Terpenoids from Euphorbia soongarica and Their Multidrug

Article pubs.acs.org/jnp

Terpenoids from Euphorbia soongarica and Their Multidrug Resistance Reversal Activity Jie Gao† and Haji A. Aisa*,†,‡ †

Key Laboratory of Chemistry of Plant Resources in Arid Regions and ‡State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new terpenoids, including five diterpenoids (1−5), three nortriterpenoids (6−8), and two triterpenoids (9, 10), and 15 known terpenoids (11−25) were isolated from an acetone extract of Euphorbia soongarica. Sooneuphoramine (1) is the first example of a euphoractine B-type diterpenoid alkaloid, while sooneuphanones A−C (6−8) are rare nortriterpenoids from the Euphorbia genus. The isolated terpenoids were tested for their cytotoxicity and multidrug resistance (MDR) reversal activity, 10 of which showed moderate cytotoxicity against the KB and KBv200 cell lines, while 11 compounds exhibited P-gp modulating potential. The triterpenoid sooneuphanone D (9) possessed a remarkable MDR reversal activity much higher than the positive control, verapamil.

T

z 524.2996 [M + H]+, calcd for C31H42NO6, 524.3012) and 13C NMR data. The preliminary inspection of its 1H (Table 1) and 13 C (Table 2) NMR data suggested the presence of a cinnamoyl group [δC 168.0 (C-1′), vinylic protons at δH 6.84 (1H, d, J = 16.2 Hz, H-2′) and 8.01 (1H, d, J = 16.2 Hz, H-3′), aromatic protons at δH 7.47 (2H, m, H-5′, H-9′) and 7.34 (3H, m, H-6′, H-7′, H-8′)] and an acetyl group [δC 170.2 (C-1″), δH 1.87 (3H, s, H3-2″)]. Further inspection of its 2D NMR spectra established a 5/6/7/3-fused-ring skeleton of a euphoractine Btype diterpenoid. The 1H−1H COSY correlation (Figure 2) between the proton of the −NH− group (δH 8.36) and H-12 (δH 5.47) and the HMBC cross-peaks from −NH− to C-12 (δC 53.4), C-11, and C-1″ and from both H-12 and H3-2″ to C-1″ confirmed the presence of an −NHCOCH3 group at C-12. Further HMBC cross-peak from H-3 (δH 6.26) to the carbonyl carbon (C-1′, δC 168.0) of the cinnamoyl group located the cinnamoyloxy group (−OCin) at C-3. In addition, the presence of the 5- and 15-OH groups was deduced from the deshielded shifts of δC 64.5 (C-5) and 86.4 (C-15) and its (+)-HRESIMS data. The 2D structure of compound 1 was thus defined and resembled that of euphoractin G,11 except for the presence of the 12-NHCOCH3 group in 1 instead of the 12-OH group in euphoractin G. The NOESY correlations (Figure 2) of 1 suggested the same relative configuration as euphoractin G. Specifically, assuming a β-orientation of HO-15 and an αorientation of H-4 based on biosynthesis considerations,11 the correlations of H-4/H-3/H-2/H-1α, H-4/H3-17/H3-20, H3-20/ −NH−/H-11, and H-11/H3-18/H-9/H-8α indicated that these

he genus Euphorbia, comprising more than 2000 species, is generally recognized as a rich source of structurally diverse terpenoids, which display a variety of bioactivities including cytotoxic, anti-inflammatory, antimicrobial, and multidrug resistance (MDR) reversal activities.1−4 Euphorbia soongarica is a perennial herb distributed in northwestern China, western Siberia to Central Asia, and Mongolia.5 Its roots have long been used for the treatment of venous hyperemia, edema, and ascites in traditional Chinese medicine. Several flavonoids, polyphenols, and triterpenoids have previously been isolated from this species,6−10 but only a few showed biological activities, while diterpenoids from E. soongarica have not been reported. During continuing investigations of bioactive constituents of the genus Euphorbia, 10 new terpenoids (Figure 1), including five diterpenoids (1−5), three nortriterpenoids (6−8), and two triterpenoids (9, 10), and 15 known terpenoids, including 13 diterpenoids (11−23), a triterpenoid (24), and a sesquiterpenoid (25), were isolated from an acetone extract of E. soongarica. All these compounds (Figure S1, Supporting Information) were isolated from E. soongarica for the first time, among which sooneuphoramine (1) is the first example of a euphoractine B-type diterpenoid alkaloid, while sooneuphanones A−C (6−8) are rare nortriterpenoids isolated from the genus Euphorbia. Herein, the isolation, structure elucidation, cytotoxicity, and MDR reversal activity of these compounds are discussed.

■

RESULTS AND DISCUSSION Sooneuphoramine (1), isolated as a white, amorphous powder, was assigned a molecular formula of C31H41NO6 with 12 indices of hydrogen deficiency, based on its (+)-HRESIMS (m/ © 2017 American Chemical Society and American Society of Pharmacognosy

Received: November 27, 2016 Published: June 7, 2017 1767

DOI: 10.1021/acs.jnatprod.6b01099 J. Nat. Prod. 2017, 80, 1767−1775

Journal of Natural Products

Article

Figure 1. Structures of the terpenoids 1−10 isolated from Euphorbia soongarica.

