Bioorganic & Medicinal Chemistry xxx (2011) xxx–xxx

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Bioorganic & Medicinal Chemistry
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Novel terpenoids of the fungus Aspergillus insuetus isolated from the Mediterranean sponge Psammocinia sp. collected along the coast of Israel
Elazar Cohen a, Liat Koch b, Kathy Myint Thu a, Yocheved Rahamim a, Yaniv Aluma c, Micha Ilan c, Oded Yarden b, Shmuel Carmeli a,⇑
a b c

Raymond and Beverly Sackler School of Chemistry and Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv Tel Aviv 69978, Israel Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv Tel Aviv 69978, Israel

a r t i c l e

i n f o

a b s t r a c t
Three novel meroterpenoids, insuetolides A–C (1–3) and four drimane sesquiterpenes, the new (E)-6-(40 hydroxy-20 -butenoyl)-strobilactone A (4) and the known 2a, 9a, 11-trihydroxy-6-oxodrim-7-ene (5), strobilactone A (6) and (E,E)-6-(60 ,70 -dihydroxy-20 ,40 -octadienoyl)-strobilactone A (7), were isolated from the EtOAc extract of the culture medium of the marine-derived fungus Aspergillus insuetus (OY-207), which was isolated from the Mediterranean sponge Psammocinia sp. The structures of the compounds were determined by spectroscopic methods. Insuetolides A-C reveal a new carbon skeleton derived from the cyclization of farnesyl and 3, 5-dimethylorsellinic acid. Compounds 1, 6, and 7 exhibited anti-fungal activity towards Neurospora crassa with MIC values of 140, 242, and 162 lM, respectively; and compounds 3, 4, and 7 exhibited mild cytotoxicity towards MOLT-4 human leukemia cells. Ó 2011 Published by Elsevier Ltd.

Article history: Received 13 January 2011 Revised 28 April 2011 Accepted 23 May 2011 Available online xxxx Keywords: Meroteroenoids Drimane sesquiterpens Marine-derived fungi Aspergillus insuetus Sponge Psammocinia sp

1. Introduction Fungi are prolific producers of diverse types of natural products including metabolites of mixed polyketide and terpene biosynthesis such as the meroterpenoids. Meroterpenoids derived from farnesyl diphosphate and the tetraketide, orsellinic acid or its derivatives, are the most abundant group of meroterpenoids found in fungi.1 The first member of this group, andibenin, was isolated from the terrestrial fungus Aspergillus variecolor.2 Biosynthetic studies using various labeled precursors revealed that andibenin is derived from mixed polyketide-terpene biosynthesis.3 Subsequently, it was found that a large number of fungal metabolites with a wide range of structural diversity are derived from this mixed biosynthetic route.1 Recently, several meroterpenoids were isolated from marine-derived Aspergillus spp. For example, tropolactones A–D were isolated from an Aspergillus sp. derived from an unidentified sponge4; asperdemin was isolated from A. versicolor collected in Sakhalin Bay5; and terretonins E and F were isolated from Aspergillus insuetus, which was in turn isolated from the Mediterranean sponge Petrosia ficifornis.6
⇑ Corresponding author. Tel.: +972 3 640 8550; fax: +972 3 640 9293.
E-mail address: (S. Carmeli). 0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.bmc.2011.05.045

As part of our ongoing research on the chemistry and chemical ecology of marine-derived fungi7,8 we have isolated a new structural group of meroterpenoids, insuetolides A-C (1–3) along with one new, 4, and three known, 5–7, drimane sesquiterpenes (see Fig. 1) from a marine-derived A. insuetus (OY-207), which was isolated from the Mediterranean sponge Psammocinia sp. collected using SCUBA diving offshore of Sdot-Yam, Israel. Insuetolides A–C (1–3) are thought to arise from the same drimane-type precursor leading to andibenin, via an additional oxidation step prior to the last step of condensation to the pentacyclic intermediate. 2. Results and discussion The fungal isolate OY-207 was cultured from the sponge sample with the use of fungicide-amended media.8 Based on amplicons obtained with the 5.8S ribosomal RNA gene internal transcribed spacers (ITS) primers (1 and 49) and b-tubulin10 gene primers, the strain was identified as A. insuetus. The fungus was mass-cultivated in Potato Dextrose Broth in stationary flasks for 20 days at 25 °C in the dark. The culture medium was extracted with ethyl acetate. The ethyl acetate extract was chromatographed using a reversed-phase open column, Sephadex LH-20 and finally a reversedphase HPLC column to afford seven natural products.

