Eurifoloids A–R, Structurally Diverse Diterpenoids from - American

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Eurifoloids A−R, Structurally Diverse Diterpenoids from Euphorbia neriifolia Jin-Xin Zhao,† Cui-Ping Liu,† Wei-Yan Qi,† Mei-Ling Han,† Ying-Shan Han,‡ Mark A Wainberg,‡ and Jian-Min Yue*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, People’s Republic of China ‡ McGill University AIDS Centre, The Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec, Canada H3T 1E2 S Supporting Information *

ABSTRACT: Eighteen new diterpenoids, named eurifoloids A−R (1−18), including ingenane (1 and 2), abietane (3−7), isopimarane (8−12), and ent-atisane (13−18) types, along with four known analogues were isolated from Euphorbia neriifolia. Eurifoloid M (13) represents a rare class of entatisane-type norditerpenoid. Eurifoloids E (5) and F (6) exhibited significant anti-HIV activities, with EC50 values of 3.58 ± 0.31 (SI = 8.6) and 7.40 ± 0.94 μM (SI = 10.3), respectively.

Euphorbia neriifolia Linn. (Euphorbiaceae) grows mainly in the tropical and subtropical regions of Asia and is cultivated as hedges in the south of China.1 Its latex is highly valued as an expectorant and purgative in Traditional Indian Medicine.2 The plant extracts were demonstrated to exhibit antihepatotoxic3 and cytotoxic activities.4 Previous chemical studies on this species afforded a number of structurally diverse compounds,5−8 but few of them showed biological activities. In continuing the search for diverse and bioactive ingredients from Euphorbiaceae,9−12 18 new compounds, eurifoloids A−R (1− 18), including ingenane (1 and 2), abietane (3−7), isopimarane (8−12), and ent-atisane (13−18) types of diterpenoids, along with four known analogues were identified from E. neriifolia. Eurifoloid M (13) represents a rare class of ent-atisane-type norditerpenoid.13 Eurifoloids E (5) and F (6) exhibited significant anti-HIV activities, with EC50 values of 3.58 ± 0.31 (SI = 8.6) and 7.40 ± 0.94 μM (SI = 10.3), respectively. Herein, the isolation, structure elucidation, and biological evaluation of the major diterpenoids are discussed.

(two oxygenated and four olefinic), and 10 quaternary (three carbonyl, two oxygenated, and four olefinic) carbons. Four double bonds and three carbonyls accounted for seven indices of hydrogen deficiency, the remaining four thus requiring 1 to be tetracyclic. An angeloyl and a tigloyl group were readily identified by 1H and 13C NMR data (Tables 1 and 4). The aforementioned data suggested that 1 was likely a diester of an ingenol diterpenoid carrying angeloyloxy and tigloyloxy residues. The presence of the angeloyloxy and tigloyloxy groups was confirmed by the HMBC correlations within two motifs and were located at C-5 and C-17 by the HMBC correlations (Figure 1A) from H-5 and H2-17 to each of the corresponding carbonyls in the ester units, respectively. The presence of hydroxy groups of C-3 (δC 80.5) and C-4 (δC 85.3) were indicated by the chemical shifts of the relevant proton and/or carbons and confirmed by the HMBC correlations from H-1 (δH 5.99) to C-3 and C-4, respectively. The only ketocarbonyl group was located at C-9 by the HMBC correlations from H-7, H-8, and H-11 to C-9. In the ROESY spectrum (Figure 1B), the cross-peaks of H217/H-11, H2-17/H-12β, H-8/H-11, and H-8/H-12β suggested that CH2-17, H-11, and H-8 were cofacial and were assigned to be β-oriented randomly. The relative configurations of the C-3, C-4, C-5, and C-10 stereocenters were identical to those of known analogues14−16 based on their similar chemical shifts and coupling patterns of the protons and carbons in the A and B rings. This was supported by the key ROESY correlation

■

RESULTS AND DISCUSSION Ingenane-Type Diterpenoids. Compound 1 was assigned a molecular formula of C30H40O7 based on the 13C NMR data and an HRESI(+)MS ion at m/z 535.2664 [M + Na]+ (calcd 535.2672), requiring 11 indices of hydrogen deficiency. Its IR spectrum (Supporting Information Figure S9) showed the presence of hydroxy (3437 cm−1), carbonyl (1711 cm−1), and olefinic (1651 cm−1) functionalities. Analysis of the 1H (Table 1) and 13C NMR (Table 4) data using DEPT and HSQC data (Supporting Information Figure S3) revealed the presence of eight methyls, two methylene (one oxygenated), 10 methine © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 10, 2014

A

dx.doi.org/10.1021/np5004752 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Data of Compounds 1−6 in CDCl3a 1b proton position

(mult., J in Hz)

3b

4b

(mult., J in Hz)

5b

(mult., J in Hz)

(mult., J in Hz)

6b (mult., J in Hz)

1 2

5.99, d (1.6)

6.11, s

3.46, m 1.66, m (2H)

3.47, m 1.64, m (2H)

3.50, dd (10.3, 5.2) 1.63, m (2H)

3

3.75, d (4.9)

4.98, br s

5 6

5.28, s

5.36, br s

7

5.86, m

5.84, m

α 1.42, dt (13.5, 3.6) β 1.27, m 1.04, dd (12.4, 1.9) α 1.60, m β 1.80, m α 1.93, m β 2.66, m

α 1.42, dt (13.6, 3.6) β 1.27, m 1.07, dd (12.3, 1.8) α 1.55, m β 1.77, m α 2.02, m β 2.47, m

