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Structure of a novel phospholipase C inhibitor, vinaxanthone (Ro 09-1450), produced by penicillium vinaceum
Tembedron Letters, Vo1.32.No.36 pp 47374740.1991 P&ted in Great Britain
00404039/9 1 $3 .OO+ .OO Pcrgamon Press plc
Structure of A Novel Phospholipase C Inhibitor, Vinaxanthone (Ro 09-1450), Produced by Penicillium Vinaceum Masahiro
Aoki, Yoshiko Itezono, Haruyoshi
Shirai, Noboru Nakayama
Akiko Sakai, Yutaka Tanaka, Akiko Yamaguchi,
Nobuo Shimma
Kazuteru Yokose* Nippon Roche Research Center, Kamakm,
Kanagawa, 247, Japan
Haruo Seto Institute of Applied Microbiology, The University
Key word: vimnthone;
&&&mvinaceum
of Tokyo,Bunkyc-ku, Tokyo, 113.Japan
Gibnan and Abbott; phospholipase C inhibiror
Abstract; Vinaxanthone is a novel phospholipase C inhibitorproduced by &&iU& vinaccunz Gilman and Abott NR6815. Its structure (MW 576, C2gH16014) has been elucidated as a polycyclic xanthone withpoly acidic functional groupsbased on various NMR studies including HMBC. COLOC, 2D-INADEQUATE and selective ID-INADEQUATE.
Phospholipase C (PLC) is an enzyme that hydrolyzes phosphatidylinositol bisphosphate (PIPi) in the cell membrane to produce diglyceride and inositol triphosphate. Since PLC plays an important role in the signal transduction cascade and the cell prolification through an enhancement of phosphatidylinositol turnover, PLC inhibitors would be expected to be cell growth inhibitors. During our screening program for PLC inhibitors from microbial origin, a novel inhibitor, vinaxanthone u, Ro 09-145O)l) was isolated from the cultured broth of Penicillium
vinaceum Gihnan and Abbott NR6815.I showed selective inhibitory activity against rat brain PLC with an IC50 being 5.4 @vl. In this paper, we describe the isolation and structure elucidation of l_based on various NMR studies including HMBC, COLOC, 2D-INADEQUATE and selective lD-INADEQUATE2). Penicillium
vinaceum NR6815, which was isolated from soilgarcelona, Spain, was cultured at 270C in a
50-liter jar fermenter containing 30 liters of a medium3) with agitation rate of 190 rpm and air flow of 30 liters/minute. After fermentation for 72 hours, the cultured broth was separated into filtrate and mycelium by centrifugation. The cultured filtrate (26 liters) was extracted with ethyl acetate at pH 2.0, and the organic layer was then extracted with water at pH 8.0. The aqueous layer was reextracted with ethyl acetate at pH 2.0. The organic layer was concentrated under reduced pressure, and the residue was triturated with MeOH. The precipitates were collected and dissolved in aq. NH4OH at 0 Oc and the solution was acidified with aq. HCl to pH 2.0. The precipitates that separated were collected and crystallized from ethyl acetate to give pure I(995 mg) as pale yellow crystals. The physico-chemical properties of 1 were as follows: MP >280 oC; IR vmax (KBr) 3400,1700,1620, 1580 cm-l; UV hax
nm (E) 230 (29,800), 280 (19,300), 320 (16,800). 380(sh.)in MeOH; 230 (29,500), 280
(16,800), 350 (16,100) in 0.01~ NaOH-MeOH. 1 showed a protonated molecular ion peak at m/z 577 in the FAB-MS spectrum. The molecular formula was determined to be C28Hl6014 based on an elemental analysis (obs. C 57.1, H 2.9; calcd. C 57.4, H 2.9 for C28Hl6014.1/2H20). 4737
The UV spectra and IR absorption bands
4139
The differences of the chemical shifts between 2 and fulvic acid dimethyl ether methyl ester are within 2.0 ppm (Fig. 2). Since 12-H (S 8.53) showed three bond CH long range couplings with C-10, C-14, C-21 and C-25 (6 132.5, 172.5, 152.0 and 199.0, respectively)in the HMRC of 1, the partial structure possessing the two units of benzopyran-4-one skeletons (Fig. 2) was determined.
