Pyridine Alkaloids From A Parthenium Hybrid

Introduction

Parthenium argentatum (guayule), Asteraceae is being developed for its natural rubber (Whiteworth and Whitehead, 1991). As an ongoing part of our investigation of secondary metabolites from guayule resin, we detected the presence of a homologous mixture of two alkaloids in the hybrid Parthenium argentatum ¥ P. tomentosa. The literature contains only a few reports on the occurrence of alkaloids in the Asteraceae family. The most common alkaloids are pyridine, piperidine, pyrrolidine, pyrrolizidine, quinoline, quinolizidine, tropane and diterpene alkaloids (Gibbis, 1974;Heywood et al., 1977). This report describes the isolation and the structural elucidation of two new pyridine alkaloids, guayulamine A and B.

Results and Discussion

During our investigation of guayule resin for antifungal agents (Maatooq et al., 1996), a bright red spot was observed on TLC after spraying with ce-0939Ð5075/2002/0300Ð0211 $ 06.00 " 2002 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D rium sulfate spray reagent. Acetylation of this material resulted in a TLC spot with a lower R f and the color reaction with Ce IV changed from red to greenish-blue. Flash column reversed phase chromatography and preparative HPLC afforded the separation of each spot (before and after acetylation) into two compounds. All compounds were soluble in aqueous HCl and precipitated upon the addition of NaOH. Furthermore, they gave a pale reddish-brown color with Dragendorff's spray reagent. In the EIMS, the parent ion peak in both compounds was even. These data indicated the possible presence of an alkaloids with even numbers of nitrogen atoms.

The analysis of the spectral data of 1 indicated its structure is (ð)-N-[4-(1-aminoethyl) phenyl]-4-[3-methylbutenylidine]-1, 4-dihydropyridine. The EIMS of 1 gave a parent ion peak at m/z of 268 (48%), while its acetate 1a gave m/z 310 (100%). The HREIMS of 1a gave m/z 310.2042 which indicated an empirical formula of C 20 H 26 N 2 O (calcd., 310.2046) and conclude that 1 should be C 18 H 24 N 2 .

The m/z 269 (10%) and 270 (1%) in 1 MSspectrum and the m/z 311 (22%) and 312 (2%), in 1a MS-spectrum, were assigned to the corresponding M+1 and M+2, respectively. These observations indicated that M+1 and M+2 are representing 20% and 2% of the parent ion peaks, respectively, in both 1 and 1a. This finding is supporting the likely presence of two nitrogen atoms in the molecule, since the calculated% of M+1 and M+2 are 20.52% and 1.91% of the parent ion peak, respectively, in both 1 and 1a (Silverstien et al., 1991). The 13 C-NMR data of 1 and 1a (Table I) indicated the likely absence of any oxygenation including acetylatable hydroxyl groups. The conversion of 1 to 1a under acetylation conditions indicated the likely presence of a free NH 2 group in 1 to give the corresponding acetamide, 1a. The 13 C-NMR spectrum of 1 displayed thirteen carbon signals. The DEPT experiment discriminated them into fourteen different signals, represented by three methyl groups at 21. symmetrical carbon atoms. This conclusion based on their high intensity and the 1 H-NMR profile.

