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Raimondalone, a sesquiterpene from a cotton interspecific hybrid
Pergamon
0031-942y94)EOO62-W
RAIMONDALONE,
Phytahrmirfry.
A SESQUITERPENE FROM INTERSPECIFIC HYBRID
Vol. 36. No. 4. pp. 95>9%. 1994 Eknn Sciena Ltd Pnnted m Great Briram 0031 9422/w S700+0.00
A COTTON
ROBERT D. STIPANOVIC, HYEONG L. KIM,+ DAVID W. ALTMAN, ALOIS A. BELL and RUSSELL J. KOHEL USDA, ARS. Southern Crops Research Laboratory, College Station, TX 77845, U.S.A.; *Texas A&M University, Department of Veterinary Physiology and Pharmacology. College Station, TX 77843, U.S.A.
(Receir;edin reuisedlorm 6 December 1993) Key Word Index-Gossypium quinone.
hirsutum; G. raimondii; Malvaceae; cotton; sesquiterpene; l,Cnaphtho-
Abstract-A new sesquiterpenoid, raimondalone (8-formyl-6,7dihydroxy-S-isopropyl-2-methoxy-3-methyl-1,4naphthoquinone), has been isolated from the leaves of a cotton plant derived from an interspecific hybrid (G. hirsutum x G. raimondii). The structure of this compound and the i3C chemical shifts of related compounds were determined using 2D HMBC NMR spectrometry.
INTRODUCTION
Cotton produces a complex assortment of sesqui- and sesterterpenoids [l] in the subepidermal pigment glands in the foliage. Gossypium species and their interspecific hybrids provide an array of plants which display novel chemistries [2, 33 and resistance characteristics which are relevant to protecting cotton from pests, including the wilt pathogen Verticillium dahliae [43 and Lepidopteran insects [S]. We have been particularly interested in hybrid between commercial Upland cotton (Gossypium hirsutum L.) and the wild species G. raimondii Ulbr. The former produces the sesquiterpenoid quinone p-hemigossypolone (2) [6] and the latter produces the sesquiterpenoid raimondal(6) [7J. We report herein the identification of a new sesquiterpenoid, which we call raimondalone, isolated from the foliage of a progeny plant obtained by self-pollination of a hexaploid parent derived from a G. hirsutum x G. raimondii hybrid. RESULTS AND Dl!XUSSlON
Raimondalone (1) gave a parent ion at m/z 304 (74%) which by high resolution mass measurement (304.09351, talc. 304.09469) indicated a molecular formula of ClgHi606. It exhibited UV absorption maxima in ethanol-HCI (log E)at 212 (4.66), 273 (4.56), 303 (4.41) and 420 (3.54) nm. The ‘H NMR showed the presence of an isopropyl group (64.17, lH, sept, J = 7.0 Hz; 6 1.37,6H, d, J =7.0 Hz), a vinyl methyl group (62.02, 3H, s) and a methoxyl group (64.0,3H, s). An aldehyde group (6 10.64, lH, s) hydrogen bonded to a phenol (612.86, lH, s) was also evident. A phenolic group accounts for the remaining proton (66.57, lH, s). The UV spectrum, the dark red colour of the crystals, and the presence of two peaks in the PHY 36:4-J
13C NMR at 6 182.3 and 187.5 indicated the presence of a quinone. The ‘HNMR was in general agreement with that of other p-naphthoquinones isolated from cotton such as hemigossypolone (2) and hemigossypolone-6methyl ether (3) [8]. Based on biosynthetic considerations, the position of the aldehyde group, isopropyl and methyl group could be established as shown in 1. The presence of a hydrogen bonded phenol group shows this group is ortho to the aldehyde. Since no aromatic protons are present, it remained only to assign the position of the quinone carbons, and the remaining phenol and methoxyl groups. Based on a DEPT experiment, and unique chemical shifts, several peaks in the 13C NMR spectrum were readily assigned. These include the isopropyl methyls (C-13, C-14,6 19.7, q) and methine(C-12.628.4, d) groups, the methoxyl group (660.5. q), a methyl group (C-l 5,695, q), the aldehyde carbon (C-l 1,6 198.3, d) and the carbon to which the aldehyde group is attached (C-8, S 115.8. s). Other assignments, such as the quinone carbonyl carbons (6 182.3 and 187.5), were more difficult to assign. In 1,4naphthoquinones, the carbonyl carbons generally occur at lower field ( - 6 187) as compared to 1,2-naphthoquinones (-6 182) [9]. but in raimondalone the quinone carbonyls appeared in both regions (6 187.5 and 182.3). These and the remaining peaks in the ’ 3C NMR spectrum were ultimately assigned using the HMBC spectrum [lo] and by comparisons to related compounds. Assignments of the naphthoquinone carbons were based on the following observations. A cross-peak from the methoxy protons to the signal at 6 156.3 identified the carbon to which the methoxy group was attached. The vinyl methyl protons showed a cross-peak to this carbon, indicating that the methoxyl group and methyl group are ortho to one another, and also correlated to peaks at 6 13 1.6 and 187.5.
