Pinolidoxin, a phytotoxic nonenolide from Ascochyta pinodes

Phyrochemistry, Vol.34,No. 4, pp. 999-1003,1993 Printedin Great Britain.

PINOLIDOXIN,

0031.9422/93 $6.00+0.00 0 1993PergamonPressLtd

A PHYTOTOXIC

NONENOLIDE

FROM ASCOCH YTA

PINODES ANTONIO EVIDENTE,ROSA LANZE~A,* RENATOCAPASSO,MAURIZIO VURRO~ and ANTONIO BOTTALICO~ Dipartimento di Scienze Chimico-Agrarie, UniversitP di Napoli ‘Federico II’, 80055 Pot&, Italy; *Dipartimento di Chimica Organica e Biologica, Universith di Napoli ‘Federico II’, 80134 Napoli, Italy; TIstituto Tossine e Micotossine da Paraaaiti Vegetali de1CNR, 70125 Bari, Italy; SIstituto di Patologia Vegetale, Universit$ di Sass@ 07100 Sassari, Italy (Received23 December 1992) Key Word Index--Ascochyta pinolidoxin.

pinodes; pea anthracnose;

fungus; phytotoxins; macrolides; nonenolides;

Abstract-Ascochyta pinodes, the causal agent of pea anthracnose, cultured on sterilized wheat, produces toxic metabolites. The main phytotoxin, named pinolidoxin, was isolated and characterized using spectral and chemical methods as 2-(2,4-hexadienoyloxy)-7,8-dihydroxy-9-propyl-5-nonen-9-olide, a new phytotoxic lo-macrolide. When assayed on host (pea) and non-host (bean) plants, pinolidoxin was highly toxic; towards brine shrimps it was only weakly toxic.

INTRODUCTION

The genus Ascochyta includes many phytopathogenic fungal species inducing severe diseases on several crops, characterized by necrotic lesion of leaves, stems and fruits. Ascochyta pinodes Jones is the causal agent of anthracnose of pea (Pisum satioum L.) causing severe lesions and necrosis of leaves and pods. Grown on sterilized wheat kernels, this fungus produces toxic metabolites. This paper describes both the isolation and the structure determination of the main phytotoxic metabolite, which is a new tetrasubstituted nonenolide. RESULTSAND

DI!SCUSSION

The crude organic culture extracts were purified by column chromatography on silica gel, as described in detail in the Experimental, to yield the main metabolite as a homogeneous oily compound. This compound, crystallized as needles, showed a high phytotoxic activity and was named pinolidoxin (I). When assayed on bean and pea leaves and on pods, using the puncture-assay method, pinolidoxin (3 x 10e2 M) caused a marked decrease of turgidity followed by the appearance of severe necrotic lesions. Tested on brine shrimps (Artemia salina) at a concentration of 5.9 x 10e4 M it showed weak mycotoxicity. Pinolidoxin had a molecular formula of C1sHa606 for a total of six unsaturations. In fact, its IR spectrum showed bands characteristic of hydroxy, olefinic and both saturated and conjugated carbonyl groups [l, 21. The presence of at least one conjugated carbonyl system was supported by the typical absorption maximum observed in the UV spectrum at 259 nm [2, 31.

The first inspection of the ‘HNMR spectrum of 1 (Table 1) showed the presence of an olefinic proton, appearing as a filled double doublet at 67.32 (H-12), which correlated in the 2D ‘H, ‘HNMR spectrum (COSY) [4] with the doublet at 65.87 (H-11), due to the other proton of the same trans-disubstituted double bond, and with one (H-13) of the two protons of the conjugated disubstituted olelinic group, both resonating as complex systems at 66.30 and 6.20; 6nally, H-12 also long-range coupled with a methyl group (Me-15), appearing as a doublet at 6 1.89 by its further coupling with the olefinic proton H-14. These findings were consistent with the four protonated olefinic carbons resonating in the ‘%NMR spectrum of 1 (Table 1) at 6145.9, 140.3, 129.7 and 118.1, which, by the correlations observed in

A. EVIDENTE et al.

1000

Table 1. ‘H and WNMR of pinolidoxin (1). The chemical shifts are in &values (ppm) from TMS

C*

6mt

1 2 3 3’

