Isolation and Structure of Cancer Cell Growth Inhibitory Tetracyclic

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Isolation and Structure of Cancer Cell Growth Inhibitory Tetracyclic Triterpenoids from the Zimbabwean Monadenium lugardae George R. Pettit,* Qinghua Ye, Delbert L. Herald, John C. Knight, Fiona Hogan, Noeleen Melody, Venugopal J. R. V. Mukku, Dennis L. Doubek, and Jean-Charles Chapuis Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, Arizona 85287-1604, United States S Supporting Information *

ABSTRACT: The Zimbabwean medicinal plant Monadenium lugardae was evaluated as a potential source of new anticancer constituents. Four new tetracyclic triterpene (1−4) were isolated, accompanied by four previously known triterpenes (5−8). Against a panel of human tumor cell lines, lugardstatins 1 (1) and 2 (2) had good cancer cell growth inhibitory activity. All of the triterpene structures (1−8) were established by 1D and 2D NMR spectrometric and HR mass spectrometric analysis.

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inhibitory constituents were concentrated in the CH2Cl2soluble fraction (ED50 0.41 μg/mL), which was subjected to bioassay-guided separation using a series of gel permeation (CH3OH) and partition (3:2 CH2Cl2−CH3OH, 8:1:1 hexane− 2-propanol−CH3OH, and 1:4:3:1 hexane−toluene−acetone− CH3OH) separations on Sephadex LH-20. Final purification via HPLC yielded the compounds lugardstatin 1 (1) (1.3 mg), lugardstatin 2 (2) (1.3 mg), (17R,20S,23E)-3β,25-dihydroxy1 3 α, 1 4 α -l a n os t a -8 ,2 3 -d ie n - 1 1- o n e ( 3 ) ( 1 . 5 m g ) , (17R,20S,23E)-3β,25-dihydroxy-5β-lanosta-8,23-dien-7-one (4) (1.5 mg), (23E)-3β,25-dihydroxytirucalla-8,23-dien-7-one (5) (1.0 mg), (25E)-3β,25-dihydroxyeupha-8,25-diene (6) (1.8 mg), (23E)-3β,25-dihydroxyeupha-8,23-diene (7) (4.0 mg), and (23E)-3β,24-dihydroxylanosta-8,23-diene (8) (3.5 mg). All of these tetracyclic triterpenoids were obtained as colorless powders.

he plant family Euphorbiaceae contains some 300 genera comprising about 7500 species of shrubs, trees, and a small number of herbaceous types. The genus Monadenium contains 47 tropical species that are found mainly in eastern Africa (tropical East Africa and the eastern Transvaal area), with a few occurring in central and southwest Africa. One of the eastern species, Monadenium lugardae, is a herbaceous tropical plant.1a Ingestion of its roots is reputed among the Zulu and Swazi people to cause sudden death,1b and the root extract is also reported to cause hallucinations and delirium.2 However, the plant has been used widely as a medicine in the eastern Transvaal area.1 The latex of M. lugardae has been found to have insecticidal properties,3a and a methanol extract showed pronounced guinea pig ileum contractions,3b but no other information related to its small-molecule components appears in the scientific literature. By employment of bioassay-guided separation using the P388 murine lymphocytic leukemia cell line and, for more advanced phases of the separations, a minipanel of human cancer cell lines, we have isolated four new tetracyclic triterpenes (1−4), two of which showed cancer cell line inhibitory activity, and four previously known triterpenes (5−8). A summary of the isolation and structural elucidation now follows. In 1982, M. lugardae was collected in Zimbabwe, and we began an evaluation for potential anticancer activity in collaboration with the U.S. National Cancer Institute (NCI). A small collection of roots and aerial parts of this plant was preserved in CH3OH and afterward extracted with CH2Cl2−CH3OH (1:1) to give 7.2 g of initial extract, which was found to be active in vivo against the NCI’s P388 lymphocytic leukemia cell line at 0.29 mg/kg (T/C 121%) and at 0.58 mg/kg, with toxicity beginning at 1.17 mg/kg (T/C 120%). After dilution with water, the CH2Cl2−CH3OH-soluble fraction was subjected to a solvent partition procedure. The P388 leukemia cell line © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The molecular formula of lugardstatin 1 (1), a colorless, crystalline solid, [α]28D −7.5 (c 0.04, CH2Cl2), was determined to be C30H50O2 on the basis of high-resolution mass spectrometry and interpretation of the NMR spectra (Tables 1 and 2). The 1H, APT, and HMQC NMR spectra of triterpene 1 showed the presence of one secondary and six tertiary methyl groups; 10 methylenes, of which one was an olefinic group; seven methines, with two of these oxygenated; and six quaternary carbons, of which two were double-bonded. The molecular formula required a tetracarbocyclic structure as well as the two olefinic units. Detailed analysis of 1H−1H COSY (Figure 1) and 13C−1H HMQC spectra of 1 enabled assignment of the structural fragments C-2−C-3, C-5−C-6− Received: February 4, 2016