Please cite this article in press as: Cohen, E.; et al. Bioorg. Med. Chem. (2011), doi:10.1016/j.bmc.2011.05.045

14.2. 3. 6b.3 s s s d. 1.2. 7b. 15 9. two carbons of a conjugated double bond.20. CH2 44.0.32 d). 5. 60 a.15. 70. qC 84. 8. Med. 11a.7. 13. 2). CH3 1. 9. 1. 11a 6a.3.3 2. Figure 1. 7. 6a. 60 b 11b.26. Chem. CH3 23. 14. 60 a.15. 1. 1. d. 12endo. 9. 7a. 100 5. 12endo. 60 a 12endo. 1717 (saturated ketone) and 1686 (unsaturated lactone) cmÀ1.3.7 5.1.8. 12exo. 33. E.43 and 1. 90 . 15 7a. qC 48. 14 6a. exo to ketone at position 40 .2.5. qC 19. 14 6a. and three quaternary.8.09. 70 .3 dddd. 13 1. 12exo 6b 12endo.37. 2. 1. 60 b 10 a. 60 a.50 and 1. 44. 15 1. 5.91 d). 60 a. 10 a. 1. 90 11a. suggesting 10° of unsaturation. Cohen et al. 11b. 60 b. 7b.45 ppm. 5. Interpretation of the COSY spectrum allowed the assignment of six isolated spin systems (see Fig.7. 1. 5. HSQC assigned the carbons to fragments a–f and to the methyl groups (see Table 1).97. dd.045 . 14. 13. 11a. 70 1. / Bioorg.5. 60 b 400 MHz for 1H and 100 MHz for From numbered H to C in row.1. was deduced from a HRESIMS protonated cluster ion at m/z 429. 70 . a b a b 2.43.8 d. 12. 100 11b. 1. qC 40. 7b. a methine a to a carbonyl (dH 3. 15 15 2. 14 10 b. 70 . 11a. 13 6a. a conjugated carboxyl.7. 60 a. a saturated carboxyl.8 a b exoc endo 54.7. 1. 90 7a.98. J (Hz) 5. 9.78. and five singlet methyl signals between 1. m 1. 10 b.0. 12endo. s 1. 7a. CH 119.9. 2. 9.3 ddd. d. 5. 12. 9. 15 1.2011.12. 60 b.6. 22. 6b 7a. The 13C NMR of 1 contained 25 carbon signals (see Table 1) assigned to a ketone.33. 9. CH 176.4 3. 60 a. 10 a. 12exo 6b.3. 13 6b. 11.4. isolated oxymethylene protons (dH 3. 7. 9. 60 b.40. 100 14 5. Please cite this article in press as: Cohen. 9. 7a. 12endo.91. CH2 32.8 m m 6a. Bioorg. 4.31. 12endo.11 dd). Insuetolides Insuetolide A (1) was isolated as a transparent glassy material. qC 33. qC CH qC CH2 dH. 12exo.2 1. CH 166.40. 11b. 149.2. dd.11. 12endo. 70 .4..0.bmc. three quaternary and one methylene oxy-carbons. 100 12exo.1016/j. Metabolites of A. 11. five methylene and five methyl carbons in the aliphatic region. 90 . 10 b.7.5.7. qC 45. 10 a.3. 100 60 a12exo. The six short fragments (a–f) could be assembled through the HMBC correlations to the entire dC.1. 15 1. qC 214.48. CH2 29. CH 21. Its molecular formula. 12exo 1. 100 9. 9. 50.12 ppm.82 d and 5. 90 10 a. 12exo. 15 5 90 7a. CH2 75. C. 14. 1. 8.8. 11b.48. 60 b. m m d. 15 1. insuetus (OY-207).85. 1. CH3 CH3 CH3 CH2 a b 83.2 E. 10 a. C25H32O6. three methine. 12. 7b NOE 2. 12.9. 10 b 5. 11a. 60 b70 . In the IR spectrum three carbonyl stretching bands were evident: 1780 (saturated c-lactone). 70 11b 12exo. 10 b. Med. eight Table 1 NMR Data of Insuetolide A (1) in CDCl3a Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 a b 20 30 40 50 60 70 80 90 100 a b c resolved protons between 2. 100 10 a. xxx (2011) xxx–xxx 2. 1. 90 11a. 70 . 13. (2011).2. 7b. Chem. mult. 12exo. 6a 6b.7 HMBCb 2.3.0. The 1H NMR spectrum measured in CDCl3 (see Table 1) revealed two vinyl protons of a conjugated double bond (dH 5. 100 12endo. s 13 11a. 60 b.2279. ddd. 60 a 60 b. 13. doi:10. 25. 12exo. 60 b 12endo. 7a. 13. 1. 14.79. dd. mult. 1. et al.98 d and 4.3 dd.

