A new cyano-substituted anthracycline metabolite from streptomyces sp. Hs-nf-1006

Anthracyclines, produced by various _Streptomyces_ species, have been proved to be important components in the treatment of cancer chemotherapy.1, 2, 3 The first clinically important

anthracycline (daunorubicin) was isolated from the pigment-producing _Streptomyces peucetius_ early in the 1960s. In the concerted effort to yield new and improved anthracyclines,

doxorubicin subsequently appeared. Although this agent differed by only one hydroxyl group from daunorubicin, it was soon realized that this minor difference was sufficient to endow the drug

with a greatly superior spectrum of activity.4 So a great effort has been made to develop various analogs and derivatives of anthracycline compounds expecting for more improved antitumor

drugs.5, 6, 7, 8 As part of our continuous screening for more secondary metabolites, we investigated the bioactive constituents of the strain _Streptomyces_ sp. HS-NF-1006 and this led to

the isolation of an interesting anthracycline analog, designated 6"-cyano-6"-deoxy-TAN-1120 (1, Figure 1), from the fermentation broth of this strain. Here, the details of

fermentation, isolation, structure characterization and bioactivity of compound 1 are described. Strain _Streptomyces_ sp. HS-NF-1006 was isolated from a soil sample collected from a

pasture, located in Hailaer, Inner Mongolia Autonomous Region, China. The strain was identified as the genus _Streptomyces_ because its 16S rRNA sequence (accession no: KU848003) exhibited a

high sequence similarity of 99.45% with that of _Streptomyces rishiriensis_ strain NBRC13407 (accession no: AB184383) and it was deposited in the Pharmaceutical Research Culture Collection,

Zhejiang Hisun Group Co., Ltd., with accession no: HS-NF-1006. This strain was incubated for 6–7 days at 28 °C on the medium containing glucose (Sinopharm Chemical Reagent Co., Ltd.,

Shanghai, China) 4 g, malt extract (Bei Jing Ao Bo Xing, Beijing, China) 10 g, yeast extract (Oxoid Ltd, Basingstoke, UK) 4 g, CoCl2·6H2O 0.005 g and agar (Becton, Dickinson and company,

Franklin Lake, NJ, USA) 18 g in 1.0 l of water, pH 7.0–7.2. The seed medium consisted of glucose 4 g, malt extract 10 g, yeast extract 4 g and CaCO3 2 g in 1.0 l water, pH 7.2–7.4. All the

media were sterilized at 121 °C for 20 min. Fermentation was carried out in a 50 l fermentor (containing 30 l of production medium; Shanghai Guoqiang Bioengineering Equipment, Shanghai,

China). The producing medium was composed of glucose 1%, soluble starch (Haiyan Liuhe Starch Chemical Co, Ltd., Haiyan, China) 4%, yeast extract 0.4%, malt extract 1%, CaCO3 0.2%, FeSO4·7H2O

0.1%, ZnSO4·7H2O 0.1%, and MnCl2·4H2O 0.1% at pH 7.2–7.4 before sterilization. The fermentation was conducted at 28 °C for 7 days by stirring at 200 r.p.m. with an aeration rate of 1000 l

of air per hour. The final 30 l of fermentation broth was centrifuged to separate mycelia and supernatant. The mycelial cake was washed with water (3 l) and subsequently extracted with MeOH

(10 l). The supernatant and the wash water were passed through a column of Diaion HP-20 resin (Mitsubushi Chemical Co, Ltd., Tokyo, Japan) equilibrating with water and then eluting with 95%

EtOH (5 l). The MeOH extract and EtOH eluent were concentrated under reduced pressure to 1 l at 45 °C, and then extracted three times using an equal volume of EtOAc. The combined organic

phase was evaporated under reduced pressure to yield a mixture (26 g). The mixture was subjected to a silica gel (Qingdao Haiyang Chemical Group, Qingdao, Shandong, China; 100–200 mesh)

column and eluted stepwise with CHCl3/MeOH (100:0, 98:2, 95:5, 90:10, 85:15 and 80:20, v/v) to give six fractions (Fr.1 to Fr.6) based on the TLC profiles. TLC was performed on silica-gel

plates (HSGF254, Yantai Chemical Industry Research Institute, Yantai, China) with solvent system of CHCl3/MeOH (9:1, v/v). The developed TLC plates were observed under a UV lamp at 254 nm,

or by heating after spraying with sulfuric acid/ethanol, 5:95 (v/v). After the Fr.3 was concentrated in vacuo, the material (1.2 g) was subjected to Sephadex LH-20 gel (GE Healthcare, Glies,

UK) column eluted with CHCl3/MeOH (1:1, v/v) and detected using TLC to afford two fractions (Fr.3-1 and Fr.3-2). The Fr.3-1 was further purified by preparative HPLC (Shimadzu LC-8A,

Shimadzu-C18, 5 μm, 250 × 20 mm inner diameter; 20 ml min−1; 220 nm/254 nm; Shimadzu, Kyoto, Japan) with a 25 min gradient program of 75–85% MeOH in H2O to obtain compound 1 (_t_R 8.2 min,

16.3 mg). 1H and 13C NMR spectra were measured with a Bruker DRX-400 (400 MHz for 1H and 100 MHz for 13C) spectrometer (Bruker, Rheinstetten, Germany). The ESIMS and HRESIMS spectra were

taken on a Q-TOF Micro LC-MS-MS mass spectrometer (Waters Co, Milford, MA, USA). Compound 1 was isolated as a red amorphous powder with [_α_]D25 +74.2 (_c_ 0.03, EtOH) and UV (EtOH) _λ_max

nm (log _ɛ_): 201 (4.22), 234 (4.25), 252 (4.12), 290 (3.71), 477 (3.68), 499 (3.70), 533 (3.50), 579 (2.89). Its molecular formula C35H40N2O12 was deduced from the pseudo molecular ion at