Table 1. 1H NMR Spectroscopic Data of Compounds 1−5 1a position 1α 1β 2 3 4 5 7α 7β 8α 8β 9 11 12 16 17 18 19 20 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 2″ −CO-NH− a

δH, mult. (J in Hz) 2.79, 1.78, 2.64, 6.26, 2.49, 5.48, 2.49, 1.52, 1.64, 1.70, 0.78, 0.81, 5.47, 1.24, 1.07, 1.03, 1.29, 1.53, 6.84, 8.01,

dd (13.2, 10.8) dd (13.8, 3.0) m t (5.4) m d (13.2) m m m m m t (9.0) t (9.0) d (7.8) s s s s d (16.2) d (16.2)

7.47, 7.34, 7.34, 7.34, 7.47, 1.87, 8.36,

m m m m m s d (9.0)

2b δH, mult. (J in Hz) 2.53, 1.46, 2.30, 4.76,

ddd (17.2, 7.6, 1.6) m m d (6.4)

5.20, 1.86, 1.58, 1.58, 1.90, 0.86, 0.90, 5.39, 0.99, 0.81, 1.00, 1.12, 1.44,

br s m m m m m m d (9.2) d (7.2) s s s s

7.89, 7.40, 7.53, 7.40, 7.89,

br br br br br

d (7.6) t (7.6) t (7.6) t (7.6) d (7.6)

3c

4b

5c

δH, mult. (J in Hz)



3.40, 1.62, 2.11, 5.66, 1.78, 3.47, 3.73,

dd (13.2, 7.8) t (13.2) m t (3.6) dd (9.6, 3.6) d (9.6) dd (10.8, 3.6)

3.51, dd (13.6, 8.4) 1.54, t (13.6) 2.21, m 5.46, t (3.2) 2.53, dd (10.8, 3.6) 5.84 d (11.2) 3.97, dd (10.8, 3.2)

2.74, ddd (18.0, 6.0, 3.0) 2.49, m 2.45, m

2.27, 1.47, 1.18, 1.53, 7.60, 1.01, 1.21, 1.21, 1.08, 1.87,

dt (14.4, 3.6) m m dd (11.4, 7.8) d (11.4) d (6.6) s s s s

2.28, 1.48, 1.05, 1.40, 7.30, 1.05, 1.49, 1.16, 1.04, 1.83,

dt (13.6, 3.2) m m dd (12.0, 8.0) d (12.0) d (6.4) s s s s

8.04, 7.45, 7.56, 7.45, 8.04,

br br br br br

8.01, 7.46, 7.58, 7.46, 8.01,

br br br br br

d (7.8) t (7.8) t (7.2) t (7.8) d (7.8)

2.83, 2.17, 2.51, 2.13, 0.94, 0.87, 0.70, 1.63, 1.19, 4.65, 1.11, 1.02, 1.03,

br d (9.0) m m m m m t (10.8) dd (10.8, 9.0) d (7.8) s, 4.16, s s s s

d (7.6) t (7.6) t (7.6) t (7.6) d (7.6)

Data were recorded in pyridine-d5 at 600 MHz. bData were recorded in CDCl3 at 400 MHz. cData were recorded in CDCl3 at 600 MHz.

protons were α-oriented, while the correlations of H-12/H319/H-8β/H-5 suggested the β-orientation of these protons. Thus, the structure of 1 was established as a rare euphoractine B-type diterpenoid alkaloid. The possible biosynthesis pathway

of 1 is as shown in Scheme 1 and may be feasible from euphoractin G and alanine. The molecular formula of sooneuphorone (2) was determined as C27H34O5 according to its (+)-HRESIMS data, 1768



Article

Table 2. 13C NMR Spectroscopic Data of Compounds 1−5 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ a

1a

2b

3c

4b

5c

δC, type

δC, type

δC, type

δC, type

δC, type

43.2, 36.5, 77.7, 55.8, 64.5, 49.1, 35.8, 19.8, 30.3, 20.9, 31.4, 53.4, 63.3, 211.3, 86.4, 17.5, 21.6, 29.2, 16.9, 14.6, 168.0, 120.2, 145.1, 135.5, 129.0, 129.5, 130.7, 129.5, 129.0, 170.2, 23.9,

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

35.6, 35.9, 78.5, 157.5, 65.3, 47.8, 32.9, 20.2, 25.1, 21.5, 29.6, 71.8, 61.1, 199.2, 137.9, 13.8, 18.3, 28.6, 16.1, 12.2, 165.9, 130.0, 130.0, 128.7, 133.5, 128.7, 130.0,

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

48.0, 38.7, 81.3, 50.7, 55.7, 64.8, 71.4, 31.4, 30.9, 26.9, 30.3, 148.0, 134.1, 197.9, 88.1, 14.0, 17.7, 29.3, 16.5, 12.8, 165.9, 130.2, 129.9, 128.8, 133.3, 128.8, 129.9,

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

47.1, 38.8, 82.9, 51.5, 119.3, 146.1, 75.2, 36.6, 31.3, 25.4, 30.2, 150.6, 132.5, 197.8, 91.3, 14.5, 19.0, 29.4, 16.5, 12.8, 166.4, 130.1, 129.8, 128.9, 133.5, 128.9, 129.8,

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

37.7, 40.8, 210.7, 141.4, 44.9, 153.2, 39.9, 26.8, 27.2, 19.9, 27.4, 42.2, 74.9, 73.6, 168.0, 17.6, 109.6, 29.0, 16.9, 21.1,

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

Data were recorded in pyridine-d5 at 150 MHz. bData were recorded in CDCl3 at 100 MHz. cData were recorded in CDCl3 at 150 MHz.

which gave a protonated molecule at m/z 439.2469 [M + H]+ (calcd for C27H35O5, 439.2484). Analysis of its NMR data revealed that 2 was also a euphoractine B-type diterpenoid featuring a Δ4(15)-double bond and was structurally related to euphoractine E.12 The difference included the C-12 acyloxy group, which changed from a cinnamoyloxy to a benzoyloxy group in 2. This deduction was confirmed by the 1H NMR signals of the aromatic protons [δH 7.89 (2H, br d, J = 7.6 Hz, H-3′, H-7′), 7.40 (2H, br t, J = 7.6 Hz, H-4′, H-6′), and 7.53 (1H, br t, J = 7.6 Hz, H-5′)], HMBC cross-peaks from H-12 (δH 5.39) to C-1′ (δC 165.9), and its (+)-HRESIMS data. The relative configuration of 2 was the same as that of euphoractine E by interpretation of the NOESY data, and the α-orientation of BzO-12 was defined via the NOESY correlations of H-5/H12/H3-19. The (+)-HRESIMS spectrum of soongalathyrone A (3) displayed a protonated molecule at m/z 455.2422 [M + H]+ (calcd for C27H35O6, 455.2434), which corresponded to a molecular formula of C27H34O6. Interpretation of its NMR data established the common 5/11/3-ring-fused 5,6-epoxylathyr-12en-14-one skeleton with a benzoyloxy group [δH 8.04 (2H, br d, J = 7.8 Hz, H-3′, H-7′), 7.45 (2H, br t, J = 7.8 Hz, H-4′, H6′), and 7.56 (1H, br t, J = 7.2 Hz, H-5′)] at C-3 (δC 81.3) and two hydroxy groups located at C-7 (δC 71.4) and C-15 (δC 88.1), which was supported by the deshielded shifts of C-7 and C-15, the HMBC cross-peak (Figure S2, Supporting

