Chengli Liu,Chenglong Yang,Yingjie Zeng,Jing Shi,Lingyu Li,Wei Li,Ruihua Jiao,Renxiang Tan,Huiming Ge*

State Key Laboratory of Pharmaceutical Biotechnology,Institute of Functional Biomolecues,School of Life Sciences,Nanjing University,Nanjing 210023,China

Keywords:Alkaloid Chartrenoline Streptomyces chartreusis 6/6/5/5-[13_TD DIFF]etracyclic nucleus Marine-derived microorganism

ABSTRACT A novel alkaloid,chartrenoline(1),featuring an unusual 6/6/5/5-tetracyclic nucleus,was isolated from a marine Streptomyces chartreusis NA02069.The structure of 1 including its absolute con fi gurations was established by extensive analyses of its high resolution mass,NMRand single-crystal X-ray diffraction data.A plausible biosynthetic pathway for chartrenoline(1)is proposed.

Marine-derived organisms have been recognized as a great reservoir for biologically active and structurally unusual secondary metabolites that have greatly impacted the quality of human life[1–6].For example,bacteria derived from marine environment have yielded several promising drug candidates such as lipopeptide antibiotic taromycin[7],anticancer agents salinosporamide A[8–10]and bryostatins[11,12].Recently,we have reported the characterization of a rare macrodilactone named streptoseomycin with potent anti-Helicobacter pyroli activity from Streptomyces seoulensis A01[13],which was originally isolated from a marine praw n,and an antitumor agent chartreusin[14],as well as antibacterial alkaloids,strepchazolins and streptazolin from a marine sediment-derived S.chartreusis NA02069 [15].Our continued chemical investigation of S.chartreusis NA02069 strain has led to the discovery of a novel alkaloid,chartrenoline(1),featuring a unique 6/6/5/5 ring system(Fig.1).Herein,we report the isolation,structure elucidation and putative biosynthetic pathw ay for 1.

Compound 1 was obtained as pale yellow crystals.The molecular formula of 1 was determined to be C18H18N2O6based on its HR-ESI-MS data(m/z 359.1237 [M+H]+,calcd.for C18H19N2O6:359.1238),indicating 11 degrees of unsaturation.Analysis of the13CNMRspectrum,together with the DEPT135 and HSQC spectra,indicated tensp2-hydribridzed carbon resonances ascribe to tw o carbonyl carbons,four ole fi nic methines and four ole fi nic carbonyl carbons;eightsp3-hydribridzed carbon signals including one methyl group,tw o methylenes,and fi ve methine carbons.The1H NMRspectrum8IF]data(Table 1)exhibited signals for a pair of 1,4-disubstituted phenyl ring at dH7.85(d,2H,J=8.9 Hz,H-14,H-16)and 7.11(d,2H,J=8.9 Hz,H-13,H-17).In addition,the1H-1H COSY spectral data revealed a partial structure of?CH2?CH2?CH–,?CH?CH?CH?OH–,and CH3?CH–,respectively;indicating the connection of C-2/C-3/C-4,C-6/C-7/C-8/8?OH,and C-11/C-10,respectively.

The gross structure of 1 was established by extensive analyses of 2D NMRspectral data,in particular with HMBCspectrum.The HMBCcorrelations of H-2(dH3.66)with C-4(dC73.6)and C-6(dC61.5),of H-3(dH2.21)with C-5(dC138.5),of H-7(dH4.72)with C-5(dC138.5)and C-9(dC138.9),of H-8(dH4.56)with C-6(dC61.5),together with the chemical shifts of dC40.4,dC61.5 and dC82.4 for C-2,C-6 and C-7,respectively,suggested the presence of a 6/5 bicyclic ring system as show n in Fig.2.The presence of a hydroxyl group substituted at C-8 wasreinforced by HMBCcorrelations of 8-OH(dH5.77)with C-7(dC82.4),C-8(dC78.7)and C-9(dC138.9).In addition,in the HMBCspectrum,H-2(dH3.66),H-6(dH4.70)and H-7(dH4.72)showed correlations to a dow n fi eld carbon C-1(dC157.4)indicated the existence of an oxazolidin-2-one ring.