_m/z_ 681.2643 [M+H]+, which indicated 17 degrees of unsaturation. The IR spectrum of 1 showed absorption bands assignable to hydroxy group (3226 cm−1) and carbonyl (1660 cm−1). The 1H NMR

spectrum (Table 1) of 1 displayed two downfield active OH proton signals at _δ_H 14.0 (1H, s) and 13.3 (1H, s), a methoxy proton signal at _δ_H 4.11 (3H, s), three doublet aliphatic methyls

at _δ_H 1.26 (3H, d, _J_=6.2 Hz), 1.30 (3H, d, _J_=6.2 Hz), 1.31 (3H, d, _J_=5.2 Hz), a 1, 2, 3-trisubstituted benzene ring system corresponding to three aromatic protons at _δ_H 8.06 (1H,

d, _J_=8.1 Hz), 7.80 (1H, t, _J_=8.1 Hz) and 7.41 (1H, d, _J_=8.1 Hz), and an acetyl proton corresponding to a singlet methyl proton signal at _δ_H 2.44 (3H, s). The 13C NMR spectrum showed

signals from 33 carbon atoms, which is incompatible with its molecular formula C35H40N2O12. The 1H and 13C NMR data assignment were supported by the 1H-1H COSY, HMBC and HMQC experiments. In

the HMBC spectrum (Figure 2), the correlation from the proton signal of C-2 (_δ_H, 7.80, _δ_C, 135.6) to C-12a (_δ_C 135.6) indicated the carbon signal at _δ_C 135.6 containing two

resonances. Careful examination of HMBC spectrum revealed a correlation between the acetyl group (_δ_H 4.11) and C-9 (_δ_C 77.0), which showed that an oxygenated quaternary carbon signal was

overlapped with the carbon signal of residual CHCl3. Combined with the DEPT experiment, the 35-carbon resonances can be categorized as 3 carbonyls (_δ_C 211.9, 187.1 and 186.8), 2 downfield

_sp_3 methines (each bonding with two oxygen atoms) (_δ_C 107.6, 101.1), 1 oxygenated _sp_3 quaternary carbon (_δ_C 77.0), 5 oxygenated _sp_3 methines (_δ_C 79.3, 79.3, 70.2, 65.1, 64.5), 1

methoxy group (_δ_C 56.7), 2 _sp_3 methines (_δ_C 54.4, 48.1), 4 _sp_3 methylenes (_δ_C 44.0, 35.0, 33.4, 32.1), 4 methyls (_δ_C 24.8, 23.6, 21.2, 17.0) and 13 carbon signals in aromatic

region. Initial inspection of both the 1H and 13C NMR spectra (Table 1) indicated compound 1 to be a member of the baumycin-group anthracycline. Comparison of the 1H and 13C NMR data of 1

with those of TAN-11209 revealed significant similarities (Table 1). Taking the molecular formula C35H40N2O12 of 1 into account, compound 1 differs from TAN-1120 by the replacement of a

hydroxy group in TAN-1120 with a cyano group. The presence of a nitrile carbon (_δ_C 117.5) is evident in the 13C NMR spectrum of 1. More detailed analysis of the 13C NMR spectrum showed a

significant upfield shift of C-6" from _δ_C 94.3 to _δ_C 54.4 when comparing the NMR spectrum of TAN-1120 with that of 1. This difference suggests that the cyano-group in 1 is located

on C-6". Application of 2D NMR techniques including 1H-1H COSY, HMQC and HMBC, the placement of the cyano-group on C-6" was definitively determined by the key correlations

described below, and shown in Figure 2. The doublet methyl proton H3-7" (_δ_H 1.31) is correlated to H-5" (_δ_H 3.92), which in turn is correlated to H-6" (_δ_H 3.66), as is

evident in the 1H-1H COSY spectrum. Furthermore, the correlated signal from H-6" to a cyano-group carbon resonance (_δ_C 117.5) was observed in the HMBC spectrum. As a consequence, the

structure of 1 was established to be 6"-cyano-6"-deoxy-TAN-1120 as shown in Figure 1. The relative configuration of 1 was attempted to be assigned as that of TAN-1120 based on the

similarities of NMR data and optical values between 1 and TAN-1120. The cytotoxicity of 1 was assayed for growth-inhibition activity _in vitro_ against two human tumor cell lines, human

hepatocellular liver carcinoma cells HepG2 and human lung tumor cells A549, according to the CCK8 colorimetric method, as reported in our previous papers10, 11 using doxorubicin and

daunorubicin as positive controls. As a result, the bioassay showed that 1 has stronger cytotoxicity against the two tumor cell lines than doxorubicin and daunorubicin (Table 2). REFERENCES

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research work was financially supported by the National Natural Science Foundation of China (31471809). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Provincial Joint Engineering Laboratory

of Biopesticide Preparation, School of Forestry & Biotechnology, Zhejiang Agricultural and Forestry University, Lin’An, China Xu Wan, An-liang Chen & Ji-dong Wang * Department of New

Drug Screening, Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou, China Hui-jun Ren, Min-na Du, Huan Qi, Hui Zhang & Ji-dong Wang Authors * Xu Wan View author publications You can also

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authors declare no conflict of interest. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Wan, X., Ren, Hj., Du, Mn. _et al._ A new cyano-substituted

anthracycline metabolite from _Streptomyces_ sp. HS-NF-1006. _J Antibiot_ 70, 219–221 (2017). https://doi.org/10.1038/ja.2016.112 Download citation * Received: 18 May 2016 * Revised: 25 June

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