Phytochemisrry, 1978, Vol 17, pp 151-152. Pergamon Press. Pnnted in England

HELIOCIDE H, AN INSECTICIDAL TERPENOID FROM GOSSYPIUM HIRSUTUM

ROBERT D. STIPANOVIC, ALOIS A. BELL, DANIEL H. O’BRIEN and MAURICE J. LUKEFAHR

U.S. Department of Agriculture, Agricultural Research Service, P.O. Drawer .J F, and Texas A & M University, College Station, TX 77840, U.S.A.

(Revised received 14 July 1977)

Key Word Index-Gossypium hirstutum: cotton: Malvaceae; terpenoid; Heliothis; heliocide H,; host plant resistance; insecticsde.

INTRODUCTION Table 1. ’ %-NMR chemical shifts of heliocide Ha*

Gossypol is the predominant sesquiterpenoid in lysige- nous glands in seeds of most Gossypium spp. [ 11. However, it is a minor terpenoid constituent in glands in foliar plant parts [2]. We previously isolated and identified the terpenoids, hemigossypolone (1) [2-4], and heliocide H, (2) [S], from the glands of leaves and bolls. We have’ now isolated a third terpenoid from young bolls, which we call heliocide H, (3). All of these compounds are toxic to Heliothis and appear to be involved in the resistance of certain varieties of cotton to this insect

[6,71.

Carbon Carbon type no.

Carbon Carbon 6 type no.

Me 25 17.6 > c=o 4 198.7 13,14 19.8 1 202.1

15 22.6 24 25.5 =C< 23 131.2.t

17 131.5t -CH,- 21 26.0

19 24.1 =CH- 18 117.0 16 35.0 22 123.7 20 37.1

I Ad 5 115.3 -CH- 12 29.0 10 135 O$

3 54.4 9 129.21

-- + 2 8 140.0

49.9 7 148.9s 6 152.25

-HC=O 11 197.9

RESULTS AND DISCUSSION

Heliocide H, (3), CZ5H3,,05 (high resolution MS), mp 128-134” (hexane), was isomeric with heliocide H,, and it had UV, IR, and mass spectra similar to those of heliocide H, [5]. The R, values of heliocide H, also were similar to those of heliocide H, in various solvent systems, but the two were separated by repeated development in solvent 1 (see Experimental).

Heliocide H, (2) was synthesized by the Diels-Alder reaction of myrcene (4) with hemigossypolone (I), and its structure was confirmed by X-ray crystal analysis. We reported that the only Diels-Alder adduct isolated was heliocide H,, which was crystallized on addition of hexane [S]. However, repeated chromatography

*Carbon-l3 shift in ppm downfield from TMS usmg central resonance of CDCI, as reference (6 = 76.9 ppm). t,$,§ Shift assignments of similar types of carbons may be interchanged.

of the mother liquors from the Diels-Alder reaction yielded a second minor adduct. This adduct and the naturally occuring heliocide H, had identical ‘H-NMR, IR, UV and mass spectra. The synthetic product had

152 Short Reports

mp 131.5135” and a mixed mp with heliocide H, of 130-135.‘.

In the t3C-NMR spectrum (Table 1). the resonances in heliocide H, at Cl6 and Cl9 (ii35.0 and 624.1, respectively) are significantly different from those in heliocrde H, ((5 31.1 and 6 26.6, respectively [5,8]). The resonances of all other carbons are essentially unchanged fAS<0.6ppm). The upheld shift for Cl6 and downfield for Cl9 agree with the structure 3 based on empirical correlations [9].

Comparison of the r3C-NMR data of hehocides H, and H, and their synthesis from hemigossypolone (I) and myrcene(4) show that heliocide H, is the isomeric Diels-Alder adduct of heliocide H,. The location of the side chain at Cl8 in heliocide H, has been proven by X-ray crystal analysis [IS]. The side chain must therefore be located at Cl7 in heliocide H,.

The en&-transition state of the Die&Alder reaction produces a c&fused ring product. The c&fused ring was proven for heliocide H, [S]. Heliocide H, apparently retains this c&fused ring as shown in 3 because it was synthesized at room temperature and did not revert to hemigossypolone and myrcene. It is therefore the product of the kinetically controlled reaction. Also, larger differences between the ‘“C-NMR spectra of heliocides H, and H, would be expected if their ring fusions were different.

(95.5 0.5; solvent 2). The first eluate (25 ml) was discarded and the next IlOml contained 500 mg of crude product, The product was further purified by TLC (solvent 2). The upper light yellow bands (R, 0.550.7) were scraped off to- gether and rechromatographed (solvent 1) The top band (R, 0 43) was heliocide H,, and the next band (R, 0 34) was helioctdes H, and H,. Hehocides HZ and H, were collected as one band, and helioctde H, was purrfted by crystalhzatton from hexane. Heliocide H, was then purified by TLC of the mother liquors in solvent 1. Fmally, heliocrde H, was crystal- lized from hexane.