C-50 presented 2J correlations with H-12endo and H-12exo.15) with C-10. Finally. Me-90 ) with C-11 (dC 33. and through those of H-11a with a quaternary oxygenated carbon (dC 83. 3J).31 bearing three large coupling constants). H-5. which presented correlations with two additional methyl groups (dH 1. E. Ring D closure was suggested based on the HMBC correlations of C-20 with H-60 b (3J) and H-70 (2J) and of C-30 with H-70 (3J). 3J). H-7a (dH 1. C-4). Me-90 exhibited an additional NOE with H-60 a (dH 1.7). Bioorg. a transparent glassy material. confirming their 1.43. Cohen et al. 3) allowed the assignment of the relative configuration of all chiral centers in 1. There was no direct evidence for the closure Figure 3. but the 10° of unsaturation and six oxygen atoms dictated by the high-resolution mass measurement.37 s. through an ether bridge. H-70 (dH 3.6. This was confirmed by the NOE of the a-oriented H-5 with H-11a (dH 1. H-11eq (dH 1. of H-12endo (dH 2. C-8). 3J) and of H-11b with C-20 (2J). C-3) and a quaternary carbon (dC 44. Me-13 and dH 1. thus confirming their b orientation and the a orientation of the cis fused ring E. The latter quaternary carbon presented additional correlations with the methine proton of fragment b (dH 2.48) with C-8 and C-9. H-60 b and H-60 a and with Me-100 .09) with C-7 and of H-11ax. the oximethine-10 was attached to the quaternary oxygenated C-20 through the 2J HMBC correlations of H-10 b and H-10 a with C-20 and the 3J correlations of C-30 with H-10 a and H-10 b and of C-70 with H-10 a. C-80 was attached to C-70 through the correlations of H-60 b and H-60 a (3J) and H-70 (2J) with C-80 .48 dd.15).E. and C-40 and C-80 were shifted upfield by ca.15. Selected NOEs confirming the relative stereochemistry of 1. Methylene-60 (in 1) was replaced by oxymethine carbon (dC 72. and the closure of the six-membered ring (ring B) was established through the 3J correlations of H-1.31) and H-7 (dH 1. and a 1–4 diaxial relationship. 1.1016/j.0.bmc. Me-13 showed NOE0 s with H-5 (J5-6b = 12. C-40 presented an additional 3J correlations with the singlet methyl (dH 1.2 Hz) and H-6a thus establishing Me-13 and H-6a as equatorial and H-5 as axial and a-oriented. H-11b exhibited a NOE with Me-90 (a-oriented and equatorial to ring C and axial to ring D) which in turn presented NOE with H-10 a confirming the cis fusion of rings C and D. H-12exo exhibited NOEs with H-60 (dH 2. C-50 . 1).40). the methylene protons of fragment e (dH 2.33 (br s). of ring C. C-5). confirmed 10° of unsaturation in 2. H-5.1. This established the structure of insuetolide A as planar 1 (Fig. Fragments established by COSY correlations.045 .2011. (2011).8. et al. Me-14). H-7a presented NOE with H-12exo and H-7b presented NOE with H-12endo.2. Insuetolide B (2). These findings establish the relative stereochemistry of insuetolide A as presented in structure 1.79). C-9) of fragment c (C-9 and C-11).0. and the signals of methyl-90 and methyl-100 were shifted downfield also presenting some changes when compared with that of 1.. H-9 displayed a NOE with axial Me-15 and H-12endo. Med. Chem.5 ppm in 2 relative to 1. presented a molecular ion of m/z 443.20) and H-70 . which confirmed the twisted boat conformation of ring D forced by the methylene (C-12) bridge between C-8 and C-50 .85) and Me-15 with the methine carbon (dC 50. The chemical shifts of the carbon and protons of Me-90 suggested that it is situated on a quaternary carbon rather than an oxygenated quaternary carbon. Med. Me-100 ).26 (Me-15). Figure suggesting that it was b-oriented and equatorial establishing methylene-11 as a-oriented and axial to ring B. Fragment b (C-5 through C-7) could be extended through the HMBC correlations of H-6 (dH 1.22 br d). The above discussion establishes the structure of the sesquiterpene part of the molecule as a drimane skeleton with an oxygenated ring A. thus confirming that methylene-12 is b-oriented and equatorial to ring B. establishing C-30 as the connecting point with C-11.7. a quaternary carbon (dH 45. The molecular formula that was calculated from the high-resolution mass measurements C25H31O7. C-40 . 6 ppm. These HMBC correlations established the sequence of carbons 40 -50 -60 -70 and C-50 as the site of the second linkage between the drimane skeleton (C-12) and the tetraketide moiety. C-10). Chem. and as the point of connection of Me-100 . The HMBC correlations of H-11ax (dH 1.32) changed multiplicity from a double doublet to a doublet. / Bioorg.12. a new acidic proton appeared at dH 3. The lactone ring E was established based on the 3J correlation of C-80 with H-10 b. which is axial in the twisted boat conformation of ring C. Me-14 showed strong NOE with Me-15 and H-6b (dH 1. suggested that the two unpaired oxygenated carbons.1) in 2. designating a twist-boat conformation of the six-membered ring B.20 and 1. further support this assignment and establish the connectivity of C-12 to C-8. H-6b exhibited NOEs with H-7a and H-7b and H-7b presented NOE with Me-15. C-30 . NOE correlations obtained from a 2D and a 1D NOE experiments (see Fig. 4J) and with the carbon of Me-90 (dC 19. ring A was established through the 4J HMBC correlation of Me-13 with C-3. 2J) and the ketone carbon (dC 214. 3-diaxial relationships.85) with a quaternary oxy-carbon (dC 75. xxx (2011) xxx–xxx 3 planar structure. 1717 and 1685 cmÀ1. The tetraketide moiety was linked to C-11 of the drimane skeleton through the correlation of a singlet methyl (dH 1.9. the protons of methylene-60 were substituted by an oxymethine (dH 4. C-8 and C-20 must be linked. doi:10. establishing the connectivity of carbons 3-2-1-10-5-4 and the substitution of C-4 by Me-13 and Me-14 and C-10 by Me-15.05. C-20 .2069 in the negative ESIMS measurement. H-5) and the singlet methyl that resonate at dH 1. a quaternary carbon (dC 48. In the IR spectrum three carbonyl stretching bands were observed: 1770. the spectrum of 2 revealed a few significant changes. The vinyl protons (H-1 and H-2) presented HMBC correlations with the unsaturated carboxyl (dC 166. and the sequence 20 -30 -40 . Finally. The 1H and 13C NMR spectra of 2 were very similar to that of 1.79. in accordance with the substitution Please cite this article in press as: Cohen. The latter methyl group presented HMBC correlations with the vinyl carbon (C-1) and the methine carbon (dC 40. one to the other. H-12endo and H-12exo (dH 1. H-60 b and H-60 a) and with H-11a. When compared with the 1H NMR spectrum of 1. C-50 and C-70 were shifted downfield by ca. Me13 and Me-14 presented HMBC correlations with a quaternary oxy-carbon (dC 84.

