Article pubs.acs.org/jnp
Chenopodolin: A Phytotoxic Unrearranged ent-Pimaradiene Diterpene Produced by Phoma chenopodicola, a Fungal Pathogen for Chenopodium album Biocontrol Alessio Cimmino,† Anna Andolfi,† Maria C. Zonno,‡ Fabiana Avolio,† Antonello Santini,§ Angela Tuzi,† Alexander Berestetskyi,⊥ Maurizio Vurro,‡ and Antonio Evidente*,† †
Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte. S. Angelo, Via Cintia 4, 80126 Napoli, Italy ‡ Istituto di Scienze delle Produzioni Alimentari del CNR, Via Amendola 122/O, 70125 Bari, Italy § Dipartimento di Farmacia, Università di Napoli Federico II, Via D. Montesano 49, 80131, Napoli, Italy ⊥ All-Russian Institute of Plant Protection, Russian Academy of Agricultural Sciences, Pushkin, Saint-Petersburg 196608, Russian Federation S Supporting Information *
ABSTRACT: A new phytotoxic unrearranged ent-pimaradiene diterpene, named chenopodolin, was isolated from the liquid culture of Phoma chenopodicola, a fungal pathogen proposed for the biological control of Chenopodium album, a common worldwide weed of arable crops such as sugar beet and maize. The structure of chenopodolin was established by spectroscopic, X-ray, and chemical methods as (1S,2S,3S,4S,5S,9R,10S,12S,13S)-1,12-acetoxy-2,3-hydroxy-6-oxopimara-7(8),15-dien-18-oic acid 2,18-lactone. At a concentration of 2 mg/mL, the toxin caused necrotic lesions on Mercurialis annua, Cirsium arvense, and Setaria viride. Five derivatives were prepared by chemical modification of chenopodolin functionalities, and some structure−activity relationships are discussed.
■
RESULTS AND DISCUSSION An organic extract of P. chenopodicola, having a high phytotoxic activity as described below, was purified by column and TLC chromatography as described in the Experimental Section, and the main metabolite was obtained as a homogeneous solid, which crystallized by slow evaporation from a MeOH−H2O (1:3) solution. As it proved to be a new naturally occurring unrearranged pimarane diterpene, it was named chenopodolin (1, Figure 1; IUPAC: 1,3-(epoxymethano)acetic acid 6-acetoxy2-hydroxy-1,4a,7-trimethyl-10,12-dione-7-vinyl1,2,3,4,4a,4b,5,6,7,8,10,10a-dodecahydrophenanthren-4-yl ester). 1 has a molecular formula of C24H30O8 deduced from its HRESIMS, with 10 hydrogen deficiencies, four of which were associated with one ketone, two acetyl, and one ester carbonyl group, and from monosubstituted and trisubstituted double bonds, as deduced from the preliminary inspection of the 1H and 13C NMR spectra (Table 1), and in agreement with the typical bands and absorption maxima observed in the IR8 and UV spectra. The latter also showed the presence of a conjugated carbonyl group.9 The other four unsaturations were due to the presence of four rings. These findings and the presence of the typical vinyl and its geminal methyl group, appearing in the 1H and 13C NMR spectra (Table 1) as a
Chenopodium album L., also known as common lambsquarters or fat hen, is a worldwide weed of arable crops such as sugar beet and maize.1 Considering the difficulties in managing this weed, of several control methods studied, biological control using pathogens was considered a suitable option. The first mycoherbicide proposed for the control of C. album was Ascochyta caulina (P. Karst.) v.d. Aa and v. Kest.2,3 Successive studies resulted in the production, purification, and chemical and biological characterization of the three main phytotoxins with interesting herbicidal properties, namely, ascaulitoxin, its aglycone, and trans-4-aminoproline.4−7 More recently, another pathogenic Sphaeropsidales, Phoma chenopodicola, was proposed as a potential mycoherbicide for the control of the same weed. Considering that leaf and stem pathogenic Sphaeropsidales are well known as toxin producers, it was considered to be worthwhile studying the production of toxic metabolites by P. chenopodicola. This article reports on (a) the isolation and the chemical characterization of the main phytotoxin produced by P. chenopodicola, named chenopodolin; (b) the preliminary studies of the biological properties of chenopodolin in comparison with its five derivatives in order to evaluate their potential to be developed as a natural and safe herbicide; and (c) the relative configuration by X-ray diffractometric analysis and the absolute configuration by applying a modified Mosher’s method. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 19, 2013