R. D. STIPAHOWC-
954
o\
et
at.
11
'CH
0
1)RI=H,R~=OCH3 2)Rt,R2=H 3)Rl=CH3 ,Rz=H 0
I \
0
I
/
ti
A
*5 Since the peak at 6 187.5 has been assigned to the quinone carbonyl, this must be C-4 and the peak at 6 f 31.6 must be C-3. The aromatic carbon C-S was assigned to the peak at 6 141.4 because of long range coupling to the isopropyl methyl protons. The methine proton of the isopropyl group showed coupling to peaks at 5 127.4 and 149.2 in addition to coupling to C-5. The chemical shift of 6 149.2 allows its assignment to the oxygenated carbon C-4, and thus C-10 is at fi 127.4. The aldehyde proton showed very strong coupling to 6115.8 confirming its assignment lo C-8 and strong coupling to 6151.8 and weak coupling to 6149.2. The peak at 6149.2 has been assigned to C-6 because of its coupling to the isopropyl methine proton and thus the remaining oxygenated aromatic carbon at d 151.8 was C-7. The remaining quinoid carbonyl (C-l) was assigned to the peak at fi 182.3. C-l is shifted upfield from its normal position as a p-quinone carbonyl because of the o-methoxyl group [l I]. C-9 is assigned to the remaining peak at b 126.4. No cross-peaks to the phenolic proton at 6 12.86 were observed. To confirm the original observation that 1,2-quinones are shifted upfirlti compared to IA-quinones, the HMBC spectra of several related compounds were examined. These spectra were used to determine or confirm the chemical shifts of ~-hemigossypolone (2) [12], ~-hemigossypolone-6-methyl ether (3) [ 121. o-hemigossypolone (4) and mansonone C and are compared to those of 1 in Table 1. The chemical shifts of the quinonoid carbonyls are shown in Table I. The carbonyls of the p-quinone appear at 6 186 or lower field and those of the o-quinone at d 183 or higher field. The HMBC spectra revealed an interesting observation (Table 2). The aldehyde protons in compounds l-4 show no coupling to C-9 but do show coupling to C-7 and C-8, and in 1-3, there is also coupling
Table 1. ‘%NMR
c 1 2
3 4 5 6 7 8 9 10 11
12 13. 14 15 OMe
6
spectral data for compounds CDCI J)
l-5
I
2
3
4*
5
182.3 156.3 13f.h 187.5 141.5 149.2 151.8
186.4 133.9 149.2 187.5 141.6 149.0 152.2 1IS.8 127.3
186.3 133.6 t 49.6 187.0 150.7 152.7 158.7 117.2 130.7 126.9 198.3 28.8 210
182.3 183.0 134.5 138.1 138.9 151.5 151.1 119.8 125.5 129.8 199.4 28.3 20.6 15.4
182.3 182.0 134.9 137.9 145.3 131.9 134.1 142.9 129.3 132.4 22.7 28.3 23.7 15.9
115.8 126.4 127.4 198.3 28.4 19.7 9.5 60.5
127.6 199.9 28.6 19.7 16.4
16.5
(in
60.8
*In acetone-d,.
