J. CHEM. SOC. PERKIN TRANS. I 1989 Petuniasterones. Part 2.' Novel Ergostane-type Steroids from Petunia hybrida Vilm. (Solanaceae) Carl A. Elliger," Mabry Benson, William F. Haddon, Robert E. Lundin, Anthony C. Waiss, Jr., and Rosalind Y. Wong U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94790 Four new compounds in the petuniasterone series were isolated from leaves and stems of Petunia hybrida. These all possess the A'74-diene-3-one structure and have a 7~oxygenated functionality. Compounds identified include the 17p-hydroxy derivative of petuniasterone A (9) and its 7-acetate (10) in addition to petuniasterone D (3), which is the orthoacetate of (22R,24R) -7a-22,24,25- tetrahydroxyergosta-1,4-dien-3-one as well as 12~-acetoxypetuniasteroneD 7-acetate (5).Seven unusually substituted ergostane-type steroids have previously been isolated from leaf and stem tissue of Petunia hybrida.' These petuniasterones appear to play a role in the defence of the plant against feeding by certain insects. We now report the isolation and structural characterization of four additional, related materials from the same source. (COSMe (1) R =H (2) R =Ac Chloroform extracts of plant material were fractionated, as previously described,' by initial separation on silica gel followed by preparative h.p.1.c. on several columns. After removal of petuniasterone A (1) and several of the other, more abundant, petuniasterones, it was possible to obtain four new compounds.The least polar of these, compound (3), which we have named petuniasterone D, is closely related to petuniasterone A (1). It has an unsubstituted orthoacetate system on its side chain, analogous to the (methy1thio)carbonyl orthoacetate at positions 22, 24, and 25 of compound (1). The U.V. spectrum of compound (3) shows a band at 246 nm which is consistent with the cross-conjugated dienone of ring and is essentially the same as that of compound (1). The i.r. spectrum shows the expected frequency (vmaX, 1 660 cm-') as in (1). The 'H n.m.r. (Table 1)and ' n.m.r. (Table 2) spectra of compounds (1) and (3) are essentially superposable except for signals associated with the pendant group of the ortho ester moiety. In com- pound (3) the orthoacetate methyl is associated with a 3-proton singlet at 6, 1.56 in the 'H n.m.r.spectrum and with 13C signals at 6, 23.5 and 117.3 (CH, and C, respectively). The corresponding values for triethyl orthoacetate are 6, 1.37 (3 H, methyl), and 6, 20.4 and 114.3 (CH, and C)." In pseudrelone B, having similar functionality, the methyl protons of the orthoacetate appeared at 6, 1.78 and the corresponding carbons were at 6, 25.8 and 117.6.s The ex- cellent correspondence of the characteristic proton couplings in compounds (1) and (3) is significant evidence that identical stereochemical configuration pertains. The n.m.r. spectral shifts and couplings in the corresponding acetates (2) and (4) confirms this point, especially with regard to the a-configuration of the 7-hydroxy substituent.Compound (5) has two acetoxy groups. One of these must be at position 72 as is indicated by comparison of the 'H n.m.r. signals of the 6~ and 6p protons in this compound with those of acetate (4). Partial methanolysis of diacetate (5) gave acetate (6) Me which has a free 7a-OH and which shows the complex and highly characteristic coupling pattern for the hydrogens attached to C-6 cf: compounds (1) and (3), Table 11. The assignment of the second acetoxy group of compound (5) to position 12 was facilitated by the observation that the I3CH, signal at 6 ca. 39 for (3) is no longer present, being replaced by the ,CH signal at 6 74.6. Confirmatory evidence is provided by the magnitude and direction of the substituent effect of the OAc group upon the chemical shifts of carbons in its vicinity.Iida et aL6 have reported A6 of ca. -6, -7, and -9 p.p.m. for carbons 9, 14, and 17 respectively and ca. +5 and +2 p.p.m. for carbons 11 and 13 in methyl 12a-acetoxy-5~-cholanoateus. the unsubstituted compound. We observe similar differences between compounds (4) and (5) or (6). The effect (+0.08 p.p.m.) reported for a 12a-acetoxy group upon the 'H chemical shift of 18-H3 'correlates satisfactorily with our data. Although the geometry of the C-19 methyl group is somewhat altered as a 144 J. CHEM. SOC. PERKIN TRANS. I 1989 Table 1. 