A New Adjacent Bis-Tetrahydrofuran Annonaceous Acetogenin from the Seeds of Uvaria chamae

• Abstract
• Materials and Methods
• References

Abstract

A novel acetogenin, joolanin (1), along with eight known acetogenins, squamocin (2), desacetyluvaricin (3), chamuvarinin (4), tripoxyrollin (5), diepoxyrollin (6), dieporeticanin-1 (7), dieporeticanin-2 (8) and dieporeticenin (9) were isolated from the seeds of Uvaria chamae (Annonaceae). Compound 1, the first adjacent bis-tetrahydrofuranic ring acetogenin bearing a ketone group at C-5, shows significant cytotoxicity towards the KB 3 - 1 cell line (IC₅₀ = 0.4 nM).

Annonaceous acetogenins are a unique class of polyketides (C₃₅ or C₃₇), with an aliphatic chain bearing 0 to 3 tetrahydrofuran (THF) rings and a terminal γ-lactone [1]. Because of their mitochondrial targeting [2], combined with potent inhibition of complex I of the respiratory chain, they show insecticidal, antiparasitic and cytotoxic activities, and have been regarded as antitumoral candidates [1]. They are suspected to be alimentary toxins causal of atypical parkinsonism [3], [4]. In our previous investigation, twelve mono- and bis-THF acetogenins were isolated from the roots of Uvaria chamae [5], [6]. We describe here the isolation and structural elucidation of a new acetogenin, joolanin (1), along with squamocin (2) [7], desacetyluvaricin (3) [8], chamuvarinin (4) [6], tripoxyrollin (5) [9], diepoxyrollin (6), dieporeticanin-1 (7), dieporeticanin-2 (8) [10], [11] and dieporeticenin (9) [11] from a cyclohexanic extract of the seeds of this species (Fig. [1]).

The CI-MS of joolanin (1) suggested the molecular formula C₃₇H₆₄O₇ ([M + H⁺], m/z = 621), confirmed by HR-ESI-MS ([M + Na]⁺, m/z = 643.4542; calcd.: 643.4550). The strong IR C = O absorption at 1756 cm^-1 and ¹H- and ¹³C-NMR signals indicated the presence of a γ-methyl-α,β-unsaturated γ-lactone [5], [6]. The absolute configuration of the C-36 stereocenter was established as S by Latypov’s method (see Supporting Information) [12]. The presence of an adjacent bis-THF ring system with two flanking hydroxy groups was evidenced by ¹H- and ¹³C-NMR [7]. Its location between C-15 and C-24 was indicated by EI-MS (Fig. [2]), and a careful comparison of ¹H-NMR data with those of model compounds [14] indicated its relative stereochemistry to be threo/trans/threo/trans/erythro or erythro/trans/threo/trans/threo, from C-15 to C-24. The IR spectrum showed an additional prominent absorption at 1715 cm^-1 assignable to a ketone group, as confirmed by the presence of a carbonyl resonance at δ = 209.3, and by the three ¹H-NMR signals at δ = 2.38 (t, J = 7.3 Hz, H-6), 2.55 (dt, J = 7.0, 14.0 Hz, H-3) and 2.70 (dt, J = 14.0 and 7.0 Hz, H-4). The EI-MS ion at m/z = 153 allowed its positioning at C-5 (Fig. [2]), in agreement with HMBC data (see Supporting Information) [14]. Only few acetogenins bearing a ketone group at C-5 have been described so far [14], [15], [16]. Joolanin (1) is the first bis-tetrahydrofuranic adjacent ring acetogenin presenting this structural feature.

The cytotoxicities of 1 to 9 were evaluated in KB 3 - 1 cells (Table [1]). Acetogenins without THF (5, 6, 7, 8 and 9) are much less active than the THF-bearing acetogenins (1, 2, 3 and 4), as previously described [1]. Squamocin (2) is more potent than joolanin (1), desacetyluvaricin (3) and chamuvarinin (4), possibly because of optimal amphiphilicity for both mitochondrial distribution and enzyme inhibition. The presence of a carbonyl group between the lactone and THF rings in 1 appears to be detrimental for activity.