171.9 st 49.8 d 29.8 t

4

21.4 t

4’ 5 6 7 8 9 10 11 12

122.8 d 132.6 d 72.9 d 73.0 d 71.3 d 166.1 s 118.1 d 145.9 d

13

129.7d

14” 15 16

140.3 d 18.7 q 33.6 t

J (Hz)

2Dr3C ‘H, LRC$

5.25 dd 2.20 m 2.00 m 2.41 m 2.20 m 5.53 br t 5.66 dd 4.44 br s 3.52 dd 5.05 td

5.6, 1.7

5.25 2.20, 2.00

15.8, 15.8, 1.4 15.8, 1.4

2.20 241

9.4, 2.5 9.4, 9.4, 26

5.66, 5.05 4.44, 1.50 7.32, 5.87

5.87 d 15.4 7.32 br dd 15.4,9.8 6.30 m 6.20 m 1.89 br d (3H) 5.5 1.78 m

1.89

1.50m

16 17 17 18

Ha*

17.4 t 13.9 q

1.33 m 1.22 m 0.87 t (3H)

7.3

*2D ‘H, ‘H (COSY) and ‘%, ‘H experiments delineated the correlation of all protons and the corresponding carbons in 1. PMultiplicities were determined by DEPT spectrum. $ Long-range correlation spectrum. “These attributions may be reversed.

the 2D r3C, ‘H NMR spectrum [4], were attributed to C12, C-14, C-13 and C-11, respectively. Using the same spectrum, the signal at 618.7 was assigned to the vinyl methyl group Me-15 Furthermore, the results from a long-range 2D 13C, ‘H NMR experiment [4] (Table 1) showed the correlations between the two oleflnic protons at 67.32 (H-12) and 5.87 (H-11) with the ester carbonyl group, resonating at 6166.1 (O=C-lo), and that of the methyl group at 61.89 (Me-15) with C-14. From these results the presence of the partial structure of a 2,4hexadienoyloxy residue as a side chain was deduced. Another partial structure, contained in 1 also as a side chain, was easily identified as a propyl residue. In fact, in following the examination of the ‘H NMR spectrum of 1 the presence of a triplet at 60.87 due to a terminal methyl group (Me-18) was noted, which in the 2D ‘H, ‘HNMR spectrum correlated to both protons of H&-17, both appearing as complex multiplets at 81.33 and 1.22, respectively; these latter, in turn, coupled with the protons of H&-16, resonating as two complex systems at 61.78 and 1.50, respectively. The correlations observed in the 2D ‘%J, ‘H NMR spectrum led to the assignment of the signals appearing at 633.6, 17.4 and 13.9 to HzC-16, HzC-17 and Me-18, respectively. Moreover, in the same 2D ‘H, ‘HNMR experiment the protons of HzC-16 also correlated with the proton (H9) of a secondary oxygenated carbon, appearing as a

