A new β-hydroxy-γ-methyl-γ-lactone bistetrahydrofuranic acetogenin, tucumanin, with the infrequent symmetrical threo/trans/threo/trans/ threo relative configuration at the tetrahydrofuran rings was isolated from Annona cherimolia (Annonaceae) seeds. The inhibitory potency on the mitochondrial complex I of acetogenins with this relative configuration (tucumanin and asimicin) was compared with that shown by the corresponding pairs with an asymmetrical threo/trans/threo/trans/erythro relative configuration (laherradurin/rolliniastatin-2, and itrabin/molvizarin). All these compounds act as selective inhibitors of mitochondrial complex 1 in the 0.18 - 1.55 nM range.Fil: Barrachina, Isabel. Universidad de Valencia; EspañaFil: Neske, Adriana. Universidad Nacional de Tucumán; ArgentinaFil: Granell, Susana. Universidad de Valencia; EspañaFil: Bermejo, Almudena. Universidad de Valencia; EspañaFil: Chahboune, Nadia. Universidad de Valencia; EspañaFil: El Aouad, Noureddine. Universidad de Valencia; EspañaFil: Alvarez, Olga. Universidad Nacional de Tucumán; ArgentinaFil: Bardon, Alicia del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Química del Noroeste. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química del Noroeste; Argentina. Universidad Nacional de Tucumán; ArgentinaFil: Zafra Polo, M. Carmen. Universidad de Valencia; Españ

