Absolute configuration and anti-tumor activity of torrubiellin B

Accep ed Manusc ip Absolu e con igu a ion and an i- umo ac i i y o o ubiellin B Ca alina F ancis Pé ez Hemphill, Geo gios Dale os, Alexand a Hamache , Ma hias Ull ich Kassack, Wenhan Lin, A ila Mándi, Tibo Ku án, Pe e P oksch PII: S0040-4039(15)00596-1 DOI: h p://dx.doi.o g/10.1016/j. e le .2015.03.126 Re e ence: TETL 46129 To appea in: Te ahed on Le e s Recei ed Da e: 11 Feb ua y 2015 Re ised Da e: 24 Ma ch 2015 Accep ed Da e: 27 Ma ch 2015 Please ci e his a icle as: Hemphill, C.F.P., Dale os, G., Hamache , A., Kassack, M.U., Lin, W., Mándi, A., Ku án, T., P oksch, P., Absolu e con igu a ion and an i- umo ac i i y o o ubiellin B, Te ahed on Le e s (2015), doi: h p://dx.doi.o g/10.1016/j. e le .2015.03.126 This is a PDF ile o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s inal o m. Please no e ha du ing he p oduc ion p ocess e o s may be disco e ed which could a ec he con en , and all legal disclaime s ha apply o he jou nal pe ain. G aphical abs ac Absolu e con igu a ion and an i- umo ac i i y o o ubiellin B Ca alina F ancis Pé ez Hemphilla, Geo gios Dale osa, Alexand a Hamache b, Ma hias Ull ich Kassackb, Wenhan Linc, A ila Mándid, Tibo Ku ánd, and Pe e P okscha,* aIns i u e o Pha maceu ical Biology and Bio echnology, Hein ich Heine Uni e si y Duesseldo , Uni e si ae ss asse 1, Geb. 26.23, 40225 Duesseldo , Ge many bIns i u e o Pha maceu ical and Medicinal Chemis y, Hein ich Heine Uni e si y Duesseldo , Uni e si ae ss asse 1, Geb. 26.23, 40225 Duesseldo , Ge many cS a e Key Labo a o y o Na u al and Biomime ic D ugs, Peking Uni e si y, 100191 Beijing, PR China dDepa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, 4010 Deb ecen, Hunga y *Co esponding au ho . Tel.: +492118114170; ax: +492118111923; email: p oksch@uni- duesseldo .de Abs ac The dime ic an h acene de i a i e o ubiellin B (1) was isola ed om he endophy ic ungus Ac emonium sp. ha had been ob ained om lea es o he Mang o e plan Sonne a ia caseola is. The absolu e con igu a ion o 1 was es ablished as (5'R,10'S,10a'R) o he i s ime on he basis o i s elec onic ci cula dich oism (ECD) spec a aided wi h TDDFT-ECD calcula ions. To ubiellin B (1) exhibi ed s ong an i- umo ac i i y when es ed in i o agains se e al solid cance cell lines including cells ha a e esis an agains he widely used cy os a ic d ug cispla in. The IC50 alues o 1 agains cispla in sensi i e and cispla in esis an cells we e in he ange o 0.2 - 2.6 µM depending on cell line in es iga ed. Keywo ds: To ubiellin B, Ac emonium sp., Absolu e con igu a ion, An i- umo ac i i y In oduc ion Endophy ic ungi a e known as a ich sou ce o s uc u ally di e se bioac i e compounds ha a e o en unp eceden ed in o he o ganisms.1-7 Endophy es which inhabi plan s ha a e subjec o se e e s ess condi ions a e especially in e es ing o biop ospec ing since he ungi a e assumed o con ibu e o he su i al o hei hos s e.g. h ough accumula ion o de ense me aboli es.8-10 We and o he s ha e epea edly shown ha Mang o e associa ed endophy es a e aluable sou ces o new bioac i e me aboli es such as he an i- umo me aboli e phomoxan hone A isola ed om he Mang o e endophy e Phomopsis longicolla which was ound o s ongly inhibi umo cells.11 Mang o es a e p one o nume ous s ess ac o s such as changing le els o salini y, pe iodical looding and a high incidence o he bi o es and o pa hogenic mic obes. Appa en ly hese ac o s ha e shaped no only he seconda y me aboli es o he hos s bu also ha o hei associa ed endophy es.12,13 In he cou se o ongoing s udies on na u al p oduc s