'H N.m.r. data' Compound H (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) 1 7.08 C7.111 7.02 d (10) 7.08 d (10) 7.08 d (10) 6.96 d (10) 6.95 d (10) 7.07 d (10) 7.02 d (10) 7.10 C7.061 7.10 d (10) 7.08 d (10) d (10) d (10)2 6.24 6.42 6.22 dd 6.25 dd 6.26 dd 6.25 dd 6.23 dd 6.25 dd 6.23 dd 6.22 6.48 6.26 dd 6.26 dd dd (10, 2) (10, 2) (10, 2) (10, 2) (10, 2) (10, 2) (10, 2) (10, 2) dd (10, 2) (10, 2) (10, 2) 4 6.13 C6.461 6.01 br d 6.14 br t 6.02 br d 6.03 br d 6.14 br t 6.00 d (2) 6.00 br s 6.14 C6.441 6.02 br s 6.00 d (2) br t (ca.2) (ca. 2) (ca. 2) (ca. 2) (ca. 2) (ca. 2) br t (ca. 2) 63 2.50 2.62 2.60 d (14) 2.48 dd 2.60 d (13) 2.60 d (14) 2.50 dd 6-H 6.03 6-H 6.04 2.50 6.623 2.60 d (14) 6-H 6.03 dd (14, 3) (14, 3) (14, 3) dd (10, 2) dd (10, 2) dd (14, 3) dd (10, 2) 6P 2.75 C2.761 2.66 d (14) 2.74 ddd 2.65 d (1 3) 2.66 d (14) 2.76 ddd 2.74 2.74 2.66 d (14) ddd (14,3,2) (14, 3, 2) (14, 3,2) br d (14) 7 4.04 C4.181 5.05 br q 4.04 br s 5.06 br q 5.08 br s 4.06 s 6.23 dd 6.22 dd 4.06 4.24 5.08 br q 6.24 dd br s (ca.3) (ca. 3) (10, 2.5) (10, 2) br s (ca. 3) (10, 2.5) 8 2.26 br t 2.30 br t 2.32 br t (10) (9) (10) 12P 2.02 dt 2.04 m 2.03 dt 2.05 m 5.08 br s 5.07 br t 2.10 dt 4.09 br s 2.10 ddd (12.5, 4) (1 2.5, 4) (3) (13, 3.5) (13, 5, 4) 20 1.78' ca. 1.8 2.36 qd (520-22 4) (520-22 4) (774)22 4.21 c4.341 4.20 dt 4.20 dt 4.18 dt 4.16 td 4.18 td 4.20 dt 4.20 ddd 4.29 ddd 4.30 ddd 4.28 ddd dt (11.5, 4) (11, 4) (11, 5) (10, 4) (8, 4) (8, 4) (11, 5) (10, 7, 4) (11, 7, 4) (11, 7, 4) (10, 7, 4) C5.141 dt (11, 4) 23 1.47 1.50 d 1.50 d ca.1.6' C1.931 dd (J22 23 4) (8) (8) (522-2, 7) (14, 11) (J22-23 10) C2.151 dd (14, 4) 1.52' (J2 2-2 3 11.5) 18-H, 0.76 0.76 s 0.76 s 0.75 s 0.83 s 0.83 s 0.80 s 0.82 s 0.97 0.97 s 1.01 s C0.671 s l.lS s 19-H3 1.23 1.25 s 1.24 s 1.25 s 1:23 s 1.22 s 1.20' s 1.20's 1.22 1.26 s 1.20's 1.24's 1.22's 21-H3 0.96 0.95 d (7) 0.96 d (7) 0.97 d (7) 0.85 d (7) 0.86 d (7) 0.98 d (7) 1.02 d (7) 0.92 0.91 d (7) 0.90 d (7) 1.091 c1.121 d (7) d (7) 26-H3 1.30 1.31 s 1.30 s 1.30 s 1.30 s 1.30 s 1.31 s 1.32 s 1.33 1.33 s 1.34 s C1.351 s C1.441 s 27-H3 1.12 1.12' s 1.16's 1.17's 1.16's 1.16's 1.17's 1.18's 1.14' 1.14's 1.14's C1.251'~ l.lS's 28-H, 1.21 1.20's 1.20's 1.19's 1.18's 1.18's 1.20's 1.18' s 1.22' 1.20's 1.20's 1.20's 1.22's COSMe 2.3 1 2.31 s 2.32 2.31 s C2.281 s C2.251 s CH2C0 3.04 + 3.04 d 3.01 + 3.02 d 2.78 d 3.10 (1413 3.07 (141, (141,C3.44 + 3.10 d d (14) 3.07 d 2.90 d 3.461 (14) C3.451 s (14) (14)each d (14)OAc 2.00 s 2.00 s 2.03 s, 2.04 s 1.98 s 2.08 s Ortho-1.56 s 1.56 s 1.54 s 1.56 s 1.56 s 1.57 s acetate C02Me 3.72 s F Values in CDCl, except in brackets 2H,pyridine: coupling constants (Hz) in parentheses. Values obtained by stepwise decoupling.'Values may be interchanged. consequence of the A-ring dienone, the small substituent effect group without elimination of the group (OH or OAc) at C-7 to (-0.02 p.p.m.) upon its chemical shift is also in agreement with yield triene (8). This lability is not unusual since these that shown by more typical steroids having 12~acetoxy groups.substituents are adjacent to a position from which a proton may It may be noted that those examples for the alternative 11-, 15, be easily abstracted.' However, shift comparisons between or 16-position of acetoxylation exhibit substantially different triene (8) and the corresponding non-hydroxylated triene (7) effects upon their respective angular methyl groups. were also in agreement with reported values for 3Csubstituent It was not possible to effect hydrolysis of the 12-acetoxy effects in the androstane series9 and for bile acids.6 lo J. CHEM. SOC. PERKIN TRANS. I 1989 145 Table 2. 