Materials and Methods

General experimental procedures: UV spectra were determined in MeOH on a Philips PU 8720 spectrophotometer. IR spectra were recorded on a Bruker Vector 22 spectrophotometer. The ¹H-NMR spectra were obtained with a Bruker AC-400 (at 400 MHz). The ¹³C-NMR spectra were obtained with a Bruker AC-200 at 50 MHz. EI-MS (70 eV) were recorded with an Automass multi spectrometer R10 - 10C, and HR-ESI-MS with a navigator spectrometer (Thermofinnigan; Courtaboeuf, France). Reversed-phase semipreparative HPLC procedures were completed with a Waters SSV injector, a Waters 590 pump, and a Waters 84 UV detector (λ = 214 nm), on a μBondapak C₁₈ 10 μm (250 × 20 mm) cartridge column, with a flow rate of 9 mL·mn^-1, and 20 mg/injection. For TLC, silica gel (Merck 60F254) and Kedde reagent were used.

Plant material: Seeds of Uvaria chamae P. Beauv. were collected in Casamance (Senegal) in August 1999. A voucher specimen (DF 127) has been deposited at the Faculty of Medicine and Pharmacy of Dakar.

Extraction and isolation: Extract and fractions were evaporated to dryness before CC and HPLC. Extraction of the dried and powdered seeds (900 g) by percolation (C₆H₁₂, 8 L, 48 h) gave a brown residue (126 g), 45 g of which were chromatographed [SiO₂ Merck (Fontenay-sous-Bois, France) 70 - 230 mesh, C₆H₁₂/EtOAc, 90 : 10 to 10 : 90] to yield 250 fractions of 250 mL each, combined to ten fractions on the basis of TLC. Fraction 8 (355 mg, TLC, SiO₂, C₆H₁₂/EtOAc, 50 : 50, Kedde reagent, R_f = 0.55) underwent CC (SiO₂ Merck 60 H 230 - 400 mesh, C₆H₁₂/CH₂Cl₂/i-PrOH, 30 : 60 : 10, 250 mL, 5 mL per fraction). Subsequent fractions 17 to 23 were chromatographed (SiO₂ as previously, C₆H₁₂/AcOEt/MeOH, 60 : 25 : 17, 200 mL, 5 mL per fraction). Subsequent fractions 21 to 29 underwent exclusion chromatography [Sephadex LH-20 Pharmacia (Uppsala, Sweden), CH₂Cl₂/MeOH, 20 : 10, 500 mL, 10 mL per fraction]. A final purification of subsequent fractions 10 to 19 by RP-HPLC [μBondapak C₁₈ 10 μm (250 × 20 mm) cartridge column, flow rate 9 mL·min^-1, 20 mg/injection, MeOH/H₂O/THF, 87 : 13 : 5] led to 21 mg of 1 (t_R = 11.5 min). Acetogenins 2 to 9 were purified similarly.