Fulvic acid dimethyl ether methyl ester
2 Vinaxanthone (1) OR
Fig. 2 Comparison of chemical shifts of vinaxanthone and fulvic acid derivative. The location of remaining one quaternary carbon (C-22) and one acetyl group (C-26 and C-27) between C-10 and C-21 could be determined by COLQC and selective lD-INADEQUATE experiments of l_(Fig. 3). The CH long range coupling between H-26 and C-22 was observed in the COLOC spectrum of 1 taken by setting the observed Ia value around 10 Hz. A spin transfer was observed with C-10, C-21 and C-27 in the selective lDINADEQUATE spectrum of 1 obtained by selective excitation of C-22 (Fig. 4).
HO
-
1 D-selective
INADEQUATE HO
-
COLOC
COOH
Vinaxanthone a, Ro 09-l 450)
OH OH
Fig. 3 Structure of vinaxanthone. Thus, the structure of vinaxanthone a, Ro 09-1450) was determined to be a novel polycyclic xamhone compound with poly acidic functions as shown in Fig. 3. Vinaxanthone showed selective inhibitory activity against PLCs from rat brain, mutine colon 26 Adenocarcinoma and murine fibroblasts NIH3T3 with 1C50k being 5.4,9.3 and 44 ph4, respectively. No inhibitory activity was obtained up to 521 plvI against phospholipase A2 (PLA2) from rabbit neutrophil.
4740
Fig. 4 Selective ID-INADEQUATE
spectrum of vinaxanthone (partial).
Acknowledgement The authors are grateful for useful discussions
with Drs. H. Ishizuka, T. Okuda
and M. Arisawa, and special thanks are due to Messrs K. Watanabe, T. Sano and S. Ohsima for large scale fermentation of Nippon Roche Research Center.
References
and Notes
1) Presented in part at the 32nd Symposium on The Chemistry of Natural Products, Chiba, Japan, Oct. 18, 1990. 2) Ouchi, S.; Kawano, S.; Imanari, T.; Kodama, Y.; Seto, H. Symposium papers of 27th Symposium of NMR Spectroscopy, 1988, pp. 243. 3) The medium consisted of glucose 2.0%, potato starch 2.0%, soya meal 2.0%, yeast extract 0.5%, CaC03 0.32%, NaClO.25%, ZnS04.7H20 0.005%, CuS04.5H20 0.0005%, MnCl2.4H20 0.0005%; pH was adjusted to 7.0. 4) Gurevich, A. I.; Karapetyan, M. G.; Kiseleva, 0. A. Chemistry of albofungin. Antibioriki 1972, 17, 717-774. 5) Kobayashi, K.; Nishino, C; Ohya, J. Actinoplanones A and B. J. Anribiotics 1988,41, 502-5 11. 6) The 13C NMR for vinaxanthone CL)(lOOMHz, DMSO-d6 6): 152.4 (C-l), 120.7 (C-2), 172.8 (C-3), 112.3 (C-4), 119.5 (C-5), 141.5 (C-6), 152.7 (C-7), 102.2 (C-8), 150.6 (C-9), 132.5 (C-10). 