Chloctanspirones A and B, novel chlorinated polyketides with an unprecedented skeleton, from marine sediment derived fungus Penicillium terrestre

Accep ed Manusc ip Ti le: Chloc anspi ones A and B, no el chlo ina ed polyke ides wi h an unp eceden ed skele on, om ma ine sedimen de i ed ungus Penicillium e es e Au ho s: Dehai Li, Li Chen, Tianjiao Zhu, Tibo Ku án, A ila Mándi, Zhimin Zhao, Jing Li, Qianqun Gu PII: S0040-4020(11)01237-3 DOI: 10.1016/j. e .2011.08.037 Re e ence: TET 22117 To appea in: Te ahed on Recei ed Da e: 1 June 2011 Re ised Da e: 29 July 2011 Accep ed Da e: 16 Augus 2011 Please ci e his a icle as: Li D, Chen L, Zhu T, Ku án T, Mándi A, Zhao Z, Li J, Gu Q. Chloc anspi ones A and B, no el chlo ina ed polyke ides wi h an unp eceden ed skele on, om ma ine sedimen de i ed ungus Penicillium e es e, Te ahed on (2011), doi: 10.1016/j. e .2011.08.037 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. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT G aphical Abs ac To c ea e you abs ac , ype o e he ins uc ions in he empla e box below. Fon s o abs ac dimensions should no be changed o al e ed. Chloc anspi ones A and B, no el chlo ina ed polyke ides wi h an unp eceden ed skele on, om ma ine sedimen de i ed ungus Penicillium e es e Dehai Li,a Li Chen,b Tianjiao Zhu,a Tibo Ku án,*c A ila Mándi,c Zhimin Zhao,a Jing Li,*a and Qianqun Gu*a aKey Labo a o y o Ma ine D ugs, Chinese Minis y o Educa ion; School o Medicine and Pha macy, Ocean Uni e si y o China, 5 Yushan Road, Qingdao, Shandong, 266003, P. R. China. bIns i u e o Biomedical and Pha maceu ical Technology, Fuzhou Uni e si y, 2 Xueyuan Road, Fuzhou,Fujian, 350002, China. cDepa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, 4010 Deb ecen, Hunga y O HO OOH O HO HO Cl O HO HO Cl 1 3 5 7 e es ol K (3): (5S,6R) e es ol L (4): (5R,6S) chloc anspi one A (1): (19R) chloc anspi one B (2): (19S) 12 3 9 14 5 6 7 815 17 19 46 Lea e his a ea blank o abs ac in o. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Te ahed on jou nal homepage: www.else ie .com Chloc anspi ones A and B, no el chlo ina ed polyke ides wi h an unp eceden ed skele on, om ma ine sedimen de i ed ungus Penicillium e es e Dehai Li,a Li Chen,b Tianjiao Zhu,a Tibo Ku án,*c A ila Mándi,c Zhimin Zhao,a Jing Li,*a and Qianqun Gu∗a aKey Labo a o y o Ma ine D ugs, Chinese Minis y o Educa ion; School o Medicine and Pha macy, Ocean Uni e si y o China, 5 Yushan Road, Qingdao, Shandong, 266003, P. R. China. bIns i u e o Biomedical and Pha maceu ical Technology, Fuzhou Uni e si y, 2 Xueyuan Road, Fuzhou,Fujian, 350002, China. cDepa men o O ganic Chemis y, Uni e si y o Deb ecen, POB 20, 4010 Deb ecen, Hunga y ——— ∗ Co esponding au ho s. Tel.: +86-532-82032065; ax: +86-532-82033054; e-mail: [email protected] (Q. Gu), ku an @ ig is.kl e.hu (T. Ku án), [email p o ec ed] (J. Li). 