Hun Zho,Meizhi Wng,Guoong Chen*,Dn HuEnqing Li,Yio Qu,Liing Zhou,Lingong Guo,Xinsheng YoHo Go*

a Institute of Traditional Chinese Medicine and Natural Products,College of Pharmacy/Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research,Jinan University,Guangzhou 510632,China

b College of Traditional Chinese Medicine,Jinan University,Guangzhou 510632,China

c Guangdong-Hongkong-Macau Institute of CNSRegeneration,Joint International Research Laboratory of CNSRegeneration,Jinan University,Guangzhou 510632,China

d State Key Laboratory of Mycology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100190,China

Key words:Meroterpenoid Dimer Diisoprenyl-cyclohexene/ane-type meroterpenoid Diels-Alder reaction Biscogniauxia sp.

ABSTRACT Dimericbiscognienynes Band C(1 and 2),tw o new diisoprenyl-cyclohexene-type meroterpenoid dimers,were isolated from Biscogniauxia sp.71-10-1-1.Their structures,including the absolute con fi gurations,were determined by spectroscopic analyses and ECD experiments.Meroterpenoids are special natural products that originate from mixed terpenoid-nonterpenoid pathway.As a member of meroterpenoid family,diisoprenyl-cyclohexene/ane-type meroterpenoids composed of tw o isoprenyl chains(C5 unit)and a cyclohexene/ane moiety(C6 unit),featuring diverse skeleton structures with w ide range of bioactivities.In these reported diisoprenyl-cyclohexene/ane-type meroterpenoids,only three dimers were identi fied.The discovery of the tw o new dimers added members of this rare class of meroterpenoids.

Meroterpenoids are broadly de fined as compounds of mixed terpenoid-nonterpenoid origin,which have attracted broad attention due to their structural complexity and remarkably w ide range of bioactivities[1,2],such as pyripyropene A(cholesterol acyltransferase-2 inhibitor)[3],territrem B(acetylcholinesterase inhibitor)[4],ascofuranone(trypanosome alternative oxidase inhibitor)[5],berkeleyacetal C(anti-in fl ammatory activity)[6],and so on.Diisoprenyl-cyclohexene/ane-type meroterpenoids,generally possessing tw o isoprenyl chains(C5 unit)attached to a cyclohexene/ane moiety(C6 unit)at ortho or meta-position,originate from the hybrid terpenoid-shikimate biosynthesis.To date,more than 50 diisoprenyl-cyclohexene/anes have been reported from fungi(such as Pestalotiopsis sp.[7–13],Isariopsis sp.[14–16],and Truncatella sp.[17])and plants(such as Ophryosporus lorentzii[18]and Cephalozia otaruensis[19]).

In our previous investigation on metabolites of a fungal strain of Biscogniauxia sp.71-10-1-1 fermented with rice,a novel diisoprenyl-cyclohexene-type meroterpenoid dimer(dimericbiscognialkyne A)with short-term memory enhancement activity was isolated(Fig.1),along with three new biscognienynes[20].Dimericbiscognialkyne A is the fi rst meroterpenoid dimer featuring hexadecahydrobenzo[kl]xanthenering system,which could derive from tw o same monomeric diisoprenyl-cyclohexene-type meroterpenoids(biscognienyne B)via aunique intermolecular redox coupling Diels-Alder adduct and anucleophilic addition reaction.In addition,the dimericbiscognialkyne A and biscognienynes possess aesthetically interesting ‘bird’-liketopologies,named ashuanhuanbirds[20].In order to obtain more diisoprenyl-cyclohexene/anetype meroterpenoid dimers,a chemical investigation on the residual fractions of this fungus was carried out,which led to the isolation of tw o new diisoprenyl-cyclohexene-type meroterpenoid dimers(dimericbiscognienynes B and C,1 and 2)(Fig.1).Herein,we describe the isolation and structural elucidation of 1 and 2.