α 1.41, dt (13.6, 3.7) β 1.27, td (13.2, 4.6) 1.06, dd (12.3, 1.7) α 1.52, m β 1.71, m α 1.92, m β 2.06, m

3.50, dd (10.4, 5.5) α 1.60, m β 1.66, m α 1.46, m β 1.35, m 1.03, dd (12.1, 2.1) α 1.60, m β 1.75, m α 1.64, m β 1.98, m

8 9 11

4.38, m

4.39, d (11.7)

2.44, m

2.42, m

12

α 1.89, m β 2.41, m 0.97, m 1.17, dd (11.9, 8.4) 1.18, s 4.31, d (12.0) 4.40, d (12.0) 1.01, d (7.0) 1.85, d (1.6) 1.57, s 6.19, qd (7.3, 1.5) 2.03, dd (7.3, 1.5) 1.97, t (1.5) 6.90, q (7.1) 1.83, dd (7.1, 1.2) 1.87, t (1.2)

α 1.87, dt (16.0, 4.8) β 2.54, m 0.89, m 1.08, dd (12.1, 8.4) 1.17, s 3.74, d (11.7) 3.81, d (11.7) 1.00, d (7.1) 1.77, s 1.54, s 6.24, q (7.3) 2.03, d (7.3) 2.02, s

α 3.69, m β 1.96, m 4.96, m

α 3.64, dd (16.5, 6.6) β 2.01, m 4.61, m

α 3.70, dd (16.6, 6.6) β 2.04, m 4.68, m

2.08, d (5.5) α 3.24, ddd (12.6, 5.3, 1.6) β 1.44, m 5.09, ddd (13.2, 5.3, 2.2)

4.68, d (5.4)

4.99, d (9.1)

6.35, s

3.76, s

1.90, d (1.9)

2.05, t (1.9)

1.81, t (1.9)

1.96, d (2.2)

0.90, s 0.86, s 1.08, s

0.90, s 0.86, s 1.08, s

0.88, s 0.84, s 1.07, s

0.91, s 0.89, s 1.11, s

1.93, d (5.4)

1.93, d (9.1)

13 14 16 17 18 19 20 3′ 4′ 5′ 3″ 4″ 5″ Ac-3 Ac-14 OH-14 a

(mult., J in Hz)

2c

2.10, s 2.19, s

Chemical shifts (ppm) referenced to solvent peak (δH 7.26).

b,c

Data were measured at 400 and 600 MHz, respectively.

Abietane-Type Diterpenoids. Compound 3 was isolated as a white, amorphous power. The 13C NMR data and an HRESI(+)MS ion at m/z 687.3878 [2 M + Na]+ (calcd 687.3873) established a molecular formula of C20H28O4 with seven indices of hydrogen deficiency. Its NMR data (Tables 1 and 4) showed typical signals for three oxygenated methines (δH 3.46, 4.68, and 4.96), two tetrasubstituted double bonds (δC 121.4, 128.3, 140.9, and 159.6), and a carbonyl group (δC 175.1). The chemical shift of the carbonyl carbon and the IR absorption bands at 1739 and 1647 cm−1 suggested the presence of an α,β-unsaturated γ-lactone moiety.17 These functionalities accounted for four out of the seven indices of hydrogen deficiency, requiring the presence of three additional rings in 3. The aforementioned data suggested that 3 was an abietane-type diterpenoid featuring an α,β-unsaturated γlactone moiety. Further analysis of NMR data revealed that the structure of 3 resembled that of phlogacantholide C,17 except for the presence of a hydroxy group at C-1 replacing the 19-OH in the latter. This deduction was verified by the HMBC spectrum (Supporting Information Figure S21), in which the key correlations from H3-18, H2-3, and H-5 to C-19 at δC 21.4 and from H3-20 and H2-2 to C-1 at δC 75.9 were observed.

between H-3 and H-5. Thus, the structure of 1 (eurifoloid A) was assigned as depicted. Compound 2, a pale gum, showed a molecular formula of C27H36O7 as determined by 13C NMR data and an HRESI(+)MS ion at m/z 495.2352 [M + Na]+ (calcd 495.2359). Analysis of its NMR data (Tables 1 and 4) revealed that the structure of 2 resembled that of 1, except for the presence of an acetyl moiety (δH 2.10, δC 172.5 and 21.2) and the concomitant absence of the tigloyl group. A hydroxy group replacing the tigloyloxy moiety of 1 was located at C-17 in 2 based on its shielded H2-17 resonances at δH 3.74 and 3.81. The acetoxy group was assigned to C-3 by the deshielded H-3 resonance at δH 4.98 and supported by a weak HMBC correlation from CH3CO to C-3 (Supporting Information Figure S13). The angeloyloxy group was subsequently located at C-5 by the chemical shifts of C-5, C-6, and C-7, which were very close to those of 1 (Table 4). The latter assignment was corroborated by the ROESY cross-peak between H-5′ and H-20 (Figure 2). The relative configuration of 2 was established as identical with that of 1 by the ROESY data, as well as their similar NMR patterns. Thus, the structure of 2 (eurifoloid B) was elucidated as shown. B

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Table 2. 1H NMR Data of Compounds 7−12 in CDCl3a 7 proton position

(mult., J in Hz)

1

4.63, dd (11.2, 4.4)

2

α 1.59, m β 1.82, m α 1.49, m β 1.43, m 1.14, dd (12.6, 2.5) α 1.62, m β 1.79, m α 1.64, m

