spectra (Fig. 2).
To confirm the assumed structure and determine its absolute configuration, a suitable crystal of compound 1, obtained from MeOH, was subjected to single crystal X-ray diffraction analysis [9-10]. On the basis of the above data, compound 1 was formed from two molecules of compound 4 through intermolecular disulfide bonds. The structure of compound 1 was assigned (Fig. 1) and the compound was named romipeptide A.
Compound 2 was obtained as a white powder with the molecular formula of C23H36O6N4S2 based on HR-ESI-MS, which was one less carbon than that (C24H36O6N4S2) of compound 4. The 13C NMR and DEPT data of compound 2 (Table 2) showed the presence of 23 carbon signals, including five methyl carbons, four methylene carbons, nine methine carbons and five carbonyl carbons. By the comparison of 13C- NMR spectra of compounds 2 and 4 (Table 2), it was found that the tri-substituted double bond of compound 4, including a quaternary carbon C-9 (δC 130.5) and an unsaturated methine C-20 (δC 128.6), disappeared in compound 2, while, an additional saturated methine carbon C-9 (δC 50.9) and a methyl crbon C-20 (δC 15.0) appeared in compound 2. The HMBC spectrum of compound 2 (Fig. 2) indicated the correlation from the saturated methine proton H-9 (δH 3.85) to C-8 (δC 170.2) and C-10 (δC 169.5) and methyl protons H3-20 (δH 1.51) to C-9 (δC 50.9) and C-8 (δC 170.2), suggesting that the methyl carbon C-20 (δC 15.0) was connected at C-9 (δC 50.9). This partial structure was confirmed by the 1H-1H COSY correlation from H3-20 (δH 1.51) to H-9 (δH 3.85) in compound 2. Based on above analysis, the chemical structure of compound 2 was very similar to that of compound 4, which the main difference between them was that the vinyl methyl group in compound 4 was replaced by a methyl group in compound 2.
To confirm the absolute configuration, a suitable crystal of compound 2, obtained from acetone, was subjected to single crystal X-ray diffraction analysis analysis. Thus, the structure of compound 2 was confidently assigned (Fig. 1) and the compound was named romipeptide B.
The other compounds 3, 4 and 5, isolated from the crude extract were identified as chromopeptide A (3)  and romidepsin (4)  and valine-leucine dipeptide (5) , respectively, by comparison of their NMR and MS data with those reported.
Compounds 1–5 were tested for cytotoxicity against three human tumor cell lines, SW620, HL-60 and A549, using the MTT assay . Compound 2 showed similar cytotoxic against SW620, HL60 and A549 cells (IC50 12.5, 6.7 and 5.7 nmol·L–1, respectively) compared with that of romidepsin (IC50 7.3, 4.9 and 5.3 nmol·L-1, respectively) (Table 3).
1D and 2D NMR spectra were obtained on Bruker Avance- 400 FT NMR spectrometer (400 MHz) (Bruker BioSpin GmBH, Rheinstetten, Germany) with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to solvent signals. HR-ESI-MS was recorded on a Waters Alliance (2695/2487) Q-Tof micro (Waters Corp., Milford, MA, USA). Single crystal X-ray diffraction (SCXRD) was recorded on a Bruker SMART APEX-II (Bruker BioSpin GmBH, Rheinstetten, Germany). Column chromatography was performed on silica gel (200−300/300−400 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China). High-perfo rmance liquid chromatography (HPLC) was employed using Elite P270 pumps and Elite UV 230 + detector (Elite Analytical Instruments Co., Ltd., Dalian China) coupled with an Agela C18 preparative column (250 mm × 30 mm, 10 μm, Venusil ASB C18, Agela Technologies Co., Ltd., Tianjin, China). Fermentation was performed in FUS-5L fermentor (National center of Bio-Engineering & Technology, Shanghai, China).Bacterial material and fermentation
The strain used in this experiment was Chromobacterium violaceum No. 968 (No. FERM BP-1968, From IPODa) [14-15]. The bacterial strain was cultured on nutrient agar (Bio-way technology Corp., Shanghai, China) at 30 ℃ for 24 h. The seeds from the above was inoculated into 250 mL erlenmeyer flask containing 30 mL sterile seed liquid medium containing 2.0% peptone and 2.0% glucose, PH 6.0, and cultured on a rotary shaker (250 r·min–1) at 30 ℃ for 16 h. Fermentation was performed in 5 L fermentor containing 3 L culture medium (3.0% glucose, 1.0% starch, 2.0% mannitol, 1.0% hydrolysis casein, 0.8% beef extract, 0.6% KH2PO4, 0.2% cysteine, 0.4% soybean oil and 0.2% CaCO3, PH 5.5). The fermentor was inoculated with 2.0% of the seed culture and maintained on a 520 rpm rotary shaker at 25 ℃ for 52 h.Isolation and purification
After the fermentation was completed, the PH of the fermentation broth was adjusted to 3.0 with hydrochloric acid, then the fermentation broth was extracted with EtOAc (5 L) and the organic layer was evaporated to dryness (14.1 g). The extract (14.1 g) was separated on a silica gel column with a gradient solvent system of cyclohexane–EtOAc (5 : 5 to 1 : 9, V/V) to give three fractions (Frs. 1–3). Fr. 1 (4.6 g) was purified on a silica gel column with a gradient solvent system of cyclohexane–isopropanol (10 : 0 to 8 : 2, V/V) to yield compounds 3 (0.03 g), 4 (1.18 g) and 5 (0.03 g). Fr. 2 (0.25 g) was purified on a preparative RP HPLC using a solvent system of CH3CN–H2O (4 : 6, V/V) to yield crude compound 1 (0.15 g). Crude compound 1 (0.15 g) was dissolved in 4 mL methanol and kept in 4 ℃ for 72 h, then crystals of compound 1 (0.05 g) were obtained. Fr. 3 (0.21 g) was purified on a silica gel column with a gradient solvent system of cyclohexane–EtOAc (3 : 7 to 10 : 0, V/V) to afford compound 2 (0.03 g).
Romipeptide A (1): colorless crystal. [α]D20 −53.9 (c 0.03, CHCl3). UV (MeOH) λmax nm (ε): end absorption. IR (KBr) υmax : 3385, 3252, 2967, 2932, 1740, 1655, 1518, 1468, 1441, 1393, 1373, 1350, 1333, 1261, 1180, 1157, 1096, 989 cm–1. MP: 190−198 ℃. 1H NMR, 13C NMR and HMBC and 1H-1H COSY, see Table 1. HR-ESI-MS m/z 1103.4020 [M + Na]+ (Calcd. for C48H72O12N8S4Na, 1103.4050).
Romipeptide B (2): colorless crystal. [α]D20 +15.7(c 0.03, CHCl3). UV (MeOH) λmax nm (ε): end absorption. IR (KBr) υmax : 3329, 3293, 2961, 2932, 2876, 1738, 1690, 1655, 1530, 1468, 1454, 1395, 1364, 1335, 1256, 1188, 1161, 1053, 1018, 970 cm–1. MP: 244−248 ℃. 1H NMR, 13C NMR and HMBC and 1H-1H COSY, see Table 2. HR-ESI-MS m/z 529.2142 [M + H]+ (Calcd. for C23H36O6N4S4, 529.2155).Cytotoxicity
The cytotoxicity of compounds 1−5 were tested in vitro using human cancer cell lines including HL60 (acute promyelocytic leukemia), SW620 (colonic carcinoma) and A549 (lung adenocarcinoma). The MTT method was used for the bioassays as described in the literature . Cisplatin was used as a positive control .
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