13CN.m.r. datau Compound Carbon (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) 1' 155.6, CH 155.2, CH 155.7, CH 155.2, CH 154.3, CH 154.7, CH 153.0, CH 152.6, CH 155.7, CH 155.3, CH 2' 127.6, CH 127.7, CH 127.6, CH 127.7, CH 128.0, CH 128.0, CH 128.1,* CH 128.2," CH 127.6, CH 127.7, CH 3 185.6, C 185.7, C 185.6, C 185.8, C 185.6, C 185.5, C 186.4, C 186.4, C 185.6, C 185.7, C 4' 127.1, CH 126.6, CH 127.1, CH 126.6, CH 126.8, CH 127.4, CH 123.7, CH 123.8, CH 127.1, CH 126.6, CH 5' 164.5, C 163.9, C 164.6, C 163.9, C 163.1, C 163.8, C 162.6, C 162.5, C 164.6, C 163.9, C 6' 41.0, CH, 37.3, CH, 41.0, CH, 37.3, CH, 37.2, CH, 40.9, CH, 138.4, CH 138.1, CH 41.0, CH, 37.3, CH, 7' 69.5, CH 72.0, CH 69.5, CH 72.0, CH 71.5, CH 69.2, CH 127.6,* CH 127.7,' CH 69.1, CH 71.7, CH 8 38.5, CH 38.3,* CH 38.5, CH 38.4,* CH 37.8, CH 39.0, CH 38.1, CH 38.2, CH 40.8, CH 39.3, CH 9 44.4, CH 45.1, CH 44.4, CH 45.2, CH 38.2, CH 37.9, CH 52.0,t CH 42.8,t CH 44.3,* CH 45.3,* CH 10 43.4,* C 43.2,t C 43.4,* C 43.2,T C 42.5, C 42.8, C 41.2, C 40.9, C 43.4 C 43.2, C 11 22.5, CH, 22.4, CH, 22.5, CH, 22.4, CH, 26.8, CH, 26.8, CH, 21.8, CH, 29.3, CH, 22.5, CH, 22.5, CH, 12' 39.0, CH, 38.8, CH, 38.9, CH, 38.8, CH, 74.6, CH 74.8, CH 39.3, CH, 72.3, CH 36.4, CH, 36.4, CH, 13 42.9,* C 43.0,t C 42.9," C 43.0,t C 45.3, C 45.2, C 43.3, C 47.4, C 48.3, C 48.3, C 14 49.9, CH 49.6, CH 49.9, CH 49.7, CH 42.7, CH 43.0, CH 48.3,T CH 41.5,T CH 44.2,* CH 44.1,* CH 15 23.8, CH, 23.8, CH, 23.8, CH, 23.8, CH, 23.0, CH, 23.1, CH, 23.8, CH, 23.2, CH, 23.1, CH, 23.3, CH, 16 27.2, CH, 27.0, CH, 27.2, CH, 27.1, CH, 26.2, CH, 26.4, CH, 27.2, CH, 26.2, CH, 33.5, CH, 33.4, CH, 17 52.0, CH 51.9, CH 52.0, CH 52.1, CH 43.9, CH 43.9, CH 53.2, CH 45.2, CH 85.1, C 84.9, C 18' 11.8, CH, 11.7, CH, 11.8, CH, 11.7, CH, 12.2, CH, 12.2, CH, 11.9, CH, 12.9, CH, 15.3, CH, 15.3, CH, 19' 18.3, CH, 18.4, CH, 18.2, CH, 18.5, CH, 18.1, CH, 18.0, CH, 20.0,: CH, 20.0,$ CH, 18.3, CH, 18.5, CH, 20' 39.8, CH 38.4," CH 39.7, CH 38.5,* CH 39.9, CH 39.7, CH 38.5, CH 39.0, CH 41.5, CH 41.7, CH 21 12.5, CH, 12.5, CH, 12.6, CH, 12.7, CH, 11.6, CH, 11.6, CH, 12.7, CH, 11.7, CH, 14.6, CH, 14.4, CH, 22 70.2, CH 70.1, CH 69.8, CH 69.8, CH 69.6, CH 69.6, CH 69.8, CH 70.4, CH 71.8, CH 71.7, CH 23' 30.3, CH, 30.3, CH, 30.2, CH, 30.3, CH, 30.1, CH, 30.1, CH, 30.2, CH, 31.8, CH, 31.5, CH, 31.3, CH, 24 82.9,t C 82.8,: C 82.5,T C 82.5,: C 82.4,* C 82.5,* C 82.5,s C 82.6,$ C 83.4,t C 83.5,t C 25 81.8,t C 81.7J C 81.3,'f C 81.3,: C Sl.l,* C 81.2,* C 81.3,s C 81.4,s C 82.1,f C 82.1,t C 26 19.9, CH, 19.9, CH, 19.9, CH, 20.0, CH, 19.9, CH, 20.0, CH, 20.5,$ CH, 20.4,: CH, 19.7, CH, 19.7, CH, 27 20.4,: CH, 20.4,s CH, 20.5,: CH, 20.5,$ CH, 20.5,T CH, 20.5,T CH, 20.7,: CH, 20.5,: CH, 20.2,: CH, 20.5,$ CH, 28 24.9,: CH, 24.9,s CH, 25.2,: CH, 25.2,4 CH, 25.1,T CH, 25.2,t CH, 25.2,: CH, 25.1,: CH, 24.8,$ CH, 24.8,$ CH, Ortho cstcr 115.3,C 115.3,C 117.3,C 117.3,C 117.3,C 117.3,C 117.3,C 117.3,C 114.9,C 115.0,C ,CH,CO 50.3m CH, 50.2, CH, 50.2, CH, 50.2, CH, l3COS 193.3, C 193.2, C 193.2, C 193.2.C SMe 12.0, CH, 12.0, CH, 12.0, CH, 12.0, CH, Acetate 170.2, C 170.3, C 170.2, C, 170.3, C 170.3, C 170.2, C Acet at c 21.0, CH, 21.1, CH, 21.1, CH,, 21.2, CH, 21.0, CH, 21.2, CH, Orthoacetate 23.5, CH, 23.5, CH, 23.4, CH, 23.5, CH, 23.5, CH, 23.5, CH, In p.p.m.from internal SiMe, for CDCl, solutions. Values with like superscripts in each column may be interchanged. Assigned by C H correlation spectroscopy in compounds (l),(2), (5),(7),(8),and (9),except for C-12 in (9). Comparative 'H n.m.r. shifts of 18-and 19-H, between trienes Compound (9)is similar to petuniasterone A (1) but bears an (7) and (8) were not definitive. We