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DOI: 10.1021/acs.jnatprod.6b00107 J. Nat. Prod. XXXX, XXX, XXX−XXX

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correlations were found as follows: H-5 to H-3, H-7α, and H-9; Me-29 to H-2β and H-6β; Me-19 to H-2β, Me-29, and Me-30; Me-18 to H-16α and H-20; Me-21 to H-12β; H-15 to H-17; and H-24 to Me-26. In a comparison of the 13C NMR data of 2 with those of 1, the only differences observed were between the signals corresponding to C-24, C-25, C-26, and C-27 with those of 2 being shifted by +0.31, −0.25, +0.50, and −0.36 ppm, respectively, which confirmed that 2 has the same configuration as 1 except at C-24. Thus, the preceding data, along with the difference in retention time obtained when cospotted with 1 on TLC and by comparison of data from similar compounds from the literature, 4,5a determined that lugardstatin 2 [2, (17S,20S,24R)-apotirucalla-14,25-diene-3β,24-diol] was the 24R-epimer of lugardstatin 1 (Figure 2). Mass spectrometric analysis of compound 3 gave the molecular formula C30H48O3. The 1H and APT NMR spectra (Table 2) of 3 showed the presence of one secondary and seven tertiary methyl groups, eight methylenes, six methines (of which one was oxygenated and two were olefinic), eight quaternary carbons (with two assigned to olefinic groups), and one carbonyl group. The seven units of unsaturation indicated by the molecular formula suggested a four-ring triterpene, which was confirmed by the NMR data. Analysis of the 1H−1H COSY and 13C−1H HMQC spectra of 3 (Figure 3) enabled assignment of fragments C-1−C-2−C-3, C-6−C-7, C-15−C16−C-17, and C-21−C-20−C-22−C-23−C-24. In the HMBC spectrum of 3 (Figure 3), 13C−1H long-range correlation signals (C-3 to H-2, -28, and -29; C-1, -5, -9, and -10 to H-19; C-3, -4, and -5 to H-28 and -29; C-14 to H-16, -17, and -18; C17 to H-18 and -21; C-24 to H-22, -26, and -27; C-25 to H-23, -26, and -27; C-12, -14, and -17 to H-18; and C-8, -13, -14, and -15 to H-30) enabled establishment of the planar structure. The large coupling constant of H-3 indicated that the hydroxy group is oriented equatorially (β) at C-3.4 In the ROESY spectrum (Figure 3), correlation signals were found between H-3 and H1α, H-5; H-7α and H-5, Me-18, Me-30; Me-19 and H-6β, Me29; Me-28 and H-6α; Me-30 and H-16α, H-17, H-20; and Me18 and H-20. The correlation of the 16α-proton with both Me18 and Me-30 suggested a cis C/D ring junction, which is rare in this series, while the correlation of Me-30 with H-17 and H20 suggested a 17R,20S-configuration. The proton resonances for H-23 and H-24 overlapped as a multiplet at 5.6 ppm in CDCl3 and were not resolved by obtaining a spectrum of 3 in deuterated benzene.5 Therefore, the geometry of the side chain was assigned as E based on both comparison of the 13C NMR resonances of analogues with similar side chains4b,5a and literature reports suggesting that most triterpenes of this type isolated from plants adopt this configuration.5b Thus, triterpene 3 was identified as (17R,20S)-3β,25-dihydroxy-13α,14α-lanosta-8,23-dien-11-one. Compound 4 was also found by mass spectrometry to have the molecular formula C30H48O3. The APT NMR spectrum of 4 exhibited 30 carbon signals corresponding to eight methyl groups, eight methylenes, six methines, and eight quaternary carbons. Resonances for a disubstituted double bond (δH 5.60, m; δc 125.3 d; and 139.5 d), an α,β-unsaturated carbonyl (δc 198.3, 138.8, and 165.4 s), and a secondary alcohol methine [δH 3.29 dd (J = 5.2, 11.2 Hz)] were evident in the 1H, APT, and HMQC NMR spectra (Tables 1 and 2). The seven units of unsaturation in total required by the molecular formula suggested a tetracarbocyclic ring system. Analysis of the 1 H−1H COSY and 13C−1H HMQC spectra of 4 (Figure 4) led to the connectivity of fragments C-1−C-2−C-3, C-5−C-6,