3 5 6 7

9 11

12 14 15

β 1.98, m 2.13, m α 2.17, ddd (12.8, 5.0, 2.0) β 1.52, m 5.09, ddd (13.6, 5.0, 2.2) 3.79, s

16

a

17 18

1.97, d (2.2) 0.93, s

19 20 Ac-1 Ac-3 Ac-18

0.92, s 1.23, s 2.11, s

8 (mult., J in Hz)

9

10

(mult., J in Hz)

11

(mult., J in Hz)

α 1.69, m β 1.17, m 1.61, m 1.71, m 3.44, dd (11.5, 4.3)

α 1.69, m β 1.18, m 1.62, m (2H)

1.32, m α 1.38, m β 1.54, m α 2.29, m

1.16, m 1.42, m (2H) α 2.27, m

β 2.01, m 1.97, m α 1.51, m

β 2.00, m 1.97, m α 1.49, m

β 1.76, m 3.55, dd (12.2, 4.1) 5.14, br s 5.75, dd (17.5, 10.8) 5.10, dd (17.5, 1.2) 5.12, dd (10.8, 1.2) 1.05, s 3.78, d (11.6) 4.19, d (11.6) 0.81, s 0.85, s

β 1.74, m 3.55, dd (12.2, 4.0) 5.13, br s 5.75, dd (17.4, 10.8) 5.09, dd (17.4, 1.2) 5.12, dd (10.8, 1.2) 1.05, s 3.42, d (10.2) 3.70, d (10.2) 0.92, s 0.86, s

3.68, dd (11.3, 4.3)

12

(mult., J in Hz)

(mult., J in Hz)

α 1.80, m β 1.35, m 1.70, m 1.83, m 5.19, m

α 1.74, m β 1.38, m 1.60, m 1.83, m 5.19, m

α 1.69, m β 1.26, m 1.57, m 1.71, m 4.52, dd (11.8, 4.1)

1.62, m α 1.02, m β 1.44, m α 2.23, ddd (14.5, 4.5, 1.6) β 2.03, m 2.02, m α 1.57, m

1.91, d (12.0) α 1.25, m β 1.53, m α 2.24, dd (4.2, 3.6) β 2.05, m 2.02, m α 1.51, m

1.10, dd (12.3, 2.5) α 1.36, m β 1.58, m α 2.28, ddd (14.4, 4.6, 2.1) β 1.99, m 1.92, m α 1.48, m

β 1.77, m 3.57, dt (12.1, 3.5) 5.16, br s 5.75, dd (17.4, 10.8)

β 1.76, m 3.57, dd (12.2, 3.9) 5.14, br s 5.75, dd (17.4, 10.8) 5.09, dd (17.4, 1.2) 5.12, dd (10.8, 1.2) 1.05, s

β 1.74, m 3.55, dd (12.2, 4.1) 5.13, br s 5.7, dd (17.4, 10.8)

1.11, s 0.88, s

1.22, s 0.86, s

0.89, s 0.82, s

1.97, s

2.00, s

2.05, s

5.09, 5.12, 1.05, 9.27,

d (17.4) d (10.8) s s

5.09, 5.11, 1.04, 0.88,

dd (17.4, 1.2) dd (10.8, 1.2) s s

2.09, s

Chemical shifts (ppm) referenced to solvent peak (δH 7.26) at 400 MHz.

those of the diacetate of phlogacantholide B (δH 6.36, δC 70.4).17 Thus, the structure of 5 (eurifoloid E) was elucidated as shown. Compound 6 (eurifoloid F) gave a molecular formula of C20H28O4 based on the 13C NMR data and an HRESI(+)MS ion at m/z 687.3878 [2 M + Na]+ (calcd 687.3873). Interpretation of its 1D (Tables 1 and 4) and 2D NMR data revealed that it was also an abietane-type diterpenoid featuring a trisubstituted epoxy group (δH 3.76, δC 56.2 and 61.6) and was structurally related to gelomulide A.18 A hydroxy group was attached to C-1 by the chemical shift of H-1 (δH 3.50) and the HMBC correlations from H-1 to C-9 and C-20 (Supporting Information Figure S46). The 8,14-epoxy group was assigned as β-oriented by the ROESY cross-peak of H-14/H-7α (Supporting Information Figure S47), which is consistent with that in gelomulide A. This assignment was supported by the biosynthetic argument18 of the epoxidation of the Δ8(14) double bond of the coexisting antiquorine A19 from the less hindered β-face, to produce compound 6. Compound 7 had a molecular formula of C22H30O5 as determined by 13C NMR data and an HRESI(+)MS ion at m/z 397.1998 [M + Na]+ (calcd 397.1991), which showed 42 mass units more than that of 6. The 1H (Table 2) and 13C NMR (Table 4) data of 7 were highly similar to those of 6, and the only difference was the presence of an additional acetyl group. The acetoxy moiety was located at C-1 by the chemical shift of H-1 at δH 4.63 and the HMBC correlation from H-1 to the