100 10 a 10 a10 b. and its 1H and COSY NMR spectra in CD3CN were measured. qC 45. qC 51. The DdBa values of the protons in the ester vicinity were calculated (see Fig. 11b.2. 2. the absolute stereochemistry of the chiral centers of insuetolide A (1) were determined as 5R. 90 11b.2.22. 90 100 14 13 1.60 R. mult.7 dt.3.25. 6 10 b. 70 .5.8. 5 15 2.81.0 3. 2. Insuetolide C (3) was isolated as a glassy material. were isolated as the major constituents of the ethyl acetate extract of the culture medium. 5.8. 15 dC. 12endo.1. 9. They were found to be identical with compounds previously isolated from two marine-derived strains of Aspergillus ustus12. 4. H-60 exhibited NOEs with H-70 .4. HSQC and HMBC data (see Table 2).9. 100 1 b. 14 6a.32.8. 1. CH2 29. 32. (2011). Bioorg. 14 7a dC.93. 12exo.bmc. CH3 4.27.47. Chem. 12.08. 1. qC 45. 12endo. CH 167. 11b. 1. dd. 12exo 7a. C. 1 1.8. 11b. 10.10R. 60 0 0 2.1. CH 21.9S.0.30 R. 22.04.91. On the basis of these arguments structure 2 was assigned to insuetolide B.6 5. CH3 7. 90 7b. J (Hz) 5. m 1. 60 11b 12endo. 13. 15 1. qC 40. s 400 MHz for 1H and 100 MHz for From numbered H to C in row. 7.30. doi:10. 15 9. 12endo. and 3. of insuetolide B (2) as 5R. 7. 7a. qC 166. Its IR spectrum exhibited stretching bands of three carbonyls: 1765.45. qC 146. d.6 HMBCb 2. t. with 11° of unsaturation.3. 11a.30 R.7 d. The rest of the NOEs presented by insuetolide B (2) were similar to that of 1. CH2 75.2.0. 70 .84. s 1. 12exo.5. 70 . 9.50 S.37.36. CH 119. et al.8.7. qC 211. 13 13. 90 . 13.9S.11-thrihydroxy-6-oxodrim-7-ene (5). strobilactone A (6) and (E. 12.82. qC 83.02 s) and the disappearance of H-70 . 5.33 br s 3.05.20 R. On the basis of these arguments structure 3 was assigned to insuetolide C. mult. 12.7.02 s 1. br d. The allocation of the conjugated double bond to position 60 and 70 of the meroterpenoid skeleton was confirmed by the HMBC correlation (see Table 2). 10 a. 51. 1.40 -octadienoyl)-strobilactone A (7). s 13 79. 12exo. 1. 6a. 7.43. 14. 4. 1. 2. thus confirming the a-orientation of 60 -OH. This was confirmed by the COSY.3 d. 2.20. 15 9.0 1. 2. Drimane sesquiterpenes The known 2a.8S.14. 6b.4.98. 50. 100 12endo. 1. 71.9. 1. 90 12exo. qC 41.27. 12endo. / Bioorg. 14. d. 11b. 100 12endo. 12. Me-100 and H-12exo.01. CH 51.47. qC CH qC CH2 dH.7 s s s d.30.3. It exhibited a sodiated molecular cluster ion (HRESIMS) of m/z 449. mult. 1.8.3 1.70 -dihydroxy-20 .38.8S. 14 6b. 9. CH2 29.4. J (Hz) 5.6.26. CH3 23. 12exo 1.70 S and those of insuetolide C (3) as 5R. uration of C-60 was determined as (R). 73. 5. qC 213.5. 5. 5. qC 48.8S. 15 2. Please cite this article in press as: Cohen. 2.44.30 R. 90 . 12exo.20 R.1.1. E. 2. s 1.8 m m m m 9.4 m d. 1719 and 1686 cmÀ1.10R. The absolute configuration of the insuetolides was established using Riguera0 s method for secondary alcohols. 60 a. 11b.7. 13. d.0. Cohen et al..20 R. 5. CH 20. 90 11a.50 S. CH3 CH3 CH3 CH2 82. 6a. CH2 S. CH 135. qC 18. 11b. 2. qC 21. 7a. 10 b. xxx (2011) xxx–xxx of C-60 in 2 by an hydroxyl. Table 2 NMR Data of Insuetolide B (2) and Insuetolide C (3) in CDCl3a Insuetolide B (2) Position 1 2 3 4 5 6a b 7a b 8 9 10 11 a b 12 exoc endo 13 14 15 10 a b 20 30 40 50 60 60 -OH 70 80 90 100 a b c Insuetolide C (3) HMBCb 2. CH 119. CH 174.50 S. 60 . 90 . 1. 4. 60 . 22.44. qC CH qC CH2 dH. 60 b. CH3 CH3 CH3 CH2 49. 5.9. m 1.1. 100 14 13 1.2. 10 b.9S. 12exo 1.5. 6b. followed by addition of Ba(ClO4)2 to saturation and measurement of the 1H and COSY NMR spectra of the MPA ester Ba+2 complex. 13 13.0.8 2. 10.9a. 1. d. qC 84. 12exo.5.8. The chemical shifts of the protons and carbons of the rest of the molecule seemed almost identical with that of 1. 11b. CH3 20.E)-6-(60 . 11a. Chem.49. 10 b. 9 90 11a.88. 60 . CH 166. 1 1.02. when compared with that of 1. 1. 1.2 m m dd.7 s s s d.13 (5 and 7) and the edible mushroom Strobilurus ohshimae14 (6).0.7 49. 14.31. 24. 14.7. 11a. 1.8. Insuetolide C (3) presented NOEs similar to those of 1 and 2. dd. 4) and the absolute config- Figure 4. 60 1. 1. In the 13C NMR spectrum of 3.1.43. 100 11a.00.2011. Med. qC 72. d.8. 12exo. a new three-substituted double bond appeared. 10. 11a.0. 12. 1. 9.6. 10.70. 12exo 6b. 11a.2. 25. 4. Med. 149. 7a. mult. 15 1. while methylene-60 and methine-70 disappeared compared to the spectrum of 70 11b 12exo.27.5 11a.7 d. exo to ketone at position 40 . 12exo.7 d. 5. 10 b. 11a. 10 a. CH2 33. 12. 90 9. 60 . Based on the relative stereochemistry established above for 1.11 The (R)-MPA ester of insuetolide B (2) was prepared. from which the molecular formula C25H30O6 was calculated. 11a 70 11a. 60 .1. 149. CH2 32. 33. 100 11a. 7. 44. 13. 10 a.5.8 5. 44. Its NMR spectra were similar to those of insuetolides A (1) and B (2). The 1H NMR spectrum of 3 in CDCl3 (see Table 2) revealed an additional conjugated vinyl proton (dH 7.6. 1.4. 7a. 5. 14. m m d.6. 1. DdBa values of the insuetolide B MPA ester Ba+2 complex.78. 10 a. 1.4 E.045 .