C-7, C-11−C-12, C-15−C-16−C-17−C-20−C-21, and C-23− C-24. In the HMBC spectrum (Figure 1), 13C−1H long-range correlation signals (C-3 to H-28 and -29; C-5 to H-7, -19, -28, and -29; C-2, -10, and -9 to H-19; C-7, -8, -9, and -14 to H-30; C-12, -14, and -17 to H-18; C-17, -20, and -22 to H-21; and C24, -27, and -25 to H-26) enabled establishment of the planar structure skeleton as a euphane- or tirucallane-type triterpene (Figure 1). The large coupling constant of H-3 indicated that the hydroxy group was oriented equatorially (β) at C-3, and both the chemical shift of Me-21 and the negative optical rotation were consistent with literature reports for tirucallanes.4a Insufficient material was available to determine the absolute configuration at C-24 using the modified Mosher’s method;4b therefore the configuration of C-24 was assigned by examination of the ROESY spectrum of 1 (Figure 1), and the following correlations were found: H-5 to H-3, H-7α, and H-9; Me-29 to H-2β and H-6β; Me-19 to H-2β, Me-29, and Me-30; Me-18 to H-16α and H-20; Me-21 to H-12β; H-15 to H-17; H23α to H-20 and H-24; and H-24 to Me-26. These data led to the assignment of lugardstatin 1 (1) as (17S,20S,24S)apotirucalla-14,25-diene-3β,24-diol. Compound 2, a colorless, amorphous solid, [α]28D −55 (c 0.02, DCM), by mass spectrometric analysis (C30H50O2) was found to be isomeric with triterpene 1. The 1H, APT, and HMQC NMR spectra of 2 showed the presence of one secondary and six tertiary methyl groups; 10 methylenes, of which one was an olefinic group; seven methines, of which two were oxygenated; and six quaternary carbons, of which two had double bonds. The 1H and 13C NMR shifts of compound 2 (Table 1 and 2) were similar to those of 1. Elucidation of the 1 H−1H COSY, HMQC, and HMBC spectra of 2 (Figure 2) revealed a planar structure identical to that of 1. This was confirmed by the ROESY spectrum of 2 (Figure 2), in which B

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Table 1. 1H NMR Spectroscopic Data for Compounds 1−4a position 1α 1β 2α 2β 3 5 6α 6β 7α 7β 9 11α 11β 12α 12β 15α 15β 16α 16β 17 18 19 20 21 22 23 24 26 27 28 29 30 a

1

2

3

0.99, m 1.64, m 1.60−1.65, m

1.00, m 1.65, m 1.58−1.62, m

0.95, m 2.62, m 1.58−1.70, m

3.20, dd (11.2, 4.8) 0.81, m 1.58, m 1.43, m 1.32, m 2.00, dt (12.8, 2.8) 1.47, m 1.38−1.42, m 1.38−1.42, m 1.91, m 1.47, m 5.27, d (2.4)

3.22,dd (11.6, 5.2) 0.82, m 1.60, m 1.45, m 1.99, dt (12.8, 3.2) 1.34,dd (12.6, 3.2) 1.49, m 1.48−1.52, m 1.48−1.52, m 1.92, m 1.47, m 5.27, d (2.8)

3.25,dd (13.8, 7.3) 1.00, m 1.73, m 1.43, m 2.37, m 2.17, m

2.18, ddd (11.4, 7, 4,) 1.96,m 1.42, m 0.99, s 0.88, s 1.62, m 0.91, d 1.38, m−1.42, m 1.65, m; 1.45, m 4.02, t (6.4) 4.93, s; 4.83, s 1.72, s 0.97, s 0.79, s 1.02, s