The relative configuration of 3 was established by a ROESY spectrum (Supporting Information Figure S22), in which the cross-peaks of H-1/H-5, H-1/H-3β, H-1/H-11β, H-3β/H3-18, and H-7β/H-14 indicated that H-1, H-14, H-5, and H3-18 were cofacial, and assigned arbitrarily in a β-orientation. The ROESY cross-peaks of H-3α/H3-19 and H-11α/H-12 revealed that H319 and H-12 were α-oriented. The structure of compound 3 was thereby assigned and named eurifoloid C. Compound 4 shared the same molecular formula C20H28O4 with 3 as determined by 13C NMR data and an HRESI(+)MS ion at m/z 687.3881 [2 M + Na]+ (calcd 687.3873). Its NMR data (Tables 1 and 4) were highly similar to those of 3, with the differences being the deshielded H-14 (ΔδH 0.31) and C-14 (ΔδC 6.1) resonances, indicating that 4 was the 14-epimer of 3. This assignment was confirmed by the ROESY correlation between H-7α and H-14 (Supporting Information Figure S31). The structure of 4 was thus assigned and named eurifoloid D. Compound 5 had a molecular formula of C22H30O5 as assigned by 13C NMR data and an HRESI(+)MS ion at m/z 375.2177 [M + H]+ (calcd 375.2171), which is 42 mass units more than that of compound 4, suggesting it was an acetylated derivative of 4. An acetyl group (δH 2.19, δC 170.7 and 20.8) and the deshielded H-14 at δH 6.35 located the acetoxy group at C-14, which was confirmed by the HMBC correlation from H14 to the carbonyl carbon of the acetyl group (Supporting Information Figure S35). H-14 was assigned as α-oriented by comparing the chemical shifts of H-14 and C-14 (δC 70.6) with C

dx.doi.org/10.1021/np5004752 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. 1H NMR Data of Compounds 13−18 proton position

13a

14a

15a

16a

17b

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

18a (mult., J in Hz)

1

α 1.89, m β 1.66, m

α 1.37, m β 1.81, m

α 1.36, m β 1.80, ddd (13.4, 7.0, 3.2)

α 1.61, m β 1.84, m

α 1.34, m β 1.96, m

2

2.37, m (2H)

α 2.35, m

α 2.34, m

α 2.35, m

α 2.22, br d (14.7)

β 2.55, m

β 2.55, ddd (16.0, 12.0, 7.0) 1.37, m 1.47, m (2H)

β 2.59, m

β 2.79, td (14.7, 5.9)

α 1.35, m β 1.93, ddd (13.5, 6.0, 2.8) α 2.30, ddd (14.8, 4.5, 2.8) β 2.79, td (14.8, 6.0)

1.35, m 1.51, m (2H)

1.33, m 1.56, m (2H)

1.34, m 1.55, m (2H)

α 2.12, m β 0.97, ddd (13.6, 12.0, 5.4) 1.36, m α 1.36, m β 1.66, m

α 1.27, m β 1.35, m

α 1.13, m β 1.43, m

α 1.17, m β 1.45, dt (13.8, 3.3)

1.31, m 1.63, m 1.84, m

1.33, m α 2.04, m β 1.21, m

1.33, m α 2.03, m β 1.21, m

4.09, d (8.0)

2.33, 1.46, 2.12, 4.18,

m m m m

1.58, 2.57, 4.86, 4.90, 1.09, 1.04,

1.61, 2.64, 4.60, 4.77, 1.09, 1.04,

d (17.3) dt (17.3, 2.0) d (2.0) d (2.0) s s

2.32, m 1.76, m 1.83, m α 2.12, m β 1.15, m 1.88, d (18.7) 2.03, d (18.7)

1.82, m α 1.49, m β 1.67, m α 1.89, m β 0.82, m 1.04, d (14.0) 1.15, d (14.0) 3.35, d (11.5) 3.48, d (11.5) 1.24, s 3.51, d (11.3) 4.05, d (11.3) 1.04, s

1.85, m α 1.52, m β 1.64, m α 1.85, m β 0.82, m 1.11, d (14.0) 1.23, d (14.0) 3.44, d (10.8) 3.58, d (10.8) 1.25, s 3.99, d (11.3) 4.70, d (11.3) 1.18, s 6.79, q (7.2) 1.76, d (7.2) 1.78, br s

5 6 7

9 11

12 13

α 2.24, m β 2.65, ddd (15.0, 4.4, 3.0) α 2.43, m β 1.07, td (13.6, 4.4) 1.89, m α 1.78, m β 2.07, ddd (13.6, 11.2, 3.7) 2.86, m 3.94, t (2.9)

14 15

2.34, m (2H)

17

4.89, s 5.06, s 1.78, d (1.2)

18 19 20 3′ 4′ 5′ HO-13 HO-14

0.98, s

1.35, m 1.50, m (2H) α 2.16, dt (13.6, 3.2) β 0.96, td (13.6, 4.6) 1.34, m α 1.48, m β 1.77, m 2.36, m 3.87, m

d (17.3) dt (17.3, 1.8) d (1.8) d (1.8) s s

1.03, s

1.03, s

1.09, s 1.06, s 1.18, s

2.76, d (2.9) 2.90, s

a

Chemical shifts (ppm) referenced to solvent peak (δH 7.26 in CDCl3) at 400 MHz. bChemical shifts (ppm) referenced to solvent peak (δH 3.31 in methanol-d4) at 400 MHz.