Information) from H-3 (δH 5.66) to C-1′ (δC 165.9), and its (+)-HRESIMS data. The aforementioned deduction established the 2D structure of 3 as an analogue of (+)-(12E,2S,3S,4R,5R,6R,9S,11S,15R)-3-benzoyloxy-5,6-epoxylathyr-12-en-15-ol-14-one,13 with an additional hydroxy group located at C-7. The NOESY correlations (Figure S3, Supporting Information) of 3 suggested the similar relative configurations of these two compounds. The β-oriented HO-15 and α-oriented H-4 were assumed as reference for lathyraneskeletoned diterpenoids on biosynthesis considerations,14 the NOESY correlations of H-4/H-3/H-2/H-1α and H-1β/H3-16 indicating the β-orientation of BzO-3 and Me-16 and the correlations of H-4/H3-17/H-7/H-8α and H-5/H-8β implying the α-orientation of H3-17 and the β-orientation of HO-7 and H-5. The 1D and 2D NMR data of soongalathyrone B (4), with a molecular formula of C27H34O5 by (+)-HRESIMS data (m/z 439.2498, calcd for C27H35O5, 439.2484), indicated it to be a derivative of 3 with the 5,6-trisubstituted double bond replacing the 5,6-oxirane moiety in 3, which was confirmed by the deshielded shifts of H-5 (δH 5.84), C-5 (δC 119.3), and C-6 (δC 146.1), as well as the HMBC cross-peaks from H-4 to C-5 and C-6 and H-5 to C-3, C-7, and C-17. The E-geometry of the Δ5(6)- and Δ12(13)-double bonds was determined by the NOESY correlations of H-4/H3-17/H-7/H-8α, H-5/H-8β/H-12, and H3-20/H-11/H3-18. 1769



Article

possessing the same 2D structure. The normal H-9α and H11α orientations of jatropholanes were assumed based on the NOESY correlations of H-9/H-11/H3-18.16 The NOESY correlations (Figure S3, Supporting Information) of H-11/H5/H3-20 confirmed the α-orientation of H-5 and Me-20, and the orientation of H-12 was shown to be β by the correlations of H3-19/H-12/H-8β. In addition, the correlations of H-12/H17b/H3-16/H-1β and H-1α/H-14/H3-20 indicated the βorientation of Me-16 and HO-14. Analyses of the 1D (Tables 3 and 4) and 2D NMR data of sooneuphanones A−C (6−8) revealed that these compounds shared the same A−D tetracyclic rings with (+)-(24S)-eupha8,25-diene-3β,24-diol-7-one (24),17 a coexisting 6/6/6/5tetracyclic euphane-type triterpenoid, with the differences being the C-17 side-chain moiety. For compound 6, with a molecular formula of C27H40O3 by (+)-HRESIMS data (m/z 413.3050 [M + H]+, calcd for C27H41O3, 413.3056), an α,βunsaturated formyl moiety [δH 9.51 (d, J = 7.8 Hz, H-24), 6.70 (dd, J = 15.6, 9.6 Hz, H-22), 6.07 (dd, J = 15.6, 7.8 Hz, H-23); δC 194.5 (C-24), 165.1 (C-22), 131.1 (C-23)] was preliminarily identified by interpretation of its 1H and 13C NMR data. The HMBC cross-peaks (Figure S2, Supporting Information) from H-24 to C-23, H-23 to C-20, H-22 to C-17, and H3-21 to C-17, C-20, and C-22 established the constitution of the C-17 side chain. For compound 7, with a molecular formula of C29H44O3 by (+)-HRESIMS data (m/z 441.3353 [M + H]+, calcd for C29H45O3, 441.3369), the differences in the C-17 side chain were rationalized by replacement of the formyl moiety in 6 with an acetyl unit in 7 as well as an additional CH2 group (δC 38.9, C-22) verified by the HMBC cross-peaks from H-17 to C-20 and C-21, H2-22 (δH 2.51 m, 2.01 m) to C-21 and C-24 (δC 132.8), H-23 (δH 6.75, ddd, J = 16.0, 8.0, 6.8 Hz) to C-25 (δC 198.7), and H3-26 (δH 2.23, s) to C-24. For compound 8, with a molecular formula of C24H36O3 via an (+)-HRESIMS ion at m/z 373.2750 [M + H]+ (calcd for C24H37O3, 373.2743), the C-17 side chain was identified as an acetyl unit, which was supported by the deshielded shifts of H-17 (δH 2.79, t, J = 9.0 Hz) and C-17 (δC 57.7) and the HMBC cross-peaks from both H-17 and H3-21 (δH 2.12, s) to C-20 (δC 210.5). The relative configurations of 6 and 7 were assigned as identical to that of 24 by comparing their NOESY spectra, coupling constants, and specific rotation. The positive specific rotations of 6 [+8 (c 0.1, MeOH)] and 7 [+7 (c 0.1, MeOH)] suggested them to be euphane-type triterpenoids rather than the tirucallane type.17−19 The NOESY correlations (Figure S3, Supporting Information) of H3-19/H3-29 and H3-28/H-3 and the large coupling constants of H-3 (δH 3.26, dd, J = 11.4, 4.2 Hz) in 6 and H-3 (δH 3.27, dd, J = 11.6, 4.4 Hz) in 7 indicated the axial α-orientation of H-3 in both compounds 6 and 7. The β-orientation of Me-30 and H-17 and α-orientation of Me-18 were assigned via the NOESY correlations of H3-19/H-11β/ H3-30, H3-30/H-17, and H-11α/H3-18. The Δ22(23) (6) and Δ23(24) (7) E-configurations were defined by the coupling constants of J22,23 (15.6 Hz) in 6 and J23,24 (16.0 Hz) in 7. For compound 8, the NOESY correlations of H3-30/H-17/H-16β indicated the β-orientation of H-17. The 13C NMR data of sooneuphanone D (9), with a molecular formula of C30H48O3 by an (+)-HRESIMS ion at m/ z 457.3662 [M + H]+ (calcd for C30H49O3, 457.3682), were highly similar to those of 24, except for slightly shielded shifts (ΔδC −0.2 to −0.4 ppm) of C-20, C-22, C-23, C-24, and C-25 (Table S2, Supporting Information). Analyses of the 2D NMR spectra and specific rotation values of compounds 9 and 24

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of sooneuphoramine (1).