to C-6. This results from the confined orientation of the aldehyde proton due to hydrogen bonding between the carbonyl and the o-phenolic group. The methyl group at C-l 1 in mansonone C (5) shows normal coupling to C-7, C-8 and C-9. Raimondalone is the first compound isolated from the group of 51 unknown UV-absorbing compounds detected in an earlier survey of Gossypium species [2]. Raimondalone was in the highest concentration in this group and was uniquely found in progeny from G. ~jrs~~~rn crossed with G. raimondii. Analysis of F,, F, and backcross progeny from a cross between a Tamcot CAMD-E line homozygous for raimondal production and the Texas
A sesquiterpene from a cotton interspecific hybrid Table 2. ‘H-l 1and 13Ccoupling as indicated in HMBC spectra in compounds l-5 Compound
6H-11
Cross-peak*
C
1
10.64
2
10.76
3
10.57
4
10.53
5
2.58
115.8(vs) 149.2 (w) 151.8(s) 115.8 (vs) 149.0 (w) 152.2 (s) 117.2 (YS) 152.7 (w) 158.7 (s) 119.8 (vs) 151.1 (s) 129.3 (s) 134.1 (s) 142.9 (s)
8 6 7 8 6 7 8 6 7 8 7 9 7 8
*vs = Very strong, s = strong, and w = weak coupling.
marker stock ‘TM-I’ showed segregation of raimondal was consistent with this character being controlled by two dominant genes with epistasis (i.e. hydroxylation at C-2 and methyiat~on~ [133. Raimondal is not found in foliar tissue of G. hirsutum, nor has hemigossypolone been found in the foliage of G. roimondii tissue [3]. Thus, it appears raimondalone results from gene and enzyme contributions from both G. ruimondii (C-2 hydroxylation and methylation) and G. hirsutum (C-4 hydroxylation). Raimondalone could be of particular importance because another unique terpenoid from this species, raimondal(6). has been shown to have the highest toxicity of any cotton terpenoid tested against Heliothis oirescens cells [S].
EXPERIMENTAL
NMR spectra were obtained in CDCI, (I,& 3 and 5) or acetone-d, (4) at 300.13 MHz (‘H) and 75.47 MHz (’ 3C), respectively. Plant material. A G. raimondii plant from accession D s _ r [ 143 was used as a male parent in an interspecific cross with G. hirsatam cv Tamcot CAMD-E. The concentrations of seven known terpenoids found in the parents and the triploid hybrid were previously reported [33. Subsequently, a hexaploid plant was derived and characterized for total terpenoids and two major compounds [2]. The accession D, _ 1 also was shown to produce very high concentrations of a unique, uncharacteri~d UVabsorbing compound [2], and a progeny plant from the self-pollinated hexaploid accumulated even higher levels of the same compound. This high producing plant was the source of all plant material used for extraction and isolation of raimondalone (1). Extraction and isolation. Freeze-dried leaves (25 g) were ground using a Wiley mill (40 mesh) and extracted with hexane-EtOAc (3: 1,400 ml) and Me,CO (360 ml). The combined extracts were evapd to dryness and chromatographed on silica gel (Baker 40-140 mesh, 155 g)
95s
using Me&O-H,0 (1: 1) as the eluent (800 ml). The Me,CO was removed by rotoevaporation and the H,O phase extracted with Et,O. After evapn, the extract was chromatographed on a second column of silica gel (50 g) using a hexane-EtOAc step gradient from 0% EtOAc to 10% EtOAc in 2% increments (125 ml increment- I, 10 mlfraction-‘). Raimondalone together with 2 were found in frs 32-42. These fractions were combined and rechromatographed by prep. HPLC using a reversed phase (C-18, 5~~ Rainin Instrument Co., Woburn, MA) column (21.4 mm i.d. x 25 cm) coupled with a guard coiumn (21.4 mm i.d. x 5 cm) of the same packing. The column was developed isocraticaily with MeOH-H,O (17: 3) at a flow rate of 4 ml min- t; 2 ml fractions were collected. Compound 2 eluted first followed by raimondalone. Purity of individual fractions was checked by