xxx (2011) xxx–xxx Table 3 NMR data of 4 in CDCl3a Position 1 ax eq 2 ax eq 3 ax eq 4 5 6 7 8 9 9-OH 10 11 12 ax eq 13 14 15 10 20 30 40 40 -OH a b 5 dC. 1. Compounds 3. (2011).59.0. 100. The N.2011. 1. which in turn reacts in a ‘penol-oxidation’ type coupling reaction with C50 (C-12) and C-50 (C-8 oxy-radical). et al.0 br d. UV spectra were recorded on an Agilent 8453 spectrophotometer. respectively. and its assembly with the terpenoid core was established by the HMBC correlation of H-6 (dH 5. 3eq. 29.0. It exhibited a pseudo-molecular cluster ion [M+Na]+ at m/z 373. 13 1ax. br s 4. gHMQC and gHMBC spectra were recorded using standard Bruker pulse sequences.2 The difference between the skeletons of the latter two and the skeleton of the insuetolides lies in ring C. 2. respectively.8 The strain was identified as A. 2. crassa (strain 74-OR23-1A) test fungus was maintained on Vogel0 s minimal medium supplemented with sucrose.17 This common precursor yields a large number of secondary metabolites with a wide range of apparent structural diversity through (i) multi oxidation steps.and di-methyl derivatives are the most frequently isolated meroterpenoids from fungi.74.13 MHz for 1H and 125. qC 44. 55% and 72%. NMR spectra were recorded on a Bruker DRX-500 spectrometer at 500. br m 1. 12.2. Its NMR spectra were similar to those of 6 and 7. such as in austin20 and the terretonins. 16.8.92. General experimental procedures Low. qC 68.04. from which a C19H26O6 formula was calculated.045 .5-dimethylorsellinic acid by farnesyl diphosphate. 12b 6. 14 1eq. mult. C. Analysis of the 1H and 13C NMR spectra. 1. 1. CH 61. CH2 17. m 13 500 MHz for 1H and 125 MHz for From numbered H to C in row. Biological material The Mediterranean sponge Psammocinia sp. 9-OH.76. was collected using SCUBA diving at a depth of 2–6 m. 14 3ax. Based on this assumption we posit a biosynthetic route to the insuetolides. as well as the COSY. 9-OH. This suggests that an additional oxidation step occurs in the biosynthesis of the insuetolides prior to the coupling of C-12 to the tetraketide moiety. CH2 24. 13. 20 . 13 2ax. 12b 1ax. 15 1ax. 2. 2ax. 5. Med. 5. and 162 lM. / Bioorg.2. CH3 18. J (Hz) 1.21 Insuetolides A–C exhibit a new skeleton of meroterpenoids closely related to that of the andilesins18 and andibenins. 12a.0 5. crassa were 140. d. mult. CH3 164. 5. 15 5 5.4 and (iii) backbone fragmentation. Cohen et al. The rest of the biosynthetic route to the insuetolides is similar to that elucidated for the andilesins. 4.0.1. CH 123.6. 15 3ax.13 MHz for 1H. 1eq. 12a. suggested a five-membered lactone ring and an unsaturated ester moieties. which is summarized in Scheme 1.5.0 4. qC 174.62 MHz for 13C. E.3. 2eq. 6 and 7 against the fungus N.96. Med. 9. for example the andilesins. It involves the biosynthesis of a drimane type sesquiterpene linked through C-11 to C-30 of the orsellinic acid. 4. HPLC separations were preformed on a Merck Hitachi (model L-6200 intelligent pump. The fungal strain OY-207 was isolated from the sponge sample using fungicide-amended media as described by Paz et al.3. 7. The polyketide unsaturated chain differed from the octyl ester of 7.2. The absolute stereochemistry of 4 was confirmed by the specific rotation that had the negative sign as the known 6 and 7. dt. br s 2. 