Scheme 1. Possible Biosynthesis Pathway of Sooneuphoramine (1)

The 1H (Table 1) and 13C (Table 2) NMR data of soongajatrophol (5), which had a molecular formula of C20H28O3 determined by an [M + H]+ ion at m/z 317.2101 (calcd for C20 H29O3, 317.2117) in the (+)-HRESIMS spectrum, showed many similarities to lagaspholone A,15 a 5/ 6/7/3-ring-fused jatropholane-type diterpenoid isolated from E. lagascae. Further analysis of its HMBC cross-peaks (Figure S2, Supporting Information) revealed them to be stereoisomers 1770



Article

Table 3. 1H NMR Spectroscopic Data of Compounds 6−10 position 1α 1β 2α 2β 3 5 6α 6β 11α 11β 12α 12β 15α 15β 16α 16β 17 18 19 20 21 22a 22b 23 24 25 26 27 28 29 30 a

6a

7b

8a

9b

10b






1.41, 1.84, 1.74, 1.67, 3.27, 1.65, 2.43, 2.37, 2.41, 2.34, 1.84, 1.95, 1.63, 2.21, 2.24, 1.80, 2.79, 0.66, 1.05,

1.39, m 1.83, dt (13.2, 3.6) 1.69, m

1.37, td (13.2, 4.4) 1.81, dt (12.8, 3.2) 1.71, m

3.26, dd (11.6, 4.4) 1.64, m 2.37, m

3.27, 1.76, 2.58, 2.35, 2.16,

1.38, 1.81, 1.74, 1.65, 3.26, 1.64, 2.42, 2.34, 2.29, 2.17, 1.47, 1.61, 1.54, 2.17, 1.43, 2.01, 1.68, 0.68, 1.03, 2.42, 1.05, 6.70,

m dt (13.2, 3.0) m m dd (11.4, 4.2) m m m m m m m m m m m m s s m d (6.6) dd (15.6, 9.6)

6.07, dd (15.6, 7.8) 9.51, d (7.8)

1.41, m 1.84, m 1.70, m 3.27, dd (11.6, 4.4) 1.65, m 2.39, m 2.39, m 2.23, m 1.74, m 1.52, 2.13, 1.37, 1.95, 1.54, 0.74, 1.04, 1.66, 0.86, 2.51, 2.01, 6.75, 6.05,

m m m m m s s m d (6.4) m m ddd (16.0, 8.0, 6.8) d (16.0)

td (13.2, 3.6) m m m dd (11.4, 4.2) m m m m m m m m m m m t (9.0) s s

2.12, s

0.98, s 0.86, s 0.95, s

1.48, 2.09, 1.33, 1.92, 1.48, 0.70, 1.02, 1.48, 0.85, 1.16,

m m m m m s s m d (7.2) m

1.48, m 3.99, t (5.8)

2.23, s 0.97, s 0.86, s 0.95, s

2.35, m 2.20, m 1.76, m

0.98, s 0.87, s 1.01, s

4.90, 1.70, 0.96, 0.85, 0.94,

s, 4.81, s s s s s

dd (11.6, 4.4) m dd (18.8, 5.2) dd (18.8, 13.6) m

1.47, m 1.67, m

2.11, d (16.0) 2.80, d (16.0) 1.95, dd (8.0, 4.4) 1.48, m 1.23, 1.73, 1.07, 0.99, 0.86, 0.98, 1.55, 0.75, 1.30, 0.92, 0.96,

m m dt (13.6, 3.6) s s s s s s s s

Data were recorded in CDCl3 at 600 MHz. bData were recorded in CDCl3 at 400 MHz.

Supporting Information) from H2-16 and H3-26 to C-15. The NOESY correlations (Figure S3, Supporting Information) of 10 suggested it to possess the same relative configuration as 7oxoisomultiflorenol. The known compounds, including 13 diterpenoids (11−23), a triterpenoid (24), and a sesquiterpenoid (25), were identified as macrorieuphorones A and B (11, 12),21 euphoractine A (13),22 euphoractine C (14),12 jolkinol A′ (15),23 latilagascene F (16),24 jolkinol B (17),14,24 jolkinol D (18),14 piscatoriol B (19),25 piscatoriol A (20),25 15β-O-benzoyl-5α-hydroxyisolathyrol (21),26 helioscopinolide B (22),27 helioscopinolide E (23),28 (+)-(24S)-eupha-8,25-diene-3β,24-diol-7-one (24),17 and litseachromolaevanes B (25),29 by comparison of the observed and reported spectroscopic data. The isolated terpenoids 1−25 were investigated for their cytotoxicities against the KB and KBv200 cell lines using an MTT assay with paclitaxel serving as the positive control. The results (Table 5) revealed that compounds 2, 4, 7, 9, 10, 12, 13, 17, 18, and 24 exhibited moderate cytotoxicity (10 μM < IC50 < 30 μM) against the KB and KBv200 cell lines, whereas other terpenoids showed low activity or were inactive (IC50 > 30 μM). Since many terpenoids isolated from the genus Euphorbia were found to exhibit MDR reversal activity by modulating Pgp-mediated drug exclusion, terpenoids 1−25 were tested for similar activity using the rhodamine-123 (Rho123) accumu-