Joolanin (1): White waxy solid (21 mg); [α]_D ²⁰: + 18° (c 0.5, CHCl₃); IR (film): ν_max = 3450, 2925, 2854, 1756, 1715, 1463, 1375, 1319, 1198, 1066, 953, 874, 722 cm^-1; UV (MeOH): λ_max (log ε) = 227.6 nm (0.95); HR-ESI-MS: m/z = 643.4542, calcd. for [M + Na]⁺ [C₃₇H₆₄O₇ + Na]⁺: 643.4550; EI-MS (40 eV): m/z = 97, 111, 153, 181, 223, 241, 291, 309, 311, 361, 379, 431, 449; ¹H-NMR (CDCl₃, 400 MHz): δ = 0.86 (t, J = 6.3 Hz, CH₃ H-34), 1.22 - 1.24 (H-8 - -13, H-26 - -33), 1.33 (m, H-25), 1.35 (m, H-14), 1.37 (d, J = 6.8 Hz, CH₃ H-37), 1.52 (m, H-7), 1.57^† (H-21_a, 22_a), 1.60^† (H-17_a, 18_a), 1.89* (H-21_b, 22_b), 1.93* (H-17_b, 18_b), 2.38 (t, J = 7.3 Hz, H-6), 2.55 (dt, J = 7.0, 14.0 Hz, H-3), 2.70 (dt, J = 7.0, 14.0 Hz, H-4), 3.37 (m, H-15), 3.83 (m, H-16, 23), 3.88 (m, H-24), 3.92 (m, H-19, 20), 4.96 (dq, J = 6.8, 1.6 Hz, H-36), 7.03 (d, J = 1.6 Hz, H-35); ¹³C NMR (CDCl₃, 50 MHz): δ = 13.9 (CH₃, C-34), 18.9 (CH₃, C-37), 19.3 (C-3), 22.5 (C-33), 23.6 (C-7), 24.4 - 29.2 (C-17, 18, 21, 22), 25.5 - 33.2 (C-8-13, 14, 25, 26-32), 39.6 (C-4), 42.8 (C-6), 71.2 (C-24), 74.0 (C-15), 77.5 (C-36), 82.1^‡ (C-20), 82.3^‡ (C-19), 82.7^‡ (C-23), 83.1^‡ (C-16), 132.5 (C-2), 150.3 (C-35), 173.0 (C-1), 209.3 (C-5); (*,^†,^‡ interchangeable).

Copies of the original spectra are obtainable from the author of correspondence.

Cytotoxic assay: Cytotoxicities [KB 3 - 1 cells (Prof. P. Couvreur, Châtenay-Malabry, France), 72 h] were evaluated as previously described [17].

References

• 1 Bermejo A, Figadère B, Zafra-Polo M -C, Barrachina I, Estornell E, Cortes D. Acetogenins from Annonaceae: Recent progress in isolation, synthesis and mechanisms of action.  Nat Prod Rep. 2005;  22 269-303
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 16 Sinz A, Matusch R, Kämpchen T, Fiedler W, Schmidt J, Santisuk T. et al . Novel acetogenins from the leaves of Dasymaschalon sootepense .  Helv Chim Acta. 1998;  81 1608-15
  CrossrefPubMedSearch in Google Scholar
• 17 Fleury C, Cotte J, Quéro A -M. Evaluation de la cytotoxicité d’un antiseptique par une microméthode photométrique.  Path Biol. 1984;  32 628-30
  PubMedSearch in Google Scholar

Docteur Pierre Champy

Laboratoire de Pharmacognosie

UMR CNRS 8076 (BioCIS)

