Caryophyllene Sesquiterpenes from the Marine-Derived Fungus

Caryophyllene Sesquiterpenes from the Marine-Derived Fungus Ascotricha sp.ZJ-M?5by the One Strain?Many Compounds Strategy Wen-Jing Wang,?,?Dan-Yi Li,?,?Yan-Chun Li,§Hui-Ming Hua,?,?En-Long Ma,*,§and Zhan-Lin Li*,?,??Key Laboratory of Structure-Based Drug Design&Discovery,Shenyang Pharmaceutical University,Ministry of Education,Shenyang 110016,People’s Republic of China

?School of Traditional Chinese Materia Media,Shenyang Pharmaceutical University,Wenhua Road103,Box49,Shenyang110016, People’s Republic of China

§School of Life Science and Biopharmaceutics,Shenyang Pharmaceutical University,Shenyang110016,People’s Republic of China *Supporting Information

marine-derived fungus Ascotricha sp.ZJ-M-5,which can produce cyclonerodiol analogues,

diketopiperazines in an eutrophic medium,was subjected to a one strain?many compounds

to produce three new caryophyllene derivatives(1?3)and the known1,3,6-trihydroxy-8-methylxanthone

medium,Czapek Dox broth with or without Mg2+.(+)-6-O-Demethylpestalotiopsin

C(2),which have a?ve-membered hemiacetal structural moiety,showed growth

K562leukemia cell lines with the lowest GI50value of6.9±0.4μM.It can be concluded

still e?ective in the discovery of novel bioactive fungal secondary metabolites.

icrobial secondary metabolites are a major source of lead

compounds used in drug development.Recently,whole genome sequencing of microbes has revealed the existence of silent pathways,which are not always expressed under standard culture conditions.These pathways allow researchers to explore the chemical diversity of microbes by physically or chemically triggering these silent gene clusters.This strategy used to increase the diversity and the yield of microorganism-derived natural products from a single strain was termed one strain?many compounds(OSMAC)by Zeeck.1One successful application is the isolation of a series of spirobisnaphthalenes and mutolide from Sphaeropsidales sp.F-24′707via modi?ca-tion of the culture media and vessels,2addition of the enzyme inhibitors ancymidole3and tricyclazole,4and exposure to UV radiation.5

Ascotricha sp.ZJ-M-5,a fungus isolated from a mud sample collected on a coastal beach in Fenghua County,Zhejiang Province,China,was found to produce cyclonerodiol derivatives,6a3,4-seco lanostane triterpenoid,ascotrichic acid B,and several diketopiperazines7when placed in a medium containing complex nutrients including yeast extract,peptone, and corn syrup.The yield of cyclonerodiol was more than23 mg/L.

During the course of this work,it

times as much MgSO4was added to the medium than was intended.Instead of replicating of the experiment,we regarded this as an opportunity to apply the OSMAC strategy to this fungus to investigate if this strain squanders the nutrients provided and requires oligotrophic conditions to produce bioactive secondary metabolites.The impact of Mg2+on the secondary metabolism of ZJ-M-5could also be examined.To avoid factors arising from complex ingredients,modi?ed Czapek Dox broth,composed of sucrose and NaNO3as the sole sources of carbon and nitrogen,respectively,and four inorganic salts including MgCl2instead of MgSO4,was selected as an oligotrophic medium.The secondary metabolites of ZJ-M-5in Czapek Dox broth without Mg2+or with Mg2+at di?erent concentrations were compared to those produced in the eutrophic medium by HPLC pro?ling.This led to the isolation of three new caryophyllene derivatives(1?3)and the known compound,1,3,6-trihydroxy-8-methylxanthone(4).The details of the OSMAC analyses,isolation and identi?cation of these compounds,and the growth inhibitory e?ects of1?3are reported here.

Received:February5,2014

Published:May30,2014

?2014American Chemical Society and

■RESULTS AND DISCUSSION The fermentation products of ZJ-M-5in the eutrophic medium,and in the modi ?ed Czapek Dox broth without MgCl 2and with MgCl 2at 0.04,0.4,0.8,2,and 4g per 100mL,were subjected to HPLC analyses.The chromatograms are displayed in Figure 1.The fungus did not biosynthesize cyclonerodiol,the major secondary metabolite of ZJ-M-5in the eutrophic medium,in the Czapek Dox broth without MgCl 2(Figure 1i).Mg 2+was found to slightly stimulate the production of cyclonerodiol (Figure 1d ?f),but the yield was signi ?cantly lower than that in the eutrophic medium (Figure 1b).The fungus synthesized (+)-6-O -demethylpestalotiopsin A (1)and (+)-6-O -demethyl-