2.18, ddd (10.8, 7.2, 3.6) 1.95, m 1.42, m 0.99, s 0.88, s 1.64, m 0.91, d (6.0) 1.55, m−1.45, m 1.65 m; 1.45 m 4.02, t (6.4) 4.92, s; 4.83, s 1.72, s 0.97, s 0.79, s 1.02, s

2.34, d (23.3) 2.52, d (23.3) 2.18, m; 2.34, m 2.18,m; 2.34, m 1.75, m 1.37, m 1.66, m 0.89, s 1.20, s 1.47, m 0.86, d (8.5) 2.20, m; 1.78, m 5.60, m 5.60, m 1.31, s 1.32, s 1.03, s 0.83, s 1.0, s

4 1.43, m 1.88, dt 1.70, m 1.76 m 3.29, dd (11.2, 5.2) 1.66, m 2.42, m 2.36, m

2.17, m 2.25, m 1.76−1.80, m 1.58, 2.12, 1.39, 2.00, 1.48, 0.74, 1.06, 1.47, 0.91, 2.20, 5.60, 5.60, 1.32, 1.32, 1.00, 0.89, 0.96,

m m m m m s s m d (6.1) m; 1.74, m m m s s s s s

Recorded in CDCl3 at 400 MHz. The assignments were based on APT, 1H−1H COSY, HMQC, and HMBC NMR experiments.

be stronger if correlations from Me-19 or H-5 to Me-18 or Me30 had been observed). The correlations between Me-30 and H-17 and between H-17 and Me-21 were indicative of a lanostane skeleton,6 as were the correlations from Me-18 to H11β and H-20 and from Me-19 to H-1β and H11β.7 From these observations and data, the structure of triterpene 4 was determined to be (20S,23E)-3β,25-dihydroxy-5β-lanosta-8,23dien-7-one. Mass spectrometric analysis also provided the molecular formula C30H48O3 for triterpene 5. The APT NMR spectrum of 5 gave carbon signals belonging to eight methyls, eight methylenes, six methines, and eight quaternary carbons. The 1 H and 13C NMR shifts of 5 were similar to those of triterpene 4, with the most significant differences being at C-17, C-18, C20, C-21, and C-22, and the 1H−1H COSY, HMQC, and HMBC spectra of 5 (Figure 5) revealed a planar structure identical to that of triterpene 4. From analysis of the NOE correlation signals (H-3 to H-2α and Me-28; H-1β to H-2β and Me-19; H-5 to H-6α and Me-28; Me-19 to H-6β, H-11β, and Me-29; Me-18 to H-11α, H-16α, and H-20; Me-30 to H-15β, H-16β, and H-17; and Me-21 to H-16α), combined with the absence of NOE correlations between Me-18 and Me-21, and from comparison of the data to those of compound 4 and its stereoisomers,4a,b compound 5 was identified as (23E)-3β,25dihydroxytirucalla-8,23-dien-7-one, which has been isolated previously from Euphorbia micractina.4b

C-15−C-16−C-17−C-20−C-21, and C-22−C-23−C-24, and the large coupling constant of H-3 indicated that the hydroxy group is oriented equatorially (β) at C-3. The HMBC spectrum (Figure 4) showed 13C−1H long-range correlations (C-4/H-2, -28, -29; C-7/H-5, -6; C-10/H-6, -19; C-13/H-11, -18, -30; C14/H-12, -15, -30; C-17/H-16, -18, -21; C-25/H-23, -24, -26, -27) that allowed assignment of the planar structural skeleton. A comparison of the 13C NMR data with those of (23E)-3β,25dihydroxyeupha-8,23-dien-7-one (kansenonol)4 showed a large degree of similarity except at C-7, C-11, C-16, C-17, C-18, C20, C-21, and C-22 [(ΔδC +2.0, −0.1, +0.2, −0.4, −0.3, +0.7, −0.9, +0.8) (ΔδC = δ4 − δref). In comparison to the signals of (24E)-3β-hydroxytirucalla-8,24-dien-7-one (epi-kansenone),4a there was also a large degree of similarity (see Table 2), whereas the 13C NMR signals of the side chain were in good agreement with those of (23E)-lanosta-8,23-diene-3β,25-diol (8).5a The optical rotation of compound 4 ([α]28D −15.5, c 0.01, CH2Cl2) was found to be opposite that of kansenonol ([α]23D +14.3, c 0.21, MeOH). In the ROESY spectrum (Figure 4) of compound 4, the following correlations were found: H-3 to H-1α, H-2α, and Me28; Me-29 to H-1β, H-5, and H-6β; Me-19 to H-1β, H-5, and H-11β; Me-18 to H-11β, H-12, H-15β, H-16β, H-20, and Me21; and Me-30 to H-15α, H-16α, and H-17. No correlation was observed between the Me-18 and Me-30 signals, indicating that the C/D ring junction is trans (although this conclusion would C