1. In oduc ion Ma ine de i ed mic oo ganisms especially ungi ha e he po en ial o p oduce compounds wi h high chemical di e si y.1 Some o hem o en p oduce di e en ypes o seconda y me aboli es jus by he al e a ion o easily accessible cul i a ion pa ame e s, which was e med OSMAC (One S ain-MAny Compounds) app oach, p o ed o be a e y success ul s a egy o inding no el seconda y me aboli es.2 In he cou se o ou con inuous sea ch o new bioac i e seconda y me aboli es om ma ine de i ed ungi, se e al new so bicillinoids ha e been isola ed om he ungus s ain Penicillium e es e.3 Di e en cul u e condi ions guided by he OSMAC app oach d ama ically changed he seconda y me aboli es o his s ain which led o he isola ion o hi een cy o oxic gen isyl alcohol de i a i es.4 In e es ingly, mos o hem we e chlo ina ed which e ealed ha al e na i e cul u e condi ions may ha e ac i a ed he halogenase enzyme and led o mo e halogena ed compounds. Fu he in es iga ion o he ex ac again esul ed in he isola ion o wo no el chlo ina ed so bicillinoids named chloc anspi ones A (1) and B (2), possessing an unp eceden ed bicyclo[2.2.2]oc ane-2-spi o cyclohexane skele on. In addi ion, hei quasi-p ecu so s e es ols K (3) and L (4), wo addi ional new chlo ina ed compounds, we e also isola ed pa allel. Up o now, chloc anspi ones A (1) and B (2) a e he i s examples o so bicillinoids con aining spi o cyclohexane moie ies and chlo ina ed so bicillinoids ha e no been epo ed ye . This pape desc ibes he isola ion and s uc u al elucida ion o he new compounds oge he wi h a en a i e biosyn he ic ou e o 1 and 2. ARTICLE INFO ABSTRACT A icle his o y: Recei ed Recei ed in e ised o m Accep ed A ailable online Keywo ds: So bicillinoids Ma ine de i ed ungus Penicillium e es e Two no el chlo ina ed so bicillinoids named chloc anspi ones A (1) and B (2), possessing an unp eceden ed bicyclo[2.2.2]oc ane-2-spi o cyclohexane skele on, oge he wi h hei quasi- p ecu so s e es ols K (3) and L (4), wo addi ional new chlo ina ed compounds, we e isola ed om a ma ine sedimen de i ed ungus Penicillium e es e. Thei s uc u es including absolu e s e eochemis ies we e elucida ed by analysis o NMR, MS da a and TDDFT CD calcula ions. The cy o oxic e ec s o 1-4 we e p elimina ily e alua ed in HL-60 and A-549 cells. Compound 1 was ac i e agains bo h HL-60 and A-549 cells wi h IC50s 9.2 and 39.7 µM espec i ely, while 2 showed weake ac i i y only agains HL-60 cells (IC50 37.8 µM). 2009 Else ie L d. All igh s ese ed . O HO OOH O HO HO Cl O HO HO Cl 1 3 5 7 e es ol K (3): (5S,6R) e es ol L (4): (5R,6S) chloc anspi one A (1): (19R) chloc anspi one B (2): (19S) 12 3 9 14 5 6 7 815 17 19 46 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Te ahed on 2 Figu e 1. S uc u es o compounds 1-4. 2. Resul s and Discussion The ungus was cul u ed and ex ac ed as disclosed in he p e ious epo .4 The ac i e c ude ex ac was ac iona ed by using silica gel column ch oma og aphy ollowed by ch oma og aphy on Sephadex LH20 and inally pu i ied by p epa a i e HPLC column o gi e 1 (13.6 mg), 2 (13.1 mg), 3 (10.3 mg) and 4 (4.9 mg). Figu e 2. a) COSY and key HMBC co ela ions o 1. b) Rele an NOE co ela ions o 1 and 2 and co esponding in e a omic dis ances in he lowes - ene gy DFT-op imized con o me o 1. Chloc anspi one A (1) was ob ained as a yellow powde . The nega i e LRESIMS exhibi ed a cha ac e is ic chlo ina ed quasi- molecula ion peak clus e a m/z 421/423 (3:1) ([M – H]–), and i s molecula o mula was de e mined as C21H23O7Cl by HRESIMS, which was in ag eemen wi h i s 1H and 13C NMR da a. 1D NMR da a showed he p esence o h ee sp3 hyb idized me hine g oups, wo o hem a ached o oxygen, i e sp2 hyb idized me hine g oups, one me hylene g oup, one allylic me hyl g