pestalotiopsin C (2)in the Czapek Dox broth without Mg 2+

(Figure 1i),as well as 4,which was also detected in the

modi ?ed Czapek Dox broth with MgCl 2(Figure 1f ?i).The presence of Mg 2+markedly inhibited the production of 1and 2and stimulated the biosynthesis of (?)-6-O -demethylpestalo-

tiopsin B (3)(Figure 1h).As the concentration of MgCl

2in the modi ?ed Czapek Dox broth increased,ZJ-M-5stopped the

production of compounds 1?4stepwise (Figure 1d).The peak at 69min (Figure 1d ?h)represents a mixture of fatty acid esters and was not investigated further and the peak at 79min (Figure 1d ?i)was not consistently observed in repeated fermentations.Additionally,highly polar secondary metabolites,mainly the diketopiperazines produced in the eutrophic medium (Figure 1b),were not found in the modi ?ed Czapek Dox broth,which simpli ?ed the isolation of compounds 1?https://www.wendangku.net/doc/ea10508287.html,pound 1,C 17H 26O 6by HRESIMS,showed the existence of four methyl singlets at δH 2.02,1.90,1.55,and 1.14,one ole ?nic proton at δH 5.88(1H,d,J =11.5Hz),and three oxymethine protons at δH 5.57(1H,dd,J =10.4,5.7Hz),4.91(1H,dd,J =11.5,5.8Hz),and 4.75(1H,dd,J =5.8,2.2Hz)in its 1H NMR spectrum (Table 1).The 13C NMR spectrum (Table 1)showed 17carbon resonances,comprising two signals for an acetyl at δC 170.3and 21.3,and 15carbons constituting a sesquiterpene sca ?old including two ole ?nic carbons at δ

C 132.1and 129.4,four oxygenated carbons at δC 97.3,80.7,74.7,and 74.2,and one distinctive carbon for a hemiacetal moiety at δC 109.6,guiding us to a related analogue

of compound 1,pestalotiopsin A.8The skeleton of 1was con ?rmed to be the same as that of pestalotiopsin A by careful comparison of their NMR data,excepting only that 1lacked a methoxy group at C-6.HSQC and HMBC experiments were used to con ?rm the planar structure of 1.The relative con ?guration and conformation of compound 1

were Figure 1.HPLC chromatograms for cyclonerodiol (a),ZJ-M-5in eutrophic medium (b),compound 3(c),ZJ-M-5in Czapek Dox with MgCl 2at 4.0(d),2.0(e),0.8(f),0.4(g),0.04((h),the peak for compound 3is marked),and 0((i),the peaks for compounds 1,2,and 4are marked)g/100mL.Although HPLC chromatogram (b)has peaks with retention times similar to compounds 1and 4,further analyses revealed that 1and 4were not

present.Table 1.NMR Data for Compounds 1and 2in Pyridine-d 5

274.2,CH 5.57,dd (10.4,5.7)74.1,CH 5.55,dd (10.2,5.8)

341.3,CH 2β2.73,t (10.4)α2.69,dd (10.4,5.7)41.4,CH 2β2.72,t (10.2),α2.67,dd (10.2,5.8)4132.1,C 131.8,C

5129.4,CH 5.88,d (11.5)129.4,CH 5.85,d (11.5)

674.7,CH 4.91,dd (11.5,5.8)72.6,CH 4.85,dd (11.5,4.9)

780.7,CH 4.75,dd (5.8,2.2)90.8,CH 4.08,dd (4.9,2.4)

868.1,CH 3.25,brd (1.6)63.4,CH 3.05,brs

939.1,CH 3.06,brt (7.4)39.2,CH 2.89,brt (7.6)

1043.1,CH 2～2.0,overlapped 43.0,CH 2～2.0,overlapped

1139.9,C 40.0,C

1227.4,CH 3 1.14,s 27.4,CH 3 1.14,s

1324.7,CH 3 1.55,s 24.8,CH 3 1.56,s

14109.6,CH 6.56,d (2.5)108.7,CH 6.52,d (1.9)