om Mang o e de i ed endophy es we in es iga ed he endophy ic ungus Ac emonium sp. ha was isola ed om lea es o he Mang o e plan Sonne a ia caseola is om he island o Hainan (P. R. China). Ou in e es in his ungus had been a oused by he s ong an i- umo ac i i y o i s c ude E OAc ex ac when es ed in i o agains solid cance cell lines including cells ha a e highly esis an agains he widely used cy os a ic d ug cispla in (se e e g ow h inhibi ion o A2780sens cells obse ed a 10 µg/mL, and o A2780CisR cells a 100 µg/mL). Resis ance o cance cells ha eme ges equen ly du ing chemo he apy is a se ious p oblem o he apy o umo pa ien s and is esponsible o he ul ima e ailu e o his he apeu ic app oach in spi e o educ ion o umo load du ing ea ly phases o ea men .14,15 The e o e, new he apeu ics ha a e able o b eak esis ance agains cu en ly employed cy os a ic d ugs a e u gen ly needed. Du ing bioassay guided ac iona ion o he ex ac o Ac emonium sp. o ubiellin B (1)16, an asymme ic an h acene de i a i e was isola ed as he ac i e ing edien ha is esponsible o he an i- umo ac i i y o he ungal ex ac . To ubiellin B had i s been epo ed om To ubiella sp. BCC 28517 and he ela i e con igu a ion o he compound had been de e mined h ough NOE expe imen s.17 Howe e , he absolu e con igu a ion o he compound was unknown so a . The de e mina ion o he absolu e con igu a ion o o ubiellin B (1) especially in ligh o i s p ominen ac i i y agains cance cells is an impo an issue du ing s uc u e de e mina ion. He e we epo o he i s ime he absolu e con igu a ion o o ubiellin B (1) oge he wi h i s p onounced an i- umo ac i i y agains cispla in sensi i e and cispla in esis an solid cance cell lines. Resul s and discussion The e hyl ace a e ex ac o he endophy ic ungus Ac emonium sp. g own on solid ice medium was submi ed o a bioassay guided ch oma og aphic sepa a ion using di e en s a iona y phases including silica gel, Sephadex LH-20 and semip epa a i e HPLC on a C18 e e sed phase column. To ubiellin B (1) was isola ed as he majo cons i uen o he ex ac (Figu e 1). Compound 1 exhibi ed a UV/Vis spec um wi h h ee abso p ion maxima a 206, 283, and 397 nm ha is ypical o a conjuga ed sys em. In he HRESIMS he pseudomolecula ion was de ec ed a m/z 541.1138 [M+H]+ and he molecula o mula was de e mined as C30H20O10. Compa ison o he MS and NMR da a o 1 wi h hose p e iously epo ed o o ubiellin B sugges ed ha hey a e iden ical.17 The s uc u e o 1 was inally con i med as o ubiellin B by ca e ul analysis o COSY, HMBC and ROESY co ela ions. In e p e a ion o he COSY and HMBC co ela ions shown in Figu e 2 e ealed wo an h acene moie ies being used ia a cyclopen ene ing. The HMBC co ela ions om H-5’ o C-4, C-3, C-10a’ and o C-11 es ablished he connec i i y o bo h monome s. The o ma ion o his a e linkage be ween C-4 and C-5’ and be ween C-3 and C-10a’ h ough C- 11 is a s uc u al ea u e ha is inhe en o o ubiellins. The linkage o he wo an h acene uni s was u he co obo a ed h ough co ela ions om H2-11 o H-2 and om H-11 o H-4’ in he ROESY spec um o 1. The ela i e con igu a ion o C-5’ and C-10’ was deduced om ROESY expe imen s (Figu e 4). H-10’ showed ROE co ela ions o H-5’ and H-11eq and u he mo e H-11eq had a co ela ion o H-2, hus indica ing he (5’S*,10’R*,10a’S*) ela i e con igu a ion o 1 as shown in Figu e 4, which is in ag eemen wi h ha p e iously epo ed.17 To ubiellin B may be iewed as a 4,5’-linked he e odime ic bia yl o 7-hyd oxyemodin and dihyd o-1,8,10- ihyd oxyan h acene-9(4H)-one, in which he o a ion along he bia yl axis is blocked by he me hylene linke be ween C-3 and C-10a. In con