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Stellettahide a, an antifungal alkaloid from a marine sponge of the genus stelletta
Temhedm Leaem.Vo1.31,No.29.p~41634164.1990 Printedin GreatBritain
STELLETTAMIDE
0040-4039/90$3.00+ .acl Pcrgamon Pressplc
A,
AN ANTIFUNGAL ALKALOID
FROM A MARINE SPONGE OF THE GENUS STELLETZ'A" HiroshiHirota:' Shigeki Matsunagat and Nobuhiro Fusetani+* Departmentof Chemistry,Facultyof Science, and +Laboratoryof Marine Biochemistry, Facultyof Agriculture, The Universityof Tokyo, Bunkyo-ku, Tokyo 113 (Japan)
Abstract:A novel antifungalalkaloid, stellettamide A (l),has been isolatedfrom a marine sponge Stelletta sp.,and itsstructurewas elucidatedmainlyby spectroscopic methods includingextensive2D NMR experiments. In the course of our search for bioactive metabolites from Japanese marine invertebrates, we found that a marine spongd) of the genus Stelletta, collected in Shikine-jima Islandof the Izu Archipelago, showed antifungal activity against Mortierella remandanus. The bioassay-guidedisolation led to the isolation of an activecompound designatedstellettamide A and itsstructure(1) was a novel alkaloid containinga farnesylmoiety and an indolizidine (= octahydroindolizine) skeletons', whichwere connectedthroughanamidebond. The CHIC& solubleportionof the 70% ethanolextractof the frozen sponge (650 g) was subjectedto flashchromatography (E, Merck silica gel 60) with CI&Cb/MeOH/I$O. The activefractions(1.4g) elutedwith C&C4/MeOH/&O
(8 : 2 : 0.15)were successively
fractionated by ODS column chromatography(YMC 70/230mesh) with I&O/MeOH. The eluent with 75% MeOH (1.2g) was furtherpurified by HPLC on ODS (YMC 005-AM)with 28% n-PrOH / 72% 20mM KH2PO4 followedby desalting on an ODS column to affordstellettamide A (1;515 mg) as a semisolid. Stellettamide A (1) had a molecularformulaof (Ces&frN~nO)&PO4which was established by elementalanalysis,4~
FAB-MS [m/e 401 (C~eHtsN20)'], taC NMR, and alp NMR data.6'IR
(3666-3200, 1670,1630,1550,1275,1225 cm-l) and ;aC NMR (8 168.9)spectraimpliedan amide linkage, whilethe other nitrogenatom was deduced to form an indolizidine ring by NMRanalysesincluding HH-COSY,CH-COSY,HMBC,HOHAHA,andINADEQUATEexperiments.The N-methyl proton signalat i 3.16 showed strong correlation signalswith be 57.6,61.0,
13'
’
4163
4164
and 73.6in HMBC spectrum,which could be assignableto C-3,C-5,and C-8a of indolizidine skeleton,respectively, from INADEQUATE experiments.Substitution of a methylenegroup at C-l was alsosecuredby the NMR experiments; the methyleneprotons(a -3.4)showed a strong correlation signalswith the amide car-bony1 carbon (8 168.9)in HMBC spectrum.Similarly, the linkageof a modifiedfarnesylmoietyto the amide carbon was alsosubstantiated from NMR data. The stereochemistry of the indolizidine portionwas deduced by NOESY spectra;the Nmethyl proton (a 3.16)correlatedwith signalsat b 2.43 (28-H),3.32 (38-H),3.43 (58H), 3.61 (5a-H),and 3.70 @s&-H), while the C-8a proton (8 3.70)with protons at