HPLC. The MeOH from those fractions containing 1 was evapd, the H,O layer was extracted with Et,O, and the organic layer evapd to dryness. The combined extracts, which contained traces of 2, were chromatographed on silica gel TLC plates (Baker G/HR) and developed with hexane-Et,O-Me&O-HCO,H(134:58:8: 1). The top band was removed and extracted with EtOAc. The product was crystallized from cyclohexane-Et,0 (with a trace of hexane), mp 125-128”. HPLC analysis showed raimondalone occurred at concentrations as high as 4 /Ig mg- * in freeze-dried leaf tissue. Raimondalone (1). EI-MS m/z (rel. int. %) 304 (74) [Ml’, 289 (lOO), 274 (17). 259 (lo), 246 (17). o-fiemigossypoione (4). Compound 4 was prepared from raimondal(6) by oxidation with FeCl,. Raimondal (29 mg) was dissolved in Me&O (3 ml) and HOAc (6 ml). A 10% solution of FeCl, (9 ml) was added dropwise over 1 min, and the soln stirred for an additional 2 min. The reaction mixture was poured into 20% H2S0, (100 ml) and extracted with Et,O. The Et,0 solution was washed successively with H,O and salt brine and dried over Na,SO,. After evapn, the product was crystallized from cyclohexane-Me&IO (mp 199-203”) to give 14mg (4). UV LL’!pl nm (log E):276 (4.12), 334 (4.09), 400 (3.46), 488 (3.18); ‘H NMR [(CD3)2CO]: 12.19 (s, OH-7), 10.53 (s, H1l), 7.85 (4. J = 1.3 Hz, H-4). 3.73 (sepc, J = 7.0 Hz, H-12), 1.97(d,J=1.3Hz,Me-15),1.43(d,J=7.0Hz,Me,-13,14) (a peak for the OH group on C-6 was not observedb EIMS m/z (ret. int. %) 276.099036 (talc. for C,sHt,O, 276.099750, lOO), 275 (26), 274.083178 (talc. for C,sH,,Os 274.084100, 93). 259.060625 (talc. for C,cHI r03 259.060625,36), 247 (19), 246.089255 (talc. for C,4H,404 246.089190, 74), 243.064813 (talc. for C~~H,~0~243.~5715,33~231(42),218(16),203(67~,149 (31). 129(25), 128(18), 127(13), 115(25), 111 (20), 109(15). Preparation of mansonone C (5). Mansonone C was prepared from ‘I-hydroxycadalene [lSJ by oxidation with diphenylseleninic anhydride [ 163. Thus, 7-hydroxycadalene (46 mg) was dissolved in THF ( *CI 2 ml) and added dropwise to a stirred suspension of benzeneseleninic anhydride (110 mg) in THF (6 ml). The suspension was heated to 50” and stirred for 1 hr. Et,0 was added and the organic phase washed with 50% satd NaHCO, and then with H20. The organic phase was dried (Na,SO*)
R. D. STIPANOVIC er al.
956
and evapd to dryness. The product was chromatographed on silica gel (J. T. Baker, 40-140 mesh) using a hexane-EtOAc step gradient from 9:l to 3:2 in 10% increments (150 ml increments _ ‘); 20 ml fractions were collected. Mansonone C eluted in frs 6-8. After recrystallization it had mp 137” (lit. 1344137” [17]). authors gratefully acknowledge the excellent technical assistance of MS Sherri Lindemann, and MS Patricia Harvey; we thank Dr Ross C. Beier for mass spectral measurements and Dr Howard J. Williams for comments on the manuscript. Acknowledgements-The
REFERENCES
1. Bell, A. A. and Stipanovic, R. D. (1978) Mycopathologia 65, 9 1. 2. Altman, D. W., Stipanovic, R. D. and Bell, A. A. (1991) Proceedings of the 43rd Beltwide Cotton Con/erences, p. 534. 3. Altman, D. W., Stipanovic, R. D. and Bell, A. A. (1990) J. Heredity 81, 447. 4. Bell, A. A., Stipanovic, R. D. and Mace, M. E. (1993) Phytochem. 5. Stipanovic,
Sot. N. Am. Newsletter
33, 21.
R. D., Elissalde, M. H., Altman, D. W. and Norman, J. 0. (1990) J. Econ. Entomol. 83, 737. 6. Gray, J. R., Mabry, T. J., Bell, A. A. and Stipanovic, R. D. ( 1976) J. Chem. Sot., Chem. Commun. 109.
7. Stipanovic, R. D., Bell, A. A. and O’Brien, D. H. (1980) Phytochemistry 19, I735 8. Bell, A. A., Stipanovic, R. D., O’Brien, D. H. and Fryxell, P. A. (1978) Phytochemistry 17, 1297. 9. Berger, St and Rieker, A. (1974) in The Chemistry of the Quinonoid Compounds (Patai, S., ed.), p. 181.