3ax. Meroterpenoids derived from 3. 16. qC 119. while those derived from orsellinic acid or its mono.01. 5 6. 1ax.2. 5.5 br d. 2ax. Based on these arguments the structure of compound 4 was established as (E)-6-(40 -hydroxy-20 -butenoyl)-strobilactone A.8 s). 30 .5. Chem. 12b. CH2 dH. 4. 40 -OH 2.2. 30 30 . It was mass cultivated in Potato Dextrose Broth in stationary flasks for 20 days at 25 °C in the dark. CH 134.14. Optical rotation values were obtained on a Jasco P-1010 polarimeter at the sodium D line (589 nm). br s 5.. CH2 33.1016/j. in the HRESIMS. 2. 13. Chem.05.5-dimethylorsellinic acid are produced via a common intermediate through the alkylation of 3. m br d. 5. COSY-45.and high-resolution ESI mass spectra were recorded on a Waters Synapt instrument. 3eq.8.76 MHz for 13C and a Bruker Avance 400 Spectrometer at 400. 1eq.3 (E)-6-(40 -hydroxy-20 -butenoyl)-strobilactone A (4) was isolated as a glassy transparent material.03.15 Please cite this article in press as: Cohen.0 dt. 13 1ax.9400 N 034°50.3.74. revealed that its bicyclic terpenoid moiety is identical to that of 7.1615. 1772 and 1715 cmÀ1. 3eq. 2. and 7 inhibited the proliferation of a MOLT-4 cell line by 51%. The structure of the hydroxyl butenoyl moiety was elucidated by interpretation of the 2D NMR data (see Table 3).16 The minimal inhibitory concentration (MIC) values of 1.19. Experimental section 3.3. The OY-207 strain was maintained on Potato Dextrose Agar. especially Penicillium and Aspergillus spp.0. Ending remarks Meroterpenoids are most often isolated from fungi and marine organisms.60. 13. The mycelium was separated from the growth medium by filtration through cheesecloth. 3. The 8.11. gTOCSY.4. and brought to the laboratory for fungal culture from this fresh material.5.0 m m dt.640 E).E. The sponge samples were sealed underwater in bags with seawater. 6. 5. gHSQC.12.5 HMBCb 2ax.4. 1eq. 5.31.bmc.3.43. 6.65. 12-terminal double bond of this common meroterpenoid-intermediate is oxidized to an epoxide. 15 7 3ax. but bacteria and higher plants produce such mixed biosynthesis products as well. HSQC and HMBC experiments of 4.74 br s) and C-10 (dC 164.39. a cyclopentane in the former two and a perhydropyran ring in the insuetolides. Biological activity The inhibitory activity of the pure compounds was assessed against the fungus Neurospora crassa15 and the MOLT-4 human leukemia cancer cell line.18 anditomins19 and tropolactones. 4. 15 9-OH. 1. 1. doi:10. qC 37. 1. IR spectra were recorded on a Bruker Vector 22 spectrometer. insuetus by amplification and direct sequencing of the fungal RNA with ITS1 and 4 and b-tubulin gene primers followed by phylogentic analysis. qC 74. model L-4200 UV–vis) and an Agilent 1100 series HPLC system. 2. Israel. CH 66.4. CH2 44. noids. for example andibenins. respectively. Bioorg.3. 7. approximately 200 m off-shore from Sdot-Yam (32°30. CH 147. CH3 32.74. Mixed polyketide-terpenoid-derived fungal natural products constitute the largest class of meroterpe- 3.2 s s s ddd.2 (ii) backbone rearrangements.0. at 50 mg/mL while 1 and 6 were not active at the same concentration. gROESY. 40 40 20 . 242. 12.0.3. The two carbonyl stretching bands which appeared in the IR spectrum.