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Table 2. 13C NMR Spectroscopic Data for Compounds 1−4a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 38.2, 27.2, 79.0, 38.9, 55.9, 18.8, 41.7, 37.6, 48.9, 37.4, 31.3, 34.3, 46.8, 165.6, 117.5, 35.0, 60.5, 19.2, 15.9, 33.5, 18.7, 31.3, 17.4, 76.4, 147.8, 110.8, 17.6, 27.9, 15.4, 27.6,

2 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 C C CH CH2 CH CH3 CH3 CH CH3 CH2 CH2 CH C CH2 CH3 CH3 CH3 CH3

38.2, 27.2, 79.0, 38.9, 56.0, 18.9, 41.7, 37.6, 48.9, 37.4, 31.2, 34.3, 46.8, 165.7, 117.5, 35.0, 60.6, 19.2, 15.9, 33.5, 18.7, 31.4, 17.4, 76.7, 147.5, 111.3, 17.2, 27.9, 15.4, 27.6,

3 CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH2 C C CH CH2 CH CH3 CH3 CH CH3 CH2 CH2 CH C CH2 CH3 CH3 CH3 CH3

34.1, 27.9, 78.9, 39.0, 51.7, 18.0, 29.7, 161.2, 139.7, 37.1, 198.9, 51.0, 27.1, 44.7, 51.0, 30.1, 50.1, 17.5, 19.8, 36.4, 18.2, 38.7, 125.0, 139.8, 70.7, 30.0, 29.9, 28.3, 15.7, 24.1,

4 CH2 CH2 CH C CH CH2 CH2 C C C C CH2 C C CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

34.6, 27.4, 78.0, 38.8, 48.2, 35.8, 198.3, 138.8, 165.4, 39.3, 23.6, 29.8, 44.6, 47.6, 31.4, 28.6, 48.5, 15.6, 18.6, 36.6, 18.7, 39.0, 125.3, 139.5, 70.7, 29.9, 30.0, 27.3, 15.1, 24.3,

CH2 CH2 CH C CH CH2 C C C C CH2 CH2 C C CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3

Figure 2. COSY, HMBC, and key NOE correlations for lugardstatin 2 (2).

Figure 3. Key HMBC, COSY, and NOE correlations for triterpene 3.

a

Recorded in CDCl3 at 100 MHz. The assignments were based on APT, HMQC, and HMBC NMR experiments.

Figure 4. Key HMBC, COSY, and NOE correlations for triterpene 4. Figure 1. Key HMBC, COSY, and NOE correlations for lugardstatin 1 (1).

spectroscopic data as the known (23E)-3β,25-dihydroxyeupha-8,23-diene, which has been isolated already from Tripetalum cymnosum (Guttiferae)5a and from C. columnaris.8 Compound 8 was identified as (23E)-3β,25-dihydroxylanosta8,23-diene, which has been isolated previously from T. cymnosum5 and from a fungus, Inonotus obliquus.9 Compounds 1−8 were evaluated against the murine P388 lymphocytic leukemia cell line and against a minipanel of human cancer cell lines in our laboratories (Table 3). While none showed activity against P388 in vitro, compounds 1 and 2 were found significantly active against the human cell lines (except for 2 against the KM20L2 line) with greater potencies

Mass spectrometric and 2D NMR (including APT) spectroscopic analyses were consistent with a structural assignment for terpene 6 of 3β,24-dihydroxyeupha-8,25-diene. The configuration at C-24 was not assigned. This compound was first described as a photo-oxygenation product of euphol5a and was later isolated from a Venezuelan collection of the fruits of Clusia columnaris Engl., a member of the family Clusiaceae (Guttiferae).8 Triterpenes 7 and 8 were also identified by high-resolution mass spectrometric and high-field 2D NMR spectroscopic analyses. Compound 7 was characterized by mass and D