Wiley, New York. 10. Bax, Ad. and Summers, M. F. (1986) J. Am. Chem. Sot. 108, 2093.
11. Stothers, J. B. (1972) in Carbon-13 NMR Spectroscopy, p. 197. Academic Press, New York. 12. O’Brien, D. H. and Stipanovic, R. D. (1978) J. Org. Chem. 43, 1105. 13. Bell, A. A., Stipanovic, R. D., Mace, M. E. and Kohel, R. J. (1993)in Genetic Engineering ofPIunt Secondary Metabolism, Recent Advances in Phytochemistry Vol. 28 (Stafford, H. A., ed.). Academic Press, New York (in press). 14. Percival, A. E. (1987) Southern Cooperatioe Res. Bull. 321, I. 15. McCormick, J. P., Shinmyozu,
T., Pachlatko, J. P., Schafer, T. R., Gardner, J. W. and Stipanovic, R. D. (1984) J. Org. Chem. 49, 34. 16. Barton, D. H. R., Brewster, A. G., Ley, S. V. and Rosenfeld, M. N. (1976) J. Chem. Sot., Chem. Commun. 985. 17. Bettolo, G. B. M., Casinovi, C. C. and Caleffi. C. (1965) Tetrahedron Letters 4857.
0031-942y94)EOO62-W
RAIMONDALONE,
Phytahrmirfry.
A SESQUITERPENE FROM INTERSPECIFIC HYBRID
Vol. 36. No. 4. pp. 95>9%. 1994 Eknn Sciena Ltd Pnnted m Great Briram 0031 9422/w S700+0.00
A COTTON
ROBERT D. STIPANOVIC, HYEONG L. KIM,+ DAVID W. ALTMAN, ALOIS A. BELL and RUSSELL J. KOHEL USDA, ARS. Southern Crops Research Laboratory, College Station, TX 77845, U.S.A.; *Texas A&M University, Department of Veterinary Physiology and Pharmacology. College Station, TX 77843, U.S.A.
(Receir;edin reuisedlorm 6 December 1993) Key Word Index-Gossypium quinone.
hirsutum; G. raimondii; Malvaceae; cotton; sesquiterpene; l,Cnaphtho-
Abstract-A new sesquiterpenoid, raimondalone (8-formyl-6,7dihydroxy-S-isopropyl-2-methoxy-3-methyl-1,4naphthoquinone), has been isolated from the leaves of a cotton plant derived from an interspecific hybrid (G. hirsutum x G. raimondii). The structure of this compound and the i3C chemical shifts of related compounds were determined using 2D HMBC NMR spectrometry.
INTRODUCTION
Cotton produces a complex assortment of sesqui- and sesterterpenoids [l] in the subepidermal pigment glands in the foliage. Gossypium species and their interspecific hybrids provide an array of plants which display novel chemistries [2, 33 and resistance characteristics which are relevant to protecting cotton from pests, including the wilt pathogen Verticillium dahliae [43 and Lepidopteran insects [S]. We have been particularly interested in hybrid between commercial Upland cotton (Gossypium hirsutum L.) and the wild species G. raimondii Ulbr. The former produces the sesquiterpenoid quinone p-hemigossypolone (2) [6] and the latter produces the sesquiterpenoid raimondal(6) [7J. We report herein the identification of a new sesquiterpenoid, which we call raimondalone, isolated from the foliage of a progeny plant obtained by self-pollination of a hexaploid parent derived from a G. hirsutum x G. raimondii hybrid. RESULTS AND Dl!XUSSlON
Raimondalone (1) gave a parent ion at m/z 304 (74%) which by high resolution mass measurement (304.09351, talc. 304.09469) indicated a molecular formula of ClgHi606. It exhibited UV absorption maxima in ethanol-HCI (log E)at 212 (4.66), 273 (4.56), 303 (4.41) and 420 (3.54) nm. The ‘H NMR showed the presence of an isopropyl group (64.17, lH, sept, J = 7.0 Hz; 6 1.37,6H, d, J =7.0 Hz), a vinyl methyl group (62.02, 3H, s) and a methoxyl group (64.0,3H, s). An aldehyde group (6 10.64, lH, s) hydrogen bonded to a phenol (612.86, lH, s) was also evident. A phenolic group accounts for the remaining proton (66.57, lH, s). The UV spectrum, the dark red colour of the crystals, and the presence of two peaks in the PHY 36:4-J