1517.2,CH 3 1.90,s 17.1,CH 3 1.85,s

16170.3,C 170.3,C

1721.3,CH 3 2.02,s 21.3,CH 3 2.02,s

1857.5,CH 3 3.56,s

established (Figure 2)in a NOESY experiment in which correlations of H-2/H-9/H 3-12/H 3-15,H 3-15/H-6,and H-6/H-9showed H-2,H-6,H-9,CH 3-12,and CH 3-15to be cofacial and randomly assigned as the α-orientation,and correlations of H-5/H-7and H-8/H 3-13suggested these protons were β-oriented.Additionally,NOEs from H-14to H-5and H-7oriented the hemiacetal proton toward the nine-instead of the four-membered ring.An in situ dimolybdenum CD method 9was used to establish the absolute con ?guration for the 6,7-diol moiety in 1.The negative Cotton e ?ect at 310nm observed in the CD spectrum (Figure 3)of the complex of 1and Mo 2(OAc)4in anhydrous DMSO permitted the assignment of the 6R and 7R con ?gurations for 1.Taking the relative con ?guration into consideration,the 1S ,2S ,6R ,7R ,8S ,9R ,and 14R con ?guration was assigned for 1,named (+)-6-O -demethylpestalotiopsin A.This con ?guration is the same as determined for (+)-pestalo-tiopsin A via total synthesis.10Compound 2was given the molecular formula of C 18H 28O 6by HRESIMS,one CH 2unit more than in https://www.wendangku.net/doc/ea10508287.html,parison of the 13C NMR data (Table 1)of 2to those of 1showed a new methoxy carbon at δC 57.5and the resonance of C-7of 2shifted down ?eld by 10.1ppm,while C-6and C-8shifted up ?eld by 2.1and 4.7ppm,respectively,indicating that etheri ?cation occurred at C-7of compound 2.The methoxy group at δH 3.56was assigned to C-7by its long-range correlation to C-7at δC 90.8observed in HMBC spectrum.Because compound 2was dextrorotary ([α]20D +92),has a shared biogenesis with 1,and exhibited the same relative con ?guration as 1as established by a NOESY experiment,it was given the trivial name of (+)-6-O -demethylpestalotiopsin C and was assigned the same absolute con ?guration as for 1.The molecular formula of compound 3was determined to be C 17H 26O 5by HRESIMS.However,34carbons were observed in the 13C NMR spectrum.The 1H NMR spectrum also indicated the existence of two equilibrating atropisomers in the ratio 1:1at room temperature,which had been reported for fuscoatrol A 11and pestalotiopsin B.8The two sets of NMR data were distinguished by HSQC,HMBC,and NOESY experi-ments and analyses of the coupling constants of protons in the same spin systems and were assigned to conformers A and B (Table 2)of compound 3,respectively.The 1H NMR signals of conformer B included two ole ?nic protons at δH 6.54(1H,s)and 6.20(1H,d,J =7.6Hz),two oxymethine protons at δH

5.64(1H,dd,J =10.4,7.6Hz)and 5.16(1H,d,J =7.6Hz),two oxymethylene protons at δ

H 4.50(1H,d,J =10.9Hz)and 4.26(1H,d,J =10.9Hz),one hydroxy at δ

H 6.82(1H,s),one acetyl at δH 2.00(3H,s),and one methyl attached to an sp 2carbon at δH 2.27(3H,s).The 13C NMR data of conformer B were similar to those of one conformer of fuscoatrol A except for the down ?eld shift of C-2by about 4ppm and the signi ?cant up ?eld shift of C-6by 10ppm,indicating that C-2of 3was acetylated and C-6was substituted with a hydroxy instead of the methoxy in fuscoatrol.These assignments were unambiguously con ?rmed by the HMBC experiment,leading to the establishment of the planar structure of compound 3.The relative con ?guration of conformers A and B was then determined using a NOESY experiment (Figure 4).Although the absolute con ?guration of fuscoatrol or pestalotiopsin B has not yet been determined,compound 3([α]D 20?150),named (?)-6-O -demethylpestalotiopsin B,was assigned the 1S ,2S ,6R ,and 9R con ?guration because of its biogenetic similarity to compounds 1and https://www.wendangku.net/doc/ea10508287.html,pound 4was identi ?ed as 1,3,6-trihydroxy-8-methyl-xanthone by 1H and 13C NMR,NOESY,and MS.The resonances of the protons adjacent to 3-OH and 6-OH were slightly more up ?eld by 0.1?0.3ppm than those in the literature.12The signals of 3-OH and 6-OH were not observed in the 1H NMR spectrum.This was attributed to the slightly alkaline environment (pH =9)of the DMSO-d 6used for NMR which favored the dissociation of the hydroxy groups at C-3and C-6.

