Antiproliferative and antimalarial sesquiterpene lactones from Piptocoma antillana from Puerto Rico.

The genus Piptocoma, a member of the Asteraceae family, was first discovered in the West Indies, and consisted initially of only three species. The genus was expanded in 2000, when Pruski described the Central and South American plant species from the genus Pollalesta as cogeneric with Piptocoma, and this raised the number of Piptocoma species to eighteen[2]. A literature search demonstrated that the only reports on the chemical constituents of the genus Piptocoma were in the last two years, describing the isolation of cytotoxic sesquiterpene lactones, phenylpropanol coumarates, and flavonoids from P. rufescens[3]. Goyazensolide (5), one of the isolated sesquiterpene lactones, was later shown to induce apoptosis in cancer cells in vitro and in vivo[4]. Our ongoing screening of extracts from the Natural Products Discovery Institute collection as part of a collaborative research program to explore the potential of the former Merck collection of extracts[5] indicated that a CH2Cl2 extract of the leaves and twigs of P. antillana exhibited significant antiproliferative activity against the A2780 human ovarian cancer cell line. We report herein the bioassay-guided isolation and structure identification of the components responsible for the antiproliferative activity, as well as the antiplasmodial activity of the same compounds. An active CH2Cl2-soluble fraction obtained from liquid-liquid partition of the extract (100 mg) was subjected to dereplication studies using size-exclusion chromatography, reverse phase HPLC coupled with bioassay, high-resolution ESIMS (HRESIMS), 1HNMR, and a database search using the online Dictionary of Natural Product (DNP). The results indicated that the extract contained at least one new bioactive compound, and so a 2.0 g sample was investigated. Fractionation of this extract yielded an antiproliferative CH2Cl2 fraction which was further subjected to size-exclusion column chromatography on Sephadex LH-20 followed by normal phase silica gel column chromatography. The most active fractions from the silica gel column were subjected to C-18 HPLC to yield two new (1, 2) and two known (3, 4) bioactive sesquiterpene lactones. Compound 4 had the molecular formula C19H20O7 as determined by high resolution electrospray ionization mass spectroscopy (HRESIMS) analysis. It was determined to be 5-epiisogoyazensolide (4) by searching its molecular formula and key 1H NMR spectroscopic features in the DNP database and comparison of its full 1H NMR spectrum with reported literature data[6]. Compound 3 was identified as 1-oxo-3,10-epoxy-8-(2-methylacryloxy)-15-acetoxy-germacra-2,4,11(13)-trien-6(12)-olide (15-acetylgoyazensolide) by comparison of its spectroscopic data with values reported in the literature[7]. Compound 1 was obtained as an amorphous white powder and had the molecular formula C20H22O7 as indicated by HRESIMS analysis (m/z: 375.1461 [M+H]+, calcd. for C20H23O7+, 375.1438). Its IR spectrum showed absorptions characteristic of an α,β-unsaturated γ-lactone (1765 and 1640cm−1) and an α,β-unsaturated ester (1710 and 1645 cm−1) functions, as well as a dihydrofuran-3-one ring (1709 and 1582 cm−1)[3a, 6c, 7a, 8]. The similarity of the IR spectroscopic data of 1 with those of 4, in conjunction with the UV absorption characteristics at λmax 208 and 270 nm, suggested that 1 was also a furan ring-containing germacranolide similar to goyazensolide or furanoheliangolide[6c, 7a, 8]. Its 1H NMR spectrum (Table 1) displayed signals for a methyl group at δH1.53 (s, 3H, CH3-14), two exocyclic methylene groups as two doublets at δH 6.21 (J= 3.3 Hz, 1H) and 5.46 (J=2.9 Hz, 1H) and two singlets at δH6.00 and 5.78 (each 1H), representing (H2-13 and H2-15), and an olefinic proton at δH5.82 (s, 1H, H-2). These signals are all very similar to those in the goyazensolide skeleton of 4. In the 13C NMR spectrum, the presence of signals attributable to a conjugated ketone carbonyl at δC204.4 ppm (C-1), a lactone carbonyl at δC169.1(C-12), the carbons of an oxygen bearing alkene at δC 106.8 (C-2) and 185.9 (C-3) and of two exocyclic alkenes at δC 133.9 (C-11), 123.8 (C-13), 134.5 (C-4), and 127.1 (C-15), and three oxygen bearing methines at δC 84.5 (C-5), 85.1 (C-6), and 