double triplet at 65.05, which, in turn, coupled with the double triplet at 63.52 (H-8); the latter also correlated with the complex system resonating at 64.44 (H-7). The signals of H-8 and H-7 by addition of D,O changed in a double doublet and a broad singlet, respectively, thus revealing the hydroxylated nature of the corresponding carbons, which in the 13C NMR spectrum appeared at the expected &values of 73.0 (C-8) and 72.9 (C-7), respectively, as well as the third oxygenated secondary carbon resonating at 671.3 (C-9). In the 2D ‘H, ‘H NMR spectrum, the multiplet of H-7 also correlated with a double doublet appearing at 65.66 (H-6), one of the two protons of the third symmetrically disubstituted double bond. In fact, the other aans-olefinic proton (H-5) resonated as a broad triplet at 65.53 by its further coupling with the protons of the adjacent methylene group (H&-4), apRearing as two complex systems at 62.41 and 2.20, respectively. The latter two protons also coupled with the multiplets resonating at 62.20 and 2.00, assigned to another methylene group (H&-3), which, in turn, correlated with the adjacent proton of a further secondary oxygenated carbon (HC-2), appearing as a double doublet at 65.25. This carbon might be the attaching point of the previously identified 2&hexadienoyloxy side chain. This partial structure was corroborated from the evidence obtained from the 2D 13C, ‘H NMR spectrum, from which the signals of the two olefinic carbons, the two methylene and that of the oxygenated secondary carbon present at 6 132.6, 122.8,29.8,27.4 and 69.8 were assigned to C-6, C-5, C-3, C-4 and C-2, respectively. Considering these results, the total of six unsaturations and the presence of another ester carbonyl group, a macrolide nature was proposed for 1.This result is in full agreement with the residual calculated unsaturation and with the chemical shiR of 171.9 ppm measured in the “C NMR spectrum for the last carbonyl group cited (O=C-1), a very typical value for a lactonic group [4]. The macrolide nature and the total structure of pinolidoxin were corroborated from the evidence deduced from a 2D “C,‘H long-range experiment [43 carried out on 1 and shown in detail in Table 1. From these results, it was clear that pinolidoxin (1) is a new tetrasubstituted IO-macrolide and may be formulated as 2-(2,4-hexadienoyloxy)-7,8-dihydroxy-9-propyl-5-nonen-9-olide. The structure assigned to the phytotoxin was supported by the peaks observed in the HR EI mass spectrum (see Experimental). From these the most significant are the peak at m/z 320.1592 (C, sH,,OJ, generated from the molecular ion (338.1744, C,sH,,O,) by loss of HzO, and those yielded from the latter by alternative losses of CsH,, C,H,CO and C,H,COO residues and observed at m/z 253.1087 (Cr3H,,0S), 225.1129 (C,,H,,O,) and 209.1186 (C1zH1,03), respectively. Moreover, the molecular ion losing C,H, and C,H, residues, by means of two alternative fragmentation mechanisms, yielded the ions at m/z 310.1409 (Ct6H2z06) and 309.1360 (ClgHz106), respectively. As expected, the 2,4-hexadienoyloxy cation represented the base peak at m/z 95.0496 (C,H,O) [2]. Moreover, the FAB mass spectrum showed only the protonated molecular ion [MH]+ at m/z 339,

Pinolidoxin from Ascochyta pinodes and a peak at m/z 321 generated by loss of a water molecule. This result was in agreement with the presence of a glycol system in 1; in fact, most probably the dienol intermediate, produced from [M]’ by loss of HzO, easily converted to the corresponding more stable u.$unsaturated ketone, ruling out a further loss of a water molecule from 1. The presence of the latter structural feature in 1 was confirmed by preparing, using the usual the 7,8-diacetyl derivative 2 ([Ml’ acetylation, =422.1926 m/z, CzzH,,O, by HR EIMS). Its ‘H NMR, as deduced also from the 2D ‘H,‘H spectrum, differed from that of 1 only in the presence of the singlets of the two acctyl groups at 62.17 and 2.02,and the downfield shifts (Asl.23 and 1.35, respectively) of H-7 and H-8, which appeared as a multiplet and a double doublet at 65.67 and 4.87, respectively. The downfield (A60.35) and the upfield shifts (A60.13, 0.33 and 0.15, respectively) observed for H-9 and for H-S, and both protons of HzC16, which resonated as a multiple& a broad triplet and two multiplets at 65.40, 5.40, 1.45 and 1.35, respectively, confirmed the a-location of the 1,Zdiol system [on C(7)C(8)] with respect to the endocyclic oletinic group [C(6) =C(S)] and to the HC-9 bearing the propyl side chain. Finally, the IR spectrum of 2 lacked hydroxyl absorptions and exhibited the typical band of an acetyl group at 1748 cm-‘. Further evidence for the presence of the glycol system and proof of its c&stereochemistry was obtained by converting 1 into the corresponding 7,8-O,O’-isopropylidene derivative 3 ([M] + = 378 m/z, by EIMS). Its ‘H NMR spectrum, compared with that of 1,showed the presence of the signals of the methyls of the isopropylidene group, appearing as two singlets at 61.53 and 1.40 and the downfield shifts (A60.21 and 0.41, respectively) of H-7 and H-8, resonating as multiplet and double doublet at 64.65 and 3.93, respectively. The ‘%NMR spectrum of 3 differed with respect to that of 1 due to the presence of the singlet and the two quartets of the carbons of the isopropylidene group at 6 109.2, 30.6 and 27.7, respectively, and the downfield shifts (A6 3.1 and 4.9, respectively) of C-7 and C-8, resonating as two doublets at 676.0 and 77.9, respectively. Hydroxyl absorptions were absent from the IR spectrum. Finally, further proof for the structure assigned to pinolidoxin was obtained by preparing the corresponding 5,6,11,12,13,14-hexahydroderivative, 4 ([Ml’ =344, by EIMS). Compared with that of 1, its ‘H NMR spectrum, corroborated by the 2D ‘H, ‘H spectrum, showed the absence of any olefinic signals and the presence of more complex signal systems in the aliphatic region between 61.6 and 1.3, and assigned to the new five methylene groups. In addition, two triplets were recorded at 62.29 and 1.32, chemical shift values characteristic of a methylene x-located to a C=O group and of a primary methyl group and therefore assigned to HzC-11 and Me-15, respectively; moreover, the multiplet of H-7 appeared shifted upfield (A60.53) at 63.91. The r3CNMR spectrum differed from that of 1 due to the absence of the signal of any oletinic carbons and the presence of five other signals in the region of ahphatic methylene groups between 6 32