Alvarez, Olga

Bardon, Alicia del Valle

Barrachina, Isabel

Bermejo, Almudena

Chahboune, Nadia

El Aouad, Noureddine

Granell, Susana

Neske, Adriana

Zafra Polo, M. Carmen

Planta Medica

English

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

Natural Products and Medicinal Plant ResearchPlanta MedicaEditorProf. Dr. Adolf NahrstedtInstitut fUr Pharmazeutische Biologieund Phytoch emie der UniversitatHittorfstra~e 560-48149 MunsterGermanyFax: +4925183-38341E-mail: nahrste@uni-muenster.deCo-EditorsProf. Dr. W. Sarz, MunsterProf. Dr. R. Bauer, GrazProf. Dr. G. Franz, RegensburgProf. Dr. M. Hamburger, BaselProf. Dr. I. Khan, Oxford, MSEditorial AdVisory BoardY. Asakawa. TokushimaL. Bohlin, UppsalaI. Calis, AnkaraS. Canigueral, BarcelonaA. M. Clark, Oxford, MSO. Guo, PekingR. Maffei Facino, MilanoS. Habtemariam, LondonG. Harnischfeger, SalzgitterW. Herz, TallahasseeM. Hesse, ZurichK. Hostettmann, LausanneP. J. Houghton, LondonP. Joseph-Nathan, Mexico CityG. Kahl, FrankfurtjMW. Kreis, ErlangenU. Matern, MarburgI. Merfort. FreiburgS. Nyiredy, BudakalaszK.-M. Oksman-Caldentey, EspooP. Potier, Gif-sur-YvetteV. Schulz, BerlinJ. F. Verbist, NantesE. J.Verspohl, MunsterA. Vlietinck, AntwerpA. Vollmar, MunchenH. Vuorela, HelsinkiH. Yamada, TokioReprint© Georg Thieme Verlag KGStuttgart· New YorkGeorg Thieme Verlag KGStuttgart· New YorkRudigerstra~e140-70469 StuttgartPostfach 3011 200-70451 StuttgartThieme New York333 Seventh AvenueNew York, NY 10001Tucumanin. a {3-Hydroxy-y-lactoneBistetrahydrofuranic Acetogeninfrom Annona cherimolia, is a PotentInhibitor of Mitochondrial Complex IIsabel Barrachina I, Adriana Neske2, Susana Granell1,Almudena Bermejo" Nadia Chahboune I, Noureddine EI Aouad l ,Olga Alvarez2, Alicia Bardon 2, M. Carmen Zafra-Polo 1AbstractAnew {3-hydroxy-y-methyl-y-lactone bistetrahydrofuranic aceto-genin, tucumanin, with the infrequent symmetrical threo/trans/threo/trans/threo relative configuration at the tetrahydrofuranrings was isolated from Annona cherimolia (Annonaceae) seeds.The inhibitory potency on the mitochondrial complex Iof aceto-genins with this relative configuration (tucumanin and asimicin)was compared with that shown by the corresponding pairs withan asymmetrical threo/trans/threo/trans/erythro relative config-uration (laherradurin/rolliniastatin-2, and itrabin/molvizarin).All these compounds act as selective inhibitors of mitochondrialcomplex I in the 0.18 -1.55 nM range."Cherimoya" is an edible fruit from a tropical tree (Annonacherimolia Mill., Annonaceae) native to Peru that is cultivatedworldwide. In previous chemical studies on the seeds of A.cherimolia cultivated in an small pseudo-tropical area in thesouth of Spain, on the coast of Granada, four monotetrahydro-furanic (mono-THF) and eleven bistetrahydrofuranic (bis-THF)acetogenins were isolated [1], [2]. Recently, three new cytotoxicmono-THF acetogenins, annocherin, annomolin, and annocheri-molin, were isolated from seeds of "cherimoya" grown in planta-tions of Southern California [31,14] .The Annonaceous acetogenins are claimed as potent inhibitors ofNADH:ubiquinone oxidoreductase (complex I) in the mitochon-drial electron transport chain [5]. Among them, the {3-hydroxy-y-methyl-y-Iactone acetogenins, a type of acetogenin only isolatedfrom the seeds of A. cherimolia, are more potent complex I inhi-bitors than any other natural acetogenin [6]. The high potency ofthis class of acetogenins led us to further investigate the compo-sition of seeds collected in Tucuman (Argentina).Affiliation: I Departamento de Farmacologia. Facliitad de Farmacia. Universi-dad de Valencia, Burjassot. Spain· 2 Departamento de Qllimica Organica. Fa-cultad de Qllimica. Universidad Nacional de Tucuman. Tllcuman. ArgentinaCorrespondence: Prof. Dr. M. Cannen Zafra-Polo . Departamento de Farmacolo-gia . Facultad de Farmacia . Avda. Vicent Andres Estelles sin· 46100 Burjassot .Valencia· Spain· Phone: +34-96-354-4944 . Fax: +34-96-354-4943 . E-mail:carmen.zafra@uv.esReceived: January 29, 2004 . Accepted: June 4. 2004Bibliography: Planta Med 2004 ; 70: 866-868 . © Georg Thieme Verlag KGStuttgart· New York· DOI1O.1055/s-2004-827237· ISSN 0032-0943Fractionation of the methanolic extract furnished 11 known acet-ogenins (almunequin, asimicin, cherimolin-1, itrabin, laherra-durin, molvizarin, motrilin, neoannonin, rolliniastatin-2, squa-mocin, squamocin-B) and a new acetogenin, tucumanin (1). Themolecular weight of 1 was established by the [M+Nar ion ob-served at m/z = 647.45183 (calcd. 