13C NMR at 6 182.3 and 187.5 indicated the presence of a quinone. The ‘HNMR was in general agreement with that of other p-naphthoquinones isolated from cotton such as hemigossypolone (2) and hemigossypolone-6methyl ether (3) [8]. Based on biosynthetic considerations, the position of the aldehyde group, isopropyl and methyl group could be established as shown in 1. The presence of a hydrogen bonded phenol group shows this group is ortho to the aldehyde. Since no aromatic protons are present, it remained only to assign the position of the quinone carbons, and the remaining phenol and methoxyl groups. Based on a DEPT experiment, and unique chemical shifts, several peaks in the 13C NMR spectrum were readily assigned. These include the isopropyl methyls (C-13, C-14,6 19.7, q) and methine(C-12.628.4, d) groups, the methoxyl group (660.5. q), a methyl group (C-l 5,695, q), the aldehyde carbon (C-l 1,6 198.3, d) and the carbon to which the aldehyde group is attached (C-8, S 115.8. s). Other assignments, such as the quinone carbonyl carbons (6 182.3 and 187.5), were more difficult to assign. In 1,4naphthoquinones, the carbonyl carbons generally occur at lower field ( - 6 187) as compared to 1,2-naphthoquinones (-6 182) [9]. but in raimondalone the quinone carbonyls appeared in both regions (6 187.5 and 182.3). These and the remaining peaks in the ’ 3C NMR spectrum were ultimately assigned using the HMBC spectrum [lo] and by comparisons to related compounds. Assignments of the naphthoquinone carbons were based on the following observations. A cross-peak from the methoxy protons to the signal at 6 156.3 identified the carbon to which the methoxy group was attached. The vinyl methyl protons showed a cross-peak to this carbon, indicating that the methoxyl group and methyl group are ortho to one another, and also correlated to peaks at 6 13 1.6 and 187.5.
R. D. STIPAHOWC-
954
o\
et
at.
11
'CH
0
1)RI=H,R~=OCH3 2)Rt,R2=H 3)Rl=CH3 ,Rz=H 0
I \
0
I
/
ti
A
*5 Since the peak at 6 187.5 has been assigned to the quinone carbonyl, this must be C-4 and the peak at 6 f 31.6 must be C-3. The aromatic carbon C-S was assigned to the peak at 6 141.4 because of long range coupling to the isopropyl methyl protons. The methine proton of the isopropyl group showed coupling to peaks at 5 127.4 and 149.2 in addition to coupling to C-5. The chemical shift of 6 149.2 allows its assignment to the oxygenated carbon C-4, and thus C-10 is at fi 127.4. The aldehyde proton showed very strong coupling to 6115.8 confirming its assignment lo C-8 and strong coupling to 6151.8 and weak coupling to 6149.2. The peak at 6149.2 has been assigned to C-6 because of its coupling to the isopropyl methine proton and thus the remaining oxygenated aromatic carbon at d 151.8 was C-7. The remaining quinoid carbonyl (C-l) was assigned to the peak at fi 182.3. C-l is shifted upfield from its normal position as a p-quinone carbonyl because of the o-methoxyl group [l I]. C-9 is assigned to the remaining peak at b 126.4. No cross-peaks to the phenolic proton at 6 12.86 were observed. To confirm the original observation that 1,2-quinones are shifted upfirlti compared to IA-quinones, the HMBC spectra of several related compounds were examined. These spectra were used to determine or confirm the chemical shifts of ~-hemigossypolone (2) [12], ~-hemigossypolone-6-methyl ether (3) [ 121. o-hemigossypolone (4) and mansonone C and are compared to those of 1 in Table 1. The chemical shifts of the quinonoid carbonyls are shown in Table I. The carbonyls of the p-quinone appear at 6 186 or lower field and those of the o-quinone at d 183 or higher field. The HMBC spectra revealed an interesting observation (Table 2). The aldehyde protons in compounds l-4 show no coupling to C-9 but do show coupling to C-7 and C-8, and in 1-3, there is also coupling
Table 1. ‘%NMR
c 1 2
3 4 5 6 7 8 9 10 11
12 13. 14 15 OMe
6
spectral data for compounds CDCI J)
l-5
I
2
3
4*
5
182.3 156.3 13f.h 187.5 141.5 149.2 151.8
186.4 133.9 149.2 187.5 141.6 149.0 152.2 1IS.8 127.3
186.3 133.6 t 49.6 187.0 150.7 152.7 158.7 117.2 130.7 126.9 198.3 28.8 210
182.3 183.0 134.5 138.1 138.9 151.5 151.1 119.8 125.5 129.8 199.4 28.3 20.6 15.4
182.3 182.0 134.9 137.9 145.3 131.9 134.1 142.9 129.3 132.4 22.7 28.3 23.7 15.9
115.8 126.4 127.4 198.3 28.4 19.7 9.5 60.5
127.6 199.9 28.6 19.7 16.4
16.5
(in
60.8
*In acetone-d,.