1001

and 18, which were assigned to the corresponding carbons, as described in the Experimental, and in agreement with the theoretical chemical shift values calculated for a suitable hexanoyloxy chain model [4, 51. Moreover the upfield shift (A6 7.4) of H&-4 at 6 20.0 and the presence of a triplet (H&-l 1) at 634.7, a typical chemical shift value for a methylene a-located with respect to a C=O group [4, 51, were observed. The IR spectrum of 4 showed the absence of C=C groups; only bands typical of hydroxyl and ester groups [ 1,2] being present; as expected, its UV spectrum lacked the strong absorption due to the extended conjugated system and exhibited only a weak band at 275 nm, characteristic of a saturated C=O group c2, 31. The 2D NOE experiment (NOESY) [4] on 1 showed a near spatial correlation between H-11 and the other olet’inic protons H-13 and/or H-14, while the same relation was noted between H-6 and H-8, and HzC-4 and HzC-16. These results agreed with the evidence obtained by an inspection of a Dreiding model of pinolidoxin, which also revealed a very great conformational freedom of the macrocyclic lactone ring as well as that of the two side chains. Moreover, a series of ‘H NOE-difference spectra (NOEDS) [5] showed a spatial proximity between Me-15, and both H-13 and H-14; as no proximity was detected between the same methyl group (Me-15) and H-12, a srans-stereochemistry was deduced for the double bond located between C-13 and C-14. In conclusion, the macrolide structure of pinolidoxin appears to be satisfactorily demonstrated. Macrolides are quite common as naturally occurring compounds and some are biologically active [68]; fungi are also known to produce macrolides [9]. EXPERIMENTAL

Mps: are uncorr.; optical rotations: CHCI,; IR and UV: neat and MeCN, respectively; ‘H and 13C NMR: CDCl,, 400 and/or 270 MHz and 100 or 67.92 MHz, respectively, using the same solvent as the int. standard. Carbon multiplicities were determined by DEPT (Distortionless Enhancement by Polarization Transfer) spectra [4]. DEPT, NOEDS, NOESY, COSY 45 (correlated spectroscopy), 2D heteronuclear and long-range heteronuclear chemical shift correlation experiments were performed using Bruker standard microprograms; EI and HR EI MS: 70 eV; FAB MS: glycerol/thioglycerol with Xe atoms at 9.5 kV. Analyt. and prep. TLC: silica gel (Merck, Kieselgel60 F,,,, 0.25 and 0.50 mm) or on reverse phase (Whatman, KC18 Fz54, 0.20 mm) plates. CC: silica gel (Merck, Kieselgel, 60,0.063-0.2 mm); solvent systems: (A) CHCI,-iso-PrOH (32.3: 1); (B) EtOH-Hz0 (2.3: 1); (C) CHCl,-iso-PrOH (19: 1); (D) n-hexane-EtOAc (5.6: 1); (E) CHCl,-iso-PrOH (9: 1). Fungus. Ascochyta pinodes Jones was isolated from infected peas near Bari (Italy) and deposited in the fungi collection of the Istituto Tossine e Micotossine da Parassiti Vegetali de1 CNR, Bari, Italy (ITEM 1094). Production, extraction and purification of pinolidoxin. (1) Wheat cultures of A. pinodes and toxin extractions