group in the latter. This assignment was confirmed by 2D NMR data, especially the HMBC data (Supporting Information Figure S60), in which the correlations from H2-18 to the carbonyl carbon of the acetyl group located an acetoxy moiety at C-18, and the correlations of H-3/C-1 and C-19 and H-12/ C-15 and C-17 attached two hydroxy groups at C-1 and C-12, respectively. The trisubstituted Δ8(14) double bond was located by the HMBC correlations of H-14/C-7, C-9, and C-12. Similarly, the terminal Δ15 double bond (δC 146.1 and 113.7) was assigned by the HMBC correlation network of H-15/C-12, C-13, and C-17; H2-16/C-13; and H3-17/C-15 and C-16. The relative configuration of 8 was established as identical to that of 3α,12α-dihydroxy-ent-8(14),15-isopimaradien-18-al20 by comparing their NMR data and was corroborated by ROESY data (Supporting Information Figure S61). The structure of eurifoloid H (8) was thus unequivocally characterized as shown. Compound 9, a white solid, displayed a molecular formula of C20H32O3 based on the 13C NMR data and an HRESI(−)MS ion at m/z 365.2330 [M + HCO2]− (calcd 365.2328). The NMR data (Tables 2 and 4) of 9 were highly similar to those of

carbonyl carbon of the acetyl group (Supporting Information Figure S53). The structure of 7 (eurifoloid G) was thus defined as shown. Isopimarane-Type Diterpenoids. Compound 8 was obtained as a white, amorphous powder and assigned a molecular formula of C22H34O4 with six indices of hydrogen deficiency according to the 13C NMR data and an HRESI(+)MS ion at m/z 747.4846 [2 M + Na]+ (calcd 747.4812). The 1 H NMR data (Tables 2 and 4) showed the existence of a vinyl group (δH 5.10, dd, J = 17.5, 1.2 Hz and 5.12, dd, J = 10.8, 1.2 Hz, H2-16; 5.75, dd, J = 17.5, 10.8 Hz, H-15), a trisubstituted double bond (δH 5.14, s; δC 136.3 and 128.1), one oxygenated methylene, two oxymethines, three methyls (δH 0.81, 0.85, and 1.05, each 3H, s), and an acetyl group (δH 2.09, s). Two double bonds and one acetyl group accounted for three out of six indices of hydrogen deficiency, and the remaining three required 8 to be tricyclic. The aforementioned evidence suggested that 8 was an isopimarane-type diterpenoid, whose structure was closely related to 3α,12α-dihydroxy-ent-8(14),15isopimaradien-18-al,20 the only difference being the presence of an acetoxymethylene group at C-4 in 8 replacing the formyl D

dx.doi.org/10.1021/np5004752 | J. Nat. Prod. XXXX, XXX, XXX−XXX

E

166.8 141.3 126.9 20.5 16.0

172.5 21.2

132.3 135.8 82.7 85.9 77.2 135.3 125.1 42.8 205.6 71.9 38.7 30.7 23.7 23.5 31.0 23.9 63.6 16.6 15.5 21.4

2a

75.9 30.7 39.4 33.3 50.2 18.5 28.7 128.3 140.9 44.2 35.6 76.3 159.6 64.9 121.4 175.1 8.7 32.9 21.4 14.6

3a 76.2 30.6 39.4 33.2 50.8 18.3 29.2 130.0 139.5 44.1 34.7 78.2 160.1 71.0 121.4 175.3 9.3 32.9 21.4 14.4

4a

170.7 20.8

75.9 30.5 39.3 33.2 50.4 18.1 28.5 126.3 141.6 44.3 34.7 78.2 156.5 70.6 120.5 174.7 8.5 32.8 21.4 14.5

5a 78.4 30.1 39.2 33.4 54.1 21.1 34.6 61.6 50.0 45.1 26.6 76.0 156.4 56.2 128.8 174.5 8.9 33.6 21.8 12.6

6a 80.9 25.0 38.8 33.3 54.5 20.9 34.1 61.5 49.4 43.5 26.5 75.3 155.8 55.9 129.4 174.2 8.9 33.5 21.8 14.1 170.6 22.0

7a

171.6 21.0

36.8 26.3 72.5 42.3 46.3 21.7 34.4 136.3 51.5 37.7 26.2 73.3 43.1 128.1 146.1 113.7 17.5 66.9 12.1 15.2

8a 36.9 27.3 77.0 42.3 48.2 22.3 34.7 136.5 51.5 37.8 26.3 73.4 43.2 128.1 146.2 113.8 17.6 72.0 11.6 15.3

9a

170.3 21.0

36.4 23.0 73.5 54.3 46.1 23.3 34.0 135.4 51.2 36.6 26.1 73.0 43.1 129.0 145.8 113.9 17.5 204.0 9.8 14.9

10b

170.5 21.3

36.4 23.6 77.3 51.5 49.5 23.9 34.2 135.9 51.5 37.2 26.2 73.3 43.2 128.7 146.0 113.9 17.7 181.4 12.1 14.9

11b

171.1 21.5

36.9 24.1 80.9 38.0 53.9 21.9 34.8 136.6 51.4 37.8 26.3 73.4 43.2 128.1 146.3 113.8 17.6 28.5 17.0 14.8

12b

18.5

34.3 33.2 198.4 129.2 161.3 24.8 29.4 47.2 49.3 39.8 25.7 45.0 75.5 217.7 43.2 141.7 111.8 11.3

13a 38.1 34.1 217.1 47.7 55.5 19.4 33.3 37.2 51.9 38.4 24.8 44.0 68.8 65.7 39.2 145.5 110.8 26.4 21.7 14.7

14b 38.1 34.2 217.3 47.7 55.6 19.3 33.1 38.6 52.2 37.3 28.1 36.7 39.6 66.1 39.3 150.6 105.4 26.4 21.7 14.5

15a

26.2 21.7 13.6

38.1 34.0 216.8 47.7 55.5 19.6 38.1 36.1 51.1 37.3 25.3 43.1 23.7 27.5 55.9 217.2

