During the course of our screening program for new anticancer agents from Okinawan marine microorganisms, a new compound, designated JBIR-150 (1), was isolated from the culture broth of Streptomyces sp. OPMA00071. Chemical and spectroscopic analyses revealed that 1 belonged to the class of 20-membered polyene macrolactam antibiotics. Herein, we report the fermentation, isolation, structure elucidation, and preliminary biological activities of 1.

Strain OPMA00071 was isolated from marine sediments collected in Okinawa prefecture, Japan. The isolated strain was identified as Streptomyces sp. on the basis on 99.9% similarity in the 16S rDNA gene sequence to Streptomyces heliomycini. The producing strain was cultivated in 50-ml test tubes, each containing 15 ml of a seed medium consisting of 1.0% starch, 1.0% Hipolypepton, 1.0% molasses, and 1.0% meat extract at pH 7.2 (adjusted before sterilization). The test tubes were shaken on a reciprocal shaker (320 r.p.m.) at 27 °C for 2 days. Aliquots (2.5 ml) of the culture were transferred into 500-ml baffled Erlenmeyer flasks filled with 100 ml of a production medium consisting of 4 g β-cyclodextrin, 0.5 g glycerol, 2 g Pharmamedia, 0.5 mg CuSO4•5H2O, 0.5 mg MnCl2•4H2O, and 0.5 mg ZnSO4•7H2O, and cultured on a rotary shaker (180 r.p.m.) at 27 °C for 5 days.

The fermentation broth (2L) was centrifuged to obtain a mycelial cake, which was extracted with acetone (250 ml × 3). The extract was concentrated in vacuo, and the residual aqueous concentrate was successively washed with EtOAc followed by extraction with n-BuOH. The n-BuOH layer was then concentrated in vacuo. The dried residue (2.29 g) was triturated in MeOH (5 ml) and the residue was filtered using a Kiriyama-rohto device (Kiriyama, Tokyo, Japan) to afford JBIR-150 (1, 190.0 mg) as a colorless amorphous powder: [α]21D 163 (c 0.01, MeOH); UV λmax nm (log ɛ): 227 (4.4), 260 (4.2), 310 (3.8), and 324 (3.7) in MeOH; IR (ATR) νmax 3300 and 1630 cm−1 (hydroxy and amide carbonyl). Its molecular formula C28H39NO3 was established by an [M+H]+ ion at m/z 438.2977 (calcd for C28H40NO3, 438.3003) in a high-resolution electrospray ionization mass spectrometry.

Because 1 was insoluble in CH3OH, CH3CN, n-hexane, EtOAc, CHCl3, n-BuOH, and water, the NMR spectra were measured in DMSO-d6. The structure determination of 1 was executed based on a series of 2D NMR analyses such as double quantum filtered COSY (DQF-COSY), gradient-enhanced heteronuclear single quantum coherence with adiabatic pulses (HSQCAD), and gradient-selected heteronuclear multiple bond correlation using adiabatic pulses (HMBCAD). The 13C and 1H NMR data for 1 are listed in Table 1.

Table 1 13C and 1H NMR spectroscopic data for JBIR-150 (1) in DMSO-d6
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The well-resolved DQF-COSY spectrum of 1 indicated two partial structures. The first one is ranging from a doublet olefinic methine proton 2-H (δH 5.79) to an olefinic methine proton 13-H (δH 6.15) through olefinic methine protons 3-H (δH 6.65), 4-H (δH 6.19), 5-H (δH 6.14), 6-H (δH 5.91), and 7-H (δH 5.44), an oxymethine proton 8-H (δH 4.53), and aliphatic methylene protons 9-H2 (δH 1.44, 1.35), an oxymethine proton 10-H (δH 3.83), aliphatic methylene protons 11-H2 (δH 2.48, 2.24) and an olefinic methine protons 12-H (δH 5.89). The second one from olefinic methine proton 15-H (δH 5.87) to terminal methyl protons 27-H3 (δH 0.84) via olefinic methine protons 16-H (δH 6.27) and 17-H (δH 5.39), methylene protons 18-H2 (δH 2.32, 1.82), a nitrogen-substituted methine proton 19-H (δH 3.74), methylene protons 20-H2 (δH 2.19), olefinic methine protons 21-H (δH 5.53), 22-H (δH 6.01), 23-H (δH 5.97), and 24-H (δH 5.57), and aliphatic methylene protons 25-H2 (δH 1.99) and 26-H2 (δH 1.34, 1.23). In the HMBC spectrum, 1H–13C long-range couplings from singlet methyl protons 28-H3 (δH 1.74) to olefinic quaternary carbon C-14 (δC 133.6) and olefinic methine carbons C-13 (δC 136.7) and C-15 (δC 130.1) connected C-13 and C-15 via C-14. 1H–13C Long-range couplings from olefinic protons H-13 and H-15 to C-28 also supported this connection. A macrolactam structure was established by the 1H–13C long-range couplings from the olefinic protons 2-H and 3-H and an amide proton (δH 7.39), which was spin coupled with 19-H, to an amide carbonyl carbon C-1 (δC 165.9). Based on the double bond equivalents of 1 and of the proton and carbon shifts, two hydroxy groups were substituted at the positions of C-8 and C-10. Thus, the gross structure of 1 was established as shown in Fig. 1.

Fig. 1
figure 1

a Structure of JBIR-150. b NMR analysis of JBIR-150. COSY: bold line; HMBC (1H to 13C): arrows, ROESY: dashed arrows

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The 2E, 4E, 6E, 12E, 14E, 16E, 21E, and 23E geometry was assigned based on the large 3JH,H values (J2,3 = J4,5 = J6,7 = J16,17 = J21,22 = J23,24 = 14–15 Hz) and ROESY correlations between 11-H2 and 13-H, 13-H and 15-H, and 15-H and 17-H.

Structurally related compounds are the 20-membered polyene macrolactams heronamide C and F and 8-deoxyheronamide C[1,2,3,4] from a marine-derived Streptomyces sp., which showed moderate cytotoxic activities. Compound 1 also exhibited cytotoxicity against human malignant mesothelioma MESO-1 and human T-lymphoma Jurkat cells [5], with IC50 values of 2.3 and 0.90 μM, respectively, and weak cytotoxic effects against human ovarian adenocarcinoma SKOV-3 at a concentration of 100 μM. Tests for further biological activities and biosynthetic studies of 1 are now underway.