have assigned the sI-additional OH on a quaternary centre as is shown by the I3C configuration to the 12-acetoxy group of compounds (5)and (6) n.m.r. spectrum (6, 85.1) and by the absence of a proton signal on the basis of the multiplicity and magnitude of the 12-H signal gerninal to this group.Acetylation of (9)yields compound (lo), (6 5.07, t, J 3 Hz) indicative of two gauche proton couplings which is also found in the plant. The 'H n.m.r. spectra of this necessitating an axial orientation (x) for the acetoxy group. pair show the same comparative features for the C-6 protons as Me TMe (7) R = H (9) R = H (8) R = OH (10) R = AC do (1) us. (Z),and (3) us. (4). Treatment of compound (10) with methoxide gives triene (11) in which the thiol ester moiety has been converted into methyl ester. The Sp hydrogen of trienone (11) appears as a triplet (6 2.32, J 10 Hz) in the lsquo;H n.m.r. rCOzMe (11) spectrum thereby confirming the presence of the axial hydrogens at C-9 and C-14.The remaining OH must therefore be attached to C-17. In pentadeuteriopyridine solvent, the lsquo;H n.m.r. spectrum of compound (9) exhibits a quartet of doublets at 6 2.36 (J 7 and 4 Hz) for 20-H. Irradiation at this position caused the 2t-H, doublet to collapse to a singlet and the 22-H signal (6 5.14) to change from a double triplet to a double doublet (J 11 and 4 Hz) in agreement with the assigned position. Decoupling of the 22-H proton by irradiation at 6 5.14 resulted in simplification of the 20-H signal to the quartet (J 7 Hz). This result is in accord with the absence of a proton at C-17 which therefore must be the position of hydroxylation. In an effort to assign the stereochemistry at C-17 we considered the magnitude of the substituent-generated proton chemical-shift difference between the C-18 methyl groups of compounds (1) and (9).The observed shift difference attributable to the 17-hydroxy group (+0.21 p.p.m.) of (9) us. (1) in CDCI, is much greater than the values reported by Page rsquo; for either x or p 17-hydroxy substituents (0.06 and 0.03 p.p.m. respectively) in this solvent. In pentadeuteriopyridine, the observed C-18 methyl signal of compound (9) is shifted 0.51 p.p.m. to lower field compared with that in compound (I), but it must be noted that the C-18 methyl group of compound (1) appears at 6, 0.67 in this solvent, which is surprisingly higher. The reported chemical-shift differences of 18-H, for C- 17 CC-and p-hydroxy substituents in pentadeuteriopyridine are 0.00 and 0.20 p.p.m.respectively.rsquo; It appears that the additional oxygenation of the side chain may be influencing the association of pyridine with the C-17 hydroxy group. Clearly the large downfield shift in pentadeuteriopyridine of 22-H for compound (9) (A6 +0.85 p.p.m.) indicates that the side chain can assume a conformation in close proximity to the 17-OH which is possibly stabilized by hydrogen bonding. Where there is no C-17 hydroxy group, i.e. structure (I), 22-H is only shifted 0.13 p.p.m. downfield in pentadeuteriopyridine. Although the large magnitude of the substituent shift for the C-18 methyl implies close proximity to the 17-OH and thereby a p orientation for that group, the perturbing influence of the side chain lends significant uncertainty to the assignment.We therefore chose to subject compound (9) to X-ray analysis. The molecular stereochemistry of compound (9) with its 17p- OH group was unequivocally determined in this manner. The established molecular conformation with the atom-numbering system used in the X-ray investigation is shown in Figure 1 and its stereoscopic view is presented in Figure 2. The final atomic co-ordinates are listed in Table 3. The asymmetric centres C(20), C(22), and C(24) have the R configuration. Molecules of J. CHEM. SOC. PERKIN TRANS. I 1989 34q3* 39 Vlsquo;y15 21 ldquo;38 Figure 1. Perspective view of compound (9) with