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Journal of Natural Products
Vol. 52, NO.4 , pp. 810-817,Jd-Aug 1989
ISOLATION, SEMI-SYNTHESIS, AND NMR SPECTRAL STUDIES OF LOLINE ALKALOIDS’ R.J. PETROSK~, S.G. YATES,D. WEISLEDER, and R.G. POWELL*
USDA , Agricultural Research Saice, Northern Regional Research Center, 1815 N. University Street, Peoria, IIlinois 61604 ABSTRhCT.-hhne, a saturated pyrrolizidine-type alkaloid, was isolated from tall fescue (Festuca armdinarea) seed infected with the endophytic fungus Acrmonium cwnophialum. Procedures are described for the efficient conversion of loline to derivatives also known to occur naturally: norloline, N-formylnorloline, N-acetylnorloline, N-methylloline, N-formylloline, and Nacetylloline. The loline alkaloids are of interest as they are suspected contributors to several disease syndromes in cattle that consume endophyte-infected tall fescue. The structure of hydroxychlorololine, a reaction product of loline with HCI, was determined, and complete ‘H- and ”Cnmr assignments for all the lolines are reported.
Occurrence of the loline alkaloids, a group ofpyrrolitidine alkaloids, has so far been restricted to the genera Lolium (1,2) and Festuca ( 3 ) (Graminae), and Adenocarpus ( 4 ) (Leguminosae). Naturally occurring loline alkaloids include loline (festucine) I l l , Nformylloline 121, N-acetylloline (lolinine) 131, N-methylloline [ 4 ] ,norloline 151, Nformylnorloline 161,N-acetylnorloline IT, and N-propionylnorloline (decorticasine) [SI. Alkaloids 1-7 have been isolated from Lolium cuneuturn, alkaloids 1-3,7 have been reported as constituents of Festuca arundinacea (tall fescue), and 8 occurs in Adenocarpus decorticans. The absolute configuration of 1 was determined by X-ray crystallography ( 5 ) , and a total synthesis of 1 has been reported (6). Partial ‘H-nmr assignments have been reported for 1 (7-9), 2 ( 7 ) ,3 ( 7 )and 5 (lo), but detailed ‘H-nmr spectra have not appeared and 13C spectra of the loline alkaloids are entirely absent in the literature. Tall fescue ( F . arundinacea) is a major forage crop of the southeastern region of the
1 2,2‘f
3
4 5 6 7 8
R=Me, R ’ = H R=Me, R’=CHO R=Me, R’=Ac R=R’=Me R=R’=H R = H , R’=CHO R = H , R’=Ac R = H . R’=EtC=O
*60:40 mixture of rotamers 2 and 2 ’ , respectively in CDCI, at ambient temperature. ‘Presented in part at the 196th American Chemical Society National Meeting, Los Angeles, CA, September 25-30, 1988. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S.Department of Agriculture over other firms or similar products not mentioned.
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United States. At least 35 million acres are under cultivation in this region ( 1 1). Over 90% of the tall fescue grown in the United States is infected, to various degrees, with Acremonium coenophialum, an endophytic fungus of the Clavicipitaceae ( 12). Studies have revealed a positive association between endophyte infection, loline alkaloid production, and resistance to insect pests such as sod webworm, Crambus spp. (13); fall armyworm, Spodoptwa frugipwdz (14); and aphids, Rhopalosipum puli and Schizaphis graminum (15). Yates et al. (16) demonstrated that 2 is toxic to the large milkweed bug, Oncopeltusfasciatus. Loline alkaloid levels in endophyte-infected tall fescue vary widely due to seasonal and environmental factors, but it is not unusual for the total of 2 and 3 to exceed 2 g/kg (17). Unfortunately, presence of the endophyte in tall fescue is also associated with several disease syndromes in cattle (18)and is responsible for losses to cattle producers of an estimated $50 to $200 million annually (11). W e report preparation of the naturally occurring loline alkaloids 2-7 from 1isolated from tall fescue seed; their nmr spectral assignments are given. The structure of hydroxychlorololine 191, a product of reaction of 1with HCI, is also reported for the first time. RESULTS AND DISCUSSION Isolation of loline El] from tall fescue and subsequent conversion of 1to loline derivatives were achieved using published procedures ( 1,7) or slight modifications thereof. However, separation problems were encountered in the preparation of norloline E51 from 1.Alkaloid 1was separated from 5 by gc after conversion to their N-formy1 derivatives. Preparative-scale separation of 1and 5 was accomplished by cc on activity-grade 111 neutral alumina. Two-dimensional homonuclear COSY and 'H/"C heteronuclear correlation nmr experiments were conducted on the series of loline alkaloids (1-7, 1-2HC1, and 9-2HCl). The 13Cand 'H assignments are reported in Tables 1 and 2. The signal at 6 33.5 was assigned to C-6 because all other carbons of loline have an adjacent heteroatom. The carbon signal at 6 34.7 and proton signal at 6 2.19 were assigned to the N-Me group. Protons on C-6 were assigned to 6 1.74 ddd and 6 1.65 dddd on the basis of 2D 'H/13C heteronuclear correlation experiments. Both protons were coupled to the protons on C-5: 6 2.77 ddd and 6 2.62 ddd. C-5 was assigned to the signal at 6 54.0. DEPT experiments confirmed that only C-3, -5, and -6 have triplet multiplicity, corresponding to two attached protons. Because C-5 and C-6 were previously assigned, the signal at 6 60.6 was assigned to C-3. The C-6 proton signal at 6 1.65, designated 6a on the structure, was coupled to the proton on C-7 (6 4.10 dd, J = 4.3 Hz). The signal at 6 8 1.0 in the carbon spectrum was assigned to C-7. H-6a was also coupled to H-6b (6 1 . 7 4 , J z 14.3 Hz), H-5a (6 2.62, J = 9 . 8 Hz), and H-5b (6 2.77,J=3.8 Hz). H-Sa was also coupled to H-5b ( J = 12.3 Hz). The H-7 also couples to H-8 (6 2.84,J = 1.9 Hz), and H-8 is further coupled to HH
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Journal ofNatural Products
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1 (6 3.03,J = 1.8 Hz). Irradiation of H-8 changes the H-7 signal (dd) to a sharp doublet showing only coupling of H-6a with H-7. The H- 1signal (apparent triplet, but actually a doublet of doublets) also changes to a sharp doublet showing only coupling of H- 1 with H-2 (6 3.7 1, J = 1.6 Hz). 'H/13C heteronuclear correlation experiments showed signals for C-8, -1, and -2 are at 6 69.0, 6 67.8, and 6 73.4, respectively. The H-2 is coupled to H-3a (6 3.10,J