A
dx.doi.org/10.1021/np400218z | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 1. 1H and 13C NMR Data of Chenopodolin (1)a position 1 2 3 4 5 6 7 8 9
double doublet (J = 17.8 and 11.2 Hz), two doublets (J = 11.2 Hz and J = 17.8 Hz), and a singlet at δ 5.87 (H-15), 5.20 (H16), 5.05 (H-16A), and 1.07 (Me-17) and at δ 139.5 (C-15), 116.7 (C-16), and 20.7 (C-17), respectively,10,11 suggested an ent-pimaradiene diterpene. Structurally it is related to sphaeropsidin A, a phytotoxin isolated from Sphaeropsis sapinea f. sp. cupressi, the causal agent of cypress canker,12 and from Diplodia mutila from oaks.13 In fact, both the vinyl and the Me17 groups were located on the same quaternary C-13 (δ 41.5) on the basis of the couplings observed in the HMBC14 spectrum (Table 1), between C-13 and Me-17; C-15 and H-12, H-16B, H2-14, and Me-17; and C-17 and H-12, H2-14, H-15, and H2-16. Furthermore, the same spectra showed the signals of HC-5 at δ 2.84 (singlet)/58.3 as observed in sphaeropsidin A and related analogues.12,15 On the basis of its couplings observed in the HMBC spectrum with H-3, H-7, H-9, and Me19, C-5 represents one of the bridgehead carbons of the junction between the A and B rings of the phenanthrene system. The other is the quaternary C-10 (δ 42.1), as shown by its couplings with H-1, H-5, and Me-20. The couplings observed in the same spectrum from C-20 to H-1 and H-9 located Me-20 at C-10. In comparing the 1H and 13C NMR spectra of chenopodolin and sphaeropsidin A,12 noteworthy differences were observed for the perhydrophenanthrene carbon skeleton and the location of the lactone ring. The latter was located in 1 between C-2 and the carboxylated C-18 on the basis of the couplings observed in the HMBC spectrum (Table 1) from C-18 to H-2, H-3, H-5, and Me-19 and from C-2 to H-1 and H-3. C-18 together with the Me-19 is bonded to the quaternary C-4 at δ 46.7, as shown by the coupling observed in the HMBC spectrum between C-4 and Me-19, and represents the head of the C-20 diterpenoid
72.8 79.0 77.0 46.7 58.3 194.5 126.5 155.6 46.0
CH CH CH C CH C CH C CH
10 11 12
42.1 C 12.0 CH2 77.1 CH
13 14
41.5 C 43.5 CH2
15
139.5 CH
16
116.7 CH2
17
20.7 CH3
18 19 20 MeCO-12 MeCO-1 MeCO-1 MeCO-12
Figure 1. Structures of chenopodolin (1); its 3-O-p-iodobenzoyl, 3-OS-MTPA, 3-O-R-MTPA, and 3-O-acetyl esters (2−5, respectively); and its 1-O-deacetyl-, 6,O,15,16-tetrahydro-, and 6,O,7,8,15,16hexahydro derivatives (6−8, respectively).
δCb
175.9 17.7 21.1 170.7 169.5 26.0 26.2
C CH3 CH3 C C CH3 CH3
δH (J in Hz)
HMBC
5.10 (1H) d (4.9) 4.58 (1H) br d (4.9) 4.14 (1H) br s
H-9, H-5, Me-20 H-1, H-3 Me-19 Me-19, H-5, H-3 H-3, H-7, H-9, Me-19 H-5, H-14B H-9, H2-14 H-9, H2-11, H2-14 H-7, H2-11, H2-14, Me20 H-1, H-5, Me-20 H-1 H-9, H2-11, H2-14, Me17 Me-17, H2-11, H2-14
2.84 (1H) s 5.86 (1H) s 2.95 (1H) m
1.46 (2H) m 4.67 (1H) dd (10.2, 4.0) 2.72 (1H) 2.26 (1H) 5.87 (1H) 11.2) 5.20 (1H) 5.05 (1H) 1.07 (3H)
d (16.0) d (16.0) dd (17.8, d (11.2) d (17.8) s
1.54 (3H) s 1.11 (3H) s
H-12, H2-14, H-16B, Me-17
H-12, H-15, H2-16, H214 H-2, H-3, H-5, Me-19 H-5 H-1, H-9 H-12, MeCO-12 H-1, MeCO-1
2.26 (3H) s 2.08 (3H) s
a The chemical shifts are in δ values (ppm) from TMS. 2D 1H,1H (COSY) and 13C,1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons. b Multiplicities were assigned by DEPT spectrum.