the crystallographic numbering scheme. Open bonds represent double bonds; small and large shaded circles represent oxygen and sulphur atoms, respectively Figure 2.Stereoscopic view of compound (9) compound (9) are weakly held together in the crystal structure by van der Waals interaction and two interniolecular hydrogen bonds 0(38) O(39) 2.94 and O(34) --O(40) 3.32 A. Experimental M.p.s were taken with a Fisher-Johns apparatus and are corrected. Optical rotations were obtained on a Perkin-Elmer model 241 automatic polarimeter at ca. 26deg;C using 1 concentration in chloroform solutions. 1.r. spectra were recorded on a Perkin-Elmer model 237 spectrophotometer and refer to chloroform solutions; U.V. spectra were taken on a Cary 2 19 spectrophotometer using solutions in methanol; rsquo;H n.m.r.spectra were obtained at 90 MHz on a Varian EM-390 instrument or at 200 MHz on a Nicolet NT-200, and ldquo;C n.m.r. spectra were taken at 50 MHz on the latter instrument. N.m.r. assignments were facilitated by decoupling methods and by use of two-dimensional proton-proton and carbon-proton correl-ation techniques.rdquo; Mass spectra were run using a VG Micromass 70/70HS instrument either by electron impact or by using ammonia chemical ionization. Listed relative abundances are those of the chemical ionization spectra. X-Ray intensities were collected with a Nicolet R3 automatic diffractometer at room temperature. Microanalyses were determined by Galbraith Enterprises, Knoxville, Tennessee. Silica gel Si60, 7amp;-230 mesh, was from E. Merck; Sephadex, LH-20, from Pharmacia Co.; h.p.1.c.columns were from Rainin J. CHEM. SOC. PERKIN TRANS. I 1989 Table 3. Atom co-ordinates (x lo4) for compound (9), with e.s.d.s in Y Y z 5 399(3) 7 908 -2 618(2) 6 122(2) 8 509(5) -2 638(2) 6 760(2) 7 528(5) -2 790(2) 6 581(2) 5 859(5) -2 885(2) 5 855(2) 5 262(5) -2 865(2) 5 709(2) 3 506(5) -2 927(2) 5 386(2) 2 853(5) -2 399(2) 4 634(2) 3 731(5) -2 291(2) 4 788(2) 5 505(5) -2 222(2) 5 156(2) 6 250(5) -2 749(2) 4 042(2) 6 386(5) -2 091(2) 3 658(2) 5 670(5) -1 583(2) 3 492(2) 3 903(5) -1 672(2) 4 281(2) 3 105(5) -1 759(2) 4 1 lO(3) 1339(5) -1 732(2) 3 451 (3) 1 202(5) -1 334(2) 3 255(2) 2 877(5) -1 138(2) 2 832(2) 3 676(6) -2 225(2) 4 525(3) 6 276(7) -3 345(2) 3 708(2) 3 362(5) -506(2) 3 869(3) 1971(6) -78(2) 3 292(2) 4 596(5) -175(2) 3 879(2) 5 607(5) 248(2) 3 566(2) 7 176(5) 441(2) 2 792(2) 7 037(6) 723(2) 2 791(3) 5 721(7) 1182(2) 2 540(3) 8 591(7) 957(2) 4 213(3) 8 183(6) 765(2) 2 562(2) 6 989(5) -317(2) 1976(2) 7 861(6) -785(2) 1 266(3) 6 820(6) -1 OOl(2) -198(3) 5 559(8) -952(3) 480( 1) 7 006(2) -609( 1) 1 250(2) 5 963(6) -1 434(2) 3 223(2) 7 944(4) -123(1) 2 216(1) 6 636(4) 195( 1) 2 793(1) 5 635(3) -582(1) 5 957(2) 2 923(4) -1 865(1) 7 414(2) 8 065(4) -2 832(2) 2 409(2) 2 845(4) -1 113(1) 0 2 228(8) 0 701(4) 1487(8) 122(3) 1411(5) 2 473( 1 1) 172(4) 717(8) 390(26) 401(7) Instruments, Alltech Associatcs, and Whatman, Inc.; solvents were h.p.1.c.grade and were pumped using an Altex/Beckman Model 110A pump.Detection was by U.V. at 254 nm using an Altex Model 150 monitor equipped with a 0.5 mm pathlength preparative cell. Plant Materid-Petunia hyhrida Vilm., commercial variety 'Royal Cascade,' was grown in outdoor beds in Albany, California. Leaf and stem material was harvested at intervals during the growing seasons of 1986 and 1987. Isolation Procedure.-This was carried out on freeze-dried plant material as previously described' by preliminary enrichment of the petuniasterone fraction on silica gel followed by preparative h.p.1.c. Columns and conditions were as follows: Rainin Dynamax Silica, 21.4 mm dia. x 250 mm with guard, 20 propan-2-01 in hexane; Alltech R-Sil C-18, 10 mm dia. x 250 mm, 30 water in acetonitrile; and Whatman Partisil-10 PAC, 9 mm dia.x 500 mm, 10 propan-2-01 in hexane. Products were eluted as indicated in Table 4. Table 4. Elution zone (ml) Compd. Dynamax silica R Sil C-18 PAC (3) 140-160 45-60 60-70 (5) 23G-265 3-3 100-120 (9) 26amp;310 28-34 2 10-240 (10) 21amp;250 2543 145-160 Compound (3), Petuniasterone D.