Faculté de Pharmacie

Université Paris Sud 11

92296 Châtenay-Malabry

France

Fax: +33 1-46 83 53 99

Email: pierre.champy@cep.u-psud.fr

References

• 1 Bermejo A, Figadère B, Zafra-Polo M -C, Barrachina I, Estornell E, Cortes D. Acetogenins from Annonaceae: Recent progress in isolation, synthesis and mechanisms of action.  Nat Prod Rep. 2005;  22 269-303
  CrossrefPubMedSearch in Google Scholar
• 2 Derbré S, Roué G, Poupon E, Susin S A, Hocquemiller R. Annonaceous acetogenins: The hydroxyl groups and THF rings are crucial structural elements for targeting the mitochondria, demonstration with the synthesis of fluorescent squamocin analogues.  Chem Biol Chem. 2005;  6 1-4
  PubMedSearch in Google Scholar
• 3 Champy P, Höglinger G U, Féger J, Gleye C, Hocquemiller R, Laurens A. et al . Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: Possible relevance for atypical parkinsonism in Guadeloupe.  J Neurochem. 2004;  88 63-9
  PubMedSearch in Google Scholar
• 4 Champy P, Melot A, Guérineau V, Gleye C, Fall D, Höglinger G U. et al . Quantification of acetogenins in Annona muricata linked to atypical parkinsonism in Guadeloupe.  Mov Disord. 2005;  20 1629-33
  CrossrefPubMedSearch in Google Scholar
• 5 Fall D, Gleye C, Franck C, Laurens A, Hocquemiller R. cis-Bullatencin, a linear acetogenin from roots of Uvaria chamae .  Nat Prod Lett. 2002;  16 315-21
  PubMedSearch in Google Scholar
• 6 Fall D, Duval R A, Gleye C, Laurens A, Hocquemiller R. Chamuvarinin, an acetogenin bearing a tetrahydropyran ring from the roots of Uvaria chamae .  J Nat Prod. 2004;  67 1041-3
  CrossrefPubMedSearch in Google Scholar
• 7 Fujimoto Y, Eguchi T, Kakinuma K, Ikekawa N, Sahai M, Gupta Y K. Squamocin, a new cytotoxic bis-tetrahydrofuran containing acetogenin from Annona squamosa .  Chem Pharm Bull. 1988;  36 4802-6
  PubMedSearch in Google Scholar
• 8 Jolad S D, Hoffmann J J, Cole J R, Barry C E, Bates R B, Linz G S. et al . Desacetyluvaricin from Uvaria acuminata, configuration of uvaricin at C-36.  J Nat Prod. 1985;  48 644-5
  PubMedSearch in Google Scholar
• 9 Sahpaz S, Figadère B, Saez J, Hocquemiller R, Cavé A, Cortes D. Tripoxyrollin, a new epoxyacetogenin from the seeds of Rollinia membranacea .  Nat Prod Lett. 1993;  2 301-8
  PubMedSearch in Google Scholar
• 10 Sahpaz S, Hocquemiller R, Cavé A, Saez J, Cortes D. Diepoxyrollin and diepomuricanin B, two new diepoxyacetogenins from Rollinia membranacea seeds.  J Nat Prod. 1997;  60 199-201
  CrossrefPubMedSearch in Google Scholar
• 11 Vu Thi T am, Phan Quan Chi H ieu, Chappe B, Roblot F, Laprévote O, Figadère B. et al . Four new acetogenins from the seeds of Annona reticulata .  Nat Prod Lett. 1994;  4 255-62
  PubMedSearch in Google Scholar
• 12 Latypov S, Franck X, Jullian J -C, Hocquemiller R, Figadère B. NMR determination of absolute configuration of butenolides of annonaceous type.  Chem Eur J. 2002;  8 5662-6
  PubMedSearch in Google Scholar
• 13 Fujimoto Y, Murasaki C, Shimada H, Nishioka S, Kakinuma K, Singh S. et al . Annonaceous acetogenins from the seeds of Annona squamosa. Non-adjacent bis-tetrahydrofuranic acetogenins.  Chem Pharm Bull. 1994;  42 1175-84
  PubMedSearch in Google Scholar
• 14 Chen Y, Chen Y R, Yu D Q. Six acetogenins from Uvaria tonkinesis .  Phytochemistry. 1996;  43 793-801
  CrossrefPubMedSearch in Google Scholar
• 15 Qin Y, Pan X, Chen R, Yu D. New annonaceous acetogenins from Uvaria boniana .  Yao Xue Xue Bao (Acta Pharm Sin). 1996;  31 381-6
  PubMedSearch in Google Scholar
• 16 Sinz A, Matusch R, Kämpchen T, Fiedler W, Schmidt J, Santisuk T. et al . Novel acetogenins from the leaves of Dasymaschalon sootepense .  Helv Chim Acta. 1998;  81 1608-15
  CrossrefPubMedSearch in Google Scholar
• 17 Fleury C, Cotte J, Quéro A -M. Evaluation de la cytotoxicité d’un antiseptique par une microméthode photométrique.  Path Biol. 1984;  32 628-30
  PubMedSearch in Google Scholar

Docteur Pierre Champy

Laboratoire de Pharmacognosie

UMR CNRS 8076 (BioCIS)

Faculté de Pharmacie

Université Paris Sud 11

92296 Châtenay-Malabry

France

Fax: +33 1-46 83 53 99

Email: pierre.champy@cep.u-psud.fr

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