as o axially chi al bia yls o an h acene-9,10-dione monome s,18 o ubiellin B has only cen al chi ali y elemen s due o he C-5’, C-10’ and C-10a’ chi ali y cen e s and he p ojec ion angle be ween he planes o he wo monome s is go e ned by he cen al chi ali y. The solu ion ECD spec a o o ubiellin B showed in ense nega i e Co on e ec (CE) a 435 nm and a posi i e one a 395 nm accompanied by shoulde s and qui e a numbe o o he o e lapping high-ene gy ECD ansi ions (Figu e 3). Fo he con igu a ional assignmen , solu ion con o ma ional analysis and TDDFT-ECD calcula ions we e pe o med. The ini ial MMFF con o ma ional analysis o he a bi a ily selec ed (5’S,10’R,10a’S)-1 esul ed in 13 con o me s, which we e eop imized a B3LYP/6-31G(d) le el in acuo. The eop imiza ion a o ded 2 con o me s (96.8% and 2.9% popula ions) abo e 1% Bol zmann-popula ion, which di e ed only in he o ien a ion o hyd ogens o he 10’-OH and 3’-Me g oups (Figu e 4). The ωC-3,C-4,C-5’,C-6’ dihed al angle, a pa ame e desc ibing he ela i e o ien a ion o he elec ic ansi ion momen s o he wo subuni s, was ound +103.17°. TDDFT-ECD calcula ions o he wo low-ene gy con o me s we e ca ied ou a 3 di e en le els o heo y and he Bol zmann-a e aged spec a we e ound mi o -image o he expe imen al ECD cu e. The PBE0/TZVP me hod showed he bes ag eemen , he esul o which is shown in Figu e 4. This allowed de e mining he absolu e con igu a ion o o ubiellin B (1) as (+)-(5’R,10’S,10a’R). The pu a i e biogene ic o ma ion o 1 and o he s uc u ally ela ed 6,7-dideshyd oxy de i a i e o ubiellin A (2) has been sugges ed o occu h ough oxida i e coupling o emodin (5), aloe-emodin (6) o o ch ysophanol (7) on he basis o he co-occu ence o hese monome s in he espec i e in es iga ed ungal ex ac s.17 A e ho ough UV and LCMS analysis o he c ude e hyl ace a e ex ac o Ac emonium sp. analyzed in his s udy ypical UV abso p ion maxima a 223, 250, 273, 293 and 442 nm, as well as a p ominen pseudomolecula ion peak a 269.5 [M-H]- we e de ec ed ha accoun o emodin, bu no signals we e ound o aloe-emodin o o ch ysophanol. These indings sugges ha 1 may biogene ically a ise om wo emodin (5) moie ies h ough subsequen educ ion and hyd oxyla ion. Compound 1 showed p onounced cy o oxic ac i i y agains se e al human solid cance cell lines including Cal27 (head-neck cance ), Kyse510 (esophageal squamous cell ca cinoma), HCC38 (b eas cance ), A2780 (o a ian cance ) and MDA-MB-231 (b eas cance ) (Table 1). The cell lines included pai s o cells ha a e ei he sensi i e (sens) o esis an (CisR) owa ds he well known cy os a ic d ug cispla in as indica ed by he signi ican ly di e en IC50 alues o cispla in. To ubiellin B (1) p o ed o be s ongly ac i e agains all cell lines in es iga ed wi h IC50 alues anging om 0.3 - 1.5 µM agains he cispla in sensi i e cells and om 0.2 - 2.6 µM agains he cispla in esis an cells, dependen on he cell line in es iga ed. Fo all cell lines in es iga ed he ac i i y o o ubiellin B (1) was supe io o ha o cispla in which makes compound 1 an in e es ing candida e o u he s udies. Expe imen al Gene al The op ical o a ion was de e mined wi h a Pe kinElme 241MC Pola ime e . The 1H, 13C and 2D NMR spec a we e eco ded in DMSO-d6 on a B uke A ance III 600 NMR spec ome e . Mass spec a we e measu ed wi h a HP110 Agilen Finnigan LCQ Deca XP The moques and high- esolu ion mass spec ome y (HRESIMS) spec a we e eco ded using a UHR-TOF maxis 4G mass spec ome e . ECD spec a we e eco ded on a J-810 spec opola ime e . HPLC analysis was pe o med wi h a HPLC sys em Dionex p580 coupled o a UVD340S pho odiode a ay de ec o ; ou