DOI: 10.1021/acs.jnatprod.6b00107 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Extraction and Isolation. A small collection of roots and aerial parts of this plant were preserved in methanol, and the extract was partitioned in 1:1 CH2Cl2−CH3OH. The CH2Cl2 fraction (7.2 g) was subjected to solvent partition separation (between n-hexane and 9:1 CH3OH−H2O followed by CH2Cl2 and 3:2 CH3OH−H2O). The final CH2Cl2 fraction (1.80 g, P388 ED50 0.41 μg/mL) proved to be the best source of murine P388 lymphocytic leukemia cell line inhibitory constituents and was subjected to P388-bioaassay-guided separation via gel permeation and partition column chromatographic procedures on Sephadex LH-20. Active fractions were separated by elution using the following sequence of solvents in succession: CH3OH, CH2Cl2− CH3OH (3:2), n-hexane−2-propanol−CH3OH (8:1:1), n-hexane− toluene−CH3OH (3:1:1), and n-hexane−toluene−acetone−CH3OH (1:4:3:1). Final separation and purification procedures were performed by repeated reversed-phase high-performance liquid chromatography (in CH3CN−H2O or CH3OH−H2O) to afford triterpenes 1 (1.3 mg), 2 (1.3 mg), 3 (1.5 mg), 4 (1.5 mg), 5 (1.0 mg), 6 (1.8 mg), 7 (4.0 mg), and 8 (3.5 mg). Lugardstatin 1 [1, (17S,20S,24S)-apotirucalla-14,25-diene-3β,24diol]: colorless crystals (CH3OH); Rf = 0.60 (5:2 hexane−ethyl acetate); mp 123−124 °C; [α]28D −7.5 (c 0.04, CH2Cl2); IR (CH2Cl2) νmax 2927, 1713, 1421, 1360, 1221, 1092, 899, 653, 614 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; (+)-HRFABMS m/z 443.39821 [M + H]+ (calcd for C30H51O2, 443.38891), 425.38113 [M + H − H2O]+ (calcd for C30H49O, 425.37835). Lugardstatin 2 [2, (17S,20S,24R)-apotirucalla-14,25-diene-3β,24diol]: colorless, amorphous powder (1.2 mg); Rf 0.55 (5:2 n-hexane− EtOAc), Rf 0.65 (12:1 CH2Cl2−acetone); mp 124−125 °C; [α]28D −55 (c 0.02, CH2Cl2); IR (CH2Cl2) νmax 3346, 2926, 2868, 2852, 2360, 2330, 1796, 1462, 1375, 1300, 1030, 993, 895, 808, 691, 635 cm−1; 1H and 13C NMR data, see Tables 1 and 2; (+)HRFABMS m/z 425.37814 [M + H − H2O]+ (calcd for C30H49O, 425.37835). (17R,20S,23E)-3β,25-Dihydroxy-13α,14α-lanosta-8,23-dien-11one (3): colorless, amorphous powder (1.5 mg); Rf 0.50 (n-hexane− EtOAc 2:1); mp 145−146 °C; [α]28D −12.5 (c 0.02, CH2Cl2); IR (CH2Cl2) νmax 3467, 3309, 2959, 2406, 1815, 1657, 1462, 1377, 1283, 1027, 977, 832, 744, 634, 611 cm−1; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRFABMS m/z 457.3619 [M + H]+ (calcd for C30H49O3, 457.3682); 439.3538 [M + H − H2O]+ (calcd for C30H47O2, 439.3576); 421.2989 [M + H − 2H2O]+ (calcd for C30H45O, 421.3470); HREIMS m/z 438.3498 [M − H2O]+ (100%), 423.3246 (31.6%), 405.3155 (21.8%). (17R,20S,23E)-3β,25-Dihydroxy-5β-lanosta-8,23-dien-7-one (4): colorless, amorphous powder (1.5 mg); [α]28D −15.5 (c 0.01, CH2Cl2); mp 143−144 °C; IR (CH2Cl2) νmax 3375, 2970, 2393, 1803, 1657, 1586, 1458, 1372, 1270, 1103, 1036, 978, 747, 652, 614 cm−1; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRAPCIMS m/ z 457.3674 [M + H]+ (calcd for C30H49O3, 457.3682). (23E)-3β,25-Dihydroxytirucalla-8,23-dien-7-one (5): colorless, amorphous powder (1.0 mg); mp 143−144 °C; [α]28D −5.5 (c 0.02, CH2Cl2) [lit.5 [α]20D −2.5 (c 0.04, CH3OH)]; IR (CH2Cl2) νmax 3365,