647.44988 for C37HGs07Na) inthe HR-LSI-MS. Absorptions at 204 nm and at 1760 cm- I in theUV and IR spectra, repectively, are in accordance with the pres-ence of a saturated y-lactone ring in 1[1] . The 1H-NMR spectrumand the 20 homo nuclear correlation (lH-IH COSY 45) of 1. provethe existence of an ABX spin system, characteristic of the threelactone ring protons of a (3-hydroxy-y-methyl-y-lactone aceto-genin [7], [8]: H-2 (rx) at {j = 2.58, H-35 ({3) at (5 = 4.19, and H-36(y) at (5 = 4.49. These protons are correlated in 20 heteronuclearNMR experiments (HMQC and HMBC) with three methine car-bons at (5 = 43.7, (573.7, and (582.4, respectively (Fig.1). The rela-tive configuration of the lactone moiety deduced from the cou-pling constants, J,,-~ = 5.5 Hz (cis) and J~- y = 1.0 Hz (trans), wasin agreement with that reported by Sibi et al. for the synthetic la-herradurin [9].The presence of an rx, rx'-dihydroxylated bis-THF system with theinfrequent symmetrical threo/trans/threo/trans/threo relativeconfiguration was deduced from IH-13C NMR heteronuclear cor-relations between the signals at (5 = 3.38 and (5 = 74.0 (two me-thine alcohol), and between the signals at (5 = 3.81 and 0 = 83.1(two methine tetrahydrofuran) [lOJ. This system is placed be-tween C-15 and C-24 based on the fragment ions observed inthe EI-MS (Fig.1). Compound 1 is the first {3-hydroxy-y-methyly-lactone adjacent bis-THF acetogenin with a threo/trans/threo/trans/ threo relative configuration.In previous studies we have shown that the inhibitory potency ofacetogenins is modulated by several structural factors. Therefore,it is relevant to compare the inhibitory potency of acetogeninswith this type of configuration (tucumanin, 1 and asimicin, 2)with that of the corresponding pairs with an asymmetricalthreo/trans/threo/trans/erythro relative configuration (laherra-durin, 3/rolliniastatin-2, 5, and itrabin, 4/molvizarin, 6). As po-tency criterion we used the ICso against the integrated NADH oxi-dase activity of beef heart open submitochondrial particles [5].[6], [12]. which evaluates the complex I activity in a more phys-iological environment. Results are shown in Table 1.The {3-hydroxyacetogenins with a threo/trans/threo/trans/erythrorelative configuration, laherradurin (3) and itrabin (4) (lCso =0.18 and 0.21 nM, respectively) are more potent inhibitors of themitochondrial complex I than the corresponding rx,{3-unsatura-ted y-Iactone acetogenins, rolliniastatin-2 (5) and molvizarin (6)(lCso = 0.51 and 1.55 nM, respectively). However, tucumanin (1)(ICso = 0.57 nM) shows an inverted tendency when compared toasimicin (2) (lcso = 0.33 nM), both with a threo/trans/threo/trans/threo relative configuration. Therefore, our data show that,indeed, the terminal y-methyl-y-Iactone ring and the relativeconfiguration at the THF system are both relevant structural fac-tors for a potent inhibition of the mitochondrial complex I, be-cause of a double binding to the active site of the enzyme towhich both moieties probably contribute.threoltranslthreoltranslthreo37 I\\1"2434HO 1: tucwnanin OH OH1 00 QH37\1 1 '- h 24342: asimicin OH OHFig. 1 lH_ 13C NMR hetero-nuclear correlations (13C inparentheses) spectral data,and significant ElMS frag-ment ions of tucumanin (1).(22.7)1.26o'~1.26OH 3.38 (3/.9)(74.0) 1.26(25.6-29.7)(18.4)threoltranslthreoltrans/erythro371JS I\,1'- 2412234/323: laherradurin (n~ 2) OH OH4: itrabin (n= 1)37/3S\\1'-34m5: roUiniaslatin-2 (n= 2) OH OH6: molyizarin (n~ I)407~435~ [4531--,--- 1m)417~,o(73.7)267~ 295~ 313 -~..---- 311(81.4) (/77.4)4.49 \ 0 (23.3)(IV) "0 1.65;1.781.12~ \I~. l-t \.~----., ~----'Y(1'f~ 1.26(15.6-19.7)319~ 347.....:':!J£- 365~ [3831----l.-- 241~ 223Table 1 Inhibitory potency of acetogenins 1- 6Compound2 3 4 5 6 rotenone {6]NADH oxidase 1(50 (nM) 0.57 ± q:07' 0.33 ± 0.03' 0.18 ± 0.02" 0.21 t 0.03' 0.5i ± 0.03' 1.551'0.17' 5.10 ± 0.09Significant difference compared with rotenone; • P< 0.001.Material and MethodsOptical rotation (Perkin-Elmer 241 polarimeter), IR spectra (Sa-tellite FTIR-Mattson 980514 spectrometer), MS (VG Auto Spec Fi-sons spectrometer), lH_ and 13C-NMR, DEPT, HMBC, HMQC, tH-1H-COSY (Varian Unity 400 MHz spectrometer) using the solventsignal as reference (CDCl) at 8 = 7.26 and 77.0). Chromatograph-ic separations were carried out by column chromatography on si-lica gel 60 H(5 - 40 ,um, 7336 Merck). Semi preparative HPLC wascarried out on a LiChroCartR 100 RP-18 column (25 x 1 em i. d.,10,um particle size), flow rate 4.5 mLjmin. using MeOH-H 20-THF. Analytical TLC was performed on Merck precoated silicagel 60 F254 plates.letter ... Planta Med 2004; 70: 866 - 868The fruits of Annona eherimo/ia Mill. ("cherimoya") were collect-ed at Yerba Buena, Tucuman, Argentina, in February 2000. Avoucher sample (No. 515092) was deposited at the Herbariumof Instituto Li 110 ofTucuman.The dried and powdered seeds of cherimoya (350 g) were percolat-ed with methanol (2 x 3 L). Evaporation of the solvent yielded acrude MeOH extract (15 g) which was further partitioned betweenCH2Cl2 (3 x 100 mL) and H20 (100 mL). The CH2CI2 layer waswashed with water, dried over anhydrous Na2S04, filtered, andevaporated in vacuum to give 9 g of a solid extract with an aceto-genin mixture, which was chromatographed on a silica gel 60 H(315 g) column, using gradients of hexane/EtOAc (90 : 10; 80: 20;70: 30; 60: 40; 50: 50; 40: 60; 20; 80 and 10: 90) giving eightfractions (F-I to F-VIII , 250 mL each). Fraction F-IH (hexane/EtOAc70: 30, 660 mg) was again chromatographed on silica gel 60 H(23 g) and eluted with CH2CI2/MeOH (95: 5) to give five subfrac-tions (50 mL each), one of them (F-III]) was applied to semipre-parative HPLC with MeOH-H20-THF (87: 13: 5) affording neoan-nonin {12 mg, Rt = 10.8 