16b 40.3 35.9 217.6 55.8 59.2 20.8 40.7 33.8 52.7 38.5 24.5 33.1 24.3 28.4 53.2 75.0 69.8 20.5 65.7 14.7

17c

168.1 128.3 138.1 14.6 12.1

39.2 34.9 213.2 52.6 57.9 20.0 39.3 33.0 51.3 37.5 23.4 32.2 23.4 27.4 52.5 74.1 69.1 20.4 66.4 14.4

18a

Chemical shifts (ppm) referenced to solvent peak (δC 77.16 in CDCl3) at 125 MHz. bData were measured in CDCl3 at 100 MHz. Chemical shifts (ppm) referenced to solvent peak (δC 77.16). cData were measured in methanol-d4 at 100 MHz. Chemical shifts (ppm) referenced to solvent peak (δH 49.00).

a

1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″

Ac-18

Ac-14

167.2 140.4 127.1 20.9 16.2 168.6 137.4 128.8 14.5 12.2

129.6 139.7 80.5 85.3 77.1 135.3 125.2 43.7 206.3 72.8 39.6 31.1 24.0 23.9 28.0 24.6 65.8 17.2 15.7 21.6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ac-1

Ac-3

1a

position

Table 4. 13C NMR Data of Compounds 1−18

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Figure 1. Selected HMBC (A) and ROESY (B) correlations of 1.

more than that of 10. Its NMR data (Tables 2 and 4) showed similarity to those of compound 10, the difference being the presence of a carboxylic group (δC 181.4) at C-4 in 11 replacing the formyl group in 10. This assignment was verified by the HMBC correlation from H3-19 to the carboxylic carbon (Supporting Information Figure S82). Thereby, the structure of 11 was established and named eurifoloid K. Compound 12, C22H34O3, was assigned as a structural analogue of 10 and 11 by analysis of the NMR data (Tables 2 and 4). A methyl group (δH 0.88, s; δC 28.5) was located at C18 in 12 by the HMBC correlations of H3-18/C-3, C-4, and C5 (Supporting Information Figure S89). The structure of 12 (eurifoloid L) was therefore defined as depicted. ent-Atisane-Type Diterpenoids. Compound 13, a white powder, gave a molecular formula of C19H24O3 on the basis of the 13C NMR data and an HRESI(+)MS ion at m/z 623.3339 [2 M + Na]+ (calcd 623.3349). The IR absorption peak at 1662 cm−1 and UV maximum at 248 nm suggested the presence of an α,β-unsaturated carbonyl group.21 Analysis of its NMR data (Tables 3 and 4) suggested that 13 was an analogue of antiquorin, a coexisting major compound.5,22 By comparing with antiquorin, the 1H NMR spectrum of 13 showed the presence of resonances for two methyls at δH 0.98 (s, H3-20) and 1.78 (d, J = 1.2 Hz, H3-18), which was one methyl less than antiquorin, suggesting the absence of CH3-19 in 13. This deduction was confirmed by the HMBC spectrum (Figure 3A), in which the persubstituted Δ4 double bond was located by the multiple correlations from H2-1, H2-6, H2-7, H-9, H3-18, and H3-20 to C-5 at δC 161.3 and from H2-6 and H3-18 to C-4 at δC 129.2. The relative configuration of 13 was assigned by the ROESY data (Figure 3B), especially by the cross-peak of H-13/ H3-20, which is identical to antiquorin from ring B through D. Thus, the structure of 13 (eurifoloid M) was established as a rare C-19-degraded atisane-type diterpenoid. Compound 14 was obtained as a white powder, and its molecular formula was determined as C20H30O3 by the 13C NMR data and an HRESI(−)MS ion at m/z 363.2169 [M + HCO2]− (calcd 363.2171). Comparison of the 1H (Table 3) and 13C (Table 4) NMR data of 14 with those of the coexisting antiquorin5,22 indicated that a hydroxy group was present at C14 in 14 replacing the C-14 carbonyl group in the latter. This assignment was confirmed by the 1H−1H COSY correlation between H-13 and H-14 (Supporting Information Figure

Figure 2. Key ROESY correlations of 2.

8 except for the shielded H2-18 resonances (ΔδH 0.36 and 0.49, respectively) and the absence of the proton and carbon signals for an acetyl group, suggesting that 9 was the deacetylated derivative of 8. This was confirmed by analysis of its HMBC spectrum (Supporting Information Figure S68). The relative configuration of 9 was assigned the same as that of 8 by comparing their NMR data. The structure of 9 (eurifoloid I) was hence elucidated as shown. Compound 10 had the molecular formula C22H32O4 as deduced from the 13C NMR data and an HRESI(+)MS ion at m/z 743.4510 [2 M + Na]+ (calcd 743.4499). Interpretation of its NMR data (Tables 2 and 4) revealed that the structure of compound 10 was closely related to 3α,12α-dihydroxy-ent8(14),15-isopimaradien-18-al,20 with the exception that H-3 was deshielded (ΔδH 1.18) and the presence of an acetyl group, indicating that an acetoxy group was attached at C-3. This conclusion was confirmed by the key HMBC correlation from H-3 to the carbonyl carbon of the acetyl group (Supporting Information Figure S75). The relative configuration of eurifoloid J (10) was assigned as identical with that of 3α,12α-dihydroxy-ent-8(14),15-isopimaradien-18-al by comparing their NMR data. The molecular formula of compound 11 was determined as C22H32O5 by 13C NMR data and an HRESI(+)MS ion at m/z 775.4388 [2 M + Na]+ (calcd 775.4397), which is 16 mass units F