precursor. The protons and carbons involved in the lactone ring resonated at the typical chemical shift values of δ 4.58/79.0 (HC-2) and 175.9 (C-18), while those of Me-19 are at δ 1.54/ 17.7.10,11 H-2, a broad doublet (J = 4.9), in the COSY spectrum14 coupled with the proton (H-1) of another adjacent secondary acetylated carbon (C-1) resonating as a doublet (J = 4.9 Hz) at δ 5.10. The last carbon of the A ring, which was assigned on the basis of the couplings observed in the HMBC spectrum, was the secondary hydroxylated C-3 resonating as a broad singlet at δ 4.14. The α,β-unsaturated ketone group system, with NMR signals appearing at δ 194.5 (C-6), 5.86/ 126.5 (singlet, C-7), and 155.6 (C-8), was located between C-5 and C-9 on the basis of the couplings observed in the HMBC spectrum of C-6 with H-5 and H-14B, C-7 with H-9 and H2-14, and C-8 with H-9, H2-11, and H2-14, respectively. C-8 and HC9, which resonated at δ 2.95 (multiplet)/46.0, appeared to be the two bridgehead carbons of the B/C ring junction. In the COSY spectrum the signal for H-9 coupled with the protons of an adjacent methylene group (H2C-11), which resonated as a multiplet at δ 1.46. The latter, in turn, coupled with the proton (H-12) of the residual secondary hydroxylated carbon, which, being acetylated, appeared as a double doublet (J = 10.2, 4.0 Hz) at δ 4.67. The residual methylene group (H2C-14) resonated as two doublets (J = 16.0 Hz) of a typical AB system at δ 2.26 and 2.72.10 The latter on the basis of the HMBC couplings above represents the closure carbon of the C ring. B
dx.doi.org/10.1021/np400218z | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
The couplings observed in the HSQC spectrum14 confirmed the conclusion above for the protonated carbons and permitted the assignment for the signals at δ 72.8, 77.0, 77.1, 43.5, and 12.0 to C-1, C-3, C-12, C-14, and C-11, respectively. The two acetyl groups observed at δ 170.7, 2.08 (singlet)/26.2 and 169.5, 2.26 (singlet)/26.0 were located at C-12 and C-1, respectively, on the basis of the HMBC couplings of their carbonyl groups with the protons of the corresponding carbons. All these results allowed the confirmation of the location of the functional groups and ring elements, and chenopodolin (1) could be formulated as 1,12-acetoxy-2,3-hydroxy-6-oxo-entpimara-7(8),15-dien-18-oic acid 2,18-lactone. This structure was supported by several other long-range couplings observed in the HMBC spectrum (Table 1) and by the HRESIMS data. Indeed, the latter spectrum showed, besides the sodiated dimeric form [2M + Na]+ at m/z 915, the potassium [M + K]+ and the sodium [M + Na]+ clusters at m/z 485 and 469.1867, respectively. In the same spectrum, recorded in the negative ion mode, the pseudomolecular ion [M − H]− at m/z 445 was observed. The relative configuration of 1 was deduced from the correlations recorded in the NOESY spectrum14 and reported in Table 2. In particular, the correlations observed between H-1
Figure 2. ORTEP view of chenopodolin (1) showing atomic labeling. Displacement ellipsoids are drawn at the 30% probability level.