min, [aI55 : + 16.2° (CHCl ], e 0.8)}; , F-IV(hexane/EtOAc, 60: 40, 2 g) was chromatographed on silica gel60 H (70 g) and eluted with CH2CI 2/EtOAc/MeOH (50: 45: 5) toyield eight subfractions (100 mL each). F-IV5 and F-IV6 by semi-preparative HPLC with MeOH-H20-THF 80: 20: 5 afforded squa-mocin {90 mg, R[ = 16.6 min, [aI55: + 14.0° (CHCI], e 0.13), squa-mocin-B (16 mg, Rr = 8.7 min, [a]55 ; + 27.6 (MeOH, e 0.2) andmotrilin (5 mg, Rr = 14.8 min, [a]b5 : + 10.8' (MeOH, c O.13)}, andasimicin {2, 5 mg, Rr = 29.4 min, [aI55 : + 11.0U (CHCl], c 0.22),molvizarin (6, 10 mg, Rr _ 13.9 min, [a155: + 10.0° (CHCI3, e 0.2)and rolliniastatin-2 (5, 5 mg, Rt = 11.3 min, [a]55 : + 12.8' (CHCl3,e 0.26 )), respectively. F-VI (hexane/EtOAc, 40: 60, 500 mg) waschromatographed on silica gel (18 g) and eluted with CH2Cl2/EtOAc/MeOH (60: 13: 10) to give four subfractions (75 mL each).One of them (F-VI2 ) was applied to semipreparative HPLC withMeOH-H20-THF (80: 20: 5) affording cherimolin-1 {IS mg,R1 = 8.9 min, [a]55 : +64.0° (MeOH, e 0.3) and almunequin(5 mg, R, = 5.8 min, laJb5: + 13.5' (CHCI3, c 0.14)}. F-VIII (hex-ane/HOAc, 10: 90, 250 mg) was chromatographed on silica gel(9 g) and eluted with CH2Cl2/HOAc/MeOH (10: 85: 5) to givethree subfractions (50 mL each). F-VlII 2 was applied to semipre-parative HPLC with MeOH-H20-THF (80: 20: 5) affording itrabin{4. 40 mg, Rr = 2.1 min. [aI55: + 21 .0' (MeOH, c 0.2), laherradurin(3,60 mg, Rt = 3.5 min, [a]55 : + 21.0° (MeOH, e0.09) and tucuma-nin (1,11 mg, Rr = 4.2 min)}.Tucumanin (1): waxy compound, [a]55: + 1.5- (EtOH, e 0.8); UV(EtOH): A max (log £) = 204 nm (3.8). IR (film ): Vmax = 3360,2923,2853,1760,1465,1052 cm- t• HR-LSI-MS: m/z = 647.45183[M + Na]+ (caled. for C37H6807Na: 647.44988). EI-MS, IH-NMR(400 MHz). and I3C-NMR (100 MHz), see Fig.1.The inhibitory potency of acetogenins as complex r inhibitorswas assayed using submitochondrial particles from beef heartas previously reported [5], [11], [12]. NADH oxidase activity wasmeasured as the aerobic oxidation of75 ,uM NADH in the absenceof external quinone substrates and other inhibitors of the re-spiratory chain. Rotenone (Sigma-Aldrich St. Louis, MO, USA)was used as the positive control. Data from four titrations wereused to determine the statistic means and the standard devia-Letter ... Planta Med 2004; 70: 866 - 868tions. Significance of differences was evaluated by the Student'st test, with P< 0.001 being regarded as significant.AclmowledgementsThis research was supported by the Spanish Direccion General delnvestigacion under grant SAF 2001 -3142. and by the Secretariadel Plan Valenciano de Investigacion Cientifica, DesarrolloTecno-logico e lnnovacion de la Generalitat Valenciana under grantGVOl-282.References1 Cave A, Figadere B, Laurens A, Cortes D. Acetogenins from Annona-ceous. In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm Ch, editors.Progress in the chemistry of Organic Natural Products: Vol.70 Wien:Springer-Verlag, 1997: p 81-2882 Zaft'a-Polo MC, Figadere B, Gallardo T, Tormo JR, Cortes D. Natural acet-ogenins from Annonaceae, synthesis and mechanisms of action. Phy-tochemistry 1998: 48: 1087 -117) Woo MH, Kim DH, Fotopoulos SS, McLaughlin JL. Annocherin and(2,4)-ci5- and trans-annocherinones, monotetrahydrofuran annonac-eous acetogenins with a C-7 carbonyl group from Annona cherimoliaseeds. J Nat Prod 1999; 62: 1250 - 54 Kim DH, Ma ES, Suk KD, SonJK, LeeJS, Woo M-H, 2001. Annomolin andannocherimolin, new cytotoxic annonaceous acetogenins fromAnnona cherimolia seeds. J Nat Prod 1996; 64: 502 - 5065 Degli Esposti M, Ghelli A, Ratta M, Cortes D, Estornell E. Natural sub-stances (acetogenins) from the family Annonaceae are powerful inhi-bitors of mitochondrial NADH dehydrogenase (complex I). Biochem J1994; 301: 161 - 7G Tormo JR, Estornell E, Gallardo T, Gonzalez MC, Cave Ad, Granell S,Cortes D, Zafra-Polo Me. y-Lactone functionalized antitumoral aceto-genins are the most potent inhibitors of mitochondrial complex I.Bioorg Med Chem Lett 2001; 11: 681 - 47 Cortes D, Myint SH, Leboeuf M, Cave A. A new type of cytotoxic aceto-genins: the tetrahydrofuranic IJ-hydroxy methyl y-lacrones. Tetrahe-dron Lett 1991 ; 32: 6133-48 Cortes D, Myint SH, Dupont B, Davoust D. Bioactive acetogenins fromseeds of Annona cherimo/ia . Phytochemistry 1993; 32: 1475 - 829 Sibi M, Lu J, Talbacka CL. Enantiospecific synthesis of trisubstitutedbutyrolactone natural products and their analogs. J Org Chem 1996;61;7848-5510 Cortes D, Figadere B, Cave A. Bis-tetrahydrofuran acetogenins fromAnnonaceae. Phytochemistry 1993: 32: 1467 - 73II Fato R, Estornell E, Di Bernardo S, Pallotti F, Parenti-Castelli G, Lenaz G.Steady-state kinetics of the reduction of coenzyme Qanalogs by com-plex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochon-dria and submitochondrial particles. Biochemistry 1996; 35: 2705 -1612 Gallardo T, Zafra-Polo MC, Tormo JR. Gonzalez MC, Franck X. EstornellE, Cortes D. Semisynthesis of antitumoral acerogenins: SAR of functio-nalized alkyl-chain bis-terrahydrofuranic acerogenins, specific inhibi-tors of mitochondrial complex I. J Med Chern 2000; 43: 4793 - 800

