Mengmeng Ln,Tongxu Cui,Kihun Xi,Guodong Cui,Yiwen Chu,Peng Fu,b,?,Weiming Zhu,b,?

aKey Laboratory of Marine Drugs,Ministry of Education of China,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China

bLaboratory for Marine Drugs and Bioproducts,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao 266237,China

cSchool of Pharmacy,Chengdu University,Chengdu 610106,China

1These authors contributed equally to this work.

ABSTRACT(±)-Pyriindolin(1)with a rare molecular backbone formed by fusing a 2,2′-bipyridine nucleus into a spiro[furan-3,3′-indoline]skeleton,was isolated from the Streptomyces albolongus EA12432.The constitution and the relative configuration of(±)-1 were determined by extensive spectroscopic analyses,13C calculation and DP4+ probability analysis.The absolute configurations of optically pure(+)-1 and(?)-1 which were obtained after a chiral high performance liquid chromatography(HPLC)separation were further identified by electronic circular dichroism(ECD)calculations.(+)- and(?)-Pyriindolins displayed moderate cytotoxicity against HCT-116 cell line with the half-maximal inhibitory concentration(IC50)values of 2.89 ± 0.17 μmol/L and 4.47 ± 0.26 μmol/L,respectively.

Keywords:Pyrisulfoxins Spiroindolone Streptomyces sp.Endophyte Cytotoxicity

Natural 2,2′-bipyridine alkaloids,represented by caerulomycins,are a class of secondary metabolites with distinctive characteristics produced by actinomycetes[1].Since the first example,caerulomycin A,was discovered from aStreptomycesstrain in 1959[2],a great number of analogs with different structural features have been isolated from actinomycetes,such as collismycins[3],pyrisulfoxins[4]and cyanogrisides[5].These compounds generally possess favorable bioactivities,including cytotoxic,antimicrobial,immunosuppressive,neuroprotective and anti-inflammatory activities[1?5].Our group has isolated dozens of 2,2′-bipyridine alkaloids from the actinomycete strainActinoalloteichus cyanogriseusWH1–2216–6 and its mutants,some of which showed significant anticancer activity[4b,5a,5b,5c,6].As part of our ongoing research on new bioactive 2,2′-bipyridine alkaloids from actinomycetes,Streptomyces albolongusEA12432 was obtained.This strain was isolated from the Chinese medicinal plantAconitum carmichaeli(Ranunculaceae)[7].From the culture of EA12432,we previously identified the new compounds pyrisulfoxin C,(±)-pyrisulfoxin D,pyrisulfoxin E and(±)-pyrisulfoxin F(Fig.S2 in Supporting information),among which(±)-pyrisulfoxin D exhibited significant cytotoxicity against a series of cancer cell lines[4b].To obtain new pyrisulfoxin analogs with small amounts,we re-fermented the strain EA12432 in corn solid media in a large-scale and investigated the different secondary metabolites.As a result,we identified a pair of enantiomers,(±)-pyriindolin(1)(Fig.1)possess a novel 2,2′-bipyridine-spiro[furan-3,3′-indoline]chimeric skeleton formed from 2,2′-bipyridine-6-carbaldehyde and 2-(7–hydroxy-2-oxoindolin-3-yl)acetic acid.

Fig.1.Structures of compounds(±)-1.