I 1.55 (76-H),-1.95 (86-H),3.13 (16-H),3.16 (N-C&), and 3.43 (58-H).Thus, the N-methyl group had equatorial configuration with respectto the six-memberedring and both hydrogens at C-l and C-8a were on the same sideas thisN-methylgroup.The stereochemistry of the C-4' methyl group togetherwith the absoluteconfiguration of 1 is under investigation. Stellettamide A (1) was not only antifungal, but also cytotoxicagainstK562 epitheliumcells(I&S 5.1 gg/mL). To our knowledge this is the firstreport of an indolizine metabolite from marine sponges.*) Acknowledgment:We thank ProfessorPaul J. Scheuer,Departmentof Chemistry, Universityof Hawaii,for readingthis manuscript.Thanks are alsodue to Professor PatriciaR. Bergquist,Department of Zoology,Universityof Auckland,for identification of the sponge.This work was partlysupportedby a Grant-in-Aid for Scientific Research from the Ministryof Education,Scienceand Cultureof Japan. ReferencesandNotes. 1) Bioactive marine metabolites Part 32. Part 31: N. Fusetani,K. Yasumuro, H. Kawai,T. Natori,L. Brinen,and J. Clardy, TetrahedronLett.,in press. 2) The sponge had a thin crustof an epizooic sponge of the genus Desmacella. 3) Reviews on indolizidine alkaloids: M. F. Grundon, Nat. Prod. Rep.,6, 523 (1989);4, 415 (1987);2, 235 (1985);1, 245 (1984);E. Gellert, J. Nat. Prod.,45, 50 (1982). 4j Trace amount (0.57%)of chlorinewas also detected.The counter anion of stellettamide A might be chlorineas the originalnaturalform, but most of chlorine might be substitutedby phosphate,which was used for purification. The presenceof phosphatewas confirmedbyionchromatographyand31PNMR. Found: C, 56.92;H, 9.27;N, 5.00%;[aID +23.10(C 5) Stellettamide A (1):semisolid; 0.3,EMH); UV (EtOH) end absorption; IR (neat)3600-3200,1670,1,630, 1550,1275,1225 cm-l; FAB-MS (glycerol, positive) m/z 401 [(Cze&5NeO)+;base peak];1H NMR (CDaOD)6 l.O5(3H,d, J = 7 Hz; 4'-CB), *1.4 (2H, m; 5'-HZ),1.55 (lH, m; 78-H).1.59 (3H, s; 8'-CH3),1.60 (3H, s; 12'-CH3),1.63 (lH, m; 8a-II), 1.66 (3H. s; 13'~Ha),*1.85 (2H, m; ~-HZ),-1.9 (lH, m; 7a-H),*1.95 (lH, m; 88-H),*2.0 (5H, m; 2a-H, 6'-H2,9'-&), 2.07 (2H, m; lO'-H2),2.32 (lH, q; 4'-H),2.43 (lH, dddd, J = 14, 9, 9, 7 Hz; 28-H).3.13 (lH, m; 18-H),3.16 (3H, s; N-CB), 3.32 (lH, ddd, J = 12, 9, 5 Hz; 38-H),*3.4 (2H, m; l-CH2),3.43 (lH, m; 58-H).3.61 (lH, br d, J = 14 Hz; 5a-H),3.70 (lH,ddd, J = 12, 6, 4 Hz; 8a6-H),3.90 (lH, ddd, J = 12, 12, 7 Hz; 3a-H),95.1 (2H,m; 7'-H and ll'-H), 5.92 (lH, dd, J = 15.5,1 Hz; 2'-H),6.70 (IH, dd, J = 15.5,8 Hz; 3,-H);13C NMR (CD3OD) b 16.1 (q; 8'-CH3),17.8 (q; 12'-(Z), 20.0 (q; 4'-CH3),21.1 (t;C-6), 21.6 (t; C-7), 23.3 (t; C-8), 25.2 (t; C-2), 25.9 (q; C-13'),26.6 (t; C-6'),27.7 (t; Clo'),37.1 (d; C-4'),37.5 (t; C-5'),39.7 (t; l-CH2), 40.4 (d; C-l), 40.6 (t; C-9'), 54.4 (q; N-CB), 57.6 (t;C-3), 61.0 (t;C-5), 73.6 (d; C-8a), 122.9 (d; C-2'),125.3 (d; C-11').125.4 (d; C-7'),132.1 (s; C-12'),136.4 (8; C-8'),151.5 (d; C-3'),168.9 (8; C-l').3'P NMR (CD3OD) a 6.28 (8). 6) D. J. Faulkner,Nat. Prod. Rep., 5, 613 (1988);4, 539 (1987);3, 1 (1986);1, 551 (1984). (peceivd in Japan 16May 1990)