Tucumanin, a β-hydroxy-γ-lactone bistetrahydrofuranic acetogenin from Annona cherimolia, is a potent inhibitor of mitochondrial complex I

CONICET Digital

Natural Products and Medicinal Plant ResearchPlanta MedicaEditorProf. Dr. Adolf NahrstedtInstitut fUr Pharmazeutische Biologieund Phytoch emie der UniversitatHittorfstra~e 560-48149 MunsterGermanyFax: +4925183-38341E-mail: nahrste@uni-muenster.deCo-EditorsProf. Dr. W. Sarz, MunsterProf. Dr. R. Bauer, GrazProf. Dr. G. Franz, RegensburgProf. Dr. M. Hamburger, BaselProf. Dr. I. Khan, Oxford, MSEditorial AdVisory BoardY. Asakawa. TokushimaL. Bohlin, UppsalaI. Calis, AnkaraS. Canigueral, BarcelonaA. M. Clark, Oxford, MSO. Guo, PekingR. Maffei Facino, MilanoS. Habtemariam, LondonG. Harnischfeger, SalzgitterW. Herz, TallahasseeM. Hesse, ZurichK. Hostettmann, LausanneP. J. Houghton, LondonP. Joseph-Nathan, Mexico CityG. Kahl, FrankfurtjMW. Kreis, ErlangenU. Matern, MarburgI. Merfort. FreiburgS. Nyiredy, BudakalaszK.-M. Oksman-Caldentey, EspooP. Potier, Gif-sur-YvetteV. Schulz, BerlinJ. F. Verbist, NantesE. J.Verspohl, MunsterA. Vlietinck, AntwerpA. Vollmar, MunchenH. Vuorela, HelsinkiH. Yamada, TokioReprint© Georg Thieme Verlag KGStuttgart· New YorkGeorg Thieme Verlag KGStuttgart· New YorkRudigerstra~e140-70469 StuttgartPostfach 3011 200-70451 StuttgartThieme New York333 Seventh AvenueNew York, NY 10001Tucumanin. a {3-Hydroxy-y-lactoneBistetrahydrofuranic Acetogeninfrom Annona cherimolia, is a PotentInhibitor of Mitochondrial Complex IIsabel Barrachina I, Adriana Neske2, Susana Granell1,Almudena Bermejo" Nadia Chahboune I, Noureddine EI Aouad l ,Olga Alvarez2, Alicia Bardon 2, M. Carmen Zafra-Polo 1AbstractAnew {3-hydroxy-y-methyl-y-lactone bistetrahydrofuranic aceto-genin, tucumanin, with the infrequent symmetrical threo/trans/threo/trans/threo relative configuration at the tetrahydrofuranrings was isolated from Annona cherimolia (Annonaceae) seeds.The inhibitory potency on the mitochondrial complex Iof aceto-genins with this relative configuration (tucumanin and asimicin)was compared with that shown by the corresponding pairs withan asymmetrical threo/trans/threo/trans/erythro relative config-uration (laherradurin/rolliniastatin-2, and itrabin/molvizarin).All these compounds act as selective inhibitors of mitochondrialcomplex I in the 0.18 -1.55 nM range."Cherimoya" is an edible fruit from a tropical tree (Annonacherimolia Mill., Annonaceae) native to Peru that is cultivatedworldwide. In previous chemical studies on the seeds of A.cherimolia cultivated in an small pseudo-tropical area in thesouth of Spain, on the coast of Granada, four monotetrahydro-furanic (mono-THF) and eleven bistetrahydrofuranic (bis-THF)acetogenins were isolated [1], [2]. Recently, three new cytotoxicmono-THF acetogenins, annocherin, annomolin, and annocheri-molin, were isolated from seeds of "cherimoya" grown in planta-tions of Southern California [31,14] .The Annonaceous acetogenins are claimed as potent inhibitors ofNADH:ubiquinone oxidoreductase (complex I) in the mitochon-drial electron transport chain [5]. Among them, the {3-hydroxy-y-methyl-y-Iactone acetogenins, a type of acetogenin only isolatedfrom the seeds of A. cherimolia, are more potent complex I inhi-bitors than any other natural acetogenin [6]. The high potency ofthis class of acetogenins led us to further investigate the compo-sition of seeds collected in Tucuman (Argentina).Affiliation: I Departamento de Farmacologia. Facliitad de Farmacia. Universi-dad de Valencia, Burjassot. Spain· 2 Departamento de Qllimica Organica. Fa-cultad de Qllimica. Universidad Nacional de Tucuman. Tllcuman. ArgentinaCorrespondence: Prof. Dr. M. Cannen Zafra-Polo . Departamento de Farmacolo-gia . Facultad de Farmacia . Avda. Vicent Andres Estelles sin· 46100 Burjassot .Valencia· Spain· Phone: +34-96-354-4944 . Fax: +34-96-354-4943 . E-mail:carmen.zafra@uv.esReceived: January 29, 2004 . Accepted: June 4. 