Figure 5. Key HMBC, COSY, and NOE correlations for compound 5.

than the previously known compounds 6−8. Triterpenes 3−5 exhibited no activity against any of the seven cell lines. The new cancer cell line inhibitory tetracyclic triterpenes 1, 2, and 6−8 were not further evaluated but may be candidates for other biological areas.10,11

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EXPERIMENTAL SECTION

General Experimental Procedures. Solvents used for column chromatography were freshly distilled. Sephadex LH-20, particle size 25−100 μm, for use in gel permeation and partition column chromatographic separations, was obtained from Pharmacia Fine Chemicals AB. The TLC plates were viewed under shortwave UV light, developed by 10% H2SO4 spray reagent, and heated at approximately 150 °C. For HPLC separations, a Phenomenex Luna (particle size 5 μm, 250 mm × 10.0 mm) C18 column or a Phenomenex Luna (particle size 5 μm, 250 mm × 4.6 mm) C18 column was used in the reversed-phase mode. For elution and detection, a Waters Delta (model 600E) solvent metering pump and a Waters 2487 dual λ absorbance detector were employed. The optical rotation values were measured using a PerkinElmer 241 polarimeter. IR spectra were recorded on an Avatar 360 FT-IR instrument with the sample prepared in a CH2Cl2 film. NMR experiments were conducted employing Varian Unity Inova 400 and 500 instruments with deuterated solvents. The HRFABMS was measured with a Kratos MS-50 mass spectrometer (University of Nebraska Midwest Center for Mass Spectrometry). Plant Material. The whole plant of Monadenium lugardae including the roots was collected in Zimbabwe in 1982 and provided by Dr. James A. Duke, Chief, Economic Botany Laboratory, Building 265, BARC-East, U.S. Department of Agriculture, Agricultural Research, Northeastern Region, Beltsville, MD, USA.

Table 3. Inhibition of the Murine P388 Lymphocytic Leukemia (ED50 μg/mL) and Human Cancer Cell Lines (GI50 μg/mL) by Triterpenes 1−8a cell lineb compound

P388

BXPC-3

MCF-7

SF-268

NCI-H460

KM20L2

DU-145

1 2 3 4 5 6 7 8

>10 >10 >10 >10 >10 >10 >10 >10

0.83 0.20 >1 >1 >1 1.5 1.8 1.8

0.79 0.68 >1 >1 >1 2.5 2.1 2.0

0.33 0.36 >1 >1 >1 1.4 2.2 1.9

0.91 0.19 >1 >1 >1 1.7 1.9 1.9

0.71 >1 >1 >1 >1 7.0 2.0 1.8

0.65 0.58 >1 >1 >1 1.5 1.9 1.9

a

DMSO was used as a vehicle in the testing. bCancer cell lines in order: murine lymphocytic leukemia (P388); pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145). E

DOI: 10.1021/acs.jnatprod.6b00107 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

2950, 1800, 1660, 1575, 1449, 1384, 1262, 1115, 1028, 747, 654, 617 cm−1; (+)-HRFABMS m/z 457.3689 [M + H]+ (calcd for C30H49O3, 457.3682), 439.3614 [M + H − H2O]+ (calcd for C30H47O2, 439.3576). (25E)-3β,25-Dihydroxyeupha-8,25-diene (6): colorless, amorphous powder (1.8 mg); mp 154−155 °C; [α]28D +6.5 (c 0.02, CH2Cl2) [lit.8 [α]D +19.1 (c 1.70, CHCl3)]; (+)-HRFABMS m/z 425.37433 [M + H − H2O]+ (calcd for C30H49O, 425.37835). (23E)-3β,25-Dihydroxyeupha-8,23-diene (7): colorless, amorphous powder; (+)-HRAPCIMS m/z 425.3816 [M + H − H2O]+ (calcd for C30H48O, 425.3783). (23E)-3β,24-Dihydroxylanosta-8,23-diene (8): colorless, amorphous powder; (+)-HRAPCIMS m/z 425.3807 [M + H − H2O]+ (calcd for C30H48O, 425.3783). Cancer Cell Line Bioassays. Inhibition of human cancer cell growth was assessed using the National Cancer Institute’s standard sulforhodamine B assay, as previously described.12 Briefly, cells in a 5% fetal bovine serum/RPMI1640 medium were inoculated in 96-well plates and incubated for 24 h. Serial dilutions of the compounds were then added. After 48 h, the plates were fixed with trichloroacetic acid, stained with sulforhodamine B, and read with an automated microplate reader. A growth inhibition of 50% (GI50, or the drug concentration causing a 50% reduction in the net protein increase) was calculated from optical density data with Immunosoft software. Mouse leukemia P388 cells13 were incubated for 24 h in a 10% horse serum/Fisher medium followed by a 48 h incubation with serial dilutions of the compounds. Cell growth inhibition (ED50) was then calculated using a Z1 Beckman/Coulter particle counter.