The 1 H-NMR of 1 demonstrated the presence of nine olefinic protons, represented by one pair of doublets at 6.58 and 6.98 ppm (two protons each), four overlapping doublets at 6.81, 6.82, 7.16 and 7.18 ppm (one proton each) and one triplet at 6.75 ppm (for one proton). The COSY correlation indicated that the doublet at 6.58 ppm is coupled to the doublet at 6.98 ppm, which were correlated to (HETCOR) the carbon signals at 129.12 and 113.92 ppm, respectively, and were assigned to (2, 6) and (3, 5)-positions of the 1, 4-dihydropyridine ring, respectively. The doublets at 6.81 and 6.82 ppm were correlated to (HETCOR) the carbon signal at 114.64 and were interacting with (COSY) the doublets at 7.16 and 7.18 ppm, which was correlated to (HETCOR) the carbon signal at 124.06 ppm, were assigned to (6ЈЈ, 2ЈЈ) and (3ЈЈ, 5ЈЈ)-positions of the 1, 4-disubstituted benzene ring. The non-protonated olefinic carbon signal at 146.29 ppm was assigned to the nitrogen bonded 1ЈЈ-position, while that at 131.95 ppm was assigned to 4ЈЈ-position, likely connected to a side chain. The 144.04 ppm signal has to be assigned to 4-position of the pyridine ring formng an exocyclic double bond. This was confirmed by the presence of only one more olefinic carbon signal at 118.52 ppm. This carbon signal was correlated to (HETCOR) the proton triplet at 6.75 ppm and was assigned to 1Ј-position. The appearance of this proton signal as a triplet indicated that its likely attachment to the methylene carbon signal at 46.90 ppm which is correlated to (HETCOR) the proton multiplets at 1.26 and 1.47 ppm. This was confirmed by COSY interactions of these positions (Fig. 1). The 1 H-NMR spectrum of 1 indicated the presence of three methyl groups doublets at 1.15, 0.94 and 0.92 ppm which were correlated to the methyl carbon signals (DEPT) at 21.06, 22.98 and 22.54 ppm, respectively. The two methin carbon signals (DEPT) at 46.90 and 25.05 ppm were correlated to (HETCOR) the protons signals at 3.48 and 1.76 ppm, respectively. This indicated the likely presence of an isopropyl group and an aminoethyl residue. The methyl proton doublet at 1.15 ppm was coupled to (COSY) the proton signal at 3.48 ppm assigned to the aminomethin proton of 1ЈЈЈ-postion. The COSY correlation (Fig. 1 indicated that the methyl doublets at 0.94 and 0.92 ppm were coupled to the proton signal at 1.76 ppm which is also coupled to the methylene protons signals at 1.26 and 1.47 ppm. This indicated that the isopropyl group is attaching to the methylene group to form the 3-methylbutenylidine residue attached to 4-position. This made it possible to confirm that the 1-aminoethyl residue is attached to 4ЈЈ-position of the benzene ring. Further structural confirmations came through the intensive use of the EIMS fragmentation pattern (Fig. 1). The m/z 211 [M-Me 2 CHCH 2 ] + confirm the presence of the 3-methylbutenylidine side

The spectroscopic data of 2 and 2a are very similar to those of 1 and 1a with a few differences. The EIMS of 2 gave a parent ion peak at 282 and 2a gave m/z 324, while the HREIMS of 2a gave 324.2198 for C 21 H 28 N 2 O (calcd., 324.2203) and conclude C 19 H 26 N 2 for 2. This indicated a difference of 14 mass units between 1 and 2. This difference could be attributed to an additional methyl or methylene group. The 13 C-NMR (Table I) and DEPT spectra supported this, where two methylene carbon signals (35.02 and 35.34 ppm) were observed for 2 (2a: 32.94 and 35.88 ppm), while only one (46.90 ppm) was observed for 1 (1a: 43.92 ppm). The location of this new methylene group was found to be adjacent to the methylene group of 1 to form 4-methylpentenylidine side chain in 2 rather than the 3-methylbutenylidine residue in 1. These two methylene carbon signals at 32.94 and 35.88 ppm were correlated to the protons multiplets at (1.24, 1.28 ppm) and (1.22, 1.41 ppm), respectively, which were coupled to each other (COSY), confirming their direct connection. The EIMS provided more structural confirmation, since, the base peak for both 1 and 2 is m/z 211, which indicated that the difference must be located in one of the cleaved fragments for each compound. For 1 this corresponds to m/z 57 [M-C 4 H 9 ] + , which is consistent with an isobutyl residue, and for 2 m/z 71 [M-C 5 H 11 ] + , for an isopentyl fragment. While the rest of the fragments are very similar in both 1 and 2. The 13 C and 1 H-NMR data of 2 and 2a are listed in Tables I and II