The growth inhibitory e ?ects of compounds 1?3against HL-60and K562cells were assayed,and 3was found to show no activity (GI

50>100μM).Compounds 1and 2exhibited higher

activities than the positive control cisplatin (13.4±1.9μM against HL-60and 19.1±2.3μM against K562)with GI 50values of 6.9±0.4μM (HL-60)and 10.1±0.9μM (K562)for 1and 8.5±0.7μM (HL-60)and 12.3±1.1μM (K562)for 2.This is the ?rst time that caryophyllene derivatives have been isolated from the Ascotricha genus.Analogues of compounds 1?3have been isolated from various fungi including Humicola fuscoatra ,11Pestalotiopsis sp.,8,13,14and Cytospora sp.9Only pestalotiopsin A has attracted attention from medicinal chemists for its in vitro cytotoxicity against P388murine

leukemia cells.10As the yields of pestalotiopsins were low and

variable in batch culture,13continuous synthetic e ?orts have been made,but

these were costly and involved stereocontrolled approaches.10,15The isolation of compounds 1?3from Ascotricha sp.ZJ-M-5creates a new opportunity for the production of pestalotiopsin derivative via a bioreactor.Analysis of the chromatograms in Figure 1showed us that ZJ-M-5produces di ?erent metabolites in the eutrophic and oligotrophic media,respectively.The di ?erence in the sources of nitrogen and carbon might be the key.Sucrose acts as

the

Figure 2.Key NOESY correlations of compound 1

.Figure 3.Conformation of the Mo 24+complex of compound 1and its ICD spectrum in anhydrous DMSO with the inherent CD subtracted.

sole carbon source and NaNO 3the sole inorganic nitrogen source in the Czapek Dox medium,while mannitol and glucose are the carbon sources in the eutrophic medium and yeast extract and peptone donate both carbon and nitrogen simultaneously.It can be conferred that the sole carbon source and the inorganic nitrogen source in Czapek Dox switched on the silent gene clusters encoding the enzymes necessary to the assembly of compound 4and the three caryophyllene derivatives.The yield of xanthone 4remained the same regardless of whether there was any Mg 2+in the Czapek Dox broth or not.Removal of Mg 2+from Czapek Dox stopped production of compound 3but stimulated synthesis of compounds 1and 2.The production of compounds 1?4was inhibited by high concentrations of Mg 2+in the modi ?ed Czapek Dox broth.The OSMAC strategy has been applied on microbes by changing the ingredients and pH of the culture media 16,17and by adding copper ions.18The results in this paper are the ?rst to describe the application of the oligotrophic Czapek Dox broth in the OSMAC approach and the alteration of secondary metabolism in fungi by Mg 2+.For ZJ-M-5,the simplest and cheapest medium results in the production of a complex bioactive sca ?old.■EXPERIMENTAL SECTION General Experimental Procedures.Optical rotations were obtained on a PerkinElmer 241MC polarimeter.CD spectra were

acquired with Bio-Logic MOS-450spectrometer.IR spectrum was

obtained on a Bruker IFS-55spectrometer (KBr pellets).NMR spectra were recorded on Bruker ARX-300or AV-600NMR spectrometers,with tetramethyl silane as the internal standard.HRESIMS was performed on a Bruker micrOTOF-Q mass spectrometer.ESIMS was conducted on an Agilent 1100mass spectrometer.Packing materials for column chromatography were silica gel (200?300mesh,Qingdao Haiyang Chemical Co.,Ltd.)and Sephadex LH-20(GE Healthcare).Isolation and Identi ?cation of the Fungus.The fungal strain,

ZJ-M-5,was isolated from a mud sample collected on the coastal beach in Fenghua County,Zhejiang Province,China.The incubation was

carried out at 28°C on potato dextrose agar (PDA)prepared by seawater,and ZJ-M-5was isolated after 9days of incubation.This

strain was submitted for authorized identi ?cation (report no.2011-

227)at the Institute of Microbiology,the Chinese Academy of

Sciences,on Aug 15,2011.The identi ?cation,established by Associate

Professor You-Zhi Wang,was performed by the morphological evaluation and the ITS1-5.8S-ITS2sequence of the rRNA.The sequence was submitted to GenBank (accession no.JX088707).The fungal strain ZJ-M-5was deposited in the China General Micro-biological Culture Collection Center (CGMCC)as CGMCC no.8278.