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(4) (a) Wang, L.-Y.; Wang, N.-L.; Yao, X.-S.; Miyata, S.; Kitanaka, S. J. Nat. Prod. 2003, 66, 630−633. (b) Xu, W.; Zhu, C.; Cheng, W.; Fan, X.; Chen, X.; Yang, S.; Guo, Y.; Ye, F.; Shi, J. J. Nat. Prod. 2009, 72, 1620−1626. (5) (a) Leong, Y.-W.; Harrison, L. J. Phytochemistry 1999, 50, 849− 857. (b) Takahashi, S.; Satoh, H.; Hongo, Y.; Koshino, H. J. Org. Chem. 2007, 72, 4578−4581. (6) Akihisa, T.; Kimura, Y.; Kokke, W. C. M. C.; Takase, S.-i.; Yasukawa, K.; Tamura, T. J. Chem. Soc., Perkin Trans. 1 1996, 2379− 2384. (7) Yang, J.-H.; Wen, J.; Du, X.; Li, X.-N.; Wang, Y.-Y.; Li, Y.; Xiao, W.-L. Tetrahedron 2010, 66, 8880−8887. (8) Compagnone, R. S.; Suarez, A. C.; Leitao, S. G.; Delle Monache, F. Rev. Bras. Farmacogn. 2008, 18, 6−10. (9) Nakamura, S.; Iwami, J.; Matsuda, H.; Mizuno, S.; Yoshikawa, M. Tetrahedron 2009, 65, 2443−2450. (10) Chen, C.; Qiang, S.; Lou, L.; Zhao, W. J. Nat. Prod. 2009, 72, 824−829. (11) Meng, D.; Qiang, S.; Lou, L.; Zhao, W. Planta Med. 2008, 74, 1741−1744. (12) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Viagro-Wolff, A.; GrayGoodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757−766. (13) Suffness, M.; Douros, J. In Methods in Cancer Research; DeVita, V. T., Jr.; Bush, H., Eds.; Academic: New York, 1979; Vol. 16, pp 73− 126.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00107. NMR spectra of compounds 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@ asu.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are pleased to extend our thanks and appreciation for the financial support provided by grant R01 CA90441 from the Division of Cancer Treatment, Diagnosis and Centers, National Cancer Institute, DHHS, and from the Arizona Biomedical Research Commission, Dr. Alec D. Keith, and the Robert B. Dalton Endowment Fund. We also appreciate the assistance provided by Dr. R. K. Pettit, as well as M. Dodson and C. A. Weber.

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REFERENCES

(1) (a) The current contribution is Antineoplastic Agents 584; for series Part 583 see: Rudy, A.; Lopez-Anton, N.; Barth, N.; Pettit, G. R.; Dirsch, V. M.; Schulze-Osthoff, K.; Rehm, M.; Prehn, J. H. M.; Vogler, M.; Fulda, S.; Vollmar, A. M.Cell Death Differ. 2008, 15, 1930− 194010.1038/cdd.2008.125. For Part 582, see: Pettit, G. R.; Meng, Y.; Pettit, R. K.; Herald, D. L.; Hogan, F.; Cichacz, Z. A. Bioorg. Med. Chem. 2010, 18, 4879−4883. (b) Watt, J. M.; Breyer-Brandwijk, M. G. The Medicinal and Poisonous Plants of Southern and Eastern Africa, 2nd ed.; Livingstone: Edinburgh, 1962; p 424. (2) De Smet, P. A. G. M. J. Ethnopharmacol. 1996, 50, 141−146. (3) (a) Gundidza, M. Planta Med. 1986, 52, 558−558. (b) Gundidza, M. Central African J. Med. 1991, 37, 141−144. F

DOI: 10.1021/acs.jnatprod.6b00107 J. Nat. Prod. XXXX, XXX, XXX−XXX