Petuniolides. Unusual ergostanoid lactones fromPetuniaspecies that inhibit insect development

摘要

J. CHEM.SOC. PERKIN TRANS. 1 1990 Petuniolides. Unusual Ergostanoid Lactones from Petunia Species that Inhibit Insect Development Carl A. Elliger," Rosalind Y. Wong, Anthony C. Waiss, Jr., and Mabry Benson Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, CA 94710, USA Four new compounds that strongly contribute to the resistance of Petunia parodii and P. integrifolia against feeding by larvae of the lepidopteran, Heliothis zea, were isolated from leaves of these plant species. These consist of the (22R,24R) -22,24,25-orthoacetates and orthopropionates derived from multiply functionalized ergostanoids in which the A-ring has lost one carbon and has been converted into a spirolactone.An epoxy group is present at position 6a,7a. Petuniolides A (11) and B (12) have a A9(11)-12a-acetoxy group whereas petuniolides C (13) and D (14) possess the corresponding allylic ketone functionality. The molecular structures of (12) and (14) were determined by X-ray crystallography. The resistance of Petunia hybrida towards attack by the polyphagous larvae of the moth Heliothis zea (Boddie) has been shown by us to be correlated with the presence of certain ergostane related allelochemicals found in the leaves of the plant. These substances, which are termed petuniasterones, occur as a diverse family of steroidal comp~unds,~-~ of which a few typical examples are shown compounds (lHlO). Although various functionalizations of the side chain and of the steroid nucleus occur within the family, the individual members all possess an 0x0 group at position-3 and an a-hydroxy or -acetoxy at position-7 on an essentially unaltered ergostane system.We have observed, however, that insect- inhibitory activity against H. zea is only significant for those compounds having a bicyclic orthoester system on the side chain. We now report the occurrence of four new compounds from Petunia species that, although related to the petuniasterones, represent a new structural type of modified ergostanoid in which ring A has undergone rearrangement with the loss of one carbon atom and formation of a spirolactone. These compounds (ll)-(14), which were isolated from the putative ancestors of P.hydrida (i.e., P. integrifolia and P. parodii), are named petuniolides in analogy with the well known ~ithanolides,~*~and we propose the numbering system shown in Figure 1 which has the advantage of preserving standard steroid numbering in the unmodified portion of the molecule. In artificial diets, the insect-inhibitory activity of the (5) R' = COCH,COSMe, R2 = H (6) R' = Ac, R2 = H (7)R' = H, R2 = H Petuniasterone C series petuniolides is greater than that of the most active petuniasterones. Petuniolides C (13) and D (14) reduce larval growth of H. zea to 50 of normal at dietary concentrations (ED,,) of about 24 mg kg-'. Petuniolides A (11) and B (12) are less inhibitory with ED50 values of ca. 10-13 mg kg-'. Chloroform extracts of plant materials were fractionated as previously described,2 and the individual petuniolides were obtained by successive preparative HPLC.Petuniolides A (11), B (12), and D (14) were found in leaves of P. integrifolia (Hook.) Schinz and Thelling (= P. violacea, Lindley) in amounts of 100, 430, and 200 ppm (dry weight basis), respectively. Petuniolide C (13) was obtained at 370 ppm from P. parodii (Steere). Petuniolide B (12) formed crystals adequate for X-ray MeI (1) R' = CH2COSMe,R2 = R3= H (2) R' = CH,COSMe, R2 = OH, R3= H (3) R' = CH,COSMe, R2 = OH, R3 = Ac (8)R' = R2 = H (4) R' = CHOHCOSMe, R2 = R3 = H (9)R' = Ac, R2 = OAC PetuniasteroneA series PetuniasteroneD series J. CHEM. SOC.PERKIN TRANS. 1 1990 18 0W-.?'OH (10) Petuniasterone F R (11) R = Me (12) R = Et RI 0 (13) R = Me (14) R = Et analysis. The molecular structure of (12) was unequivocally established and is shown in Figure 2 with the atom numbering system used in the X-ray investigation. Figure 3 presents a stereoscopic view of its molecular conformation, and the final atomic co-ordinates and their estimated standard deviations (in parentheses) are listed in Table 3. Carbons 5, 13, 17,22, and 24 possess the (R)-configuration, and carbons 6,7,8,10,12,14, and 20 have the (9-configuration. The 'H and 13C NMR spectra of (12) are consistent with this structure (Tables 1 and 2). Their assignments, based upon the positions and multiplicities of the observed signals compared with spectra of the petuniasterones2-5 were facilitated by 'H-'H and I3C-'H correlation spectroscopy * and by decoupling experiments. A complex set of proton signals was observed at tiH 2.0-2.2 and 2.5-2.8 in all the compounds in the series, and was assigned to the spirolactone methylenes (Table 1); however, their complexity precluded detailed analysis. In the 13C NMR spectrum, the lactone carbonyl signal appeared at 6c 175.6 with the methylene carbons at positions-3 and -4 at 6c 28.6 and 29.2 respectively. The proton signals at 6H-63.19 (d, J4 Hz)and tiH-, 3.31 (dd, J 4, 1.5) were assigned to the 6,7-epoxy functionality.Signals at 6c 55.1 and 53.8 were correlated with positions 6 and 7 respectively.By comparison, the oxirane carbons of epoxycyclohexane appear at 6c 51.9.9 The vinyl signal at 6H5.79, Figure 1. Petuniolide numbering. -1 9 n18 "39 Figure 2. Perspective view of compound (12) with the crystallographic numbering scheme. Open bonds represent double bonds, and shaded circles represent oxygen atoms. which appears as a broadened doublet as a result of long-range coupling to H-8 was assigned to H-11. H-12 is observed as a sharp doublet at 6H4.96 and C-12 is at 6c 73.2. Even though (12) has a 9-11 double bond and a 12-acetoxy substituent, the position of the 18-methyl group resonance (6, 0.71) is essentially unshifted compared to compound (1). Just such a small substituent effect is expected for an a-oriented 12-acetoxy group." NMR signals associated with the side chain and the orthopropionate ester were similar to those previously rep~rted.