Further Dihydrochalcones from Crotolaria ramosissima

Crotolaria ramosissima is a weed plant belonging to the family Leguminoseae. It is a much branched sticky woody herb. The flowers are yellow tinged with purple. It is distributed in some places of Tamilnadu and coastal areas of Andrha Pradesh. The plant contains waxes, and, when flowering, a yellow exudate appears in the inflorescences. Recently, we published the corrected structure of crotaramosmin (1) from crotolaria ramosissima which appeared to be a dihydrochalcone, a class of compound which is rare as a plant secondary metabolite. Except 1, only one other instance of a dihydrochalcone possessing a chromenyl moiety has been reported in the literature. This is a synthetic compound and has an unsubstituted phenyl ring (see Scheme 1: X = Y = H). Our continued interest on crotolaria ramosissima led to the identification of two more dihydrochalcones, crotaramin (2) and crotin (3) (Scheme 1).


Introduction
Crotolaria ramosissima is a weed plant belonging to the family Leguminoseae.It is a much branched sticky woody herb.The flowers are yellow tinged with purple.It is distributed in some places of Tamilnadu and coastal areas of Andrha Pradesh.The plant contains waxes, and, when flowering, a yellow exudate appears in the inflorescences.Recently 1 , we published the corrected structure of crotaramosmin (1) from crotolaria ramosissima which appeared to be a dihydrochalcone, a class of compound which is rare as a plant secondary metabolite.Except 1, only one other instance of a dihydrochalcone possessing a chromenyl moiety has been reported in the literature 2 .This is a synthetic compound and has an unsubstituted phenyl ring (see Scheme 1: X = Y = H).Our continued interest on crotolaria ramosissima led to the identification of two more dihydrochalcones, crotaramin (2) and crotin (3) (Scheme 1).

Results and Discussion
The identification of the two new dihydrochalcones was based on extensive 1D and 2D NMR spectroscopy as described before 3,4 .The 1 H and 13 C chemical shifts are collected in Table 1.The assignments were confirmed by DEPT experiments which allowed the differentiation between the CH n (n = 0-3) fragments and further by HMQC experiments providing the assignment of the hydrogens to the directly attached carbon atoms.Long-range 1 H, 13 C correlations (via more than one bond; HMBC, optimized to a 13 C, 1 H coupling constant of 7 Hz) afforded the unequivocal assignment of the quaternary carbons and the connectivity within the carbon framework.For example, C-5' and C-9' gave rise to very close 13 C signals, δ = 159.7 and 159.6, respectively.Their assignment could be accomplished by an HMBC peak correlating the 159.6-signal to those of the methyl protons H-1" and H-2".Only in one case, C-2 and C-5 of 3, the signals were so close that the resolution in the 13 C domain of the HMBC spectrum was not good enough for a safe assignment.
NOESY experiments were helpful in corroborating the bicyclic chromene structure because they ascertained the spatial proximity between H-8' and the methyl protons H-1" and H-2" (compare Scheme 2) so that a benzene ring carrying a prenyl side chain could be excluded.
It turned out that 2 is the 4-O methyl ether of 1 whereas an extra hydroxyl group in 3-position is present in 3, as compared to 1.Both dihydrochalcones have not yet been described in the literature.

Experimental
The plant material was collected in november 1997 near the city of Hyderabad.It was identified by Dr. V.S. Raju and Dr. Ragan, Department of Botany, Kakatiya University, Warangal.A voucher specimen is deposited in the Department of Botany.
The fresh plant material (2 kg) was extracted with petroleum ether in a soxhlet extractor.The extract was concentrated under reduced pressure to give a dark yellow syrupy liquid.The concentrate was dissolved in MeOH and kept in a refrigerator for a week.The separated waxy material was filtered off, and this procedure was repeated several times to remove a maximum amount of wax.The methanolic solution (2 g) was chromatographed over silica gel (100-200 mesh) and eluted with petrol ether with increasing quantities of ethyl acetate.The fractions obtained from petrol ether-ethyl acetate (95:5) showed a single spot on TLC.These fractions were collected and evaporated to give a colourless solid which yielded colourless needles (120 mg) of crotaramin (2) on recrystallization from methanol; m.p. 62 °C.
Further elution of the column with petrol ether/ethyl acetate (85:15) fractions showed another single spot on TLC.These fractions were separated and concentrated to get a pale yellow solid which was recrystallized with methanol and yielded pale yellow prisms (100 mg) of crotin (3); m.p. 105°.
Both compounds were optically inactive and gave a dark green ferric reaction.Crotin (3) gave a red coloration with aqueous KOH.The UV spectra were recorded on a Shimadzu UV-240 and the IR spectra on a Nicolet-740 spectrophotometer.The NMR spectra were obtained on a Bruker DRX-500 spectrometer (500 MHz 1 H and 125 MHz 13 C) in CDCl 3 ; for the experimental conditions see ref. 3. Electron-impact mass spectra were recorded on a Finnigan MAT-312.

Table 1 .
1H and 13 C chemical shifts of the dihydrochalcones 2 and 3.