The culture was extracted thrice with EtOAc,and the organic solvent was evaporated to dryness under vacuum to afford a crude extract(43.2 g).Then the crude extract was subjected to silica gel CC(4 cm?15 cm)using cyclohexane-MeOH(100:0 and 0:100,v/v)to afford a cyclohexane extract(C,12.5 g)and a MeOH extract(w,21.9 g).The MeOH extract(w,21.9 g)was separated by ODS CC(4 cm?30 cm)eluting with MeOH-H2O(20:80,50:50,70:30,and 100:0,v/v)to afford 4 fractions(w 1–w 4).Fraction w 3(3.1 g)was further subjected to ODS(4 cm?45 cm)MPLC eluted with a gradient of MeOH-H2O(20:80 to 100:0,v/v)for 420 min at a fl ow rate of 20 m L/min to afford fractions w 3-1–w 3-9.Fraction w 3-8(673.2 mg)was subjected to silica gel CCusing cyclohexane-ethyl acetate(100:0 to 0:100,v/v)to afford fractions w 3-8-1–w 3-8-8.Fraction w 3-8-3(47.3 mg)was subjected to preparative HPLCusing MeCN-H2O(50:50,v/v)at a fl ow rate of 3 m L/min to yield 1(tR:24.3 min,2.1 mg)and 2(tR:28.7 min,5.3 mg).

Fig.1.Chemical structures of 1,2,and dimericbiscognialkyne A.

Dimericbiscognienyne B(1):Colorless oil;[a]2D7.1?123.6(c 0.25,MeOH);UV(MeOH,nm)lmax(log e):205(4.86),224(4.59),285(4.30);IR(KBr,cm?1):nmax3421,2969,2920,2363,1647,1616,1540,1457,1377,1089;ECD lnm(D e)(c 9.7?10?4mol/L,MeOH):213(+4.74);ESI-MS(positive):m/z 545[M+Na]+;HR-ESI-MS(positive):m/z 545.2886 [M+Na]+(calcd.for C32H42O6Na:545.2879),1H and13CNMR data can be found in Table 1.

Dimericbiscognienyne C(2):Colorless oil;[a]D27.1+51.6(c 0.5,MeOH);UV(MeOH,nm)lmax(log e):224(4.43),315(3.34);IR(KBr,cm–1):nmax3442,2965,2924,2360,1718,1668,1437,1376,1276,1089;ECD lnm(D e)(c 6.3?10?4mol/L,MeOH):213(+6.70);ESIMS(positive):m/z 543[M+Na]+;HR-ESI-MS(positive):m/z 521.2899[M+H]+(calcd.for C32H41O6:521.2903);1H and13C NMR data can be found in Table 1.

Dimericbiscognienyne B(1)was obtained as a colorless oil.The molecular formula of 1 was established as C32H42O6(12 degrees of unsaturation)from its HR-ESI-MS(m/z 545.2886[M+Na]+,calcd.for C32H42O6Na:545.2879),which was 2 Da more than dimericbiscognienyne A.The13C NMR data showed 32 carbon signals(Table 1).Combined with data from the DEPT 135 experiment,these carbons can be categorized as ten aromatic or ole fi nic carbons(dC135.7,134.6,132.9,132.7,125.8,125.6,122.6,119.0,118.2,116.8),tw o sp quaternary carbons(dC88.4,86.1),four sp3quaternary carbons(dC94.9,73.5,64.2,48.1),six sp3methine carbons(dC75.2,66.9,65.8,60.1,41.7,37.6),four sp3methylene carbons(dC40.5,38.1,31.1,26.3),and six methyl carbons(dC26.0,25.8,23.9,23.5,18.1,18.0).The1H NMRdata(Table 1)of 1 revealed the characteristic signalsof six ole fi nic or aromatic protons[dH5.62(br s,1 H),5.47(br s,1 H),5.25(br t,1H,J=7.5 Hz),5.19(1 H),5.17(1 H),and 5.03(br t,1H,J=7.5 Hz)]and six methyls[dH1.78(br s,3 H),1.74(br s,3 H),1.73(br s,3 H),1.71(br s,3 H),1.64(br s,3 H),1.64(br s,3 H)].Except for the disappearence of one oxygenated methine signal[dC59.4(C-30);dH3.21(H-30)],one additional methylene signal[dC40.5(C-30);dH2.32(Ha-30),1.32(Hb-30)],and the obvious down field shift of C-20(dC73.5),the NMRdata of 1 are similar to those of dimericbiscognienyne A[20],which indicated that 1 should have the same skeleton as dimericbiscognienyne A,and 1 is a deoxidized derivative of dimericbiscognienyne A at C-30.All the proton resonances were associated to the directly attached carbon atoms through the HSQC experiment(Table 1 and Supporting information).The key1H-1H COSY correlations(Table S1 in Supporting information)of H-30a with H-30b/H-40/H-50,H-30b with H-30a/H-40,and H-40with H-30a/H-30b/H-50and the HMBCcross-peaks(Table S1)from H-30a/H-30b to C-10/C-20/C-40/C-50,and from H-50to C-10/C-30/C-70con fi rmed the above deduction.The w hole planar structure of 1 was established by the analysis of 2D NMR(Table S1),and the assignments of all proton and carbon resonances are show n in Table 1.