2004Bibliography: Planta Med 2004 ; 70: 866-868 . © Georg Thieme Verlag KGStuttgart· New York· DOI1O.1055/s-2004-827237· ISSN 0032-0943Fractionation of the methanolic extract furnished 11 known acet-ogenins (almunequin, asimicin, cherimolin-1, itrabin, laherra-durin, molvizarin, motrilin, neoannonin, rolliniastatin-2, squa-mocin, squamocin-B) and a new acetogenin, tucumanin (1). Themolecular weight of 1 was established by the [M+Nar ion ob-served at m/z = 647.45183 (calcd. 647.44988 for C37HGs07Na) inthe HR-LSI-MS. Absorptions at 204 nm and at 1760 cm- I in theUV and IR spectra, repectively, are in accordance with the pres-ence of a saturated y-lactone ring in 1[1] . The 1H-NMR spectrumand the 20 homo nuclear correlation (lH-IH COSY 45) of 1. provethe existence of an ABX spin system, characteristic of the threelactone ring protons of a (3-hydroxy-y-methyl-y-lactone aceto-genin [7], [8]: H-2 (rx) at {j = 2.58, H-35 ({3) at (5 = 4.19, and H-36(y) at (5 = 4.49. These protons are correlated in 20 heteronuclearNMR experiments (HMQC and HMBC) with three methine car-bons at (5 = 43.7, (573.7, and (582.4, respectively (Fig.1). The rela-tive configuration of the lactone moiety deduced from the cou-pling constants, J,,-~ = 5.5 Hz (cis) and J~- y = 1.0 Hz (trans), wasin agreement with that reported by Sibi et al. for the synthetic la-herradurin [9].The presence of an rx, rx'-dihydroxylated bis-THF system with theinfrequent symmetrical threo/trans/threo/trans/threo relativeconfiguration was deduced from IH-13C NMR heteronuclear cor-relations between the signals at (5 = 3.38 and (5 = 74.0 (two me-thine alcohol), and between the signals at (5 = 3.81 and 0 = 83.1(two methine tetrahydrofuran) [lOJ. This system is placed be-tween C-15 and C-24 based on the fragment ions observed inthe EI-MS (Fig.1). Compound 1 is the first {3-hydroxy-y-methyly-lactone adjacent bis-THF acetogenin with a threo/trans/threo/trans/ threo relative configuration.In previous studies we have shown that the inhibitory potency ofacetogenins is modulated by several structural factors. Therefore,it is relevant to compare the inhibitory potency of acetogeninswith this type of configuration (tucumanin, 1 and asimicin, 2)with that of the corresponding pairs with an asymmetricalthreo/trans/threo/trans/erythro relative configuration (laherra-durin, 3/rolliniastatin-2, 5, and itrabin, 4/molvizarin, 6). As po-tency criterion we used the ICso against the integrated NADH oxi-dase activity of beef heart open submitochondrial particles [5].[6], [12]. which evaluates the complex I activity in a more phys-iological environment. Results are shown in Table 1.The {3-hydroxyacetogenins with a threo/trans/threo/trans/erythrorelative configuration, laherradurin (3) and itrabin (4) (lCso =0.18 and 0.21 nM, respectively) are more potent inhibitors of themitochondrial complex I than the corresponding rx,{3-unsatura-ted y-Iactone acetogenins, rolliniastatin-2 (5) and molvizarin (6)(lCso = 0.51 and 1.55 nM, respectively). However, tucumanin (1)(ICso = 0.57 nM) shows an inverted tendency when compared toasimicin (2) (lcso = 0.33 nM), both with a threo/trans/threo/trans/threo relative configuration. Therefore, our data show that,indeed, the terminal y-methyl-y-Iactone ring and the relativeconfiguration at the THF system are both relevant structural fac-tors for a potent inhibition of the mitochondrial complex I, be-cause of a double binding to the active site of the enzyme towhich both moieties probably contribute.threoltranslthreoltranslthreo37 I\\1" 2434HO 1: tucwnanin OH OH1 00 QH37\1 1 '- h 24342: asimicin OH OHFig. 1 lH_ 13C NMR hetero-nuclear correlations (13C inparentheses) spectral data,and significant ElMS frag-ment ions of tucumanin (1).