oup, wo me hyl g oups a ached o qua e na y ca bons, and nine qua e na y ca bons. Figu e 3. DFT op imized geome ies o he h ee lowes -ene gy con o me s (99.5%) o (1R,4S,5S,8R,18S,19R)-1. The 1,3-pen adienyl moie y o he so byl g oup om C-10 o C-14 was indica ed by he cha ac e is ic p o on chemical shi s 3,5 a δH 6.41, 7.12, 6.32, 6.21 and 1.84 ppm, which we e also con i med by he COSY and HMBC spec a (Table 1). The E con igu a ions o he wo double bonds in he so byl esidue was de e mined ia he la ge coupling cons an s (3J12,13 = 15.1 Hz, 3J10,11 = 14.6 Hz) obse ed. The p esence o bicyclo[2.2.2]oc ane moie y was sugges ed by he compa ison o 1D NMR da a o 1 wi h hose o known so bicillin de i a i es 5,6 and con i med by he HMBC co ela ions om H-4 (δH = 3.49 ppm) o C-2 (δC = 198.3 ppm), C-3 (δC = 109.3 ppm), C-5 (δC = 74.8 ppm), C-6 (δC = 210.2 ppm), C-7 (δC = 38.0 ppm) and C-8 (δC = 57.6 ppm), and om H-7 (δH = 1.75, 2.57 ppm) o C-1 (δC = 59.4 ppm), C-2, C-4 (δC = 45.2 ppm), C-6 and C-8. The posi ions o CH3-1, CH3-5 and OH-5 we e assigned acco ding o he HMBC co ela ions be ween CH3-1 (δH = 1.10 ppm) and C-1, C-2, C-6 and C-7, be ween CH3-5 (δH = 1.07 ppm) and C-4, C-5 and C-6, and be ween OH-5 (δH = 6.37 ppm) and C-4, C-5 and C-6. The connec ion o C-3 and C-10 ia C-9 was deduced om HMBC co ela ions o OH-9 o C-3 and C-9, and o H-10 o C-3 and C- 9. The dihyd oxycyclohexenone moie y was es ablished on he basis o COSY co ela ions (OH-18/H-18/H-19/OH-19) and HMBC co ela ions be ween OH-18 and C-17, C-18 and C-19, be ween H-17 and C-15, C-16 and C-19, be ween OH-19 and C- 8, and be ween H-19 and C-15. Wi h C21H23O7 accoun ed o , he plana s uc u e was de e mined by in oducing he chlo ine a om o C-16 in acco dance wi h he molecula o mula and chemical shi o C-16 (δC = 128.3 ppm). Selec i e g adien NOE da a and analysis o 3JH,H p o on coupling cons an s we e used o de e mine he ela i e con igu a ion. The b idged ing sys em is only possible i H-4 and CH3-1 a e o ien ed equa o ially. NOE enhancemen o OH-5 was obse ed by i adia ion o H-19 (δH = 4.66 ppm), showing ha OH-5 was o ien ed owa ds he cyclohexene and C-19 was cis o C-5. Wi h hese indings, he ela i e con igu a ion o his pa o he molecule was designa ed as (1R*,4S*,5S*,8R*). The NOE enhancemen s o H-4 and H-18 upon i adia ion o he H-19 esonance, as well as enhancemen s o H-4 and H-19 upon i adia ion o he H-18 esonance (δH = 5.03 ppm) indica ed he H-18 and H-19 o be loca ed on he same side o cyclohexene and o be o ien ed owa ds he so byl side chain. The cis con igu a ion o H-18 and H-19 was also suppo ed by he small coupling cons an measu ed o 3J18,19 (3.2 Hz). Based on hese e idences, he ela i e con igu a ion o 1 was p oposed as (1R*, 4S*, 5S*, 8R*, 18S*, 19R*). The in e a omic dis ances o p o ons in he lowes -ene gy con o me we e all in ag eemen wi h he obse ed NOE e ec s (Figu e 2b). The CD spec um o 1 showed in ense posi i e Co on E ec s (CEs) a 356 and 248 nm and nega i e ones a 306 and 219 nm. In o de o de e mine he absolu e con igu a ion, he TDDFT CD calcula ions we e ca ied ou on he compu ed solu ion con o me s. An MMFF con o ma ional sea ch ollowed by DFT op imiza ion a o ded h ee sligh ly di e en con o me s (65.4, 27.2 and 6.9% popula ions) abo e 0.5% popula ion (Figu e 3). In all he h