-M.p. 209-21 2 "C (from heptane-EtOAc); a(h/nm) +47.7"(589), +49.6"(578), +55.3" (546), +79.4" (436), and +38.9" (365); v,,,, 3 450br (OH) and 1 660 cm-I (conj. CO); h,,,, 246 nm (log E 4.18); m/z485 (MH', 86) and 467 (MH' -H20, 12) (Found: C, 74.75; H, 9.0. C30H4405 requires MH', 485; C, 74.34; H, 9.15). Compound (5), 12a-AcetoxypetuniasteroneD 7-Acetate.-M.p. 241-243 "C (from heptane-EtOAc); x(h/nm) +60.3" (589), +62.6" (578), +69.8" (546), +105.8" (436), and +97.6" (365); v,,,.1 730 (OAc) and 1665 cm-' (conj. CO); Amax, 244 nm (loge 4.15);mlz 585 (MH+, 18), 525 (MH' -HOAc, 19), and 465 (MH' -2 HOAc, 6) (Found: C, 69.9; H, 8.8. C,,H4,0, requires MH', 585; C, 69.84; H, 8.27). Compound (9), 17P-HydroxypetuniusteroneA.-M.p. 145-147 "C with gas evolution (from heptane-EtOAc); x(h/nm +44.2" (589), +46.1" (578), +52.0" (546), +83.6" (436), and +82.6" (365); vmax,3 475br (OH), 1 685 (COSMe), and 1 665 cm-I (conj. CO); I,,,. 242 nm (log E 4.20); m/r 575 (MH', 13:;) and 557 (MH' -H20, 21) (Found: C, 66.4; H, 7.3; S, 5.5. C3,H4,07S requires MH', 575; C, 66.87; H, 8.07; S, 5.58). Compound (lo), 17p-Hydroxypetuniusterone A 7-Acetute.-a(h/nm) +12.4" (589), +12.7" (578), +13.5" (546), +14.9" (436), and -35.9" (365); vmaX,3 500br (OH), 1730 (ester), 1 685 (COSMe), and 1 665 cm-' (conj.CO); h,,,. 243 nm (log E 4.15); m/z 617 (MH', 84), 599 (MH' -H,O, 63), and 557 (MH' -HOAc, 5.3) (C34H4,O8S requires MH', 617). Petuniasterone A Acetate (2).-Petuniasterone A (1) (56 mg) was dissolved in acetic anhydride (5 ml) and the mixture was warmed at 100 "C for 15 h. Most of the Ac,O was removed at 80deg;C on a rotary evaporator and MeOH (5 ml) was added. After being kept for 1 h at room temperature, the mixture was taken to dryness and the residue redissolved in CH,C12 (1 ml). Chromatography of this solution on the PAC column gave pure acetate (2) (50 mg), retn.vol. 55-65 ml (10 propan-2-01- hexane), m/z 601 (MH', 83) and 541 (MH' -HOAc, 14) (C34H4807S requires MH', 601). Petuniasterone D Acetate (4).-Petuniasterone D (3) was converted into the acetate as described above. H.p.1.c. on the PAC column gave pure acetate (4), retn. vol. 50-58 ml (10 propan-2-ol-hexane). Acetylation of Compound (9).-Conversion of compound (9) to the 7-acetate was carried out as described above. H.p.1.c. on the RSil C-18 column, retn. vol. 3amp;38 ml (30 water-MeCN) gave pure monoacetate that was chromatographically and spectroscopically identical with compound (10). Conversion of Dienone (3) into Trienone (7).-Petuniasterone D (3) (27 mg) was dissolved in pyridine (1.0 ml) and methanesulphonyl chloride (0.1 ml) was added.The mixture was warmed to -50 "C for 15 min, and the development of a slight brown colour was observed. Most of the pyridine was 148 removed under reduced pressure and the remaining material was dissolved in MeOH (25 ml). After evaporation of MeOH, the crude product was dissolved in CH,CI, (1.0 ml) and the solution was filtered through a 0.5 pm PTFE filter. H.p.1.c. on the PAC column gave mainly one substance, retn. vol. 110-1 30 ml (10 propan-2-ol-hexane), showing a 'H n.m.r. signal at 6 3.00 (CDC1,; mesyl ester). To the mesyl ester was added 0.5~ NaOMe-MeOH (1 ml) and this mixture was warmed briefly to reflux then kept for 1 h at ambient temperature. Acetic acid (0.025 ml) was added and the solution was taken to dryness.The mixture was redissolved in CH,Cl, (1.0 ml), and the solution was filtered through a 0.5 pm PTFE filter and chromatographed on the PAC column to give pure trienone (7), retn. vol. 38-46 ml (10 propan-2-ol-hexane) (13 mg); h,,,, 223, 257, and 301 nm; m/z 467 (MH', 97) (C30H4104 requires MH', 467). Treutment of Diucetate (5) with Na0Me.