136.1 ( C-11), 126.7 (C-12), 120.5 (C-13), 172.5 (C-14), 109.9 (C-15), 119.5 (C-16), 140.9 (C-17), 152.0 (C-18), 102.1 (C-19), 150.2 (C-20), 152.0 (C-21), 133.2 (C-22), 167.3 (C-23), 29.0 (C-24), 199.0 (C-25), 32.0 (C-26j, 201.1 (C27), 167.3 (C-28). 7) The 1H NMR for vinaxanthone (1) ( 4OOMHz, DMSO-d6, 6): 8.18 (IH, s, H-l), 6.96 (IH, s, 8-H), 8.53 (lH, s, 12-H), 6.94 (lH, s, 24-H), 2.54 (3H, s, 24-H), 2.55( 3H, s, 26-H), 11.42 (IH, s, S-OH), 9.42 (lH, s, 7-OH ), 11.68 (lH, s, 17-OH), 9.42 (lH, s, 18-OH), 13.02 (lH, s, 23-COGH), 12.77 (IH, s, 28CGOH). 8) The lH NMR for 2 ( 4OOMHz, CDCl3, 6): 7.84 (lH, s, H-l), 6.96 (lH, s, 8-H), 8.62 (lH, s, 12-H), 6.95 (lH, S, 24-H), 2.59 (3H, S, 24-H), 2.44 ( 3H, s, 26-H), 3.91 (3H, s, 6-OMe), 4.03 (3H, s, 7-OMe ), 3.89 (3H, s, 17-OMe), 4.01 (3H, s, 18-OMe), 3.94 (3H, s, 23COGMe), 4.07 (3H, s, 28COOMe). 9) The L3C NMR for 2 (lOOMHz, CDC13, 6): 153.8 (C-l), 121.4 (C-2), 173.6 (C-3), 114.0 (C-4), 126.3 (C5), 144.0 (C-6), 159.3 (C-7), 106.6 (C-8), 154.2 (C-9), 132.6 (C-lo), 136.3 ( C-11), 126.8 (C-12), 120.4 (C-13), 173.4 (C-14), 111.6 (C-15), 126.9 (C-16), 144.6 (C-17), 158.2 (C-18). 101.4 (C-19), 153.5 (C-20), 152.6 (C-21), 133.9 (C-22), 166.7 (C-23), 28.7 (C-24), 199.0 (C-25), 32.3 (C-26), 201.1 (C-27), 166.7 (C28), 53.0 (2C, 23-COOMe* and 28-COOMe*), 56.5 (2C, 7-OMe and 18-OMe), 62.3 (2C, 6-OMe and 17OMe). LO) Itsuo, K.; Hutchinson, C. R.; Vining, L. C. The biosynthesis of fulvic acid. Terrahedron Left. 1981,22, 493-496.
(Received in Japan 10 April 1991)
00404039/9 1 $3 .OO+ .OO Pcrgamon Press plc
Structure of A Novel Phospholipase C Inhibitor, Vinaxanthone (Ro 09-1450), Produced by Penicillium Vinaceum Masahiro
Aoki, Yoshiko Itezono, Haruyoshi
Shirai, Noboru Nakayama
Akiko Sakai, Yutaka Tanaka, Akiko Yamaguchi,
Nobuo Shimma
Kazuteru Yokose* Nippon Roche Research Center, Kamakm,
Kanagawa, 247, Japan
Haruo Seto Institute of Applied Microbiology, The University
Key word: vimnthone;
&&&mvinaceum
of Tokyo,Bunkyc-ku, Tokyo, 113.Japan
Gibnan and Abbott; phospholipase C inhibiror
Abstract; Vinaxanthone is a novel phospholipase C inhibitorproduced by &&iU& vinaccunz Gilman and Abott NR6815. Its structure (MW 576, C2gH16014) has been elucidated as a polycyclic xanthone withpoly acidic functional groupsbased on various NMR studies including HMBC. COLOC, 2D-INADEQUATE and selective ID-INADEQUATE.