ee con o me s, he OH-19 had axial and he OH-18 had equa o ial o ien a ion in ag eemen wi h he measu ed coupling cons an s, and OH-5 and OH-9 we e hyd ogen bonded o ca bonyl g oups. Then he CD spec a o each con o me we e calcula ed by a ious unc ionals (B3LYP, BH&HLYP, PBE0) and TZVP basis se . The Bol zmann-weigh ed CD spec a o he O HO OOH O HO HO Cl 1 HMBC a) O HO O H O HO H Cl H HO 1 O HO O H O HO HO Cl H H 2 b) COSY 2.73 3.29 2.76 2.39 2.38 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 (1R,4S,5S,8R,18S,19R)-enan iome ep oduced well he expe imen al CD cu e wi h PBE0/TZVP gi ing he bes ag eemen (Figu e 4). Thus he absolu e con igu a ion o 1 was de e mined unambiguously as (+)-(1R,4S,5S,8R,18S,19R). Table 1. NMR da a o 1 and 2 (600 and 150 MHz, TMS, δ ppm) 1 2 No. δCa δH (J in Hz)a HMBC (H→C)a COSYa δCa δH (J in Hz)a δH (J in Hz)b 1 59.4 C 59.6 C 2 198.3 C 198.7 C 3 109.3 C 109.9 C 4 45.2 CH 3.49 (1H, s) 2, 3, 5, 6, 7, 8, 9, CH3-5 45.8 CH 3.55 (1H, s) 3.53 (1H, s) 5 74.8 C 74.3 C 6 210.2 C 209.8 C a: 1.75 (1H, d, 14.0) 1, 2, 6, 8, 15, 19 7b a: 2.04 (1H, d, 14.0) 2.14 (1H, d, 13.7) 7 38.0 CH2 b: 2.57 (1H, d, 14.0) 1, 2, 4, 6, 8, 15, CH3-1 7a 33.8 CH2 b: 2.44 (1H, d, 14.0) 2.61 (1H, d, 13.7) 8 57.6 C 57.0 C 9 166.6 C 166.1 C 10 118.5 CH 6.41 (1H, d, 14.6) 3, 9, 11, 12 11 118.5 CH 6.42 (1H, d, 14.9) 6.20 (1H, d, 14.6) 11 141.8 CH 7.12 (1H, dd, 14.6, 10.7) 9 10, 12 141.7 CH 7.11 (1H, dd, 14.9, 11.0) 7.21 (1H, dd, 14.6, 11.4) 12 130.9 CH 6.32 (1H, dd, 15.1, 10.7) 11, 13 11, 13 130.9 CH 6.30 (1H, dd, 15.1, 11.0) 6.33 (1H, dd, 14.6, 11.4) 13 139.1 CH 6.21 (1H, dq, 15.1, 6.4) 11, 12 12, 14 139.1 CH 6.21 (1H, dq, 15.1, 6.4) 6.16 (1H, dq, 14.6, 6.6) 14 18.7 CH3 1.84 (3H, d, 6.4) 12, 13 13 18.7 CH3 1.84 (3H, d, 6.4) 1.88 (3H, d, 6.6) 15 193.5 C 192.8 C 16 128.3 C 128.5 C 17 145.8 CH 6.83 (1H, dd, 2.3, 2.2) 15, 16, 19 19 147.8 CH 7.09 (1H, d, 2.3) 7.08 (1H, d, 2.2) 18 66.7 CH 5.03 (1H, ddd, 8.3, 3.2, 2.3) 16, 17 18-OH, 19 69.5 CH 4.78 (1H, b . dd, 5.9, 5.4) 4.87 (1H, m) 19 76.6 CH 4.66 (1H, b .dd, 5.5, 3.2) 15, 17 17, 18, 19-OH 74.1 CH 3.44 (1H, b . dd, 6.9, 5.4) 3.47 (1H, d, 8.4) Me-1 12.1 CH3 1.10 (3H, s) 1, 2, 6, 7 12.2 CH3 1.10 (3H, s) 1.21 (3H, s) Me-5 24.7 CH3 1.07 (3H, s) 4, 5, 6 25.0 CH3 1.01 (3H, s) 1.13 (3H, s) OH-5 6.37 (1H, s) 4, 5, 6 7.29 (1H, s) OH-9 13.77 (1H, s) 3, 9 13.68 (1H, s) OH-18 5.43 (1H, d, 8.3) 17, 18, 19 18 6.01 (1H, d, 5.9) OH-19 5.34 (1H, d, 5.5) 8, 19 19 7.52 (1H, d, 6.9) a Reco ded in DMSO-d6. b Reco ded in CD3OD. Table 2. NMR da a o 3 and 4 (600 and 150 MHz, TMS, δ ppm) 3 4 No. δCa δH (J in Hz) a HMBC (H→C) a COSYa δCa δH (J in Hz) a δH (J in Hz) b 1 192.6 C 193.0 C 2 129.6 C 131.2 C 3 148.7 CH 7.07 (1H, d, 2.3) 1, 2, 5 4 142.7 CH 7.04 (1H, dd, 5.5, 1.4) 6.99 (1H, d, 4.8) 4 72.3 CH 4.26 (1H, ddd, 8.3, 6.0, 2.3) 2, 5 4-OH, 3, 5 68.0 CH 4.18 (1H, b .dd, 6.0, 5.5) 4.31 (1H, dd, 4.8, 3.3) 5 76.5 CH 3.32 (1H, ddd, 8.3, 5.5, 2.3) 4 5-OH, 4, 6 74.1 CH 3.82 (1H, m) 3.95 (1H, dd, 3.3, 2.9) 6 47.8 CH 2.49 (1H, dq, 6.9, 2.3) 1, 4, 5, 7 5, 7 43.4 CH 2.99 (1H, dq, 6.9, 2.7) 3.03 (1H, dq, 7.0, 2.9) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Te ahed on 4 7 11.1 CH3 1.11 (3H, d, 6.9) 1, 5, 6 6 11.1 CH3 1.09 (3H, d, 6.9) 1.20 (3H, d, 7.0) 4-OH 5.76 (1H, d, 6.0) 3, 4 4 5.72 (1H, d, 6.0) 5-OH 5.61 (1H, d, 5.5) 