-Compound (5) (20 mg) was warmed at reflux for 30 min with 0.5~NaOMe-MeOH (1 ml). Acetic acid (0.05ml) was added and the mixture was evaporated. The residue was dissolved in CH,Cl, (1 ml), and the solution was filtered through a 0.5 pm PTFE filter and chromatographed on a Whatman M-9 silica column, 9 mm dia. x 50 cm, with 20 propan-2-ol-hexane to give two products (6), retn. vol.52-62 ml and (S), retn. vol. 82-100 ml. Compound (6) resulted from methanolysis of the C-7 acetate, and had h,,,, 245 nm (log E 4.15); nz/z 543 (MH+, 15) and 483 (MH' -HOAc, 8.0) (C3,H4@, requires MH', 543). Compound (S), m.p. 270 "C (decomp.) (from hexane-Pr'OH), is the trienone resulting from elimination of the 7-acetate and methanolysis of the 12-acetoxy group; it had h,,,, 224,258, and 302 nm; nz/z 483 (MH', 49) (C30H4205 requires MH', 483). Treutment of Acetate (10) with Na0Me.-Compound (10) (20 mg) was treated with 0.5~NaOMe-MeOH (1 ml) as in the above case. After work-up, the mixture was chromatographed on the M-9 silica column to give two components. retn. vols. 58-66 ml (11) and 108-128 ml (20 propan-2-ol-hexane). The material of greater retention volume results from methanolysis of the C-7 acetate to the alcohol with simultaneous formation of methyl ester on the side chain, and had m/z 559 (MH', 11) and 541 (MH' -H,O, 9.2) (C32H4h08 requires MH+, 559).Compound (11) is the trienone resulting from elimination of the 7-acetoxy group with concomitant conversion of methyl thiolester into methyl ester; it had h,,,. 225, 251, and 298 nm; nzjr 541 (MH+, 53) and 523 (MHf -H,O, 8.5) (C32H440, requires MH', 541). Crystal Duta.-Compound (9) with solvent (ethyl acetate) of crystallization, C3,H,,0,S~0.5C4H,0,: M = 618.9, monoclinic, space group 12; a = 16.932(9), b = 8.514(3), c = 22.600( 12) A, = 98.79(3)", V = 3 219.7 A3, Z = 4, D, = 1.28 g crnp3,Do = 1.30 g ~m-~, F(OO0) = 1 335.9, ~(CU-K,) = 12.6 cm-'; colourless prismatic crystals were obtained from ethyl acetate by slow evaporation.Dutci Collection and Structure Rejineme1zt.-Intensity data were measured on a Nicolet R3 diffractometer with graphite- monochromatized Cu-K, radiation (h 1.5418 A) by the 8-20 scan technique with variable scan speed (4-30" min-') at room temperature. The lattice constants were refined by least-squares fit to setting angles of 20 independent reflections measured on the diffractometer. Intensity data were recorded as space group C2 with lattice constants u = 26.087, b = 8.514, c = 22.600 A, and p = 140.10". Two standard reflections were monitored periodically for crystal and instrument stability; no significant change in their intensities was noted during the course of the experiment. Intensity data were corrected for background, J.CHEM. SOC. PERKIN TRANS. I 1989 Lorentz, and polarization factors,'* but not for absorption or secondary extinction. The unit-cell dimensions and all hkl indices were transformed to space group 12; the crystal structure was 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 SHELXTL13 program package on a Nova-3 computer. The function minimized was Crv(IF,I -IFc1)2,where w = 021FoI + 0.001lFo12-1.Scattering factors were from 'International Tables for X-ray Crystallography,' l4 those of oxygen and sulphur being corrected for anomalous dispersion.Least-squares refinement of atomic parameters of the 40 non-hydrogen atoms with anisotropic temperature factors converged at R = 0.15 1. Inclusion of 46 hydrogen atoms, whose positions were calculated, and of hydroxy groups, which were located on subsequent difference Fourier maps, in the structure-factor calculation with isotropic temperature factors and constraint on their positional parameters, reduced the R index to 0.141. Both the n.m.r. analysis and the observed density indicate that a solvate is present in the crystal structure. The n.m.r. spectrum of crystalline (9) dissolved in CHC1, indicated that