established their same 2D structure, which further confirmed the same configurations of the stereocenters in the A, B, C, and D rings as well as C-20 in the side chain. Thus, the difference between 9 and 24 could only be the absolute configuration of C-24. The results of a repeated 13C NMR experiment in acetone-d6 (Table S3, Supporting Information) revealed that the chemical shifts of 24 were completely consistent with those of (+)-(24S)-eupha-8,25-diene-3β,24-diol-7-one,17 while compound 9 differed from (+)-(24S)-eupha-8,25-diene-3β,24-diol7-one in the minor shielded chemical shifts of C-20 (Δδ −0.22 ppm), C-23 (Δδ −0.20 ppm), and C-24 (Δδ −0.34 ppm), which finalized compound 9 as a C-24 epimer of 24. The 1H NMR data (Table 3) of soonoleanone (10), with a molecular formula of C30H46O3 by (+)-HRESIMS (m/z 455.3544 [M + H]+, calcd for C30H47O3, 455.3525), revealed the presence of an oxymethine group [δH 3.27 (1H, dd, J = 11.6, 4.4 Hz)] and eight tertiary methyl groups (δH 1.55, 1.30, 0.99, 0.98, 0.96, 0.92, 0.86, 0.75). Analysis of its 2D NMR data suggested a 6/6/6/6/6-pentacyclic skeleton of the D:C-friedooleanane-type triterpenoid, with an α,β-unsaturated carbonyl unit located at C-8−C-9 of ring B. Comparison of the NMR data of 10 with those of 7-oxoisomultiflorenol,20 a known D:Cfriedo-oleanane-type triterpenoid, indicated that they were related, except for the presence of a carbonyl group at C-15 (δC 210.5) in 10 replacing the 15-CH2 in the latter, which was demonstrated by the HMBC cross-peaks (Figure S2, 1771



Article

Table 4. 13C NMR Spectroscopic Data of Compounds 6−10 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 a

6a

7b

8a

9b

10b

δC, type

δC, type

δC, type

δC, type

δC, type

34.8, 27.6, 78.2, 39.0, 48.5, 35.9, 198.4, 138.7, 165.6, 39.6, 23.7, 29.4, 45.0, 47.6, 31.5, 28.1, 49.2, 16.4, 18.8, 41.2, 20.5, 165.1, 131.1, 194.5,

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

34.8, 27.6, 78.2, 39.1, 48.5, 36.0, 198.4, 139.0, 165.3, 39.5, 23.8, 30.4, 44.9, 47.9, 31.5, 28.7, 48.6, 16.2, 18.8, 35.8, 19.6, 38.9, 147.4, 132.8, 198.7, 27.1,

27.5, CH3 15.3, CH3 24.5, CH3

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

34.8, 27.6, 78.2, 39.1, 48.4, 35.9, 198.2, 138.3, 165.2, 39.6, 23.9, 29.3, 46.4, 47.9, 31.8, 23.1, 57.7, 17.5, 18.8, 210.5, 31.7,

27.5, CH3 15.3, CH3 24.6, CH3

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

34.8, 27.6, 78.2, 39.0, 48.5, 36.0, 198.5, 139.1, 165.6, 39.5, 23.9, 30.1, 44.8, 47.9, 31.6, 28.8, 48.4, 16.0, 18.8, 35.8, 19.2, 31.2, 31.6, 76.4, 148.0, 111.2, 17.7, 27.5, 15.3, 24.6,

27.5, CH3 15.3, CH3 24.7, CH3

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

34.0, 27.4, 78.3, 39.0, 47.4, 35.7, 197.9, 135.1, 163.3, 39.4, 22.3, 28.6, 42.2, 55.1, 210.5, 53.9, 37.1, 43.8, 34.7, 28.3, 34.7, 37.9, 27.4, 15.2, 18.5, 21.8, 18.8, 31.8, 32.2, 35.0,

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

Data were recorded in CDCl3 at 150 MHz. bData were recorded in CDCl3 at 100 MHz.

Table 5. Cytotoxicities of Compounds 1−25 against the KB and KBv200 Cell Linesa

Table 6. Inhibitory Effects of P-gp-Mediated Drug Efflux by Compounds 1−25 in the KBv200 Cell Linea

IC50 (μM) sample

KB

KBv200

2 4 7 9 10 11 12 13 14 17 18 21 22 23 24 paclitaxel

13.54 21.42 21.63 16.08 10.34 >30 26.58 10.94 20.11 19.84 25.87 27.25 20.78 22.44 25.18 0.0036

16.13 20.61 22.94 14.55 11.95 29.20 29.04 10.61 >30 18.20 19.87 >30 >30 >30 19.63 2.117

sample

FAR

sample

FAR

1 2 3 4 5 6 7 8 9 10 11 12 13

1.51 1.79 0.44 0.43 0.76 1.25 1.30 0.98 5.23 0.95 0.94 0.97 1.47

14 15 16 17 18 19 20 21 22 23 24 25 varapamil

0.63 0.35 1.30 3.61 0.66 0.70 1.27 1.13 0.49 0.45 2.91 0.40 2.25

a The concentration of compounds 1−25 and varapamil was 10 μM. FAR (fluorescence activity ratio) values were calculated on the basis of the equation given in the Experimental Section.

a

Compounds that were not shown in this table did not exhibit cytotoxicity (IC50 > 30 μM).

1 but lower than 2, whereas compounds 9, 17, and 24 exhibited potent P-gp-modulating potential (FAR = 5.23, 3.61, 2.91, respectively), among which the lathyrane-type diterpenoid 17 (jolkinol B) was previously regarded as a strong MDR1/P-gp modulator.24,30 It is interesting to note that the euphane-type triterpenoid 9 exhibits the highest MDR reversal activity (FAR

lation assay. As shown in Table 6, compounds 1, 2, 6, 7, 13, 16, 20, and 21 could slightly increase the intracellular accumulation of Rho 123 with a fluorescence activity ratio (FAR) higher than 1772