forms a five-membered ring (D in Figure 2). The saturated rings A and C adopt a chair conformation, while ring B, which contains a double bond, is in a twisted conformation with C5 and C10 pointing up and down from the plane formed by C6/ C7/C8/C9. Ring A is trans fused with the B cyclohexene ring through C5 and C10. These conformational characteristics resulted in an overall molecular flat shape. The five-membered D ring is in the envelope conformation, with the C3 atom flipped away from the C2/O3/C18/C4 plane. Nine stereogenic centers are present in the molecule, at the C1, C2, C3, C4, C5, C9, C10, C12, and C13 atoms, respectively, whose relative configurations are S*, S*, S*, S*, S*, R*, S*, S*, and S*. The absolute configuration assignment from X-ray data was not possible because of the weak anomalous scattering. The two acetyl groups at the C1 (ring A) and C12 (ring C) carbon atoms are in the axial and equatorial orientation, respectively. The ethylene moiety at C13 (ring C) is in the axial orientation. The crystal packing is characterized by strong intermolecular head to tail hydrogen bonding involving the hydroxyl H atom and carbonyl oxygen atom of the γ-lactone moiety [O1H···O4i 0.97, 2.723(6) Å, 2σ(I). Minimum and maximum residual electronic density was −0.238 and 0.257 e Å−3. Crystal data: formula C24H30O8, formula weight 446.48 g mol−1, orthorhombic P212121, a = 9.629(2) Å, b = 10.0340(8) Å, c = 24.848(5) Å, α = β = γ = 90°, 11 502 collected reflections, 2411 unique reflections. Phytotoxic Activity. The culture filtrate, the fractions obtained by the extraction and purification processes described above, and chenopodolin and derivatives were assayed by the leaf puncture assay, as already described.32 Pure metabolites were assayed on detached leaves of Mercurialis annua, Cirsium arvense, and Setaria viride (two dicot and one monocot plant species, respectively) by applying a droplet (10 μL) of the main metabolite or its derivatives (previously dissolved in methanol) at a final concentration of 20 μg/droplet. Droplets of the same solutions were injected into the leaves of the same species below the epidermis by using modified medical pliers for hypodermal injections, having two rubber plugs to gently hold the leaf, one of them bearing the tip of a syringe needle, which allows solution injection into the leaf mesophyll. After injection, plants were kept in the greenhouse under natural light conditions for 5−7 days, with daily checks for symptom appearance.32 Solutions of methanol without any metabolite were applied as described above and used as negative control. Antimicrobial Activity. The antimicrobial activity was tested against three microorganisms by using an agar diffusion assay according to the protocol already described.33 The antifungal activity was tested on Geotrichum candidum grown on PDA, whereas the antibacterial activity was assayed against Bacillus subtilis (a Grampositive bacterium) grown on TGYA (tryptic glucose yeast agar, Biolife) and Escherichia coli (a Gram-negative bacterium), grown on F
dx.doi.org/10.1021/np400218z | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
LB agar (Sigma). Up to 100 μg of each metabolite was applied per disc. Three replications were performed for each compound. One day after the application, the eventual presence of an inhibition halo of the microbial growth was visually assessed. Droplets of methanol without the metabolites were applied as described above and used as negative control.
■
(12) Evidente, A.; Sparapano, L.; Motta, A.; Giordano, F.; Fierro, O.; Frisullo, S. Phytochemistry 1996, 42, 1541−1546. (13) Evidente, A.; Sparapano, L.; Fierro, O.; Bruno, G.; Motta, A. Phytochemistry 1997, 45, 705−713. (14) Berger, S.; Braun, S. 200 and More Basic NMR Experiments: a Practical Course, 1st ed.; Wiley-VCH: Weinheim, 2004. (15) Sparapano, L.; Bruno, G.; Fierro, O.; Evidente, A. Phytochemistry 2004, 65, 189−198. (16) Ohtani, I.; Kusumi, T.; Ishitsuka, M. O.; Kakisawa, H. Tetrahedron Lett. 1989, 30, 3147−3150. (17) Lallemand, B.; Masi, M.; Maddau, L.; De Lorenzi, M.; Dam, R.; Cimmino, A.; Moreno y Banuls, L.; Andolfi, A.; Kiss, R.; Mathieu, V.; Evidente, A. Phytochem. Lett. 2012, 5, 