5, 6 5 5.43 (1H, d, 4.1) a Reco ded in DMSO-d6. b Reco ded in CD3OD. Figu e 4. Expe imen al CD spec um o 1 in MeOH compa ed wi h he PBE0/TZVP Bol zmann-weigh ed spec um calcula ed o he h ee lowes - ene gy con o me s o he (1R,4S,5S,8R,18S,19R)-enan iome . Ba s indica e he calcula ed o a o y s eng hs o con o me A. An accu a e mass analysis o 2 showed he same C21H23O7Cl molecula o mula as ha o 1. I s NMR da a (Table 1), in pa icula he COSY and HMBC co ela ions, e ealed ha 2 and 1 sha e he same plana s uc u e implying ha hey only di e in he con igu a ion o chi al cen e s. NOE enhancemen o OH-5 was obse ed upon i adia ion o OH-19, which showed ha he C-5 hyd oxyl g oup o ien ed owa ds he cyclohexenone moie y in 1. Mo eo e , NOE e ec s be ween H-4 and H-18 was also indica ing ha H-18 o ien ed owa ds he so byl side chain. Howe e , he ans ela i e con igu a ion o H-18 and H-19 was sugges ed by he 8.4 Hz coupling cons an o 3J18,19 measu ed in CD3OD, which was accompanied by chemical shi di e ences o C-7, C-18 and C-19. The CD spec um o 2 was nea ly iden ical wi h ha o 1, which p o ed ha he absolu e con igu a ion o he so bicillin moie y was he same as ha o 1. The e o e compound 2 is he C-19 epime o 1 and i s absolu e con igu a ion was de e mined as (+)-(1R,4S,5S,8R,18S,19S) and named as chloc anspi one B. Te es ol K (3) was ob ained as an oil. The molecula o mula (C7H9O3Cl) was de e mined by NMR da a and i s HRESIMS spec um showing an [M - H]- peak a m/z 175.0165. A de ailed analysis o he 1H-NMR and 13C-NMR da a o 3 indica ed he p esence o one me hyl g oup, ou me hines, wo qua e na y ca bons and wo hyd oxyl g oups (Table 2). The COSY expe imen eadily es ablished he ca bon skele on om C-3 o C-7 as well as he posi ions o wo hyd oxyl g oups. Fu he mo e, he HMBC co ela ions be ween H-7 and C-1, be ween H-3 and C-1, and be ween H-4 and C-2 comple ed he plana s uc u e o 3 by he connec i i y o C-2 o C-6 ia C-1. Figu e 5. DFT op imized geome ies o he ou lowes -ene gy con o me s (94.1%) o (4S,5S,6R)-3. The ωO-1,C-1,C-3,C-3 o sional angle o con o me s A-D a e −178.6°, −177.5°, −179.7°, −179.4°, espec i ely. The analysis o 3JH,H coupling cons an s was used o deduce he ela i e con igu a ion. The small coupling cons an o 3J3,4 (2.3 Hz) indica ed ha he ωH-3,C-3,C-4,H-4 dihed al angle is close o 90° wi h an axial H-4. On he o he hand, he la ge coupling cons an measu ed o 3J4,5 (8.3 Hz) e ealed ha H-5 was also axial. In addi ion, he equa o ial o ien a ion o H-6 was deduced om he small alue o he 3J5,6 (2.3 Hz). Thus he ela i e con igu a ion o 3 was de e mined as (4S*,5S*,6R*). The absolu e con igu a ion o 3 was es ablished by TDDFT CD calcula ion. Al hough qui e a numbe o semiempi ical ules we e epo ed o co ela e he s e eochemis y o cyclohexenone de i a i es wi h cha ac e is ic CD bands, he subs i u ion pa e n o 3 wi h chi ali y cen e s did no allow a sa e con igu a ional assignmen .7,8 Compound 3 showed a posi i e n-π* band be ween 370 and 290 nm, a posi i e π-π* band (I band) a 245 nm, a nega i e π-π* band a 216 nm (II band made up wo π-π* ansi ions) and a nega i e π-π ansi ion below 200 nm (III band).7 An MMFF con o ma ional sea ch o 3 ollowed by DFT op imiza ion a o ded ou majo con o me s wi h 43.5%, 28.8%, 14.4% and 7.4% popula ions (Figu e 5). In all he