Table 1 NMR data of 1 and 2 in CDCl3.

The relative con fi guration of 1 wasdeduced by comparison with dimericbiscognienyne A,which was previously established by X-ray crystallography analysis[20].Their planar structures can be divided into tw o parts(A and B),and the planar structures of part A in 1 and dimericbiscognienyne A were identical.Because the13C chemical shifts of part A in 1 were substantially identical with those of dimericbiscognienyne A[20],the relative con fi guration of part A in 1 was considered as the same as that of dimericbiscognienyne A,which wasassigned as1R*,2S*,3S*,4R*,5S*,6R*,70R*.In part Bof 1,the large value of3JHb-30,H-40(9.5 Hz)indicated that H-40was on the pseudoaxial orientation in the cyclohexene ring(C10–C60),and Hb-30was on the opposite axial orientation in the cyclohexene ring(C10–C60)of 1(Fig.2).In the NOESYexperiment of 1,the observed correlations between H-10and Hb-30/Ha-120/Hb-120demonstrated that H-10,Hb-30,and C-120were on the same face in the cyclohexene ring(C10–C60)of 1(Fig.2 and Table S1).On the basis of the analyses of3JHb-30,H-40and the NOESYcorrelations,the relative con fi guration of the part B in 1 was assigned as 10R*,20S*,40S*(Fig.2).Combined with the above deductions and the key NOESYcorrelation between H-10and H-70(Fig.2 and Table S1),the w hole relative con fi guration of 1 was assigned as 1R*,2S*,3S*,4R*,5S*,6R*,10R*,20S*,40S*,70R*(Fig.2).

Fig.2.Key NOESY or ROESY correlations of 1 and 2.

Fig.3.ECD spectra of 1,2 and dimericbiscognialkyne A.

Schem e 1.Plausible biosynthetic pathw ays of 1 and 2.

Dimericbiscognienyne C(2)was obtained as a colorless oil.The molecular formula of 2 was established as C32H40O6(13 degrees of unsaturation)from its HR-ESI-MS(m/z 521.2899[M+H]+,calcd.for C32H41O6:521.2903),which was 2 Da less than dimericbiscognienyne B(1).The13CNMRdata showed 32 carbon signals(Table 1).Combined with data from the DEPT 135 experiment,these carbons can be categorized as eleven sp2carbons[including one carbonyl(dC197.0)],tw o sp quaternary carbons,four sp3quaternary carbons[including three oxygenated ones(dC94.9,75.4,64.2)],fi ve sp3methine carbons[including three oxygenated ones(dC75.0,66.7,60.4)],four sp3methylene carbons,and six methyl carbons.The1H NMRdata(Table 1)of 2 revealed that the characteristic signals of six ole fi nic or aromatic protons[dH5.95(br s,1 H),5.44(br s,1 H),5.20(1 H),5.18(2 H),5.05(br t,1H,J=6.9 Hz)],and six methyls[dH1.76(br s,3 H),1.76(br s,3 H),1.73(br s,3 H),1.73(br s,3 H),1.65(br s,3 H),1.64(br s,3 H)].Except for the lacking of an oxygenated methine signal[dC65.8(C-40);dH4.56(H-40)]and the appearance of a carbonyl[dC197.0(C-40)],the NMR data of 2 highly resem ble those of 1(Table 1),which indicated that 2 is an oxidation product of 1 at C-40.The detailed analyses of 2D NMR(Table S2 in Supporting information)established the w hole planar structure of 2,and the assignments of all proton and carbon resonances are show n in Table 1.