72.3 (C-8), supported the preliminary structural assignment[7c]. In the HMBC experiment, protons at δH6.00and 5.78 correlated with both C-3 (δC 185.9) and C-5 (δC 84.5), which allowed us to assign the two olefinic protons to be those of H2-15 (Fig. 2). In the same manner, the other pair of the exocylic methylene protons at δH 6.21 and 5.46 were determined to be those of H2-13 due to their correlations with δC169.1(C-12) and δC44.8(C-7). The 1H and 13C NMR spectra of 1 also revealed the expected signals from the goyazensolide methacrylate side chain, namely for a methyl group at δH1.82 (s, 3H, H-4'), two olefinic proton signals at δH 5.95 and 5.53 (m, 1H for each, H-3'), and four carbon resonances at δC 166.9 (C-1'), 135.4 (C-2'), 126.4 (C-3'), and 18.0 (C-4')[7c]. This assignment was confirmed by the HMBC correlations (Fig. 2) between the proton signals of H-4' with both C-1' and C-3'. The acyl group was located at C-8 by the long range HMBC cross peak observed between the signals at δH4.31 (ddd, J=11.8, 1.4, 1.3 Hz, 1H, H-8) and δC 166.9 (C-1'). The1H spectrum of 1 contained an additional proton signal at δH3.73 (s, 3H) as compared with the spectrum of 4. A carbon signal at δC57.2 and a deshielded proton signal at δH 4.23 (H-5) confirmed the presence of a methoxyl group at C-5 in 1 as compared with the hydroxyl group of 4. The HMBC correlation between δH3.73 (s, 3H) and δC 84.5 (C-5) substantiated the placement of the methoxyl group at the C-5 position in 1. The complete assignments of all protons and carbons of 1 (Table 1) were accomplished by further interpretation of the HMBC and NOESY spectra. The relative configurations at C-5, C-6, C-7, C-8, and C-10 of 1 were suggested by the analysis of a NOESY experiment (Fig. 2). The absolute configuration at C-7 was determined to be R by the negative Cotton effects at 225 and 268 nm observed in its CD spectrum. This is consistent with previous studies demonstrating that sesquiterpene lactones with a C-7, C-6 trans-fused α-methylene-γ-lactone moiety display negative Cotton effects in the range of 216–225 and 252–271 nm in their CD spectra, arising from the π→π * and n→π* transitions of the lactone ring, respectively[9]. Compound 1 was thus assigned as (5S,6S,7R,8S,10R)-1-oxo-3,10-epoxy-5-methoxy-8-(2-methylacryloxy)germacra-2,4(15),11(13)-trien-6,12-olide[10], or 5-O-methyl-5-epiisogoyazensolide. Figure 2 Key HMBC and NOESY correlations of 1 and 2. Table 1 1H and 13C NMR data for compounds 1 and 2. Compound 2 was isolated as an amorphous white powder with a molecular formula of C20H22O7 based on its HRESIMS spectrum. The close similarity of the UV, IR,1H and 13C NMR spectroscopic data of 2 with those of 1 and 3 demonstrated that all three compounds contain the same goyazensolide skeleton with a methacrylate side chain at the C-8 position. Comparison of the 1H NMR spectroscopic data of 2 with those of 3 indicated that the major differences between them were the lack of signals for the 15-acetyl group in 3 and the addition of signals at δH 3.40 (s, 3H) and δC 58.5 (15-OMe) for an O-methyl group. HMBC correlations from the H-15 with the methoxy carbon at δC 58.5 and from the methoxy protons δH 3.40 (s, 3H) with the deshielded oxygen bearing methylene carbon at δC 72.7 (C-15) confirmed that the methoxy group was at C-15. As in the case of 1, all proton and carbon signals of 2 were assigned by interpretation of the data obtained from HMBC and NOESY experiments (Table 1). In addition, comparison of the CD and NOESY spectrum of 2 with 1 indicated that both compounds have the same absolute configurations at their C6, C7, C8, and C10 stereogenic centers. Therefore, compound 2 was determined to be (6S,7R,8S,10R)-1-oxo-3,10-epoxy-15-methoxy-8-(2-methylacryloxy)germacra-2,4(5),11(13)-trien-6,12-olide[7a], or 15-O-methylgoyazensolide. Compounds 1–4 were evaluated for their antiproliferative activity against the A2780 human ovarian cancer cell line. They showed micromolar activities with half-maximum inhibitory concentration (IC50) values of 1.5±0.5,0.6 ±0.3, 1.62 ±0.05, and1.56 ±0.04 µM, respectively. All the compounds were further evaluated for their antimalarial activity against P. falciparum Dd2 (a chloroquine/mefloquine-resistant strain), and showed IC50 values of 6.2 ± 0.5, 2.2 ± 0.5, 8.0±0.4, and 9.0 ± 0.6 µM, respectively.