ou con o me s, H-4 and H-5 we e axial, while H-6 had equa o ial o ien a ion in acco dance wi h he measu ed coupling cons an s. The con o me s di e ed mainly in he o ien a ion o he hyd oxyl p o ons. The cyclohexenone ing had an en elop con o ma ion wi h C-5 ou o he plane (Figu e 6) and he enone ch omopho e was nea ly plana (ωO-1,C-1,C-3,C-3 o sional angle was close o 180°). The Bol zmann-weigh ed a e age o he CD spec a calcula ed o he ou con o me s o (4S,5S,6R)-3 wi h h ee unc ionals ep oduced well he π-π* ansi ions (I-III band). The bes ag eemen was ob ained wi h BH&HLYP/TZVP shown in Figu e 7, which allowed de e mining he absolu e con igu a ion o 3 as (+)-(4S,5S,6R) . Figu e 6. a) Schema ic ep esen a ion o he p e e ed en elop con o ma ion o (4S,5S,6R)-3 iewed om he di ec ion o he double bond. b) P e e ed en elop con o ma ion o (4S,5R,6S)-4. c) S uc u e o he lowes -ene gy con o me o (4S,5S,6R)-3. Te es ol L (4) was ob ained as an oil. The HRESIMS o 4 and NMR da a sugges ed ha i was an isome o 3, wi h a molecula o mula o C7H9O3Cl. The compa ison o he 1D NMR da a o 4 (Table 2) wi h hose o 3 a o ded he same plana s uc u e as 3. The NOESY co ela ions be ween CH3-6 and OH- 4, as well as be ween CH3-6 and OH-5, sugges ed ha CH3-6 and OH-4 loca ed on he same side o he ing and hey a e axial, while OH-5 had an equa o ial o ien a ion. This was also con i med by he small 3JH,H coupling cons an o he me hine O Me CCCC H Cl H OH HHOH O Me CCCC H Cl OH H HHOH a) b) c) MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 p o ons measu ed in CD3OD (3J5,6 = 2.9 Hz, 3J4,5 = 3.3 Hz). Thus 4 had an all-cis ela i e con igu a ion, i.e. (4S*,5R*,6S*). The CD spec a o 4 showed a nega i e n-π* band be ween 370 and 290 nm and a posi i e π-π* band a 192 nm (III band), i.e. opposi e o hose o 3. This sugges ed ha he en elop con o ma ion (helici y) o he cyclohexenone ing was in e ed as shown in Figu e 6b. By in oducing he OH-4 and CH3-6 in axial and OH-5 in equa o ial posi ion in o his con o me , he absolu e con igu a ion o 4 could be deduced as (+)-(4S,5R,6S). Figu e 7. Expe imen al CD spec um o 3 compa ed wi h he BH&HLYP/TZVP CD spec um calcula ed o he ou lowes -ene gy con o me s o (4S,5S,6R)-3. Ba s indica e he calcula ed o a o y s eng hs o con o me A (Figu e 5). A possible biosyn he ic ou e o 1 and 2 was p oposed (Figu e 8). The me hyl g oups o he chlo ina ed p ecu so s 3 and 4 we e oxidized and wi h a subsequen dehyd a ion gene a ed a double bond was p oduced. By a Diels-Alde eac ion, his exocyclic double bond eac ed wi h he diene moie y o he uns able in e media e (5S)-so bicillinols,9 o med om so bicillin which was also isola ed om his ungus, o o m he spi o ca bon C-8. The absolu e con igu a ions o compounds 1 and 2 we e in ag eemen wi h he p ecu so s (3 o 2 and 4 o 1 espec i ely). The isola ion o 3 and 4 indica ed ha he chlo ina ion s ep p eceded he Diels-Alde eac ion. Figu e 8. A possible biosyn he ic ou e o chloc anspi ones A (1) and B (2) Mos o he epo ed so bicillin polyme s we e p oduced in he addi ion o wo o h ee so bicillin de i a i es.5,10 Na u al Diels- Alde p oduc s o so bicillinol wi h a dienophile no ela ed o he so bicillinoids we e qui e a e in na u e, and only a ew examplesha e been epo ed.6,11 I is he i s case ha his kind o p ecu so s (3 and 4) ha e been isola ed oge he wi h he Diels-Alde p oduc s (1 and 2). To he bes o ou knowledge, 1 and 2 ep esen he i s na u al p oduc s con aining bicyclo[2.2.2]oc ane-2-spi ocyclohexenone moie y. Table 3. Cy o oxici ies o 1-4 on cance cell lines cy o oxici y (IC50, µM) compound HL-60 A-549 1 9.2 39.7 2 37.8 >100 3 >100 >100 4 >100 >100 The cy o oxici ies o 1–4 we e p elimina ily e alua ed in HL- 60 and A-549 cells (Table 3) by he MTT me hod.4 Compound 1 was ac i e agains bo h cells wi h IC50s 9.2 and 39.7 µM epec i ely, while 2 showed weake ac i i y only agains HL-60 cells (IC50 37.8 µM). In e es ingly, he in e sion o con igu a ion a C-19 a ec ed he ac i i ies o 1 and 2 signi ican ly sugges ing ha al hough he cyclohexenone moie y was no he pha macopho e (3 and 4 showed no ac i i ies), i s s e eochemis y in luenced he obse ed ac i i y. 3. Expe imen al Sec ion 3.1 Gene al Expe imen al P ocedu es. Op ical o a ions we e ob ained on a JASCO P-1020 digi al pola ime e . UV spec a we e eco ded on a Beckman DU® 640 spec opho ome e . CD spec a we e measu ed on a J-810 CD spec opola ime e . IR spec a we e eco ded on a NICOLET NEXUS 470 spec opho ome e in KB discs. 1H, 13C NMR and DEPT spec a and 2D NMR we e eco ded on a JEOL JNM-ECP 600 spec ome e and BRUKER AV-400 spec ome e using TMS as an in e nal s anda d and chemical shi s we e eco ded as δ alues. ESIMS we e measu ed on a Q-TOF ULTIMA GLOBAL GAA076 LC mass spec ome e . Semip epa a i e HPLC was pe o med using an ODS column [YMC-pack ODS (A), 10 × 250 mm, 5 µm, 4 mL/min]. 3.2 Fungal Ma e ial. The ungus P. e es e was isola ed om ma ine sedimen s collec ed in Jiaozhou Bay, China. I was iden i ied by P o esso C. X. Fang, China Cen e o Type Cul u e Collec ion (iden i ica ion numbe : 2004021), and he ouche specimen is p ese ed in he China Cen e o Type Cul u e Collec ion (pa en deposi a y numbe : CCTCC M 204077). Wo king s ocks we e p epa ed on Po a o Dex ose aga slan s s o ed a 4 °C. 3.3 Fe men a ion and Ex ac ion. The ungus was cul u ed unde s a ic condi ions a 24 °C o 30 days in 200 1000-mL conical lasks con aining he liquid medium (300 mL/ lask) composed o soy one (0.1%), soluble s a ch (1.0%), and seawa e (60%) as desc ip ed p e iously.4 The e men ed whole b o h (60 L) in he modi ied cul u e condi ion was il e ed h ough cheese clo h o sepa a e i in o supe na an and mycelia. The o me was ex ac ed h ee imes wi h E OAc o gi e an E OAc solu ion and concen a ed unde educed p essu e o gi e a c ude ex ac (20.0 g). 3.4 Pu i ica ion. The c ude ex ac (20.0 g) was pu i ied by a silica gel (200~300 mesh) column packed in pe oleum e he . The column was elu ed by g adien a ios o pe oleum e he – CHCl3 and hen wi h CHCl3–MeOH. The ac i e ac ion (Inhibi o y Ra e >50% a a conce a ion o 100 µg/ml) ha was elu ed wi h CHCl3–MeOH (50:1) was u he sepa a ed in o h ee sub ac ions by Sephadex LH-20 using CHCl3–MeOH (1:1) as he elu ing sol en . Sub ac ion 1 was u he pu i ied by PHPLC (62% MeOH, 4.0 mL/min), o gi e 1 (13.6 mg) and 2 (13.1 mg). The ac i e ac ion elu ed wi h CHCl3–MeOH (100:1) was u he sepa a ed in o ou sub ac ions by Sephadex LH-20 using CHCl3–MeOH (1:1) as he elu ing sol en . Sub ac ion 4 was O Cl HO HO O HO OOH H O HO HO Cl 4/3 So bicillinol Diels-Alde OH O O HO s O HO HO Cl [O] O HO HO Cl OH 