~In the IR spectrum, compound (12) exhibits two carbonyl bands: 1 775 cm-' corresponding to the spirolactone and 1 725 cm-' associated with the acetate at position 12.The UV absorption maximum at 206 nm is consistent with the A9(I1) isolated double bond.' 2a The spectroscopic properties of petuniolide A (11) were very similar to those of (12). The elemental composition of (11) compared with that of (12) indicated only the difference of one -CH2- group. In the 'H NMR spectrum of (ll),the signals associated with the pendant orthopropionate system of the side chain (CH,, 6 0.99 and CH2, 6 1.80) in (12) are absent, and a new methyl singlet appears at 6 1.56. Similarly, the 13C NMR spectrum of (11) does not show the orthopropionate CH3 and CH2 signals at 7.7 and 29.3 respectively which are observed in (12) but does exhibit a CH, peak at 23.5.Additionally, the 13C signal at 6c 118.6 of (12) now appears at 6 117.3. These spectral changes show that an orthoacetate moiety is attached to the side chain of (ll), and are in full agreement with the spectra of similar orthoacetates in the petuniasterone D ~eries.~ Petuniolides C (13) and D (14) showed lactone IR absorption at 1775 cm-' similar to that of (11) and (12); however, no acetate band (1 725 cm-') was observed, and a new carbonyl band at 1680 cm-' was present. Elemental compositions of (13) and (14) differed from those of (11) and (12) respectively by the loss of C2H40.UV absorption at cu. 234 nm was consistent with an a$-unsaturated ketone, suggesting the presence of a A9~11~-12-oxo-system.'2bIn the NMR spectra of (13) and (14), no resonances associated with an acetoxy group were observed, and the signal at 6,4.96 for H-12 of the allylic acetates was not present. The signal at 6, 73.2 (C-12) was missing, and a new carbonyl resonance at 6c 204.4 appeared in each case. Additionally, olefinic signals in (13) and (14) now were found at 6c 155.4 (C) and 128.0 (CH), which, together with the above, indicate a conjugated olefinic ketone', at position 12. J. CHEM. SOC. PERKIN TRANS. 1 1990 Figure 3. Stereoscopic view of compound (12).Table 1. 'H NMR data.* Compound 3-H2 ca. 2.5-2.8m ca. 2.5-2.8m ca. 2.5-2.8m ca. 2.5-2.8m 4-H 2 ca. 2.0-2.2m ca. 2.0-2.2m ca. 2.2m ca. 2.2m 6-H 7-H 8-H 10-H 11-H 12-H 14-H 3.20d (4) 3.29dd (4, 1.5) 2.26br d (10) 2.58q (7) 5.79br d (5) 4.96d (5) ca. 2.Om 3.19d (4) 3.31dd (4, 1.5) 2.26br d (10) 2.59q (7) 5.78br d (5) 4.96d (5) ca. 2.Om 3.29d (4) 3.35br d (4) 2.72m 2.78m 5.84br s ca. 2.2m - 3.29d (4) 3.35br d (4) 2.72m 2.78m 5.84br s ca. 2.2m - 15-Hz ca. 1.4 and 2.1 m" ca. 1.4 and 2.lm" 1.6 and 1.9m" 1.6 and 1.9m" 16-Hz ca. 1.6 and 1.9m" ca. 1.6 and 1.9m " 1.6 and 2.Om " 1.6 and 2.0m" 17-H ca. 1.95m ca. 1.95m ca. 1.9m ca. 1.9m 18-H 0.71s 0.71s 1.oos 1.01s 19-H, 20-H ca. 1.8m 1.06d (7) 1.06d (7) ca.1.8m 1.15d (7) ca. 1.7m 1.16d (7) ca. 1.7m 21-H, 22-H 23-H2 26-H, 27-H, 0.85d (6) 4.21 td (8,4) 1.53d (8) 1.33s 1.18~~ 0.85d (6) 4.21td (8,4) 1.54d (8) 1.34s 1.17~~ 1.08d (7) 4.25dt (1 1.5,4) 1.48dd (14,5) and 1.80br d (14) 1.31s 1.18~~ 1.08d (7) 4.25dt (1 1.5,4) 1.50dd (14, 5) and 1.78m 1.33s 1.17~~ 28-H3 1.22s 1.21sb 1.22s 1.22s Orthoacetate 1.56s - 1.56s - Orthopropionate CH* 1.80m 1.82m CH3 OAc -2.07s 0.99t (7.5) 2.06s 0.99t (7.5) - * 6 values in CDCl,; coupling constants (Hz) in parentheses. Assignments are by decoupling and correlation techniques. Values with identical superscripts in each column may be interchanged. Confirmatory evidence for this position of functionalization above indicates that compound (13) possesses orthoacetate was provided by the proton at position-11 which appeared at functionality and (14) has an