to C-6. This results from the confined orientation of the aldehyde proton due to hydrogen bonding between the carbonyl and the o-phenolic group. The methyl group at C-l 1 in mansonone C (5) shows normal coupling to C-7, C-8 and C-9. Raimondalone is the first compound isolated from the group of 51 unknown UV-absorbing compounds detected in an earlier survey of Gossypium species [2]. Raimondalone was in the highest concentration in this group and was uniquely found in progeny from G. ~jrs~~~rn crossed with G. raimondii. Analysis of F,, F, and backcross progeny from a cross between a Tamcot CAMD-E line homozygous for raimondal production and the Texas
A sesquiterpene from a cotton interspecific hybrid Table 2. ‘H-l 1and 13Ccoupling as indicated in HMBC spectra in compounds l-5 Compound
6H-11
Cross-peak*
C
1
10.64
2
10.76
3
10.57
4
10.53
5
2.58
115.8(vs) 149.2 (w) 151.8(s) 115.8 (vs) 149.0 (w) 152.2 (s) 117.2 (YS) 152.7 (w) 158.7 (s) 119.8 (vs) 151.1 (s) 129.3 (s) 134.1 (s) 142.9 (s)
8 6 7 8 6 7 8 6 7 8 7 9 7 8
*vs = Very strong, s = strong, and w = weak coupling.
marker stock ‘TM-I’ showed segregation of raimondal was consistent with this character being controlled by two dominant genes with epistasis (i.e. hydroxylation at C-2 and methyiat~on~ [133. Raimondal is not found in foliar tissue of G. hirsutum, nor has hemigossypolone been found in the foliage of G. roimondii tissue [3]. Thus, it appears raimondalone results from gene and enzyme contributions from both G. ruimondii (C-2 hydroxylation and methylation) and G. hirsutum (C-4 hydroxylation). Raimondalone could be of particular importance because another unique terpenoid from this species, raimondal(6). has been shown to have the highest toxicity of any cotton terpenoid tested against Heliothis oirescens cells [S].
EXPERIMENTAL
NMR spectra were obtained in CDCI, (I,& 3 and 5) or acetone-d, (4) at 300.13 MHz (‘H) and 75.47 MHz (’ 3C), respectively. Plant material. A G. raimondii plant from accession D s _ r [ 143 was used as a male parent in an interspecific cross with G. hirsatam cv Tamcot CAMD-E. The concentrations of seven known terpenoids found in the parents and the triploid hybrid were previously reported [33. Subsequently, a hexaploid plant was derived and characterized for total terpenoids and two major compounds [2]. The accession D, _ 1 also was shown to produce very high concentrations of a unique, uncharacteri~d UVabsorbing compound [2], and a progeny plant from the self-pollinated hexaploid accumulated even higher levels of the same compound. This high producing plant was the source of all plant material used for extraction and isolation of raimondalone (1). Extraction and isolation. Freeze-dried leaves (25 g) were ground using a Wiley mill (40 mesh) and extracted with hexane-EtOAc (3: 1,400 ml) and Me,CO (360 ml). The combined extracts were evapd to dryness and chromatographed on silica gel (Baker 40-140 mesh, 155 g)
95s