UV (MeOH) kmax (log e) 203 (3.05.35. to obtain three pure compounds: 2 (tR 53. IR (CHCl3): 2977. C-18.2 mg.2011.6 E. 2939. CHCl3). The crude extract exhibited anti-fungal activity against N. 3.3. eluted with MeOH/water 7:3). Fraction 6 (297 mg) was separated on a HPLC column (YMC Pack ODS-A.2277). 1717. 1. 1H and 13C NMR (see Table 2). xxx (2011) xxx–xxx Scheme 1. The EtOAc-extract (1.2069 [MÀH]À. 443. respectively. 21. Please cite this article in press as: Cohen.2070). 2. 5 lm.07).2 mm  250 mm. 1715 cmÀ1.11% yield of crude extract) and 4 (tR 37.1. 1.29 g). Proposed biosynthetic route to the insuetolides. 3. 0. flow rate 5 mL/min. and on a Hibar Lichospher 60 RP-Select B (25 mm  250 mm. CHCl3).4.2279 [M+H]+.2 mg. UV detection.02) nm. 1686 cmÀ1. Chem.56% yield of crude extract). 0. 2.9 min. 2950. eluent acetonitrile/ water. eluent MeOH/water. HRESIMS m/z 443. eluent MeOH/water 3:2.6 min. 1719. IR (CHCl3): 2990.2 min. 1686 cmÀ1.3. et al. Insuetolide A (1) Glassy material. Fractions 6 and 9–12 exhibited the strongest anti-fungal activity and were thus farther separated. 0. ½aŠ26 D À209 (c 0. 449.18% yield of crude extract). flow rate 5 mL/min. / Bioorg. 0.1940). (2011). 2928.1 mg.5 min. 1H and 13C NMR (see Table 1).3.64% yield of crude extract) and 5 (tR 27.bmc.56% yield of crude extract). 1H and 13C NMR (see Table 2). 20 mm  250 mm. 3:1) to afford 1 (tR 14. after evaporation of the solvent.78% yield of crude extract) and 6 (tR 47. 248 (2. 1772. 1780.5 min. 429. Insuetolide B (2) Glassy material. CHCl3).045 .1941 [M+Na]+. (calcd for C25H31O7. Fraction 3 was separated on an HPLC column (Phenomenex Gemini-NX.3.5 min. 3.77).2 mg.4 mg. 3. Isolation procedure Extraction of the mycelium-free culture medium (4 L) with EtOAc (2  4 L) afforded.1016/j. 0. HRESIMS m/z 429.29 g) was separated on an open column C-18 reversed-phase material (YMC ODS. 1:1) to give in fractions 1 and 4 semi-pure compounds that were further purified on the same column (fraction 1. 3 (tR 49. 7.05.32) nm. flow rate 5 mL/min. 21. 3. Fraction 4 (30 mg) was further separated on the same semi-preparative HPLC column eluted with 9:1 MeOH/water.99) nm. 0. E. Cohen et al. The united fractions 9–12 from the initial separation (530 mg) were further separated on a Sephadex LH-20 column (CHCl3/MeOH 7:3) to yield two anti-fungal active fractions. doi:10. UV (MeOH) kmax (log e) 206 (3. to afford 7 (tR 11.17.3. 25 g) eluted with solvent of decreasing polarity from water through MeOH (10% steps of MeOH each fraction). (calcd for C25H30O6Na. (calcd for C25H33O6. Med. UV detection. the crude extract (1. (E)-6-(40 -hydroxy-20 -butenoyl)-strobilactone A (4) Glassy material.8 min.2. 1765. HRESIMS m/z 449. UV detection. 7.2 mg. IR (CHCl3): 2980.3 mg.. Med. ½aŠ26 D À98 (c 0. 1780. 10 lm.17% yield of crude extract).90) nm. fraction 3 (40 mg) and fraction 4 (30 mg).3. 10. ½aŠ25 D À212 (c 0. IR (CHCl3): 2979. 1686 cmÀ1. Bioorg. Insuetolide C (3) Glassy material. CHCl3). 10 lm. UV (MeOH) kmax (log e) 203 (4. 2929.15. UV (MeOH) kmax (log e) 214 (4. 302 (3. Chem. ½aŠ30 D À68 (c 0. crassa at a concentration of 1 mg/mL. 1717.