orthopropionate group.The 6, 5.84 as a broadened singlet showing coupling to H-8. remaining features in the NMR spectra of (13) and (14)CH3-18 in (13) and (14) exhibits a substituent-induced compared to those of (11) and (12) and to those of the chemical-shift difference in the 'H NMR of +0.30 ppm which petuniasterones did not provide sufficient information for the agrees closely with the value reported for an unsaturated ketone assignment of further stereochemical details. at position-12 of steroids." Methyl groups at positions 19 and The molecular structure and absolute stereochemistry of (14) 21 also show shifts to lower field for their proton resonances, were determined by X-ray crystallography (Figures 4 and 5).but in other respects the NMR spectra of (13) and (14) are quite Atomic co-ordinate data are given in Table 4. Compound (14) similar to those of (11) and (12) and show signals ascribable to and by comparison (13) have the same absolute configuration the oxirane and lactone moieties. Evidence analogous to that as compounds (11) and (12). In the molecular structures of both J. CHEM. SOC. PERKIN TRANS. 1 1990 Table 2. I3C NMR data.* ~~~~~~ Compound ~~ 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Orthoester Acetate Acetate Orthoacetate Orthopropionate Orthopropionate 175.7, C 28.6,CH2 29.2,CH2 85.8, C 55.1, CH 53.8,CH 36.5,CH 139.9, C 41.4, CH 123.0, CH 73.2, CH 44.3, c 42.2,"CH 24.0; CH, 27.0: CH, 43.3," CH 10.9, CH, 14.4, CH, 38.1, CH 11.1, CH, 69.7, CH 30.1, CH, 82.5,' C 81.2,' C 20.0, CH, 20.6," CH, 25.2,6CH3 117.3, C 21.3, CH, 170.3, C 23.5,CH3 -- 175.6, C 28.6, CH, 29.2, CH, 85.8, C 55.2, CH 53.8, CH 36.6,CH 139.9, C 41.4, CH 123.0, CH 73.2, CH 44.3, c 42.2PCH 24.1,"CH2 27.1; CH, 43.3,"CH 10.9, CH, 14.4, CH, 38.2, CH 11.1, CH, 69.5, CH 30.4, CH, 82.1,' C 81.0,' C 20.2, CH, 20.6," CH, 25.4,"CH3 118.6, C 21.2, CH, 170.2, C 7.7, CH, 29.3,CH2 -175.2, C 28.5, CH, 28.8,CH2 85.3, C 55.4,CH 53.3,CH 37.7,CH 155.4, C 41.4, CH 128.0, CH 202.4, C 53.8, C 49.7,CH 23.8,"CH2 27.5,"CH2 43.4,CH 10.1, CH, 13.7, CH, 39.4, CH 13.3, CH, 70.1, CH 30.4, CH, 82.5: C 81.4: C 20.0, CH, 20.4,' CH, 25.1,' CH, 117.2, C --23.5,CH3 --175.2, C 28.5, CH, 28.8,CH2 85.3, C 55.5,CH 53.3,CH 37.7,CH 155.4,C 41.4, CH 128.0, CH 202.4, C 53.8, C 49.8,CH 23.8," CH, 27.4,"CH2 43.5, CH 10.2, CH, 13.7, CH, 39.5,CH 13.4, CH, 69.9,CH 30.8, CH, 82.1: C 81.2; C 20.1, CH, 20.5,' CH, 25.3,'CH3 118.7, C ---7.7,CH3 29.3,CH2 * In ppm from internal SiMe, for CDC1, solutions.Values with identical superscripts in each column may be interchanged. Assignments were facilitated by C-H correlation spectroscopy. 19 Figure 4. Perspective view of compound (14) with the crystallographic numbering scheme. Open bonds represent double bonds, and shaded circles represent oxygen atoms. (12) and (14) the dihedral angle between the normals of the two best least-squares ring planes defined by (0-1, C-2, C-3, C-4, and C-5) and (C-5, C-6, C-7, C-8, C-9, and C-10) is nearly orthogonal: petuniolide B, 93.0" and petuniolide D, 83.4'.ExperimentalM.p.s were taken with a Thomas-Hoover apparatus and are corrected, Optical rotations were obtained for chloroform solutions on a Perkin-Elmer Model 241 automatic polarimeter at ca. 26 "C. IR spectra