1002

A. EVIDENTE et al.

were performed as described [lo]. The brown oily CHICI, extract (337 mg), obtained from 1 kg of dry wheat culture, was fractionated by CC eluted with solvent A, producing 8 groups of homogeneous frs. The residue (194.2 mg) left from the 6th group, containing the main metabolite (R, 0.30 and 0.50 by TLC on silica gel eluent A and on reverse phase eluent B, respectively) was further purified by CC, also eluted with solvent A, yielding 4 groups of homogeneous frs: the second and the third produced pinolidoxin (1) as a homogeneous oily compound (110.8 mgkg- ’ dry wt) which was crystallized as needles from n-hexane at - 20’: mp 4547”; [z];’ + 142.9 (c 0.31); UV I.,,, nm (log E):259 (4.34); IR v,,,_ cm - ‘: 3442, 1718, 1645, 1618, 1244, 1185; ‘H and 13CNMR spectra: Table 1; HR EIMS, m/z (rel. int.): 338.1744 (C18Hr606. calcd338.1730, 1.6)[M]+,336.1602(C,,H,,O,,0.34)[M -2H]+, 320.1592 (C,,H,,O,, 0.3) [M -H,O]+, 310.1409 (Cl,H2r0,, 0.5) CM-C,H,]+, 309.1360 (C,,H,,O,, 0.3) [M-C,H,]+, 269.1004 (Ci3H,,06,1.3) [M-2H-C,H,]+,267.0892 (C,,H,,O,,l.4) [M-4H 253.1087 (C13H,70s,3.9) [M - H,O --C&l+, 251.0940 (C,3H,,0,,1.0) [M-2H-H,O -GH,l+, [M-2H-C,H, -C&l +, 241.0745 (C11H,30,,8.8) -C,H,]+, 239.0555 (C,,H,r0,,2.4) [M-4H -C,H, 235.0927 (Cl 3H, s0,,,0.3) [M - 2 x H,O -C,bl+, 225.1129 (ClLH,,04. 6.7) [M-H,0 -GH,l+, -&H,CO]+, 209.1186 (C,,H,,0,,0.6) [M-H,0 -CsH,COO] +, 207.0998 (C,,H,,0,,0.5) [M-2H -H,O-C,H,COO] +, 95.0496 (C,H,O,lOO) [C,H,CO] +. 7,8-O,O’-Diacetylpinolidoxin (2). Pinolidoxin (1, 8 mg) was acetylated with pyridine (300 ~1) and Ac,O (300 ~1) at room temp. overnight. The oily residue, left by the reaction work-up, was purified by prep. TLC (silica gel, eluent D) to give 2 as an homogeneous compound (7 mg): [a];‘+ 104 (c 0.79); UV I.,,,,, nm (log E): 259 (4.32); IR v,,, cm-‘: 1748, 1718, 1646, 1618, 1239, 1220; ‘HNMR, 6: differed from that of 1 in the following signal systems: 5.67(lH,m,H-7),5.4O(lH,brt,J,~,=J,~,=l5.7Hr_H5), 5.40 (I H, m, H-9). 4.87 (I H, dd, J,,, = 2.3 Hz and J,,, =7.9 Hz, H-8), 2.17 and 2.02 (3H each, s, two AC), 1.45 (lH, m, H-16) 1.35 (IH, m, H-16); HR EIMS, m/z (rel. int.): 422.1926 (Cr2H3,,08, calcd 422.1941,2.0) [M] +, 380.1863 (C,,H,,07,1.2) [M-CH,CO]+, 362.1763 (C,cHz60,, 1.6) [M -HOAc]+, 320.161 I (C,sHz,,Os, 2.0) [M -CH&OHOAc] +, 302.1503 (ClBHz204, 0.5) [M - 2 x HOAc] +, 277.1060 (ClgH1705, 1.7) [M -CH&O - HOAc-C,H,] +, 235.0979 (C13H,50,,6.9) [M-2xHOAc-CsH,]+, 208.1092(C,,H,,0,,3.1) [M -HOAc -AcO-CSH,CO]+,95.0499(C6H,0,100)[M -C,H,COJ+. 7,8-O,O’-lsopropylidenepinolidoxin (3). Pinolidoxin ( 1, 14 mg) in dry Me&o (9 ml) was stirred with dry CuSO, (530 mg) under reflux for 2 hr. The mixt. was filtered and evapd to give an oily residue, which was purified by prep. TLC (silica gel, CHCI,) to yield 3 as a homogeneous oil (12.5 mg): [cr]y + 102.6 (c 0.96); UV i,,, nm (log E): 259 (3.93); IR v,,, cm -I: 1732, 1646, 1618, 1381, 1241, 1221; ‘H NMR, 6: differed from that of 1 in the following signal systems: 4.65 (IH, m, H-7), 3.93 (lH, dd, J,, 8 =4.2 Hz and J8,9= 10.9 Hz, H-8), 1.53 and 1.40 (3H each, s, O-