Phospholipase C (PLC) is an enzyme that hydrolyzes phosphatidylinositol bisphosphate (PIPi) in the cell membrane to produce diglyceride and inositol triphosphate. Since PLC plays an important role in the signal transduction cascade and the cell prolification through an enhancement of phosphatidylinositol turnover, PLC inhibitors would be expected to be cell growth inhibitors. During our screening program for PLC inhibitors from microbial origin, a novel inhibitor, vinaxanthone u, Ro 09-145O)l) was isolated from the cultured broth of Penicillium
vinaceum Gihnan and Abbott NR6815.I showed selective inhibitory activity against rat brain PLC with an IC50 being 5.4 @vl. In this paper, we describe the isolation and structure elucidation of l_based on various NMR studies including HMBC, COLOC, 2D-INADEQUATE and selective lD-INADEQUATE2). Penicillium
vinaceum NR6815, which was isolated from soilgarcelona, Spain, was cultured at 270C in a
50-liter jar fermenter containing 30 liters of a medium3) with agitation rate of 190 rpm and air flow of 30 liters/minute. After fermentation for 72 hours, the cultured broth was separated into filtrate and mycelium by centrifugation. The cultured filtrate (26 liters) was extracted with ethyl acetate at pH 2.0, and the organic layer was then extracted with water at pH 8.0. The aqueous layer was reextracted with ethyl acetate at pH 2.0. The organic layer was concentrated under reduced pressure, and the residue was triturated with MeOH. The precipitates were collected and dissolved in aq. NH4OH at 0 Oc and the solution was acidified with aq. HCl to pH 2.0. The precipitates that separated were collected and crystallized from ethyl acetate to give pure I(995 mg) as pale yellow crystals. The physico-chemical properties of 1 were as follows: MP >280 oC; IR vmax (KBr) 3400,1700,1620, 1580 cm-l; UV hax
nm (E) 230 (29,800), 280 (19,300), 320 (16,800). 380(sh.)in MeOH; 230 (29,500), 280
(16,800), 350 (16,100) in 0.01~ NaOH-MeOH. 1 showed a protonated molecular ion peak at m/z 577 in the FAB-MS spectrum. The molecular formula was determined to be C28Hl6014 based on an elemental analysis (obs. C 57.1, H 2.9; calcd. C 57.4, H 2.9 for C28Hl6014.1/2H20). 4737
The UV spectra and IR absorption bands
4139
The differences of the chemical shifts between 2 and fulvic acid dimethyl ether methyl ester are within 2.0 ppm (Fig. 2). Since 12-H (S 8.53) showed three bond CH long range couplings with C-10, C-14, C-21 and C-25 (6 132.5, 172.5, 152.0 and 199.0, respectively)in the HMRC of 1, the partial structure possessing the two units of benzopyran-4-one skeletons (Fig. 2) was determined.
Fulvic acid dimethyl ether methyl ester
2 Vinaxanthone (1) OR
Fig. 2 Comparison of chemical shifts of vinaxanthone and fulvic acid derivative. The location of remaining one quaternary carbon (C-22) and one acetyl group (C-26 and C-27) between C-10 and C-21 could be determined by COLQC and selective lD-INADEQUATE experiments of l_(Fig. 3). The CH long range coupling between H-26 and C-22 was observed in the COLOC spectrum of 1 taken by setting the observed Ia value around 10 Hz. A spin transfer was observed with C-10, C-21 and C-27 in the selective lDINADEQUATE spectrum of 1 obtained by selective excitation of C-22 (Fig. 4).
HO
-
1 D-selective
INADEQUATE HO
-
COLOC
COOH
Vinaxanthone a, Ro 09-l 450)
OH OH
Fig. 3 Structure of vinaxanthone. Thus, the structure of vinaxanthone a, Ro 09-1450) was determined to be a novel polycyclic xamhone compound with poly acidic functions as shown in Fig. 3. Vinaxanthone showed selective inhibitory activity against PLCs from rat brain, mutine colon 26 Adenocarcinoma and murine fibroblasts NIH3T3 with 1C50k being 5.4,9.3 and 44 ph4, respectively. No inhibitory activity was obtained up to 521 plvI against phospholipase A2 (PLA2) from rabbit neutrophil.
4740
Fig. 4 Selective ID-INADEQUATE
spectrum of vinaxanthone (partial).
Acknowledgement The authors are grateful for useful discussions
with Drs. H. Ishizuka, T. Okuda
and M. Arisawa, and special thanks are due to Messrs K. Watanabe, T. Sano and S. Ohsima for large scale fermentation of Nippon Roche Research Center.