were recorded on a Perkin-Elmer Model 237 spectrophotometer and refer to ch:oroform solutions; UV spectra were taken on Cary 219 and Hewlett-Packard 8451A spectrophotometers using methanol solutions; 'H NMR spectra were obtained in CDCl, at 90 MHz on a Varian EM- 390 instrument or at 200 MHz on a Nicolet NT-200, and "C NMR spectra were taken at 50 MHz on the latter instrument. NMR assignments were facilitated by decoupling methods and by the use of two-dimensional proton-proton and carbon- proton correlation techniques8 Mass spectra were obtained using a VG Micromass 70/70 HS instrument either by electron impact or using ammonia chemical ionization.X-Ray intensities were collected with a Nicolet R3 automatic diffractometer at room temperature. HPLC columns were from Rainin Instruments, Alltech Associates and IBM, Inc. Solvents were HPLC grade and were pumped using an Altex-Beckman Model 110A pump. Detection was by UV spectroscopy either at 254 nm with an Altex Model 150 monitor or at lower wavelengths with a Beckman Model 165 variable wavelength detector. Bulk RP-18 preparative column packing was obtained from Waters Chromatography Division.Insect Bioassays.-Solutions containing materials for bio- assay were evaporated on to cellulose powder. The powder was mixed thoroughly and incorporated into modified Berger-diet premix.14 The test diets were divided into ten portions, placed in individual plastic containers, and newly hatched larvae of Heliothis zea were added. The insects were maintained at 26 "C J. CHEM.SOC. PERKIN TRANS. 1 1990 Figure 5. Stereoscopic view of compound (14). Table 3. Petuniolide B atomic co-ordinates (x lo4) with e.s.d.s in Plant Material.-Petunia parodii (Steere) seeds were obtained paren theses. from the National Seed Storage Laboratory, Colorado State University, Fort Collins, CO, USA. Petunia integrifolia (Hook.)Atom x Y 2 seeds were from the Northeastern Plant Introduction Station, 691(3) 1171 8 695(2) US Dept.of Agriculture, Geneva, New York. Leaf material and 1342(5) -142(7) 8 705(3) seed of P.integrifolia were also supplied by Ing. Agr. Hugo A. 2 698(4) 200(6) 8 853(3) Cordo, Biological Control of Weeds Laboratory, Hurlingham, 2 683(4) 1823(5) 9 194(3) Buenos Aires Prov., Argentina. Plants were grown in a 1 517(4) 2 480(5) 8 779(3) greenhouse and in outdoor beds in Albany, California. Leaf 1781(4) 3 087(5) 7 919(3) material was harvested at intervals between December 1987 1088(4) 4 380(5) 7 574(3) and June 1988. 8(4) 5 080(5) 8 039(2) -298(4) 4 235(5) 8 833(3) Isolation Procedure.-Freeze-dried leaf material (typically 803(4) 3 631(5) 9 332(3) -1451(4) 4 036(5) 9 112(3) 100-300 g) was successively ground with chloroform (3 x 1 400 -2 614(4) 4 581(5) 8 61 l(3) ml) using a Tekmar SD-45homogenizer at maximum speed -2 310(4) 5 725(5) 7 941(2) followed by filtration. After evaporation of the combined filtrate -1 134(4) 5 186(5) 7 479(2) under reduced pressure, the resulting green oil (ca.10 of -1054(4) 6 206(6) 6 698(3) original dry wt.) was suspended in boiling acetonitrile (ca. 10-15 -2 423(4) 6 572(6) 6 475(3) volumes) with stirring for 1 h. Upon cooling to 5"C, the -3 244(4) 5 904(5) 7 199(3) solution was easily decanted from waxy, solid material. -2 083(4) 7 254(5) 8 360(3) Approximately 50 of the original extract remained in solution. 