A New DrimaneSesquiterpenoid from Cultures of Fungus Psathyrellacandolleana

LIXue-mei，YIN Xia，LIZheng-hui，FENG Tao* ，LIU Ji-kai* State Key Laboratory of Phytochemistry and Plant Resources in West China，Kunming Institute of Botany，Chinese Academy of Sciences，Kunming 6500，China; Yunnan Agriculture University，Kunming 6500，China; University of Chinese Academy of Sciences，Beijing 00049，China

李学梅1，2，尹 霞1，3，李正辉1，冯 涛1* ，刘吉开1*

1中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室，昆明650201;2云南农业大学，昆明650201;3中国科学院大学，北京100049

Introduction

Searching for new and bioactive products from higher fungi has been a long-term job of our group．Recently，more and more terpenoids，especiallysesquiterpenoids have been found from both fruiting bodies and their fermentation broths．For instance，trefolane A［1］and conosilane A［2］are two novel sesquiterpenoids from cultures of Tremellafoliacea and Conocybesiliginea，respectively，while irlactins A-D are also four ring-rearranged carbon skeletons from cultures of Irpexlacteus［3］．Boledulins A-C were three non-isoprenoidbotryanesesquiterpenoids that have been isolated from cultures of basidiomycete Boletus edulis，and boledulin A showed certain cytotoxicities against five human cancer cell lines［4］．In addition，a number of toxic triterpenoids with a raretrioxaspiroketal backbone have been obtained from French mushroom Tricholomaterreum，which declared another poisonousmushroom(T．terreum)in Europe［5］．In this paper，we report a new drimanesesquiterpenoid，namely15-hydroxydrimenol(1)，along with a knowngermacranesesquiterpenoid1α-hydroxy-bisabola-2，10-dien-4-one(2)(Fig．1)，from cultures of fungus Psathyrellacandolleana．

Fig.1 Chem icalstructures of compounds 1 and 2

M aterials and M ethods

General experimental procedures

Optical rotationsweremeasured on a Jasco-P-1020 polarimeter(Horiba，Kyoto，Japan)．IR spectra were ob-tained by using a Bruker Tensor 27 FT-IR spectrometer(Bruker，Karlsruher，Germany)with KBr pellets．NMR spectra were acquired with instrument of an Avance III 600(Bruker，Karlsruher，Germany)．HR-EI-MS were measured on a Waters Autospec Premier P776 mass spectrometer(Waters，Milford，MA，USA)．Preparative HPLC was performed on an Agilent 1100 series with a Zorbax SB-C18(5 μm，9．4 × 150 mm)column(Agilent Technologies，Santa Clara，CA，USA)．Silica gel(200-300 mesh，Qingdao Marine Chemical Inc．，Qingdao，China)，was used for column chromatography．Fractions were monitored by TLC(Qingdao Marine Chemical Inc．，Qingdao，China)and spots visualized by heating silica gel plates immersed in vanillin-H2SO4in EtOH．

Fungalm aterial and culture

The fungus Psathyrella candolleana(Pers．)Fr．was collected from Kunming Institute of Botany in Yunnan Province，People’s Republic of China，in 2003．The fungus was identified by Prof．Zhu-Liang Yang at the Kunming Institute of Botany．A specimen (No．KIB20030828)was deposited at Kunming Institute of Botany，Chinese Academy of Sciences．

Extraction and isolation

The culture broth(21 L)was filtered，and the filtrate was extracted with ethyl acetate(20 L ×3)，while the mycelium was extracted three times with CHCl3-MeOH(1∶1)．The EtOAc layer together with the mycelium extractwas concentrated under reduced pressure to give a crude extract(10 g)，and the latter was applied to a silica gel column eluted with a gradient of CHCl3/MeOH(1∶0 → 0∶1)to obtain five fractions(1-5)(This part has been described in a previous report［6］)．Fraction 2(800 mg)was subjected to silica gel eluted with CHCl3/Me2CO(10∶1)to give three subfractions(3a-3c)．Fraction 3c was separated by semipreparative HPLC(MeCN/H2O，3∶7 → 4∶6，15 mins)to give 1(3 mg)and 2(1 mg)．

15-Hydroxydrimenol(1)

Colorless oil;［α］16D+6．8(c2．1 MeOH);IR(KBr)υmax3423，2924，1630 cm-1;1H NMR(600 MHz)and13C NMR(150 MHz)data(CDCl3)，see Table 1;HREI-MS m/z 238．1933(calcd for C15H26O2［M］+，238．1933)．

1α-Hydroxy-bisabola-2，10-dien-4-one(2)