OSMAC Analyses.The eutrophic medium (100mL)was prepared by dissolving 2.0g of D -mannitol,2.0g of D -glucose,0.5g of yeast extract,1.0g of peptone,0.05g of KH 2PO 4,0.03g of MgSO 4,0.1g of corn syrup,and 3.3g of sea salt in 100mL of distilled H 2O.To prevent the formation of H 2O insoluble Mg(OH)2during autoclaving,0.04g of solid MgCl 2was irradiated under UV light for 3h and then added into 100mL of the autoclaved broth containing 3.0g of sucrose,0.3g of NaNO 3,0.1g of K 2HPO 4,0.05g of KCl,and 0.001g of FeSO 4to a ?ord the modi ?ed Czapek Dox broth as the oligotrophic medium.Additionally,Czapek Dox broth without MgCl

2and the modi ?ed ones with

0.4,0.8,2,and 4g MgCl 2per 100mL were prepared.The spores

of ZJ-M-5were inoculated into 100mL of these seven di ?erent media

and cultivated at 28°C on a shaking platform for 7days.The fermented broth was ?ltered through cheesecloth,and the supernatant was partitioned three times with an equal volume of EtOAc.The residue was dissolved in 2mL of MeOH and 10μL was subjected to HPLC analysis,which was performed on Shimadzu instrument with a diode array detector (SPD-M20A)and LC-20AB pump equipped with

an Inertsil ODS-SP column (4.6mm ×150mm,5μm).The detection wavelength for the chromatogram was 210nm.The mobile phase comprised CH

3CN (A)and H 2O (B),and the gradient program was 0?20min (10%?20%A),20?50min (20%?50%A),50?75min (50%?90%A),and 75?85min (90%A).

Fermentation and Isolation.The spores of ZJ-M-5on PDA

plates were inoculated into 150mL of Czapek Dox broth

without

Table 2.NMR Data for Compound 3in Pyridine-d 5

277.0,CH 5.60,dd (11.2,4.1)80.2,CH 5.64,dd (10.4,7.6)

340.8,CH 2β3.00,t (10.9),α2.56,dd (10.4,4.1)36.1,CH 2β3.23,dd (13.6,10.4),α2.14,dd (13.6,7.6)4128.5,C 139.4,C

5134.3,CH 5.93,d (10.1)131.0,CH 6.20,d (7.6)

667.0,CH 5.33,brd (10.1)66.7,CH 5.16,d (7.6)

7143.1,CH 6.64,d (1.5)143.2,CH 6.54,s

8137.4,C 134.5,C

941.6,CH 3.44,dd (10.6,7.8)46.7,CH 3.56,m

1034.0,CH 2β2.32,dd (12.4,7.8),α1.73,dd (12.4,10.6)36.4,CH 2β2.50,t (10.9),α1.63,dd (10.6,8.7)1142.1,C 41.0,C

1227.5,CH 3 1.18,s 24.6,CH 3 1.25,s

1325.2,CH 3 1.42,s 25.6,CH 3 1.30,s

1466.4,CH 2 4.66,d (11.2)66.8,CH 2 4.50,d (10.9)

1517.4,CH 3 1.96,s 25.2,CH 3 2.27,s

16170.3,C 170.3,C

1721.4,CH 3 1.96,s 21.4,CH 3 2.00

1-OH 6.28,s 6.82,

s

Figure 4.Key NOESY correlations of the two atropisomers of compound 3.

MgCl 2in a 500mL ?ask,and 77?asks were incubated on a shaking platform at 28°C for 7days.The combined fermentation broth of

11.55L was ?ltered through cheesecloth,and the supernatant was

partitioned with butanol (11.55L)three times to a ?ord a light-yellow gum (39g).The separation of the butanol extract was carried out by

silica gel column

chromatography (45mm ×240mm)eluting with petroleum ether (PE)?acetone (7:1,5:1,3:1,1:1,each 2.1L,v/v)followed by acetone (2.1L)and MeOH (2.1L)to a ?ord six fractions

(Fr1?Fr6).