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

(±)-Pyriindolin(1)was obtained as a yellow amorphous powder.The molecular formula was established as C23H19N3O5S according to its high resolution electrospray ionization mass spectroscopy(HRESIMS)peak(Fig.S3 in Supporting information)atm/z450.1107[M + H]+(calcd.for C23H20N3O5S,450.1118).The13C nuclear magnetic resonance(NMR)spectra(Figs.S6 and S7 in Supporting information)showed 23 carbon signals which were classified by heteronuclear single quantum correlation(HSQC)(Fig.S8 in Supporting information)as two carbonyls,nine non-protonated carbons(one sp3and eight sp2ones),eight sp2-methines,one sp3-methine,one methylene and two methyls(one methoxyl).The1H NMR spectra(Figs.S4 and S5 in Supporting information)showed four coupled signals atδH8.35(H-3′,d,J=8.0 Hz),8.04(H-4′,dd,J=8.0,7.7 Hz),7.54(H-5′,dd,J=7.7,4.7 Hz)and 8.76(H-6′,d,J=4.7 Hz)(Table 1),suggesting the presence of a 2-substituted pyridine ring system.One methoxy signal(δH4.02,s)and one methylthio signal(δH1.77,s)were observed in the1H NMR spectra(Figs.S4 and S5).Careful comparison of the NMR data with those of pyrisulfoxins E and F[4b]indicated that pyriindolin(1)also contained a 4–methoxy-5-methylthio-2,2′-bipyridine structure,which could be confirmed by the key correlation spectroscopy(COSY)correlations of H-3′/H-4′/H-5′/H-6′(Fig.2 and Fig.S9 in Supporting information),and the key heteronuclear multiple bond correlation(HMBC)correlations of H-4′ to C-2′(δC154.1),H-3 to C-2′/C-5(δC120.6),4–OCH3to C-4(δC166.9)and 5-SCH3to C-5(Fig.2 and Fig.S10 in Supporting information).In addition,the proton signals atδH5.19(H-4′′,d,J=7.6 Hz),6.40(H-5′′,dd,J=7.6,8.2 Hz)and 6.61(H-6′′,d,J=8.2 Hz)(Table 1)were assigned to a vicinal trisubstituted benzene ring,which was further supported by the COSY correlations of H-4′′/H-5′′/H-6′′(Fig.2 and Fig.S9),and the key HMBC correlations of H-4′′ to C-7′′a(δC130.3)and H-5′′to C-3′′a(δC126.6)and C-7′′(δC141.5)(Fig.2 and Fig.S10).The HMBC correlations of H-7(δH6.40,s)to C-2′′(δC175.7)/C-3′′a/C-9′′(δC179.1)and H-8′′ to C-7(δC79.7)/C-2′′/C-3′′(δC54.1)/C-3′′a/C-9′′(Fig.2 and Fig.S10)indicated that the vicinal trisubstituted benzene ring was further fused to form a 7′′–hydroxy-7H-spiro[furan-3′′,3′′-indoline]?2′′,9′′(4H)–dione skeleton moiety.This moiety and the 2,2?-bipyridine unit were connected to form compound 1 through the C–C sigma bond between C-6 and C-7,confirmed by the HMBC correlation of H-7 to C-5(Fig.2 and Fig.S10).

Table 1 1H(500 MHz)and 13C(125 MHz)NMR data for(±)-pyriindolin(1)in DMSO-d6.

Fig.3.Experimental and calculated ECD curves for(±)-1.

The relative configuration of 1 was assigned by the NOESY correlations of H-8′′a(δH3.20,d)to H-3′ and H-4′′(Fig.2 and Fig.S11 in Supporting information).Furthermore,δCvalues of two plausible epimers,(7S,3′′R)-1 and(7R,3′′R)-1(Fig.S12 in Supporting information),were calculated at the B3LYP/6–311++G(2d,p)level[8].The DP4+ probability analysis(Fig.S12)[9]supported the relative configuration of 1 as(7S?,3′′R?).To determine its absolute configuration,we measured the electronic circular dichroism(ECD)spectrum.The result showed that compound 1 was not optically active,indicating pyriindolin(1)as a racemic mixture.This was supported by a chiral high performance liquid chromatography(HPLC)analysis(Fig.S1 in Supporting information).Then(±)-1 was successfully resolvedviaa chiral HPLC separation into two optically pure enantiomers,(+)-1 and(?)-1.To determine their absolute configurations,the predicted ECD spectrum was obtained by the TDDFT[B3LYP/6–31G(d)]method[10].The measured ECD spectrum of(+)-1 matched well with the calculated ECD curve of(7S,3′′R)-1(Fig.3).Thus,the absolute configurations of(+)- and(?)-pyriindolins were unambiguously assigned as(7S,3′′R)and(7R,3′′S).

The plausible biosynthetic pathways for(±)-1 were postulated(Scheme 1).Tryptophan was utilized to form the intermediate,indole-3-acetic acid(a),which further underwent oxidation to form intermediate,2-(7–hydroxy-2-oxoindolin-3-yl)acetic acid(b).Meanwhile,another biosynthetic precursor,4–methoxy-5-methylthio-2,2′-bipyridine-6-carbaldehyde(c)was formed from lysine,malonyl-CoA,cysteine and leucine[11].The two precursors b and c underwent an intermolecular aldol condensation followed by an intramolecular esterification to generate the racemic products,(±)-pyriindolin(1).

Scheme 1.The postulated biosynthesis of(±)-1.

Compounds(+)-1 and(?)-1 were evaluated for cytotoxicity against the cancer cell lines HCT-116,A549,Hela and K562.(+)-1 and(?)-1 exhibited inhibitory activity against HCT-116 cell line with the IC50values of 2.89 ± 0.17 μmol/L and 4.47 ± 0.26 μmol/L,respectively,while no obvious cytotoxic activity was observed for other cell lines at the concentration of 10 μmol/L.

In summary,we identified a pair of enantiomers containing a novel 2,2′-bipyridine-spiro[furan-3,3′-indoline]chimeric skeleton,(+)- and(?)-pyriindolins.The formation mechanism of this spirocyclic scaffold could provide objects for the study of condensation reactions involving aldehyde in the biosynthesis of chimeric natural products.

Declaration of competing interest

The authors declare no competing financial interest.

Acknowledgments

This work was financially supported by the National Key Research and Development Program of China(No.2019YFC0312504)and the National Natural Science Foundation of China(Nos.41876172,U1906213).

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.cclet.2021.05.028.