Article

semipreparative HPLC (CH3CN−H2O, 28:72) from Fr. 6C2, and compounds 3 (3 mg), 5 (2 mg), and 18 (2 mg) were isolated from Fr. 6C5 by semipreparative HPLC (CH3CN−H2O, 45:55). Similarly, a semipreparative HPLC separation was performed to yield 19 (2 mg) and 20 (2 mg) from Fr. 6D3 (CH3CN−H2O, 40:60) and 21 (2 mg) from Fr. 6D5 (CH3CN−H2O, 50:50). Fr. 7 (14.8 g) was separated by silica gel (600 g, 200−300 mesh using a stepwise gradient of n-hexane−CHCl3−Me2CO, 100:9:1 to 0:0:1) CC to yield eight subfractions (7A−7H), among which Fr. 7F was applied to a Sephadex LH-20 gel column (130 × 3.0 cm, MeOH) followed by a LiChroprep RP-18 gel column (H2O−MeOH, 70:30 to 0:100) and silica gel (60 g, 200−300 mesh) CC to obtain 16 (380 mg) and subfractions 7F2E2−7F2E4. Subfractions 7F2E2 and 7F2E3 were separately chromatographed over a semipreparative HPLC (CH3CN− H2O, 50:50) to afford 4 (5 mg), 17 (11 mg), and 23 (1.5 mg) from 7F2E2 and 6 (1.8 mg) and 22 (7 mg) from 7F2E3. Fr. 7G was chromatographed over a Sephadex LH-20 gel column (130 × 2.5 cm, MeOH) followed by a silica gel (100 g, 300−400 mesh, n-hexane− EtOAc, 10:1 to 0:1) CC to afford subfractions 7G3A−7G3H. Compound 14 (31 mg) was isolated by a LiChroprep RP-18 gel (H2O−MeOH, 50:50 to 0:100) CC followed by a semipreparative HPLC (CH3CN−H2O, 35:65) from Fr. 7G3D. Subfraction 7G3E was subjected to a LiChroprep RP-18 gel column (H2O−MeOH, 50:50 to 0:100) to give 7G3E1−7G3E6. A semipreparative HPLC separation was further conducted to yield compounds 1 (8 mg), 8 (1.5 mg), and 13 (5 mg) from Fr. 7G3E3 (CH3CN−H2O, 45:55) and compounds 7 (5 mg), 9 (15 mg), 10 (9 mg), and 24 (14 mg) from Fr. 7G3E4 (CH3CN−H2O, 47:53). Fr. 7H was applied to silica gel (100 g, 300− 400 mesh, n-hexane−Me2CO, 10:1 to 0:1) CC followed by a LiChroprep RP-18 gel (H2O−MeOH, 50:50 to 0:100) CC and then purified by a semipreparative HPLC separation (CH3CN−H2O, 47:53) to give 2 (5 mg), 11 (19 mg), and 12 (21 mg). Fr. 8 (5.6 g) was subjected to CC over a Sephadex LH-20 gel column (130 × 2.5 cm, MeOH) to afford eight subfractions (8A−8H), among which Fr. 8B was further applied to repeated flash columns over LiChroprep RP-18 gel (H2O−MeOH, 50:50 to 0:100) and silica gel (n-hexane−Me2CO, 100:1 to 0:1) to obtain subfractions 8B3A− 8B3F. Compound 15 (4 mg) was isolated from Fr. 8B3D by semipreparative HPLC separation (CH3CN−H2O, 40:60). Sooneuphoramine (1): white, amorphous powder; [α]20D +1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.23), 277 (4.29) nm; ECD (MeOH) 203 (Δε −10.54), 260 (Δε −1.43), 311 (Δε +4.49) nm; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRESIMS m/z 524.2996 [M + H]+ (calcd for C31H42NO6, 524.3012). Sooneuphorone (2): colorless, oil; [α]20D −31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.01) nm; ECD (MeOH) 250 (Δε −7.37), 333 (Δε +0.67) nm; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRESIMS m/z 439.2469 [M + H]+ (calcd for C27H35O5, 439.2484). Soongalathyrone A (3): white, amorphous powder; [α]20D +44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.21), 267 (4.19) nm; ECD (MeOH) 202 (Δε −2.71), 231 (Δε −8.34), 272 (Δε +8.15), 348 (Δε +0.53) nm; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRESIMS m/z 455.2422 [M + H]+ (cacld for C27H35O6, 455.2434). Soongalathyrone B (4): white, amorphous powder; [α]20D +90 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.20), 274 (4.13) nm; ECD (MeOH) 216 (Δε −16.13), 237 (Δε −14.58), 278 (Δε +16.85), 345 (Δε +2.53) nm; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRESIMS m/z 439.2498 [M + H]+ (calcd for C27H35O5, 439.2484). Soongajatrophol (5): white, amorphous powder; [α]20D −76 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.88) nm; ECD (MeOH) 231 (Δε −12.86) nm; 1H and 13C NMR data, see Tables 1 and 2; (+)-HRESIMS m/z 317.2101 [M + H]+; calcd for C20H29O3, 317.2117). Sooneuphanone A (6): white, amorphous powder; [α]20D +8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (3.94) nm; ECD (MeOH) 219 (Δε +1.37), 247 (Δε −0.27), 280 (Δε +1.34), 337 (Δε +0.65)

= 5.23), while its 24S isomer (24, FAR = 2.91) was less active. Additionally, a notable decrease of activity was observed in the nortriterpenoids 7, 6, and 8 (FAR = 1.30, 1.25, 0.98, respectively), which differed from 9 and 24 in the structure of the C-17 side chain. These results indicated that the structure of the C-17 side chain of the euphane-type triterpenoids play an important role in the inhibition of the P-gp-mediated drug exclusion. To further evaluate the MDR reversal activity of sooneuphanone D (9), the IC50 values of 9 in combination with the known anticancer agent navelbine were tested against KBv200 cell lines that overexpressed P-gp. As shown in Table 7, the Table 7. Multidrug Resistance Reversal Activity of Compound 9 against the KBv200 Cell Line sample navelbine navelbine navelbine navelbine navelbine a

+ 1 μM 9 + 3 μM 9 + 10 μM 9 +10 μM verapamil

IC50 (μM)

RFa

9.6 0.302 0.0426 3200 259.46

Reversal fold (RF) = IC50 (navelbine)/IC50 (navelbine+compound).

IC50 values for navelbine in combination with compound 9 (1, 3, and 10 μM) significantly decreased with a dose-dependent effect, indicating that compound 9 exhibited a remarkable MDR reversal activity, compared with the positive drug (verapamil).