ine de ec ion was a 235, 254, 280, and 340 nm. The sepa a ion column was p e illed wi h Eu osphe e-10 C18 using a linea g adien o 0.1% aqueous HCOOH and MeOH wi h a low a e o 1mL/min. Final pu i ica ion was pe o med ia semi-p epa a i e HPLC using a linea g adien o 0.1% aqueous TFA and MeOH o e a Eu osphe e-100 C18 RP column (5µm; 300x8mm) wi h a low a e o 5mL/min using a Me ck Hi achi HPLC sys em (I-7400 UV de ec o ; L-7100 pump). Column ch oma og aphy included Silica gel 60M (Mache ey.Nagel) and Sephadex LH20 (Sigma). P ecoa ed TLC pla es (Silica gel 60 F254) we e used o moni o ac ions; de ec ion was unde UV a 254 and 366 nm. Sol en s we e dis illed be o e use, and spec al g ade sol en s we e used o spec oscopic measu emen s. Compu a ional sec ion Mixed o sional/low mode con o ma ional sea ches we e ca ied ou by means o he Mac omodel 9.9.22319 so wa e using Me ck Molecula Fo ce Field (MMFF) wi h implici sol en model o chlo o o m applying a 42 kJ/mol ene gy window. Geome y eop imiza ions o he esul an con o me s [B3LYP/6-31G(d) le el in acuo] and TDDFT calcula ions we e pe o med wi h Gaussian 0920 using a ious unc ionals (B3LYP, BH&HLYP, PBE0) and TZVP basis se . ECD spec a we e gene a ed as he sum o Gaussians21 wi h 2400 cm–1 hal -heigh wid h (co esponding o ca. 29 nm a 350 nm), using dipole- eloci y compu ed o a ional s eng hs. Bol zmann dis ibu ions we e es ima ed om he ZPVE co ec ed B3LYP/6-31G(d) ene gies. The MOLEKEL22 so wa e package was used o isualiza ion o he esul s. Fungal ma e ial Ac emonium sp. was isola ed om lea es o he mang o e plan Sonne a ia caseola is, collec ed in Oc obe 2005 in Dong Zhai Gang Mang o e Ga den, Hainan, P. R. China, and cul i a ed in la ge-scale ollowing he espec i e p o ocol desc ibed in he li e a u e.23 The ungal s ain could be iden i ied a e DNA ex ac ion and ampli ica ion and a e sequencing o o s ITS egion acco ding o he li e a u e.24 The sequence da a was submi ed o GenBank, accession numbe FR822815.1. Ex ac ion and isola ion A e 4 weeks o cul i a ion he ungi we e ex ac ed wi h E OAc. The d ied c ude ex ac (1g) was submi ed o VLC sepa a ion. The las ac ion (0.1% me hanolic TFA) con aining 1 was u he sepa a ed o e a Sephadex LH-20 column a o ding F1-F10 ac ions. F ac ions 6 and 7 we e combined and pu i ied using semip epa a i e HPLC wi h an elu ing g adien o MeOH/0.1% aqueous TFA o yield 1 (20 mg). Bioassay Figu e legends Figu e 1. S uc u e o o ubiellin B (1). Figu e 2. Key COSY (bold lines) and HMBC (a ows) co ela ions o o ubiellin B (1). Figu e 3. Expe imen al spec um o 1 in MeCN and PBE0/TZVP-calcula ed ECD spec um o he wo low-ene gy con o me s (> 1%) o (5’S,10’R,10a’S)-1 in acuo. Ba s ep esen he calcula ed o a ional s eng h alues o he lowes -ene gy con o me . Figu e 4. Key long- ange ROE co ela ions shown on he o e lapped lowes -ene gy compu ed con o me s o (5’R,10’S,10a’R)-1. Figu e 1. S uc u e o o ubiellin B (1). Figu e 2. Key COSY (bold lines) and HMBC (a ows) co ela ions o o ubiellin B (1). Figu e 3. Expe imen al spec um o 1 in MeCN and PBE0/TZVP-calcula ed ECD spec um o he wo low-ene gy con o me s (> 1%) o (5’S,10’R,10a’S)-1 in acuo. Ba s ep esen he calcula ed o a ional s eng h alues o he lowes -ene gy con o me . Figu e 4. Key long- ange ROE co ela ions shown on he o e lapped low-ene gy compu ed con o me s o (5’R,10’S,10a’R)-1. Highligh s • A dime ic an h acene de i a i e was isola ed om he endophy e Ac emonium sp. • The absolu e con igu a ion o o ubiellin B was es ablished by ECD calcula ions. • To ubiellin B showed s ong an i- umo ac i i y agains se e al cance cell lines.