(22.7)1.26o'~1.26OH 3.38 (3/.9)(74.0) 1.26(25.6-29.7)(18.4)threoltranslthreoltrans/erythro371JS I\,1'- 2412234/323: laherradurin (n~ 2) OH OH4: itrabin (n= 1)37/3S\\1'-34m5: roUiniaslatin-2 (n= 2) OH OH6: molyizarin (n~ I)407~435~ [4531--,--- 1m)417~,o(73.7)267~ 295~ 313 -~..---- 311(81.4) (/77.4)4.49 \ 0 (23.3)(IV) "0 1.65;1.781.12~ \I~. l-t \.~----., ~----'Y(1'f~ 1.26(15.6-19.7)319~ 347.....:':!J£- 365~ [3831----l.-- 241~ 223Table 1 Inhibitory potency of acetogenins 1- 6Compound2 3 4 5 6 rotenone {6]NADH oxidase 1(50 (nM) 0.57 ± q:07' 0.33 ± 0.03' 0.18 ± 0.02" 0.21 t 0.03' 0.5i ± 0.03' 1.551'0.17' 5.10 ± 0.09Significant difference compared with rotenone; • P< 0.001.Material and MethodsOptical rotation (Perkin-Elmer 241 polarimeter), IR spectra (Sa-tellite FTIR-Mattson 980514 spectrometer), MS (VG Auto Spec Fi-sons spectrometer), lH_ and 13C-NMR, DEPT, HMBC, HMQC, tH-1H-COSY (Varian Unity 400 MHz spectrometer) using the solventsignal as reference (CDCl) at 8 = 7.26 and 77.0). Chromatograph-ic separations were carried out by column chromatography on si-lica gel 60 H(5 - 40 ,um, 7336 Merck). Semi preparative HPLC wascarried out on a LiChroCartR 100 RP-18 column (25 x 1 em i. d.,10,um particle size), flow rate 4.5 mLjmin. using MeOH-H 20-THF. Analytical TLC was performed on Merck precoated silicagel 60 F254 plates.letter ... Planta Med 2004; 70: 866 - 868The fruits of Annona eherimo/ia Mill. ("cherimoya") were collect-ed at Yerba Buena, Tucuman, Argentina, in February 2000. Avoucher sample (No. 515092) was deposited at the Herbariumof Instituto Li 110 ofTucuman.The dried and powdered seeds of cherimoya (350 g) were percolat-ed with methanol (2 x 3 L). Evaporation of the solvent yielded acrude MeOH extract (15 g) which was further partitioned betweenCH2Cl2 (3 x 100 mL) and H20 (100 mL). The CH2CI2 layer waswashed with water, dried over anhydrous Na2S04, filtered, andevaporated in vacuum to give 9 g of a solid extract with an aceto-genin mixture, which was chromatographed on a silica gel 60 H(315 g) column, using gradients of hexane/EtOAc (90 : 10; 80: 20;70: 30; 60: 40; 50: 50; 40: 60; 20; 80 and 10: 90) giving eightfractions (F-I to F-VIII , 250 mL each). Fraction F-IH (hexane/EtOAc70: 30, 660 mg) was again chromatographed on silica gel 60 H(23 g) and eluted with CH2CI2/MeOH (95: 5) to give five subfrac-tions (50 mL each), one of them (F-III]) was applied to semipre-parative HPLC with MeOH-H20-THF (87: 13: 5) affording neoan-nonin {12 mg, Rt = 10.8 min, [aI55 : + 16.2° (CHCl ], e 0.8)}; , F-IV(hexane/EtOAc, 60: 40, 2 g) was chromatographed on silica gel60 H (70 g) and eluted with CH2CI 2/EtOAc/MeOH (50: 45: 5) toyield eight subfractions (100 mL each). F-IV5 and F-IV6 by semi-preparative HPLC with MeOH-H20-THF 80: 20: 5 afforded squa-mocin {90 mg, R[ = 16.6 min, [aI55: + 14.0° (CHCI], e 0.13), squa-mocin-B (16 mg, Rr = 8.7 min, [a]55 ; + 27.6 (MeOH, e 0.2) andmotrilin (5 mg, Rr = 14.8 min, [a]b5 : + 10.8' (MeOH, c O.13)}, andasimicin {2, 5 mg, Rr = 29.4 min, [aI55 : + 11.0U (CHCl], c 0.22),molvizarin (6, 10 mg, Rr _ 13.9 min, [a155: + 10.0° (CHCI3, e 0.2)and rolliniastatin-2 (5, 5 mg, Rt = 11.3 min, [a]55 : + 12.8' (CHCl3,e 0.26 )), respectively. F-VI (hexane/EtOAc, 40: 60, 500 mg) waschromatographed on silica gel (18 g) and eluted with CH2Cl2/EtOAc/MeOH (60: 13: 10) to give four subfractions (75 mL each).One of them (F-VI2 ) was applied to semipreparative HPLC withMeOH-H20-THF (80: 20: 5) affording cherimolin-1 {IS mg,R1 = 8.9 min, [a]55 : +64.0° (MeOH, e 0.3) and almunequin(5 mg, R, = 5.8 min, laJb5: + 13.5' (CHCI3, c 0.14)}. F-VIII (hex-ane/HOAc, 10: 90, 250 mg) was chromatographed on silica gel(9 g) and eluted with CH2Cl2/HOAc/MeOH (10: 85: 5) to givethree subfractions (50 mL each). F-VlII 2 was applied to semipre-parative HPLC with MeOH-H20-THF (80: 20: 5) affording itrabin{4. 