References
and Notes
1) Presented in part at the 32nd Symposium on The Chemistry of Natural Products, Chiba, Japan, Oct. 18, 1990. 2) Ouchi, S.; Kawano, S.; Imanari, T.; Kodama, Y.; Seto, H. Symposium papers of 27th Symposium of NMR Spectroscopy, 1988, pp. 243. 3) The medium consisted of glucose 2.0%, potato starch 2.0%, soya meal 2.0%, yeast extract 0.5%, CaC03 0.32%, NaClO.25%, ZnS04.7H20 0.005%, CuS04.5H20 0.0005%, MnCl2.4H20 0.0005%; pH was adjusted to 7.0. 4) Gurevich, A. I.; Karapetyan, M. G.; Kiseleva, 0. A. Chemistry of albofungin. Antibioriki 1972, 17, 717-774. 5) Kobayashi, K.; Nishino, C; Ohya, J. Actinoplanones A and B. J. Anribiotics 1988,41, 502-5 11. 6) The 13C NMR for vinaxanthone CL)(lOOMHz, DMSO-d6 6): 152.4 (C-l), 120.7 (C-2), 172.8 (C-3), 112.3 (C-4), 119.5 (C-5), 141.5 (C-6), 152.7 (C-7), 102.2 (C-8), 150.6 (C-9), 132.5 (C-10). 136.1 ( C-11), 126.7 (C-12), 120.5 (C-13), 172.5 (C-14), 109.9 (C-15), 119.5 (C-16), 140.9 (C-17), 152.0 (C-18), 102.1 (C-19), 150.2 (C-20), 152.0 (C-21), 133.2 (C-22), 167.3 (C-23), 29.0 (C-24), 199.0 (C-25), 32.0 (C-26j, 201.1 (C27), 167.3 (C-28). 7) The 1H NMR for vinaxanthone (1) ( 4OOMHz, DMSO-d6, 6): 8.18 (IH, s, H-l), 6.96 (IH, s, 8-H), 8.53 (lH, s, 12-H), 6.94 (lH, s, 24-H), 2.54 (3H, s, 24-H), 2.55( 3H, s, 26-H), 11.42 (IH, s, S-OH), 9.42 (lH, s, 7-OH ), 11.68 (lH, s, 17-OH), 9.42 (lH, s, 18-OH), 13.02 (lH, s, 23-COGH), 12.77 (IH, s, 28CGOH). 8) The lH NMR for 2 ( 4OOMHz, CDCl3, 6): 7.84 (lH, s, H-l), 6.96 (lH, s, 8-H), 8.62 (lH, s, 12-H), 6.95 (lH, S, 24-H), 2.59 (3H, S, 24-H), 2.44 ( 3H, s, 26-H), 3.91 (3H, s, 6-OMe), 4.03 (3H, s, 7-OMe ), 3.89 (3H, s, 17-OMe), 4.01 (3H, s, 18-OMe), 3.94 (3H, s, 23COGMe), 4.07 (3H, s, 28COOMe). 9) The L3C NMR for 2 (lOOMHz, CDC13, 6): 153.8 (C-l), 121.4 (C-2), 173.6 (C-3), 114.0 (C-4), 126.3 (C5), 144.0 (C-6), 159.3 (C-7), 106.6 (C-8), 154.2 (C-9), 132.6 (C-lo), 136.3 ( C-11), 126.8 (C-12), 120.4 (C-13), 173.4 (C-14), 111.6 (C-15), 126.9 (C-16), 144.6 (C-17), 158.2 (C-18). 101.4 (C-19), 153.5 (C-20), 152.6 (C-21), 133.9 (C-22), 166.7 (C-23), 28.7 (C-24), 199.0 (C-25), 32.3 (C-26), 201.1 (C-27), 166.7 (C28), 53.0 (2C, 23-COOMe* and 28-COOMe*), 56.5 (2C, 7-OMe and 18-OMe), 62.3 (2C, 6-OMe and 17OMe). LO) Itsuo, K.; Hutchinson, C. R.; Vining, L. C. The biosynthesis of fulvic acid. Terrahedron Left. 1981,22, 493-496.
(Received in Japan 10 April 1991)