Molecular formula C15H20O;colorless oil;-72．2(c1．2，MeOH);1H NMR(600 MHz，CDCl3)δ:4．38(1H，m，H-1)，6．78(1H，br d，J=5．7 Hz，H-2)，2．45(1H，m，H-5a)，2．45(1H，m，H-5b)，1．79(1H，m，H-6)，1．67(1H，m，H-7)，1．59(1H，m，H-8a)，1．20(1H，m，H-8b)，2．05(1H，m，H-9a)，1．95(1H，m，H-9b)，5．12(1H，t，J=7 Hz，H-10)，1．69(3H，s，H-15)，1．67(2H，s，H-12)，1．59(3H，s，H-13)，0．93(3H，d，J=6．3 Hz，H-14);13C NMR(150 MHz，CDCl3)δ:200．2(s)，145．3(d)，136．0(s)，131．4(s)，125．7(d)，64．5(d)，44．9(d)，37．4(t)，34.4(t)，33．8(d)，25．9(q)，25．8(t)，17．7(q)，16．9(q)，15．7(q)．

Structural identification

Compound 1 was isolated as a colorless oil．Itsmolecular formula were established as C15H26O2on the basis of ion peak at m/z238．1993 in HR-EI-MS(calcd for C15H26O2［M］+，238．1933)．IR absorption band at 3423 cm-1indicated the presence of hydroxy group(s)．In1H NMR spectrum(Table 1)，threemethyls as singletswere readily identified(δH0．84，0．89，and 1.78，each 3H)．In addition，two oxygenated methylenescan also be established(δH3．12，3．38，3．75，and 3．87)．The13C NMR data(Table 1)，in combination with HSQC data，identified fifteen carbon resonances corresponding to threemethyls，sixmethylenes(two oxygenated)，threemethines(one olefinic)，and three quaternary carbons．Analysis of these data suggested that 1 was similar to drimenol，a known compound discovered in 1950s，which suggested that 1 possessed a drimanesesquiterpenoid skeleton．The key difference between 1 and drimenol was that onemethyl at C-4 was oxygenated into an oxymethylene(δH3．12 and 3．38 each 1H，d，J=10．8 Hz;δC71．4，t)，as supported by HMBC correlations from δH3．12 and 3．38 to δC37．4(s，C-4)and 17．9(q，C-14)(Fig．2)．Detailed analysis of other 2D NMR data(HMBC，1H-1H COSY)suggested that the other parts of1 were the same to those of drimenol(Fig．2)．In the ROESY spectrum，the correlation between H-15 and Me-13 suggested that Me-15 rather than Me-14 was oxygenated into an oxymethylene．Detailed analysis of2D NMR data suggested that the other parts of1 were the same to those of drimenol(Fig．2)．Therefore，compound 1 was established as 15-hydroxydrimenol，as shown in Fig．1．

Fig.2 Key 2D NMR correlations of 1

Table 1 NMR spectroscopic data of com pound 1(CDCl3，600 MHz，δ in ppm，J in Hz)

Compound 2 was isolated as a colorless oil．Its NMR data were closely related to those of 1-hydroxy-bisabola-2，10-dien-4-one．The optical rotation=-72．2(c1.2，MeOH)suggested that compound 2 should be 1(-hydroxy-bisabola-2，10-dien-4-one．［6］

1 Ding JH，Feng T，Li ZH，et al．Trefolane A，a sesquiterpenoid with a new skeleton from cultures of the basidiomycete Tremellafoliacea．Org Lett，2012，14:4976-4978．

2 Yang XY，Feng T，Li ZH，et al．Conosilane A，an unprecedented sesquiterpene from the cultures of basidiomycete Conocybesiliginea．Org Lett，2012，14:5382-5384．

3 Ding JH，Feng T，Cui BK，et al．Novel sesquiterpenoids from cultures of the basidiomycete Irpexlacteus．Tetrahedron Lett，2013，54:2651-2654．

4 Feng T，Li ZH，Dong ZJ，et al．Non-isoprenoidbotryanesesquiterpenoids from basidiomycete Boletus edulis and their cytotoxic activity．Nat Prod Bioprospect，2011，1:29-32．

5 Yin X，Feng T，Shang JH，et al．Chemical and toxicological investigations of a previously unknown poisonous European mushroom Tricholomaterreum．Chem-Eur J，2014，20:7001-7009．

6 Castro V，Tamayo-Castillo G，Hakupovic J．Sesquiterpenelacrones and other constitutents from Caleaprunifolia and C．pckii．Phytochemistry，1989，28:2415-2418．