770−775. (18) Evidente, A.; Venturi, V.; Masi, M.; Degrassi, G.; Cimmino, A.; Maddau, L.; Andolfi, A. J. Nat. Prod. 2011, 74, 2520−2525. (19) Wang, X. N.; Bashyal, B. P.; Wijeratne, E. M. K.; U’Ren, J. M.; Liu, M. X.; Gunatilaka, M. K.; Arnold, A. E.; Gunatilaka, A. A. L. J. Nat. Prod. 2011, 74, 2052−2061. (20) Evidente, A.; Sparapano, L.; Andolfi, A.; Bruno, G.; Motta, A. Aust. J. Chem. 2003, 56, 615−619. (21) Evidente, A.; Sparapano, L.; Bruno, G.; Motta, A. Phytochemistry 2002, 59, 817−823. (22) Sekido, H.; Akatsuka, T. Agric. Biol. Chem. 1987, 51, 1967− 1971. (23) Akatsuka, T.; Kodama, O.; Sekido, H.; Kono, Y.; Takeuchi, S. Agric. Biol. Chem. 1985, 49, 1689−1694. (24) Kono, Y.; Takeuchi, S.; Kodama, O.; Akatsuka, T. Agric. Biol. Chem. 1984, 48, 253−255. (25) Akatsuka, T.; Kodama, O.; Kato, H.; Kono, Y.; Takeuchi, S. Agric. Biol. Chem. 1983, 47, 445−447. (26) Cartwright, D.; Langcage, P.; Pryce, D. R. J.; Leworthy, P. Nature 1977, 267, 511−513. (27) Kato, T.; Kabuto, T.; Sasaki, N.; Tsunagwa, M.; Aizawa, H.; Fujita, K.; Kato, Y.; Kitahara, Y.; Takahashi, N. Tetrahedron Lett. 1973, 39, 3861−3864. (28) Kato, T.; Aizawa, H.; Tsunakawa, M.; Sasaki, N.; Kitahara, Y.; Takahashi, N. J. Chem. Soc., Perkin Trans. 1 1977, 250−254. (29) Pinkerton, F.; Strobel, G. A. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 4007−4011. (30) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 1999, 32, 115−119. (31) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122. (32) Evidente, A.; Andolfi, A.; Vurro, M.; Fracchiolla, M.; Zonno, M. C.; Motta, A. Phytochemistry 2005, 66, 715−721. (33) Bottalico, A.; Capasso, R.; Evidente, A.; Randazzo, G.; Vurro, M. Phytochemistry 1990, 29, 93−96.
ASSOCIATED CONTENT
S Supporting Information *
Spectra of 1, cif data file, and Table SI-1 are available free of charge via the Internet at http://pubs.acs.org. Crystallographic data for the structure 1 have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 908251. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/ retrieving.html or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (internat.) +44-1223/336-033.
■
AUTHOR INFORMATION
Corresponding Author
*(A.E.) Tel: +39 081 2539178. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The NMR spectra were recorded in the laboratory of the CERMANU Centre, Università di Napoli Federico II, Portici, Italy, by P. Mazzei, whose contribution is gratefully acknowledged. Authors are also grateful to “Centro Regionale di Competenza Nuove Tecnologie per le Attività Produttive” (CRdC-NTAP) of the Campania Governorate and to “Centro Interdipartimentale di Metodologie Chimico Fisiche” (CIMCF) of the Università di Napoli Federico II, for X-ray facilities. A.E. is associated with “Istituto di Chimica Biomolecolare del CNR”, Pozzuoli, Italy.
■
REFERENCES
(1) Holm, L. G.; Pluckett, D. L.; Pancho, J. V.; Herberger, J. P. The World’s Worst Weeds (Distribution and Biology);University Press of Hawaii: Honoloulou, HI, 1977; pp 84−91. (2) Kempenaar, C. Studies on the Biological Control of Chenopodium album by Ascochyta caulina. Doctoral Thesis, University of Wageningen, The Netherlands, 1995. (3) Kempenaar, C.; Horsten, P. J. F. M.; Scheepens, P. C. Eur. J. Plant Pathol. 1996, 102, 143−153. (4) Evidente, A.; Capasso, R.; Cutignano, A.; Taglialatela-Scafati, O.; Vurro, M.; Zonno, M. C.; Motta, A. Phytochemistry 1998, 48, 1131− 1137. (5) Evidente, A.; Andolfi, A.; Vurro, M.; Zonno, M. C. Phytochem. Anal. 2001, 12, 383−387. (6) Evidente, A.; Andolfi, A.; Vurro, M.; Zonno, M. C.; Motta, A. Phytochemistry 2000, 53, 231−237. (7) Bassarello, C.; Bifulco, G.; Evidente, A.; Riccio, R.; GomezPaloma, L. Tetrahedron Lett. 2001, 42, 8611−8613. (8) Nakanishi, K.; Solomon, P. H. Infrared Absorption Spectroscopy, 2nd ed.; Holden Day: Oakland, CA, 1977; pp 17−44. (9) Scott, A. I. Interpretation of Ultraviolet Spectra of Natural Products; Pergamon Press LTD: Oxford, 1964; pp 45−89. (10) Pretsch, E.; Bühlmann, P.; Affolter, C. Structure Determination of Organic Compounds−Tables of Spectral Data, 3rd ed.; Springer-Verlag: Berlin, 2000; pp 161−243. (11) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH: Weinheim, 1987; pp 183−280. G
dx.doi.org/10.1021/np400218z | J. Nat. Prod. XXXX, XXX, XXX−XXX