1676(4) 4 931(5) 9 631(3) This solution was evaporated and redissolved in four volumes of-4 438(4) 6 828(5) 7 348(3) -5 250(4) 6 196(6) 8 062(3) acetonitrile for application to a 50 mm x 250 mm column of 25 -5 182(4) 7 002(5) 6 515(3) p RP-18 packing.Elution with acetonitrile gave a zone of active -5 767(4) 5 532(5) 6 187(3) material (elution volume 375-900 ml) containing numerous -6 895(4) 5 814(5) 5 615(3) petuniasterones as well as the petuniolides reported herein. All -6 612(4) 6 845(5) 4 848(3) chlorophyll and considerable amounts of less polar lipid -5 388(4) 6 529(6) 4 396(3) material were removed in this way to yield a mixture -7 670(4) 6 921(6) 4 211(3) representing about 4 of the original plant weight. Further -7 575(5) 4 374(5) 5 406(3) fractionation was accomplished by preparative HPLC.-7 057(4) 8 199(5) 6 085(3) Columns and conditions are as follows: Dynamax C-18, -7889(5) 9 497(6) 6 298(3) 21.4 x 250 mm + guard column (30 water in acetonitrile); R-8 985(5) 9 734(7) 5 737(3) -6 533(3) 8 325(3) 5 274(2) Sil C-18, 10 mm x 250 mm (30 water in acetonitrile); and -7 728(3) 6 809(3) 6 071(2) IBM Cyano, 10 mm x 250 mm (20 propan-2-01 in hexane). -6 122(3) 8 132(3) 6 690(2) Results are given in Table 5. -3 139(3) 3 225(3) 8 215(2) -3 787(4) 2 270(6) 8 694(4) Compound (ll), petuniolide A, m.p. 232-235 "C (from-4 094( 5) 889(6) 8 225(4) MeOH); N (h/nm) (589) +99", (578) + 104", (546) + 118",-4 040(4) -1 2 521(5) 9 424(2) (436) +209", (365) +32l0; v,,, 1 775 (lactone) and 1 725867(4) 344(4) 8 617(3) 883(3) 2 844(4) 7 276(2) cm-' (acetate); A,,, 206 nm (log E 3.75); m/z 562.3370 (MNH,+, 3779, 545 (MH+, 4), and 485 (MH' -C2H402, 63).C31H48N08 requires 562.3379. for ten days, and their weights were determined and com- Compound (12), petuniolide B, m.p. 249-252 "C (frompared with those of control subjects maintained on diets con- MeOH-H20); a (h/nm) (589) + 113O, (578) + 117O, (546) taining as additive only the standard quantity of cellulose + 134O, (436) +237", (365) +390"; v,,, 1 775 (lactone) and powder. 1 725 cm-' (acetate); h,,, 206 nm (log E 3.63); m/z (EI) 558.3220 Table 4. Petuniolide D: atomic co-ordinates (x lo4) with e.s.d.s in parentheses. Atom x Y Z 12 218(3) 13 368(5) 14 504(5) 13 752(4) 12 426(3) 12 612(4) 11 593(3) 10 342(3) 10 028(4) 11 196(6) 7 357 7 326(6) 8 072(7) 9 055(5) 8 125(5) 6 747( 5) 6 444w 7 445(5) 8 331(4) 9 213(5) 10 482( 1) 10 984(2) 10 611(3) 9 929(3) 9 736(2) 9 151(2) 8 454(2) 8 285(2) 9 007(2) 9 483(2) 8 783(3) 7 609(3) 7 802(3) 8 425(4) 7 748(5) 7 363(5) 9 195(2) 8 707(2) 7 854(2) 9 119(3) 9 152(4) 7 657(3) 6 808(3) 7 914(4) 11 554(5) 5 494(3) 4 631(4) 4 691(3) 4 259(4) 3 084(3) 3 269(4) 6 386(5) 5 596(7) 5 209( 7) 6 142(5) 8 945(5) 10 681(6) 6 813(5) 7 804(6) 5 434(5) 4 102(5) 3 124(5) 2 324(6) 7 954(2) 7 148(2) 6 860(2) 7 412(2) 7 384(2) 8 991(3) 6 947(2) 7 450(2) 6 511(2) 7 036(2) 6 660(2) 5 858(2) 4 625(4) 1601(7) 5 819(2) 2 163(5) 1 153(9) 5 541(3) 2 553(4) 2 017(6) 7 264(3) 2 5W4) 1416(4) 1 818(4) 3 128(2) 2 034(2) 3 538(2) 6 580(2) 11 673(3) 13 422(5) 5 058(6) 6 774(9) 3 805(5) 4 297(4) 6 185(4) 7 469(4) 5 420(3) 6 721(6) 5 94003) 5 763(2) 5 253(3) 