11). Please cite this article in press as: Cohen. Seco..12 3. J. Ilan. H.1413 [M+Na]+. 1761. References and notes 3. Wang..3. Clardy.. Top. 2982. Hong. Edrada-Ebel. 2a. 2010. 3590. F.. T.2011. Nat. doi:10. J. Yarden. Soc.. Riguera.40 -octadienoyl)-strobilactone A (7) Glassy material. M. T... 1704 cmÀ1.21. L.. 852. Stud. A. Cox. The Mass Spectrometry Laboratory of The School of Chemistry.bmc.2044 [M+Na]+. P. King.045 .. R.. W. J. T... J. J.. Aluma. S. J. W.12 3. for the mass spectra measurements. F.. Sninisky. Med. 15. J.. E. 1976. Commun. Chem. Gellfand. J. P. 2. C.. Davis. 3. White.. Johnstone. A. J. M. Prod.. Muller. Cueto. Huijpers. T. (calcd for C19H26O6Na. Houbraken. 2004. Chem. Chem. J. Wary. CHCl3).. C. Ahmed. Fung. T. 533. et al. P..1627). Lessinger... H. 13.1615 [M+Na]+. King. E. The crude extract. Sklarz. Kalinovsky. Corner. 4013.05. ½aŠ21 D À164 (c 0. Bioorg. J. Yasuda. Kirksey.. Soc. K.. R. Sadler. R.. Komon-Zelazowaska. W. growth was assessed and the MIC determined. 16. W. P.5. McIntyre. Shiono. Druzhinina.. J.70 -dihydroxy-20 . Oxford University Press: Oxford. Prod. S. Conidia of N. W.E.. R. 72.. M.. Dunn. S. M. 1585. 1348. 72. 2005.41) nm. 4852. Y. Flescher. Chem. T.. G. S. E. 2000. Prod. P. 18. Lin.. Simpson. Curr. J. Cole.1016/j.1416). Avila. O. D. J.. R.. C... HRESIMS m/z 373.. Ilan. 59. T. Z. Aveskamp. O. Proksch.. Cohen et al. Y.. M. Chem. T. G. Nat.. Mycol.. in the online version. Y. in duplicates. J.. Seifert. J. 42. Z. M. (calcd for C15H22O4Na. 49. UV (MeOH) kmax (log e) 204 (3. (calcd for C23H32O7Na. Bull. 72. S. Murayama. A. A..045. 2997. 1. R. 2009.05. The cytotoxicity assay was performed as described in Rotem et al.16 Acknowledgments We thank Noam Tal. I.. K. Smith.. Ein-Gil.. 1976. Org. Simpson. F. Cabedo.. 1984. K. Supplementary data Supplementary data (1D and 2D NMR data and HR-MS of compounds 1–4. CHCl3). 3. 1773. In PCR Protocol: a guide to Methods and Applications. Springer. N. Samson. 1585. fractions or compounds were dissolved at various concentrations in Vogel0 s minimal medium with sucrose15 and 1 mL aliquots were distributed into each well of a 24-well tissue culture dish. Walkinshaw... J.. P. 1063. J. Gonzalez-Mas. Russ. Chem. A. Cutler. M. Strobilactone A (6) Glassy material. 289. H. R. A.3. Liu. 2009. Identical in all respect with the published data. S. 62. J. Chem.... E... Bruns. D. Carmeli. Sklarz.E)-6-(60 .. Chem.. Ebel. 9a. Med. 4. J. Naturforsch. 1990. 270. N. R.61) nm. Wang. Commun. 373. J. 17. / Bioorg.05. H. A. 1981..7. Dunn.2011. Bioassays The anti-fungal activity of the crude extract. J. N. 17. 2009. B.. are included.4. 1. W. Jensen.. Yarden. Ed. J.. 2007. (E. 8. Yurchenko. Kuznetsova.) associated with this article can be found. I.. 2009. 98. Smetanina. Funakoshi. Blickstein. 14. 2951.. Johnstone. Lopez-Gresa. R. Chexal. Chem. Chem.. Vasques. M. A. Eds. 195.. Koseki. 65. R. Neurospora: Contributions of a Model Organism. Shaklai. xxx (2011) xxx–xxx 1 7 H and 13C NMR (see Table 3). 26. Ciavatta. Dmitrenok. 3019. O. 6478. Schniderman. B. (2011)... J. W. B: Chem. This study was supported by the Israel Science Foundation grant (ISF 996/06). R. ISME J... 2009.. A.2046). A. Carmeli. Am. R.. Diversity 2010. Nat. Identical in all respect with the published data. Int.. B. HRESIMS m/z 443. Cole. Ueda. T. I. 261 (4. pp 315–322. Srawfer. M. Pawlik.. 914. Z. W. Rotem. Hiramatsu. V.. IR (CHCl3): 3680. W. W. Dorner... M.. Fingrut. 443. Following 12 h of incubation at 34 °C. J. K.35. Org. Fenical. Sci.. R. P. 2002. White. Pivkin. J. V. J. 1719 cmÀ1. R. 1998.. 1978. P. C. Lee. 5. W. T. 11-Thrihydroxy-6-oxodrim-7-ene (5) Glassy material. 20. 175. K. J. F. J. Simpson.. Heyfets.1016/j. fractions and pure compounds was assessed in liquid medium. M. D. T. Miao. 67. S. Identical in all respect with the published data. ½aŠ21 D À155 (c 0. Nat. Academic Press: New York. 9. Y. Quinoa. T.. Scott. A.13 3. A. Draeger. L. ½aŠ25 À65 (c 0. Tel Aviv University. A. H. 1826. M. E. Commun. M.. E. J.. M.. Lu.. 1979.6.. 19. IR (CHCl3): 3610. H. 752. M. J. 3017. Tetrahedron 1997.. Frisvad. crassa (107) were used to inoculate each well... UV (MeOH) kmax (log e) 201 (4. Tylor. Soc. W. Simpson.. C. A. Prod. T. Soc. Cancer Res. Phytochemistry 2006.. H. Chem. J.. 12. 53. CHCl3). 3550. ESIMS m/z 269 D [M+H]+. Chem... A. 6. 67.. HRESIMS m/z 289. 7. Chem. MacMillan. R. Zhu. Rep. Liu. B. Primo. Geris. J. Garcia. 11. T. S.3.. O. J. at doi:10. Springer. Innis. Schulz. 44. 21. Paz.bmc. 4579. 10. R. G.

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