C(Me),-0); “C NMR, 6: differed from that of 1 in the following signal systems: 109.2 (s, 0-C(Me),-0), 77.9 (d, C-8), 76.0 (d, C-7). 30.6 and 27.7 (q, 0-C(Me),-0); EIMS, m/z (rel. int.): 378 [M]’ (O.l), 363 [M-Me]+ (0.3X 320 [M - MeCOMe] + (0.6). 293 [M - H,O -C,H,] + (0.5), 283 [M-C,H,CO]+ (I), 235 [M-H20-CsH,MeCOMe]’ (3), 225 [M-C,H,CO-MeCOMe]+ (I), 111 [C,H,COO]+ (4). 95 [C,H,COJ+ (lCO), 67

CC,H:l+ (25). 5,6,11,12,13,14-Hexahydropinolidoxin (4). Pinolidoxin (1, 30 mg) in MeOH (6 ml) was added to an H,presaturated PtO, (30 mg) suspension in the same solvent (6 ml) and hydrogenated at room temp. and atm. pres. under stirring. After 3 hr, the reaction was stopped by filtration, evapd and the residue purified by prep. TLC (silica gel, eluent E) to give 4 as a pure oil (21.7 mg): [a];’ +27.7 (c 0.31); UV A,,, nm (log E): 275 (sh), ~220; IR v,,, cm- ‘: 3419, 1734, 1717. 1457, 1377, 1260, 1160, ‘H NMR, 6: differed from that of I in the following signal systems: 3.91 (lH, m, H-7), 3.79 (IH, td, J8,9=J9.16 =9.0 Hz and J 9.16’_- 2 .5 Hz, H-9). 3.30 (I H, m, H-8), 2.29 7.0 Hz, H-l I), 1.90 (2H, m, H-3 and H-6), (2H, t, J,,.,z= 1.70 (IH, m, H-3’), 1.6&1.30 (llH, m, 2H-4, 2H-5. H-6, 2H-12, 2H-13, 2H-14) 1.32 (3H, t, J,,.,,=7.0 Hz,, Me15); 13C NMR, 6: differed from that of 1 in the following signal systems: 34.7 (t, C-l 1), 31.2 (1, C-13) 27.7 (t. C-6), 25.2&C-12),24.8(t,C-5). 22.3(r,C-14),2O.O(f,C-4), 14.1 (q, C-15); EIMS, mjz (rel. int.): 344 CM]’ (3). 327 [M -OH-J+ (1.5), 308 [M-2x H,O]+ (0.3). 271 [M-OH -C,Ha]+ (1.8), 256 [M-OH-C5H,,]+ (0.4), 254 [M -H,0-C,H,2]+ (ll),245[M -C,H,,CO]+ (7.5),241 [M-OH-C5H,,-Me]+ (8.5),229 [M-C,H,,COO]+ (3.7), 115 [CSHl,COO]+ (16). 99 [CsH,,CO]+ (ICO), 71 C&Hi ,I + (84). Biological methods. The phytotoxic and the mycotoxic activity were assayed as described [I 1J.

Acknowledgements-This investigation was supported in part by grants from the Italian Ministry of University and Scientific and Technological Research and in part by the National Research Council, special ad hoc program ‘Chimica Fine II’, subproject 3. Mass spectral data were provided by ‘Servizio di Spettrometria di Massa del CNR-Universiti di Napoli’, the assistance of the staff is gratefully acknowledged. The authors thank the ‘Centro di Metodologie Chimico-Fisiche dell’ Universita di Napoli ‘Federico II” for NMR spectra.

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