4 534(3) 5 357(1) 6 391(2) 6 049(2) 8 981(1) 9 129(1) 11 607(2) (M', 2).C32H4608 requires 558.3192; m/z (CI) 576 (MNH4', 1273,559 (MH', 6) and 499 (MH' -CZH402,32). Compound (13), petuniolide C, m.p. 235-238 "C (from EtOAc-heptane); a (h/nm) (589) +7", (578) +7", (546) +3", (436) -60deg;, (365) -699'; v,,, 1 775 (lactone) and 1 680 cm-' (conjugated CO); A,,, 233 nm (log E 4.09); m/z 501.2831 (MH', 100) and 457 (MH' -C02). C29H4107 requires 501.2852. Compound (14), petuniolide D, m.p. 2W206"C (from MeOH); a (hlnm) (589) +9", (578) +8", (546) +6", (436) -50", (365) -651"; v,,, 1 775 (lactone) and 1 680 cm-' (conjugated CO); A,,, 234 nm (log E 4.14); m/z 515.3001 (MH', 100).C30H4307 requires 515.3008. Crystal Structure of Compound (12).-Petuniolide B, C32H4608, M = 558.8, monoclinic, space group P2,, a = 10.739(1), b = 8.824(1), c = 15.903(2) A, p = 90.26(1)", U = 1 507.1 A', D, = 1.23 g ~m-~, = 2, F(0oO) = 603.9, ~(CU- 2 K,) = 6.72 cm-'. Final R = 0.053 (361 parameters), R, = 0.056 for 2 779 unique reflections with lFol 2 3alF01 in the range 3" 28 114", average parameter shift is 0.050, and difference Fourier synthesis excursions are within amp; 0.6 eA-3. Crystals were obtained from methanol. * Supplementary data (see section 5.6.3 of Instructions for Authors in the January issue). For both (12) and (14), a complete list of final atomic bond lengths and angles, anisotropic thermal parameters for the non- hydrogen atoms, and positional parameters for the hydrogen atoms have been deposited at the Cambridge Crystallographic Data Centre.J. CHEM. SOC. PERKIN TRANS. 1 1990 Table 5. Elution zone (ml). Compound Dynamax C18 R Sil C18 Cyano (11) 160-220 33-40 90-100 (12) 2-300 43-53 85-95 (13) 1W140 25-35 135-150 (14) 160-220 30-38 13Ck140 Crystal Structure of Compound (14).-Petuniolide D, C30H4207, M = 514.7, monoclinic, space group P2', a = 10.066(3), b = 8.192(2), c = 17.076(3) A, p = 97.94(2)', U = 1394.6 A3, D, = 1.23 g ~m-~, Z = 2, F(O00) = 555.9, ~(CU-K,) = 6.60 cm-'. Final R = 0.059 (334 parameters), R, = 0.074 for 2 754 unique reflections with IFo 2 3olF01 in the range 3" 29 114", average parameter shift is 0.080, and difference Fourier synthesis excursions are within amp;0.4 eA-3.Crystals were obtained from methanol by slow evaporation. Data collection and Structure Re$nement.-Intensity data were collected on a Nicolet R3 diffractometer with graphite monochromatized Cu-K, radiation (h = 1.5418 A)by the 0-28 scan technique with variable scan speed (4-30deg;/min) at room temperature. The intensity data were corrected for background and Lorentz-polarization effects,' but not for absorption. In the final cycles of refinement of (14), a secondary extinction correction (0.0593) was included to minimize the discrepancy between lFol and IFcI of the most intense reflections and led to a significant improvement in R.The crystal structures were solved by direct methods. Atomic co-ordinates, thermal parameters, and scale factors were refined by a 'blocked-cascade' full-matrix least-squares procedure with the SHELXTL l6 program package. The function minimized was Zco(~Fo~-IFcl)*, whereo = a21Fol+ 0.0011F012-1. Scattering factors were from 'International Tables for X-ray Crystallography'; those