STELLETTAMIDE
0040-4039/90$3.00+ .acl Pcrgamon Pressplc
A,
AN ANTIFUNGAL ALKALOID
FROM A MARINE SPONGE OF THE GENUS STELLETZ'A" HiroshiHirota:' Shigeki Matsunagat and Nobuhiro Fusetani+* Departmentof Chemistry,Facultyof Science, and +Laboratoryof Marine Biochemistry, Facultyof Agriculture, The Universityof Tokyo, Bunkyo-ku, Tokyo 113 (Japan)
Abstract:A novel antifungalalkaloid, stellettamide A (l),has been isolatedfrom a marine sponge Stelletta sp.,and itsstructurewas elucidatedmainlyby spectroscopic methods includingextensive2D NMR experiments. In the course of our search for bioactive metabolites from Japanese marine invertebrates, we found that a marine spongd) of the genus Stelletta, collected in Shikine-jima Islandof the Izu Archipelago, showed antifungal activity against Mortierella remandanus. The bioassay-guidedisolation led to the isolation of an activecompound designatedstellettamide A and itsstructure(1) was a novel alkaloid containinga farnesylmoiety and an indolizidine (= octahydroindolizine) skeletons', whichwere connectedthroughanamidebond. The CHIC& solubleportionof the 70% ethanolextractof the frozen sponge (650 g) was subjectedto flashchromatography (E, Merck silica gel 60) with CI&Cb/MeOH/I$O. The activefractions(1.4g) elutedwith C&C4/MeOH/&O
(8 : 2 : 0.15)were successively
fractionated by ODS column chromatography(YMC 70/230mesh) with I&O/MeOH. The eluent with 75% MeOH (1.2g) was furtherpurified by HPLC on ODS (YMC 005-AM)with 28% n-PrOH / 72% 20mM KH2PO4 followedby desalting on an ODS column to affordstellettamide A (1;515 mg) as a semisolid. Stellettamide A (1) had a molecularformulaof (Ces&frN~nO)&PO4which was established by elementalanalysis,4~
FAB-MS [m/e 401 (C~eHtsN20)'], taC NMR, and alp NMR data.6'IR
(3666-3200, 1670,1630,1550,1275,1225 cm-l) and ;aC NMR (8 168.9)spectraimpliedan amide linkage, whilethe other nitrogenatom was deduced to form an indolizidine ring by NMRanalysesincluding HH-COSY,CH-COSY,HMBC,HOHAHA,andINADEQUATEexperiments.The N-methyl proton signalat i 3.16 showed strong correlation signalswith be 57.6,61.0,
13'
’
4163
4164
and 73.6in HMBC spectrum,which could be assignableto C-3,C-5,and C-8a of indolizidine skeleton,respectively, from INADEQUATE experiments.Substitution of a methylenegroup at C-l was alsosecuredby the NMR experiments; the methyleneprotons(a -3.4)showed a strong correlation signalswith the amide car-bony1 carbon (8 168.9)in HMBC spectrum.Similarly, the linkageof a modifiedfarnesylmoietyto the amide carbon was alsosubstantiated from NMR data. The stereochemistry of the indolizidine portionwas deduced by NOESY spectra;the Nmethyl proton (a 3.16)correlatedwith signalsat b 2.43 (28-H),3.32 (38-H),3.43 (58H), 3.61 (5a-H),and 3.70 @s&-H), while the C-8a proton (8 3.70)with protons at I 1.55 (76-H),-1.95 (86-H),3.13 (16-H),3.16 (N-C&), and 3.43 (58-H).Thus, the N-methyl group had equatorial configuration with respectto the six-memberedring and both hydrogens at C-l and C-8a were on the same sideas thisN-methylgroup.The stereochemistry of the C-4' methyl group togetherwith the absoluteconfiguration of 1 is under investigation. Stellettamide A (1) was not only antifungal, but also cytotoxicagainstK562 epitheliumcells(I&S 5.1 gg/mL). To our knowledge this is the firstreport of an indolizine metabolite from marine sponges.