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Chart 1

C-11, C-12, C-14, and C-16. The β-orientation of 14-OH was defined by the key ROESY cross-peak between H-14 and H3-20 (Supporting Information Figure S115). Compound 16 gave a molecular formula of C19H28O2 as determined by 13C NMR data and an HREIMS ion at m/z 288.2086 [M]+ (calcd 288.2089). Analysis of its NMR data (Tables 3 and 4) showed that it shared the same A and B rings with compounds 14 and 15. A ketocarbonyl carbon at δC 217.2 was located at C-16 by the correlations from H2-11, H2-13, and H 2 -15 to C-16 in the HMBC spectrum (Supporting Information Figure S123), which presumably resulted from the degradation of the exocyclic Δ16 double bond in compounds 13−15. The relative configuration of 16 (eurifoloid P) was established by the ROESY spectrum (Supporting Information Figure S124), in which the cross-peaks of H-6α/H14α and H-7α/H-14α were observed (Figure 4A) to confirm the relationship of the C and D rings. Compound 17 had the molecular formula C20H32O4 on the basis of 13C NMR data and an HRESI(−)MS ion at m/z 381.2272 [M + HCO2]− (calcd 382.2277). Its NMR data (Tables 3 and 4) showed many similarities to those of the coexisting ent-16α,17-dihydroxyatisan-3-one,23 with the only difference being the presence of a hydroxymethyl group at C-4 in 17 replacing the CH3-4 in the latter. This was corroborated by the correlations from H2-19 to C-3, C-4, C-5, and C-18 in the HMBC spectrum (Supporting Information Figure S132). In the ROESY spectrum (Figure 4B), the correlation of H2-19/ H3-20 indicated that 19-CH2OH was α-oriented. The other stereocenters in 17 were assigned as identical to those of ent16α,17-dihydroxyatisan-3-one by comparing their NMR data and analyzing the ROESY spectrum (Figure 4B). The structure of 17, named eurifoloid Q, was thus elucidated as shown. Compound 18 possessed a molecular formula of C25H38O5 by 13C NMR data and an HRESI(+)MS ion at m/z 419.2807 [M + H]+ (calcd 419.2797). Analysis of the NMR data (Tables

Figure 3. Selected HMBC (A) and ROESY (B) correlations of 13.

S104). The strong ROESY cross-peaks of H-13/H3-20 and H14/H3-20 (Supporting Information Figure S106) revealed that both 13-OH and 14-OH were β-oriented. Therefore, the structure of 14 (eurifoloid N) was established as shown. Compound 15 (eurifoloid O) was determined to have a molecular formula of C20H30O2 by 13C NMR data and an HREIMS ion at m/z 302.2241 [M]+ (calcd 302.2246). Its NMR data (Tables 3 and 4) were highly similar to those of 14, and the only differences revealed the absence of 13-OH in 15, indicated by the HMBC correlations (Supporting Information Figure S114) from H2-13 at δH 1.46 and 2.12 (each 1H, m) to G

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internal standard. EIMS and HREIMS were carried out on a Finnigan MAT-95 mass spectrometer. ESIMS and HRESIMS were made on a Bruker Daltonics Esquire 3000 Plus instrument and a WatersMicromass Q-TOF Ultima Global mass spectrometer, respectively. Silica gel (300−400 mesh), C18 reverse-phased silica gel (150−200 mesh, Merck), MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries, Ltd.), and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography. Precoated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for TLC. Semipreparative HPLC was performed on a Waters 1525 pump with a Waters 2489 detector and a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, Japan). All solvents used for column chromatography were of analytical grade (Shanghai Chemical Reagents Company, Ltd.), and solvents used for HPLC were of HPLC grade (J&K Scientific Ltd.). Plant Material. The twigs and leaves of Euphorbia neriifolia Linn. were collected in August 2010 from Yunnan Province, People’s Republic of China, and were authenticated by Professor You-Kai Xu of Xishuangbanna Topical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (accession number: EuN-2010-1Y) has been deposited in Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The air-dried powder of the twigs and leaves of E. neriifolia (3.5 kg) was extracted with 95% EtOH three times (each 10 L) at room temperature to give a crude extract (200 g). The extract was partitioned between EtOAc and H2O to obtain the EtOAc-soluble fraction (133 g), which was subjected to passage over a column of D101-macroporous absorption resin eluted with EtOH/ H2O (30%, 80%, and 100%) to obtain a major fraction (72 g) as monitored by TLC. This fraction was separated on an MCI gel column (MeOH/H2O, 50% to 100%) to afford six fractions, A−F. Fraction B (6.6 g) was subjected to a silica gel column, eluted with DCM/MeOH (200:1 to 10:1), to yield seven factions (B1−B7). Fraction B3 was chromatographed over a column of C18 reversed-phase silica gel (MeOH/H2O, 50% to 100%) to give six subfractions (B3a−B3f). Fraction B3b was purified by semipreparative HPLC with the mobile phase of 45% CH3CN in H2O to give compounds 3 (2 mg) and 4 (3 mg), and similarly, fraction B3d yielded compound 11 (12 mg). Fraction B3c was subjected to a silica gel column eluted with CHCl3/ MeOH (180:1 to 100:1) to give ent-16α,17-dihydroxyatisan-3-one (151 mg). Fraction B1 was separated over a column of C18 reversedphase silica gel (MeOH/H2O, 50% to 60%) to obtain three subfractions (B1a−B1c). Fraction B1b was subjected to a silica gel column eluted with petroleum ether/EtOAc (8:1 to 6:1) to afford antiquorin (133 mg) and minor compound 13 (2 mg). Fraction B4 was chromatographed on a silica gel column (CHCl3/MeOH, 150:1 to 100:1) to give a fraction, which was further resolved by semipreparative HPLC to yield compound 9 (4 mg). Fraction C was separated over a column of silica gel (petroleum ether/acetone, 20:1 to 1:1) to obtain two major fractions, C1 and C2. Fraction C1 was chromatographed on a column of C18 reversed-phase silica gel (MeOH/H2O, 40% to 100%) to give six components, and each was then purified by semipreparative HPLC with the mobile phase of 50% CH3CN in H2O to yield compounds 2 (2 mg), 5 (12 mg), 6 (8 mg), 8 (3 mg), 14 (11 mg), and 18 (1 mg), respectively. Fraction C2 was separated by semipreparative HPLC (50% CH3CN in H2O) to yield antiquorin A (6 mg) and 3α,12α-dihydroxy-ent-8(14),15-isopimaradien-18-al (3 mg). In similar procedures, fraction D yielded compounds 1 (3 mg), 7 (4 mg), 10 (8 mg), 12 (15 mg), 15 (12 mg), and 16 (14 mg), and the higher polar fraction A gave compound 17 (18 mg). Eurifoloid A (1): pale gum; [α]27D +7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 214 (4.65) nm; IR (KBr) νmax 3437, 2956, 2925, 2856, 1711, 1651, 1456, 1381, 1259, 1228, 1149, 1038 cm−1; 1H NMR (CDCl3) see Table 1 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 535.3 [M + Na]+, 551.1 [M + K]+; ESI(−)MS m/z 557.6 [M + HCO2]−; HRESI(+)MS m/z 535.2664 [M + Na]+ (calcd for C30H40O7Na, 535.2672). Eurifoloid B (2): pale gum; [α]27D +27 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 209 (4.34) nm; IR (KBr) νmax 3419, 2958, 2925,