1/2 [-H2O] So bicillin OH HO O [O] OH HO O O MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Te ahed on 6 subjec ed o Sephadex LH-20 (CHCl3–MeOH, 1:1), hen u he pu i ied by PHPLC (20% MeOH, 4.0 mL/min) o gi e 3 (10.3 mg) and 4 (4.9 mg). Chloc anspi one A (1): yellow powde ; [α]D +304 (c = 0.1, MeOH); ECD (MeOH), λ [nm] (∆ ε ), c = 1.18 × 10−3): 356 (17.6), 306 (−22.0), 274sh (−2.8), 248 (15.9), 219 (−7.6); IR (KB ) νmax 3419, 1735, 1701, 1612 cm-1; UV (MeOH) λmax nm (log ε): 245 (3.67), 359 (3.98); 1H NMR and 13C NMR da a see able 1; HRESIMS m/z: 421.1068 [M – H]– (calcd o C21H22O7Cl, 421.1054). Chloc anspi one B (2): yellow powde ; [α]D +110 (c = 0.1, MeOH); ECD (MeOH), λ [nm] (∆ ε ), c = 1.18 × 10−3): 362sh (24.9), 345 (27.6), 306 (−32.1), 273sh (−3.8), 249 (18.9), 221 (−10.0); IR (KB ) νmax 3459, 1735, 1700, 1607 cm-1; UV (MeOH) λmax nm (log ε): 247 (3.68), 359 (3.97); 1H NMR and 13C NMR da a see able 1; HRESIMS m/z: 421.1062 [M – H]– (calcd o C21H22O7Cl, 421.1054). Te es ol K (3): colo less oil; [α]D +89 (c = 0.1, MeOH); ECD (MeOH), λ [nm] (∆ ε ), c = 2.831 × 10−3): 326 (0.4), 245 (6.4), 216 (−3.8), 193 (−4.6); IR (KB ) νmax 3360, 1696, 1613 cm- 1; UV (MeOH) λmax nm (log ε): 240 (3.52); 1H NMR and 13C NMR da a see able 2; HRESIMS m/z: 175.0165 [M - H]- (calcd o C7H8O3Cl, 175.0162). Te es ol L (4): colo less oil; [α]D +49 (c = 0.1, MeOH); ECD (MeOH), λ [nm] (∆ ε ), c = 2.831 × 10−3): 311 (−0.3), 248 (5.7), 220 (−3.5), 192 (1.0); IR (KB ) νmax 3337, 1696, 1612 cm-1; UV (MeOH) λmax nm (log ε): 238 (3.54); 1H NMR and 13C NMR da a see able 2; HRESIMS m/z: 175.0159 [M - H]- (calcd o C7H8O3Cl, 175.0162). 3.5 Biological Assays. Cy o oxic ac i i y was e alua ed by he MTT me hod. CDDP (cis-diamminedichlo opla inum) was used as posi i e con ol. The IC50 alues we e ob ained using he Bliss me hod. 3.6 Compu a ional Sec ion. Con o ma ional sea ches we e ca ied ou by means o he Mac omodel 9.7.21112 so wa e using Me ck Molecula Fo ce Field (MMFF) wi h implici sol en model o chlo o o m. In each con o ma ional sea ch he maximum numbe o s eps we e se o 30000. Geome y eop imiza ions a B3LYP/6-31G(d) le el o heo y ollowed by TDDFT calcula ions using a ious unc ionals (B3LYP, BH&HLYP, PBE0) and TZVP basis se we e pe o med by he Gaussian 0313 package. Bol zman dis ibu ions we e es ima ed om he ZPVE co ec ed B3LYP/6-31G(d) ene gies. CD spec a we e gene a ed as he sum o Gaussians14 wi h 3000 cm–1 hal - heigh wid h (co esponding o ca. 19 nm a 250 nm), using dipole- eloci y compu ed o a ional s eng hs. The MOLEKEL14 so wa e package was used o isualiza ion o he esul s. Acknowledgmen s This wo k was inancially suppo ed by he Chinese Na ional Science Fund (No. 30973627), he Shandong P o incial Na u al Science Fund (No. ZR2009CZ016), he P omo i e esea ch und o excellen young and middle-aged scien isi s o Shandong P o ince (No. BS2010HZ027), he specialized esea ch und o he doc o al p og am o highe educa ion (No. 20100132120026), he Public P ojec s o S a e Oceanic Adminis a ion (No. 2010418022-3) and he P og am o Changjiang Schola s and Inno a i e Resea ch Team in Uni e si y (No. IRT0944). The cy o oxici y assays we e pe o med a he Shanghai Ins i u e o Ma e ia Medica, Chinese Academy o Sciences. T. 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