40 mg, Rr = 2.1 min. [aI55: + 21 .0' (MeOH, c 0.2), laherradurin(3,60 mg, Rt = 3.5 min, [a]55 : + 21.0° (MeOH, e0.09) and tucuma-nin (1,11 mg, Rr = 4.2 min)}.Tucumanin (1): waxy compound, [a]55: + 1.5- (EtOH, e 0.8); UV(EtOH): A max (log £) = 204 nm (3.8). IR (film ): Vmax = 3360,2923,2853,1760,1465,1052 cm- t• HR-LSI-MS: m/z = 647.45183[M + Na]+ (caled. for C37H6807Na: 647.44988). EI-MS, IH-NMR(400 MHz). and I3C-NMR (100 MHz), see Fig.1.The inhibitory potency of acetogenins as complex r inhibitorswas assayed using submitochondrial particles from beef heartas previously reported [5], [11], [12]. NADH oxidase activity wasmeasured as the aerobic oxidation of75 ,uM NADH in the absenceof external quinone substrates and other inhibitors of the re-spiratory chain. Rotenone (Sigma-Aldrich St. Louis, MO, USA)was used as the positive control. Data from four titrations wereused to determine the statistic means and the standard devia-Letter ... Planta Med 2004; 70: 866 - 868tions. Significance of differences was evaluated by the Student'st test, with P< 0.001 being regarded as significant.AclmowledgementsThis research was supported by the Spanish Direccion General delnvestigacion under grant SAF 2001 -3142. and by the Secretariadel Plan Valenciano de Investigacion Cientifica, DesarrolloTecno-logico e lnnovacion de la Generalitat Valenciana under grantGVOl-282.References1 Cave A, Figadere B, Laurens A, Cortes D. Acetogenins from Annona-ceous. In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm Ch, editors.Progress in the chemistry of Organic Natural Products: Vol.70 Wien:Springer-Verlag, 1997: p 81-2882 Zaft'a-Polo MC, Figadere B, Gallardo T, Tormo JR, Cortes D. Natural acet-ogenins from Annonaceae, synthesis and mechanisms of action. Phy-tochemistry 1998: 48: 1087 -117) Woo MH, Kim DH, Fotopoulos SS, McLaughlin JL. Annocherin and(2,4)-ci5- and trans-annocherinones, monotetrahydrofuran annonac-eous acetogenins with a C-7 carbonyl group from Annona cherimoliaseeds. J Nat Prod 1999; 62: 1250 - 54 Kim DH, Ma ES, Suk KD, SonJK, LeeJS, Woo M-H, 2001. Annomolin andannocherimolin, new cytotoxic annonaceous acetogenins fromAnnona cherimolia seeds. J Nat Prod 1996; 64: 502 - 5065 Degli Esposti M, Ghelli A, Ratta M, Cortes D, Estornell E. Natural sub-stances (acetogenins) from the family Annonaceae are powerful inhi-bitors of mitochondrial NADH dehydrogenase (complex I). Biochem J1994; 301: 161 - 7G Tormo JR, Estornell E, Gallardo T, Gonzalez MC, Cave Ad, Granell S,Cortes D, Zafra-Polo Me. y-Lactone functionalized antitumoral aceto-genins are the most potent inhibitors of mitochondrial complex I.Bioorg Med Chem Lett 2001; 11: 681 - 47 Cortes D, Myint SH, Leboeuf M, Cave A. A new type of cytotoxic aceto-genins: the tetrahydrofuranic IJ-hydroxy methyl y-lacrones. Tetrahe-dron Lett 1991 ; 32: 6133-48 Cortes D, Myint SH, Dupont B, Davoust D. Bioactive acetogenins fromseeds of Annona cherimo/ia . Phytochemistry 1993; 32: 1475 - 829 Sibi M, Lu J, Talbacka CL. Enantiospecific synthesis of trisubstitutedbutyrolactone natural products and their analogs. J Org Chem 1996;61;7848-5510 Cortes D, Figadere B, Cave A. Bis-tetrahydrofuran acetogenins fromAnnonaceae. Phytochemistry 1993: 32: 1467 - 73II Fato R, Estornell E, Di Bernardo S, Pallotti F, Parenti-Castelli G, Lenaz G.Steady-state kinetics of the reduction of coenzyme Qanalogs by com-plex I (NADH:ubiquinone oxidoreductase) in bovine heart mitochon-dria and submitochondrial particles. Biochemistry 1996; 35: 2705 -1612 Gallardo T, Zafra-Polo MC, Tormo JR. Gonzalez MC, Franck X. EstornellE, Cortes D. Semisynthesis of antitumoral acerogenins: SAR of functio-nalized alkyl-chain bis-terrahydrofuranic acerogenins, specific inhibi-tors of mitochondrial complex I. J Med Chern 2000; 43: 4793 - 800

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Tucumanin, a β-hydroxy-γ-lactone bistetrahydrofuranic acetogenin from Annona cherimolia, is a potent inhibitor of mitochondrial complex I

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