Fr2(PE ?acetone 5:1,75mg)was subjected to preparative

thin-layer chromatography (PTLC)developed with PE ?acetone (1:1,

v/v,R f =0.4),followed by Sephadex LH-20column chromatography

to a ?ord compound 4(10mg).Compound 2(15mg)was

recrystallized from Fr3(PE ?acetone 3:1,128mg)in the mixture of

MeOH and EtOAc.Recrystallization of Fr4(PE ?

acetone

1:1,194

mg)in MeOH

and EtOAc a ?orded compound 1(80mg).The fermentation broth (14.85L)of ZJ-M-5in the modi ?ed Czapek Dox

broth with MgCl 2(0.4g/100mL)after 7days of

cultivation at 28°C

was partitioned

three times with an equal volume of EtOAc.The

extract (1.1g)was chromatographed on silica gel (18mm ×45mm)eluting with a step gradient of PE ?acetone (10:1,7:1,5:1,3:1,1:1,each 60mL,v/v),acetone (60mL),and MeOH (60mL),successively,yielding seven fractions (Fr1?Fr7).Compound 3(35

mg)was recrystallized from Fr4(PE ?acetone 3:1,164mg)in MeOH and EtOAc.

(+)-6-O-Demethylpestalotiopsin A (1).Colorless needles

(MeOH ?EtOAc);mp 193?195°C;[α]D 20+103(c 1.0,MeOH);IR (KBr)νmax 3355,2981,2936,2870,1738,1239,1036,998cm ?1;1H (400MHz)and 13C NMR (150MHz),see Table 1;HRESIMS m /z 349.1607[M +Na]+(calcd for C 17H 26NaO 6,349.1627).(+)-6-O-Demethylpestalotiopsin C (2).Colorless needles

(MeOH ?EtOAc);mp 179?181°C;[α]D 20

+92(c 0.2,MeOH);IR (KBr)νmax 3412,2956,2928,2869,1738,1261,1240,1017,984cm ?1;1H (600MHz)and 13C NMR (100MHz),see Table 1;HRESIMS m /z 363.1796[M +Na]+(calcd for C 18H 28NaO 6,349.1784).

(?)-6-O-Demethylpestalotiopsin B (3).Colorless needles (MeOH-EtOAc);mp 230?232°C;[α]D 20?150(c 0.5,MeOH);IR (KBr)νmax 3423,3321,2979,2936,2881,1734,1247cm ?1;1H (400MHz)and 13C NMR (150MHz),see Table 2;ESIMS m /z 333.0[M +Na]+,345.5[M +Cl]?;HRESIMS m /z 333.1673[M +Na]+(calcd for C 17H 26NaO 5,333.1678).Induced Circular Dichroism of Compound https://www.wendangku.net/doc/ea10508287.html,ing a method described in the literature,19

1.7mg of compound 1was dissolved in 4

mL of anhydrous DMSO,of which 2mL were used for the

measurement of the CD of 1.Mo 2(OAc)4(1.4mg)was added into the

remaining 2mL solution.The CD spectrum of the complex was recorded 10min later,and the inherent CD spectrum was subtracted.CD (c 1.3mM,DMSO)λmax (Δε)274(+0.37),310(?3.65),357(?0.98),377(?1.08)nm.Biological Assay.The growth inhibitory assay was performed as described previously.20Human leukemia HL-60and K562cells (American Type

Culture Collection)were cultured in RPMI-1640medium (Gibco)with 10%fetal bovine serum,100IU/mL penicillin,

100μg/mL streptomycin,and 1mmol/L L

-glutamine.Cell

suspensions (3×104cells/mL)of 1mL were seeded into 24-well plates,followed by the addition of test samples in DMSO at ?ve

concentrations (1,3,10,30,and 100μM).After coincubation for 72h,

0.4%trypan blue was added and the cell viability was determined by

hemocytometer within 3min.The growth inhibitory e ?ects of the

tested compounds were expressed as GI 50values calculated using

LOGIT method,and the results are representative of three individual experiments.Cisplatin (99.0%,Jiutai Pharmaceutical Co.,Ltd.)was

used as a positive control.■ASSOCIATED CONTENT

*Supporting Information

Identi ?cation report for ZJ-M-5,and ESIMS and NMR spectra of compounds 1?4.This material is available free of charge via the Internet at https://www.wendangku.net/doc/ea10508287.html,.

AUTHOR INFORMATION Corresponding Authors *

For Z.-L.L.:phone,+86-024-********;E-mail,lzl1030@https://www.wendangku.net/doc/ea10508287.html,.*For E.-L.M.:E-mail,maenlong@https://www.wendangku.net/doc/ea10508287.html,.

Notes

The authors declare no competing ?nancial interest.■ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no.21002064)and Program for

Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University.We thank LetPub (https://www.wendangku.net/doc/ea10508287.html,)for its linguistic assistance during the preparation of this manuscript.■REFERENCES

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