there is half a molecule of ethyl acetate per formula unit of (9). The subsequent difference Fourier map revealed a cluster of four residual peaks, C(41), C(42), C(43), and C(44) with significantly high electron densities of 6.0,4.2,3.3, and 1.6 e A-3,respectively. These four peaks are incompatible with the geometry of possible solvate molecules; however, the distances C(41)-C(42) and C(42)-C(44) of 1.34 and 1.13 A, respectively, are close to those for C-0 and C=O.Inclusion of these peaks, with isotropic temperature factors and assuming that they were all carbon atoms, in the least-squares refinement reduced the R value to 0.063 and R, = 0.071 (375 parameters for 3 049 unique reflections with IF, 2 3olFoI in the range 3" I28 I 110"). At convergence, the four solvent atoms exhibit extremely high thermal vibrational motions, the average parameter shifts were less than 0.090, and the final difference Fourier synthesis excursion are within 0.7 e k3.The first five residual peaks are within the range (0.7-0.5 e k3)and (1.1-0.4 A) from the four solvent atoms.The structure refinement indicated that solvent of crystallization is present in the crystal structure but failed to establish the definite crystallographic position of the complete solvate molecule from the X-ray data presumably owing to poor intensity data. Crystals of compound (9),crystallized from ethyl acetate, appeared opaque and slightly fractured; nevertheless, they seemed to remain stable during data collection. Attempts to improve crystal quality by recrystallization from a variety of solvents were unsuccessful.There are no obvious intermolecular hydrogen bonds between the parent and solvent molecules; this perhaps accounts for the fact that the ethyl acetate molecules are partially disordered. An attempt to determine the absolute configuration by change of chirality in the structure refinement did not change R and R, significantly.* Acknowledgements We thank Ms. S. C. Witt for collection of n.m.r. data and Mr. R. England for mass spectral determinations. * Supplementary data (sce section 5.6.3 of Instructions for Authors, in the January issue). Tables of bond lengths and angles, anisotropic thermal parameters with their estimated standard deviations for the non-hydrogen atoms, and positional and thermal parameters of hydrogen atoms have been deposited at the Cambridge Crystallographic Data Centre.References 1 For Part 1, see C. A. Elliger, M. E. Benson, W. F. Haddon, R. E. Lundin, A. C. Waiss, Jr., and R. Y. Wong, J. Cliem. SOC.,Perkin Truns. 1, 1988, 711. J. CHEM. SOC. PERKIN TRANS. I 1989 2 A. I. Scott, lsquo;Interpretation of the Ultraviolet Spectra of Natural Products,rsquo; Pergamon Press, Oxford, 1964, p. 406. 3 lsquo;The Sadtler Standard N.M.R. Spectra,rsquo; Sadtler Research Labs., Inc., Philadelphia, 1974, p. 17336141. 4 lsquo;The Sadtler Standard Carbon-1 3 N.M.R. Spectra,rsquo; Sadtler Research Labs., Inc., Philadelphia, 1976, p. 718C. 5 D. A. H. Taylor, Pliyrochemistry, 1979, 18, 1574. 6 T. Iida, T. Tamura, T. Matsumoto, and F. C. Chang, Org. Mugn. Reson., 1983, 21, 305. 7 J. E. Page. Annu. Rep. NMR Spectrosc., 1970, 3, 149. 8 T. H. Lowry and K. S. Richardson, lsquo;Mechanism and Theory in Organic Chemistry,rsquo; Harper and Row, New York, 1976, pp. 358- 362. 9 J. W. Blunt and J. B. Stothers, Org. Mugn. Reson., 1977, 9, 439. 10 D. V. Waterhous, S. Barnes, and D. D. Muccio, J. Lipid Res., 1985, 26, 1068. 11 W. McFarlane and D. S. Rycroft, Annu. Rep. NMR Spectrosc., 1985, 16, 293. 12 Nicolet XTL Operation Manual, 1980, Nicolet Analytical Instru- ments Inc., 10041 Bubb Road, Cupertino, CA 95014, U.S.A. 13 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. 14 lsquo;International Tables for X-ray Crystallography,rsquo; Kynoch Press, Birmingham, 1974, vol. 4. Received 17th November 1987; Paper 712033
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