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired with an Autopol VI automatic polarimeter (Rudolph Research Analytical, NJ, USA). UV spectra were recorded in MeOH on a UV-2550 UV−visible spectrophotometer (Shimadzu, Japan), and a Chirascan spectropolarimeter (Applied Photophysics, UK) was used to obtain ECD spectra. Measurement of NMR spectra was performed using a Varian INOVA 400 or 600 MHz NMR spectrometer (Varian, CA, USA), in CDCl3, acetone-d6, or pyridine-d5. (+)-HRESIMS data were determined on a QSTAR Elite LC-MS/MS spectrometer (AB Sciex, MA, USA). Semipreparative HPLC separations were carried out with a Dionex P680 HPG/2 pump equipped with a UVD170U detector (Thermo Scientific, MA, USA), using an XSelect CSH Prep C18 (5 μm, 10 × 250 mm) column (Waters, MA, USA). Silica gel (Qingdao Marine Chemical Ltd., Qingdao, P. R. China), Sephadex LH-20 gel (GE Healthcare, Sweden), and LiChroprep RP-18 gel (20− 45 μm, Merck, Germany) were used to perform column chromatography (CC). Plant Material. Euphorbia soongarica Boiss. was collected in July 2013 from Aletai, Xinjiang, China, and authenticated by Prof. Yin Feng of Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. A voucher specimen (XTIPC-ESG-130704) has been deposited at Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. Extraction and Isolation. A sample of the whole plant of E. soongarica (10.0 kg) was extracted with acetone (9 × 40 L) at room temperature (each time for 24 h), and the solvent evaporated to yield a crude extract (600 g), which was partitioned between cyclohexane and CH3CN. The CH3CN-soluble extract (140 g) was subjected to silica gel CC (2200 g, 100−200 mesh) eluting with a gradient of n-hexane− EtOAc (75:1 to 0:1) to give 12 fractions (Fr. 1−12). Fractionation of Fr. 6 (10.2 g) was conducted on another silica gel CC (400 g, 300−400 mesh using a gradient of n-hexane−EtOAc, 40:1 to 0:1) to afford six subfractions (6A−6F). Fractions 6C and 6D were subjected to flash chromatography on a LiChroprep RP-18 gel column (H2O−MeOH, 50:50 to 0:100) to afford subfractions 6C1−6C6 and 6D1−6D7, respectively. Compound 25 (4 mg) was obtained by 1773



■

nm; 1H and 13C NMR data, see Tables 3 and 4; (+)-HRESIMS m/z 413.3050 [M + H]+ (calcd for C27H41O3, 413.3056). Sooneuphanone B (7): white, amorphous powder; [α]20D +7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (3.96) nm; ECD (MeOH) 233 (Δε +0.75), 257 (Δε −1.13), 284 (Δε +0.49), 335 (Δε +1.09) nm; 1H and 13C NMR data, see Tables 3 and 4; (+)-HRESIMS m/z 441.3353 [M + H]+ (calcd for C29H45O3, 441.3369). Sooneuphanone C (8): white, amorphous powder; [α]20D −24 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 252 (3.89) nm; ECD (MeOH) 257 (Δε −2.75), 287 (Δε −1.41), 339 (Δε +1.35) nm; 1H and 13C NMR data, see Tables 3 and 4; (+)-HRESIMS m/z 373.2750 [M + H]+ (calcd for C24H37O3, 373.2743). Sooneuphanone D (9): white, amorphous powder; [α]20D +15 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 254 (3.97) nm; ECD (MeOH) 220 (Δε −2.73), 260 (Δε −2.44), 338 (Δε +1.86) nm; 1H and 13C NMR data, see Tables 3 and 4; (+)-HRESIMS m/z 457.3662 [M + H]+ (calcd for C30H49O3, 457.3682). Soonoleanone (10): white, amorphous powder; [α]20D +1 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 245 (3.96) nm; ECD (MeOH) 209 (Δε −4.22), 254 (Δε −4.76), 284 (Δε +0.53), 334 (Δε +0.74) nm; 1H and 13C NMR data, see Tables 3 and 4; (+)-HRESIMS m/z 455.3544 [M + H]+ (calcd for C30H47O3, 455.3525). Cytotoxicity and MDR Reversal Activity Assays. MTT assays were used to measure the cytotoxicities of the isolated terpenoids against the human epidermoid carcinoma cell line KB and its VCRselected P-gp-overexpressing cell line KBv200 as previously described.21 Briefly, cells were seeded into 96-well plates at 2500 cells/well and incubated for 12 h (37 °C, 5% CO2). Different concentrations of paclitaxel (Biocompounds Pharmaceutical Inc., Shanghai, P. R. China) and isolated terpenoids were added and incubated for 72 h, after which 20 μL of MTT (5 mg/mL) was added to each well and incubated for another 1 h. After removing the medium, 100 μL of DMSO was added to dissolve the formazan product, and the absorbance at 570 nm was measured to calculate the percentage of cell growth inhibition. The MDR reversal effect of compound 9 was measured using the same method. Briefly, various concentrations of navelbine (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μM) in the absence or presence of compound 9 (1, 3, 10 μM) or verapamil (10 μM, Shanghai Aladdin Bio-Chem Technology Co., LTD, Shanghai, P. R. China) were added into wells, which were seeded with KBv200 cells, respectively, and incubated for 72 h for MTT assays. The IC50 values for navelbine and navelbine in combination with compound 9 were obtained by calculation of the percentage of cell growth inhibition. Inhibition of P-gp-Mediated Drug Exclusion Assays. A Rho123 efflux assay was performed by flow cytometry using Rho123 to emit fluorescence according to the protocols reported previously.21 Briefly, KBv200 cells were seeded into 24-well plates (1.5 × 105/mL, 1 mL/well) for overnight attachment, after which compounds 1−25 (10 μM in DMSO) and verapamil were added and incubated for 4 h (37 °C, 5% CO2). Next, 10 μM Rho123 was added to each well and incubated for additional 1 h; then the compounds were washed twice and resuspended in ice-cold phosphate-buffered saline for flow cytometric analysis. The fluorescence activity ratio was calculated via the following equation: FAR = (Fsample treated − FDMSO control)/ (Fsample untreated − FDMSO control).