*) Acknowledgment:We thank ProfessorPaul J. Scheuer,Departmentof Chemistry, Universityof Hawaii,for readingthis manuscript.Thanks are alsodue to Professor PatriciaR. Bergquist,Department of Zoology,Universityof Auckland,for identification of the sponge.This work was partlysupportedby a Grant-in-Aid for Scientific Research from the Ministryof Education,Scienceand Cultureof Japan. ReferencesandNotes. 1) Bioactive marine metabolites Part 32. Part 31: N. Fusetani,K. Yasumuro, H. Kawai,T. Natori,L. Brinen,and J. Clardy, TetrahedronLett.,in press. 2) The sponge had a thin crustof an epizooic sponge of the genus Desmacella. 3) Reviews on indolizidine alkaloids: M. F. Grundon, Nat. Prod. Rep.,6, 523 (1989);4, 415 (1987);2, 235 (1985);1, 245 (1984);E. Gellert, J. Nat. Prod.,45, 50 (1982). 4j Trace amount (0.57%)of chlorinewas also detected.The counter anion of stellettamide A might be chlorineas the originalnaturalform, but most of chlorine might be substitutedby phosphate,which was used for purification. The presenceof phosphatewas confirmedbyionchromatographyand31PNMR. Found: C, 56.92;H, 9.27;N, 5.00%;[aID +23.10(C 5) Stellettamide A (1):semisolid; 0.3,EMH); UV (EtOH) end absorption; IR (neat)3600-3200,1670,1,630, 1550,1275,1225 cm-l; FAB-MS (glycerol, positive) m/z 401 [(Cze&5NeO)+;base peak];1H NMR (CDaOD)6 l.O5(3H,d, J = 7 Hz; 4'-CB), *1.4 (2H, m; 5'-HZ),1.55 (lH, m; 78-H).1.59 (3H, s; 8'-CH3),1.60 (3H, s; 12'-CH3),1.63 (lH, m; 8a-II), 1.66 (3H. s; 13'~Ha),*1.85 (2H, m; ~-HZ),-1.9 (lH, m; 7a-H),*1.95 (lH, m; 88-H),*2.0 (5H, m; 2a-H, 6'-H2,9'-&), 2.07 (2H, m; lO'-H2),2.32 (lH, q; 4'-H),2.43 (lH, dddd, J = 14, 9, 9, 7 Hz; 28-H).3.13 (lH, m; 18-H),3.16 (3H, s; N-CB), 3.32 (lH, ddd, J = 12, 9, 5 Hz; 38-H),*3.4 (2H, m; l-CH2),3.43 (lH, m; 58-H).3.61 (lH, br d, J = 14 Hz; 5a-H),3.70 (lH,ddd, J = 12, 6, 4 Hz; 8a6-H),3.90 (lH, ddd, J = 12, 12, 7 Hz; 3a-H),95.1 (2H,m; 7'-H and ll'-H), 5.92 (lH, dd, J = 15.5,1 Hz; 2'-H),6.70 (IH, dd, J = 15.5,8 Hz; 3,-H);13C NMR (CD3OD) b 16.1 (q; 8'-CH3),17.8 (q; 12'-(Z), 20.0 (q; 4'-CH3),21.1 (t;C-6), 21.6 (t; C-7), 23.3 (t; C-8), 25.2 (t; C-2), 25.9 (q; C-13'),26.6 (t; C-6'),27.7 (t; Clo'),37.1 (d; C-4'),37.5 (t; C-5'),39.7 (t; l-CH2), 40.4 (d; C-l), 40.6 (t; C-9'), 54.4 (q; N-CB), 57.6 (t;C-3), 61.0 (t;C-5), 73.6 (d; C-8a), 122.9 (d; C-2'),125.3 (d; C-11').125.4 (d; C-7'),132.1 (s; C-12'),136.4 (8; C-8'),151.5 (d; C-3'),168.9 (8; C-l').3'P NMR (CD3OD) a 6.28 (8). 6) D. J. Faulkner,Nat. Prod. Rep., 5, 613 (1988);4, 539 (1987);3, 1 (1986);1, 551 (1984). (peceivd in Japan 16May 1990)