Spectra of heliocide H,. UV ;$$J’( (2.) 356 (-1700) 308 (sir). 272 (37700) nm. IR .v”,f 3300, 1695. 1682, 1633 cm-’ MS (Probe = 90’),m.‘r I”,,) 410 (28. C2,H,,0,) 383 (15), 382 (55) 314 (23). 313 (91, C,,HZ,O,). 297 (19). 275 (15). 274 (17). 259 (19), 135 (19). 105 (35) 93 (23). 91 (78), 79 fl8). 77 (20). 69 (100). ‘H-NMR (CDCl,).a 1.35 i3H. s’). 130 (6H. d). 1.55 (3H. hs), 1.63 (3H. hs), 2.0-2.8 (1H, nr). 2.02 (-1H. ,n), 2.96 (IH. r). 3.66 (lH, sept), 5.05 (IH, m), 5.42 (lH, m). 6 50 (IH, s. exchanged with D,O). 10.42 (1H. s), 12.92 (IH. s, exchanged with D,O) Acknowledgements-We thank J. K. Cormsh, M. E. Bearden, and G. W. Tribble for excellent technical assistance. and Dr. Ron Grigsby for high resolution mass measurements.

REFERENCES

EXPERIMENTAL

Extractton and purtfication. Young bolls with bracts were collected in the field, frozen at - 15’. lyophtlized. and ground to a powder in a blender. The powder (IOOg) was extracted successively with hexan+EtOAccH,O I I .3.0.4) (500 ml). hexane-EtOAc (1 3) I1 I.), and Et,0 (1 1.). The extracts were filtered through a short column of St gel and the solvent evapd. The residue was dissolved m EtOAc-hexane (0.1.1; 500 ml) and extracted with MeOH- ‘Q,, aq. Na,CO, (19.1; 4 x 200 ml). A 50% satd soln of NaCl (1.81.) was added to the MeOH extract. the pH was adjusted to 5, EtOH (50ml) was added,

1. Stipanovic. R. D., Bell, A. A., Mace, M. E. and Howell, C. R. (1975) Phytochemistry 14. 1077.

2. Bell, A. A. and Stipanovic, R. D. (1976) in Beltwdr Cotton Prod. Rex Co& Proc. Las Vegas. Nev pp 52-54. Nattonal Cotton Council. Memphis, Tenn.

3. Stipanovtc, R. D ~ Bell. A. A. and Lukefahr. M. J. (1976) m BeltGde Cotton Prod. Res. ConJI, Proc. Las Vegas. Nev. p. 91. National Cotton Council. Memphts. Tenn.

4. Gray, J. R., Mabry. T. J.. Bell, A A.. Sttpanovrc, R. D. and Lukefahr. M. J (1976) J. Chern. Sot. C’hern Co~nmun. 109.

5. Sttpanovic. R. D., Bell, A. A., O‘Brten, D. H and Lukefahr, M. J. (1977) Tetrahedron Letter? 567.

6. Seaman, F., Lukefahr, M. J. and Mabry. T. J. Il977)in Beltwide Cotton Prod. Res. Conj. Proc. Atlanta. GA pp. 102-103. National Cotton Council, Memphis, Tenn.

and the soln extracted wrth EtOAc (3 x 200 ml). The EtOAc fractron was evapd and the MeOH-2% aq. Na,CO,

7. Lukefahr, M. J.. Stipanovic. R: D.. Bell. A. A. and Gray. J R. (1977) in Beltwide Cotton Prod. Res Cotz/.. Proc.. Atlanta,

extractton procedure was repeated. The resulting dark 011 GA, pp. 97-100. National Cotton Council, Memphis. Ten. was dtssolved in C,H,. adsorbed onto Si gel (3&g. Mallin- 8. Assignments for C21 and Cl9. and Cl and C4,- have been ckrodt CC-4). and chromatographed on St gel (20s). The product was eluted with hexane--EtOAc-HOAc (90: 10:0.5.

reversed since the earlier report on helroctde H, (see ref.

m. solvent I), and 5 ml fractions were collected. The fractions 9. Levy, G. C and Nelson G. L. (1977) Ctrrhorl-13 Nuclecrr (numbers 10-13) containing the heliocides were chromato- graphed on Si gel (72g) eluting with CHCl,-HCO,H

Magnetic Resonance for Organrc Chemists, Chap 3. Wrley- Interscience, New York