■

Article

AUTHOR INFORMATION

Corresponding Author

*Tel (H. A. Aisa): +86-991-3835679. Fax: +86-991-3835679. E-mail: [email protected]. ORCID

Haji A. Aisa: 0000-0003-4652-6879 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge the International Cooperation and Exchange of the National Natural Science Foundation of China (No. 31110103908) for financially supporting this work, and Prof. X.-W. Zhang of East China Normal University for bioactivity assays.

■

REFERENCES

(1) Shi, Q. W.; Su, X. H.; Kiyota, H. Chem. Rev. 2008, 108, 4295− 4327. (2) Vasas, A.; Rédei, D.; Csupor, D.; Molnár, J.; Hohmann, J. Eur. J. Org. Chem. 2012, 2012, 5115−5130. (3) Vasas, A.; Hohmann, J. Chem. Rev. 2014, 114, 8579−8612. (4) Durán-Peña, M. J.; Botubol Ares, J. M.; Collado, I. G.; Hernández-Galán, R. Nat. Prod. Rep. 2014, 31, 940−952. (5) Ed.ial Committee of Flora of China, Chinese Academy of Sciences. Flora of China. Science Press: Beijing, 1997; Vol. 44, pp 75. (6) Ding, Y. L.; Jia, Z. J.; Zhu, Z. Q. Chem. J. Chin. Univ. 1989, 10, 1129−1130. (7) Shi, X. H.; Qiu, L.; Kong, L. Y. Chin. Tradit. Herbal Drugs 2006, 09, 1313−1315. (8) Lin, J.; An, N.; Liu, C. Y.; Xu, L. Z. Chin. Tradit. Herbal Drugs 2008, 04, 497−499. (9) Shi, X. H.; Xu, D. R.; Kong, L. Y. Chin. Tradit. Herbal Drugs 2009, 05, 686−689. (10) Shi, X. H.; Luo, J. G.; Kong, L. Y. J. Asian Nat. Prod. Res. 2009, 11, 49−53. (11) Tian, Y.; Xu, W.; Zhu, C.; Lin, S.; Guo, Y.; Shi, J. J. Nat. Prod. 2013, 76, 1039−1046. (12) Shi, J. G.; Jia, Z. J. Phytochemistry 1995, 38, 1445−1447. (13) Tian, Y.; Xu, W.; Zhu, C.; Lin, S.; Li, Y.; Xiong, L.; Wang, S.; Wang, L.; Yang, Y.; Guo, Y.; Sun, H.; Wang, X.; Shi, J. J. Nat. Prod. 2011, 74, 1221−1229. (14) Uemura, D.; Nobuhara, K.; Nakayama, Y.; Shizuri, Y.; Hirata, Y. Tetrahedron Lett. 1976, 50, 4593−4596. (15) Duarte, N.; Ferreira, M. J. U. Org. Lett. 2007, 9, 489−492. (16) Yang, D. S.; Zhang, Y. L.; Peng, W. B.; Wang, L. Y.; Li, Z. L.; Wang, X.; Liu, K. C.; Yang, Y. P.; Li, H. L.; Li, X. L. J. Nat. Prod. 2013, 76, 265−269. (17) 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. (18) Wang, L. Y.; Wang, N. L.; Yao, X. S.; Miyata, S.; Kitanaka, S. J. Nat. Prod. 2003, 66, 630−633. (19) Wang, S.; Liang, H.; Zhao, Y.; Wang, G.; Yao, H.; Kasimu, R.; Wu, Z.; Li, Y.; Huang, J.; Wang, J. Fitoterapia 2016, 108, 33−40. (20) Akihisa, T.; Yasukawa, K.; Kimura, Y.; Takido, M.; Kokke, W.; Tamura, T. Chem. Pharm. Bull. 1994, 42, 1101−1105. (21) Gao, J.; Chen, Q.; Liu, Y.; Xin, X.; Yili, A.; Aisa, H. A. Phytochemistry 2016, 122, 246−253. (22) Shi, J. G.; Jia, Z. J.; Yang, L. Phytochemistry 1993, 32, 208−210. (23) Valente, C.; Pedro, M.; Ascenso, J. R.; Abreu, P. M.; Nascimento, M. S. J.; Ferreira, M. J. U. Planta Med. 2004, 70, 244− 249. (24) Duarte, N.; Varga, A.; Cherepnev, G.; Radics, R.; Molnar, J.; Ferreira, M. J. U. Bioorg. Med. Chem. 2007, 15, 546−554. (25) Reis, M. A.; Paterna, A.; Mónico, A.; Molnar, J.; Lage, H.; Ferreira, M. J. U. Planta Med. 2014, 80, 1739−1745.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01099. 1D and 2D NMR spectra and (+)-HRESIMS data of compounds 1−10, 13C NMR (in acetone-d6) data of 9 and 24, structures of the isolated terpenoids (1−25), and key HMBC and NOESY correlations of 3, 5, 6, 10 (PDF) 1774



Article

(26) Shi, J. G.; Jia, Z. J.; Yang, L. Planta Med. 1994, 60, 588−589. (27) Crespi-Perellino, N.; Garofano, L.; Arlandini, E.; Pinciroli, V.; Minghetti, A.; Vincieri, F. F.; Danieli, B. J. Nat. Prod. 1996, 59, 773− 776. (28) Borghi, D.; Baumer, L.; Ballabio, M.; Arlandini, E.; Perellino, N. C.; Minghetti, A.; Vincieri, F. F. J. Nat. Prod. 1991, 54, 1503−1508. (29) Zhang, H. J.; Tan, G. T.; Santarsiero, B. D.; Mesecar, A. D.; Van Hung, N.; Cuong, N. M.; Soejarto, D. D.; Pezzuto, J. M.; Fong, H. H. S. J. Nat. Prod. 2003, 66, 609−615. (30) Lage, H.; Duarte, N.; Coburger, C.; Hilgeroth, A.; Ferreira, M. J. U. Phytomedicine 2010, 17, 441−448.

1775


Terpenoids from Euphorbia soongarica and Their Multidrug

Recommend Documents