using Me&O-H,0 (1: 1) as the eluent (800 ml). The Me,CO was removed by rotoevaporation and the H,O phase extracted with Et,O. After evapn, the extract was chromatographed on a second column of silica gel (50 g) using a hexane-EtOAc step gradient from 0% EtOAc to 10% EtOAc in 2% increments (125 ml increment- I, 10 mlfraction-‘). Raimondalone together with 2 were found in frs 32-42. These fractions were combined and rechromatographed by prep. HPLC using a reversed phase (C-18, 5~~ Rainin Instrument Co., Woburn, MA) column (21.4 mm i.d. x 25 cm) coupled with a guard coiumn (21.4 mm i.d. x 5 cm) of the same packing. The column was developed isocraticaily with MeOH-H,O (17: 3) at a flow rate of 4 ml min- t; 2 ml fractions were collected. Compound 2 eluted first followed by raimondalone. Purity of individual fractions was checked by HPLC. The MeOH from those fractions containing 1 was evapd, the H,O layer was extracted with Et,O, and the organic layer evapd to dryness. The combined extracts, which contained traces of 2, were chromatographed on silica gel TLC plates (Baker G/HR) and developed with hexane-Et,O-Me&O-HCO,H(134:58:8: 1). The top band was removed and extracted with EtOAc. The product was crystallized from cyclohexane-Et,0 (with a trace of hexane), mp 125-128”. HPLC analysis showed raimondalone occurred at concentrations as high as 4 /Ig mg- * in freeze-dried leaf tissue. Raimondalone (1). EI-MS m/z (rel. int. %) 304 (74) [Ml’, 289 (lOO), 274 (17). 259 (lo), 246 (17). o-fiemigossypoione (4). Compound 4 was prepared from raimondal(6) by oxidation with FeCl,. Raimondal (29 mg) was dissolved in Me&O (3 ml) and HOAc (6 ml). A 10% solution of FeCl, (9 ml) was added dropwise over 1 min, and the soln stirred for an additional 2 min. The reaction mixture was poured into 20% H2S0, (100 ml) and extracted with Et,O. The Et,0 solution was washed successively with H,O and salt brine and dried over Na,SO,. After evapn, the product was crystallized from cyclohexane-Me&IO (mp 199-203”) to give 14mg (4). UV LL’!pl nm (log E):276 (4.12), 334 (4.09), 400 (3.46), 488 (3.18); ‘H NMR [(CD3)2CO]: 12.19 (s, OH-7), 10.53 (s, H1l), 7.85 (4. J = 1.3 Hz, H-4). 3.73 (sepc, J = 7.0 Hz, H-12), 1.97(d,J=1.3Hz,Me-15),1.43(d,J=7.0Hz,Me,-13,14) (a peak for the OH group on C-6 was not observedb EIMS m/z (ret. int. %) 276.099036 (talc. for C,sHt,O, 276.099750, lOO), 275 (26), 274.083178 (talc. for C,sH,,Os 274.084100, 93). 259.060625 (talc. for C,cHI r03 259.060625,36), 247 (19), 246.089255 (talc. for C,4H,404 246.089190, 74), 243.064813 (talc. for C~~H,~0~243.~5715,33~231(42),218(16),203(67~,149 (31). 129(25), 128(18), 127(13), 115(25), 111 (20), 109(15). Preparation of mansonone C (5). Mansonone C was prepared from ‘I-hydroxycadalene [lSJ by oxidation with diphenylseleninic anhydride [ 163. Thus, 7-hydroxycadalene (46 mg) was dissolved in THF ( *CI 2 ml) and added dropwise to a stirred suspension of benzeneseleninic anhydride (110 mg) in THF (6 ml). The suspension was heated to 50” and stirred for 1 hr. Et,0 was added and the organic phase washed with 50% satd NaHCO, and then with H20. The organic phase was dried (Na,SO*)
R. D. STIPANOVIC er al.
956
and evapd to dryness. The product was chromatographed on silica gel (J. T. Baker, 40-140 mesh) using a hexane-EtOAc step gradient from 9:l to 3:2 in 10% increments (150 ml increments _ ‘); 20 ml fractions were collected. Mansonone C eluted in frs 6-8. After recrystallization it had mp 137” (lit. 1344137” [17]). authors gratefully acknowledge the excellent technical assistance of MS Sherri Lindemann, and MS Patricia Harvey; we thank Dr Ross C. Beier for mass spectral measurements and Dr Howard J. Williams for comments on the manuscript. Acknowledgements-The
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