Fig.1.Structure of chartrenoline(1).

Table 11H and 13CNMRspectral data for 1.

Fig.2.Key 2D NMR correlations of 1.

The HMBCcorrelationsof H-11(dH1.09)with C-9(dC138.9),and of H-10(dH4.66)with C-5(dC138.5)and C-8(dC78.7)revealed that C-10(dC53.1)was connected to C-9(dC138.9).Moreover,a benzoic acid partial structure was elucidated by the HMBCcorrelation of H-14/16(dH7.85)with C-18(dC167.5).The HMBCcorrelation between H-10(dH4.66)and C-12(dC151.6),together with the chemical shift for C-10(dC53.1),revealed the benzoic acid group and C-10 was connected through a nitrogen atom.The only remaining oxygen atom was substituted at C-4 as revealed by chemical shifts of H-4(dH5.00)and C-4(dC73.6).Finally,ow ing to the shortage of any more double bonds,the last unsaturation degree could be ascribe to an oxazine ring through generating an O??Nbond.The proposed structure was further supported by the1H-15N HMBCcorrelations of H-10 and H-11 with N-20,and of H-3 with N-19.

Fig.3.X-ray crystallographic analysis of compound 1.

Compound 1 contained fi ve chiral centers,which were difficult to be fully solved by analysis of its NMR data.Fortunately,a high quality crystal suitable for X-ray diffraction was obtained from CH2Cl2-methanol(1/1,v/v)solvent mixture.The subsequent X-ray crystallographic data collected in Cu Kα radiation at low temperature with a fl ack parameter of?0.07(14)con fi rmed the above proposed planar structure(Fig.3),and further established the absolute con fi guration of 1 as show n in Fig.1.Compound 1 was therefore determined as an unusual 6/6/5/5-tetracyclic alkaloid with a novel skeleton.

Structurally,compound 1 is actually related to streptazolin[16],which has been isolated from the same strain w hen fermented using different medium[15].Therefore,a plausible biosynthetic pathw ay for 1 is proposed as show n in Scheme 1.Streptazolin is proposed to be biosynthesized through bacterial type Ipolyketide synthases(PKSs)[17].The C-terminal thioester reductase domain in PKSis responsible for release of the assembled polyketide chain as an aldehyde,and its subsequent reductive amination and cyclization gives streptazolin [17,18].Streptazolin can then undergo an intermolecular Diels-Alder reaction with 4-nitrosobenzoic acid to give 1[19].

Compound 1 was evaluated for its antibacterial activities against four pathogenic bacteria including Staphylococcus aureus CMCC 26003,Bacillus subtilis CICC 10283,Escherichia coli ATCC 25922,and Pseudomonas aeruginosa CICC 10351.However,compound 1 is almost inactive to them at the concentration of 128 m mol/L.

In conclusion,a novel alkaloid,chartrenoline(1),was isolated from a marine actinobacterium S.chartreusis NA02069.The structure of 1 possessing a novel skeleton with a fused 6/6/5/5 ring system was unambiguously established by extensive analysis of its spectroscopic data including HR-ESI-MS,NMR,as well as single crystal X-ray diffraction data.Furthermore,the proposed biosynthetic pathway starting from a know n alkaloid streptazolin was proposed.Our work presented here sets the stage for future biosynthetic study.Ack now led gm ents

This work was fi nancially supported by the National Natural Science Foundation of China(Nos.81522042,21572100,81773591,81421091, 81500059, 81673333, 21672101, 21761142001,21661140001,J1210026,and J1103512),Natural Science Foundation of Jiangsu Province(No.BK20151397),and Fundamental Research Funds for the Central Universities(No.020814380092).

Appendix A.Supp lementary data

Schem e 1.Proposed biosynthetic pathw ay for chartrenoline(1).

Supplementary material related to this article can be found,in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.10.030.