of oxygen were corrected for anomalous dispersion. Positions of all non-hydrogen atoms were refined anisotropically, and all hydrogen positions were estimated but verified in subsequent difference Fourier maps and included at invariant idealized values in the respective structure-factor calculation. The absolute configurations of both (12) and (14) were determined by least-squares refinement of the parameters of both enantiomers in each structure, giving a ratio of the two final R, values of 1.014 and 1.026 for (12) and (14) respectively.According to Hamilton's statistical test," the enantiomer with the lower R, value has a probability of being correct to a significance level better than 0.5.* Acknowledgements We thank Mr. Hugo A. Cordo for furnishing plant material, Dr. W. F. Haddon and Mr. Roger England for obtaining mass spectral data, and Ms. S. C. Witt for determining NMR spectra. References 1 C. A. Elliger and A. C. Waiss, Jr. 'Insect Growth Inhibitors from Petunia and Other Solanaceous Plants,' in 'Pesticides of Plant Origin,' eds.J. T. Arnason, B. J. R. Philogene, and P. Morand, ACS Symposium Series No. 387, American Chemical Society, Washington, DC, 1989, pp. 188-205. 2 C. A. Elliger, M. E. Benson, W. F. Haddon, R.E. Lundin, A. C. Waiss, Jr., and R.Y.Wong, J. Chem. SOC.,Perkin Trans. 1,1988,711. 3 C. A. Elliger, M. E. Benson, W. F. Haddon, R.E. Lundin, A. C. Waiss, Jr., and R.Y. Wong, J. Chem. SOC.,Perkin Trans. I, 1989,143. 4 C. A. Elliger, M. Benson, R. E. Lundin, and A. C. Waiss, Jr., Phytochemistry, 1988,27,3597. 5 C. A. Elliger, A. C. Waiss, Jr., R. Y. Wong, and M. Benson, Phytochemistry, in the press. J. CHEM. SOC. PERKIN TRANS. 1 1990 6 E. Glotter, I. Kirson, D. Lavie, and A. Abraham, in lsquo;The Withanolides-A Group of Natural Steroids,rsquo; in lsquo;Bioorganic Chemistry,rsquo; vol.11, ed. E. E. van Tamelen, Academic Press, New York, 1978, pp. 57-95. 7 I. Kirson and E. Glotter, J. Nat. Prod., 1981,44,633. 8 W. McFarlane and D. S. Rycroft, Annu. Rep. NMR Spectrosc., 1985, 16,633. 9 lsquo;The Sadtler Standard Spectra,rsquo; Sadtler Research Labs., Inc., Philadelphia, 1978, p. 4033 C. 10 J. E. Page, Annu. Rep. NMR Spectrosc., 1970,3,149. 11 L. J. Bellamy, lsquo;The Infrared Spectra of Complex Molecules,rsquo; 3rd edn., Chapman and Hall, London, 1975, p. 212. 12 A. I. Scott, lsquo;Interpretation of the Ultraviolet Spectra of Natural Products,rsquo; Pergamon Press, Oxford, 1964, (a)p. 375; (b)p. 401. 13 G. C. Levy, R. L. Lichter, and G. L. Nelson, lsquo;Carbon-13 Nuclear Magnetic Resonance Spectroscopy,rsquo; 2nd edn., Wiley, New York, 1980. 531 14 C. A. Elliger, Y. Wong, B. G. Chan, and A. C. Waiss, Jr., J. Chem. Ecol., 1981,7,753. 15 Nicolet XTL Operation Manual, 1980, Nicolet Analytical Instruments Inc., 10041 Bubb Road, Cupertino, CA 95014, USA. 16 G. M. Sheldrick, SHELXTL, lsquo;An integrated System for Solving, Refining and Displaying Crystal Structures from Diffraction Data,rsquo; University of Gottingen, Federal Republic of Germany, 1981. 17 lsquo;International Tables for X-ray Crystallography,rsquo; Kynoch Press, Birmingham, 1974, vol. 4. 18 W.C. Hamilton, Acta Crystallogr., 1965, 18,502. Paper 9/0 1 189G Received 20th March 1989 Accepted 9th August 1989