The planar structures of part A in 2 and 1 were also identical.Because the13C chemical shifts of part A in 2 were substantially identical with those of 1[20],the relative con fi guration of part A in 2 was considered as the same as that of 1,which was assigned as 1R*,2S*,3S*,4R*,5S*,6R*,70R*.The ROESYcorrelations of between H-5 and 1?OH,and between H-4 and H-70in 2 were consistent with the above deduction(Fig.2 and Table S2).In addition,the observed ROESY correlations between H-10and Hb-30/Ha-120/Hb-120demonstrated that H-10,Hb-30,and C-120were on the same face in the cyclohexene ring(C10–C60)of 2(Fig.2 and Table S2),and the relative con fi guration of part B in 2 was assigned as 10R*,20S*.Combined with the above deductions and the key ROESY correlation between H-10and H-70(Fig.2 and Table S2),the w hole relative con fi guration of 2 was assigned as 1R*,2S*,3S*,4R*,5S*,6R*,10R*,20S*,70R*(Fig.2).

The ECD curves(Fig.3)of 1 and 2 were similar to that of dimericbiscognienyne A,which suggested that 1 and 2 shared the same obsolute con fi gurations with dimericbiscognienyne A.Therefore,the obsolute con fi gurations of 1 and 2 were assigned as1R,2S,3S,4R,5S,6R,10R,20S,40S,70R,and 1R,2S,3S,4R,5S,6R,10R,20S,70R,respectively.

Since the fi rst report on the diisoprenyl-cyclohexene/ane-type meroterpenoids from Beauveria felina SANK 13682 in 1983[21],more than 50 diisoprenyl-cyclohexene/ane-type meroterpenoids have been reported,most of which are monomeric(C16).Only three dimers have been reported,including pestalofones B and C with unique 2-cyclohexylspiro[5.5]undecane ring system(Pestalotiopsis sp.)[12]and dimericbiscognialkyne A with unusual hexadecahydrobenzo[kl]xanthene ring system(Biscogniauxia sp.)[20].In our chemical investigation,tw o new diisoprenyl-cyclohexene-type meroterpenoid dimers(dimericbiscognialkynes B and C)were obtained,which derive from tw o different monomeric diisoprenyl-cyclohexene-type meroterpenoids(biscognienynes A and B[20])via a unique intermolecular redox coupling Diels-Alder adduct and a nucleophilic addition reaction(Scheme 1,Figs.S1 and S2).The result of this study added the members of the rare class of diisoprenyl-cyclohexene/ane-type meroterpenoid dimers.

Acknow ledgments

This work was fi nancially supported by grantsfrom the National Natural Science Foundation of China(No.3171101305),Chang Jiang Scholars Program(Hao Gao,2017)from the Ministry of Education of China,the Guangdong Natural Science Funds for Distinguished Young Scholar(No.2017A03036027),Guangdong Special Support Program(No.2016TX03R28),Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme(Hao Gao,2014),Pearl River Nova Program of Guangzhou(No.201610010021),and K.C.Wong Education Foundation(Hao Gao,2016).

Appendix A.Supplem entary data

Supplementary material related to thisarticlecan befound,in the online version,at doi:https://doi.org/10.1016/j.cclet.2018.05.019.