Figure 4. Key ROESY correlations of 16 (A) and 17 (B).

3 and 4) revealed that its structure was closely related to that of 17, except for the presence of a tigloyl moiety. The tigloyloxy group was located at C-19 by the deshielded H2-19 resonances (ΔδH 0.48 and 0.65) as compared with those of 17, and this assignment was confirmed by the correlations from H2-19 to the carbonyl carbon of the tigloyl unit in the HMBC spectrum (Supporting Information Figure S140). The relative configuration of 18 was established as identical to that of 17, due to their similar NMR patterns and presumed biosynthetic similarities. The structure of 18 (eurifoloid R) was thus assigned as shown. Compounds 13−18 were tentatively assigned as ent-atisane diterpenoids by biosynthetic considerations and their negative specific rotations.24 Biogenetically, diterpenoids 13−18 were related with the coexisting ent-16α,17-dihydroxyatisan-3-one, whose absolute configuration was determined by single-crystal X-ray crystallography and which also showed a negative specific rotation.23 Four known diterpenoids, 3α,12α-dihydroxy-ent-8(14),15isopimaradien-18-al,20 antiquorine A,19 antiquorin,5,22 and ent16α,17-dihydroxyatisan-3-one,23 were also isolated from this plant. Their structures were identified by spectroscopic analysis and/or by comparison of the data with those reported. Acquired immunodeficiency syndrome (AIDS) is one of the most fatal diseases worldwide, and the continuing reports of drug resistance require the development of new, more potent anti-HIV agents.25 Eurifoloids E (5) and F (6) were tested in vitro for anti-HIV activity on HIV-1 NL 4-3 infected MT4 cells26 and showed significant activities, with EC50 values of 3.58 ± 0.31 (SI = 8.6) and 7.40 ± 0.94 μM (SI = 10.3), respectively. The rest of the major compounds 10−12 and 14− 17 were also evaluated for their in vitro inhibition against the XBP1 mRNA splicing11 and NF-κB signaling pathway,27 which are two therapeutic targets in cancer treatment, as well as for inhibitory activity against the PTP1B enzyme,28 but none of them were active.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured at room temperature with a PerkinElmer 341 polarimeter. UV spectra were acquired on a Shimadzu UV-2550 UV−visible spectrophotometer. IR spectra were recorded on a PerkinElmer 577 spectrometer with KBr disks. NMR spectra were measured on a Bruker AM-400 or AM-500 NMR spectrometer with the solvents as H

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2856, 1718, 1666, 1646, 1455, 1379, 1230, 1143, 1022 cm−1; 1H NMR (CDCl3) see Table 1 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 495.2 [M + Na]+, 511.1 [M + K]+; ESI(−)MS m/z 517.3 [M + HCO2]−; HRESI(+)MS m/z 495.2352 [M + Na]+ (calcd for C27H36O7Na, 495.2359). Eurifoloid C (3): white, amorphous powder; [α]27D −147 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 215 (4.33) nm; IR (KBr) νmax 3423, 2956, 2923, 2852, 1739, 1647, 1468, 1387, 1284, 1093, 1014, 602 cm−1; 1H NMR (CDCl3) see Table 1 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 371.1 [M + K]+, 687.4 [2 M + Na]+; ESI(−)MS m/z 331.3 [M − H]−; HRESI(+)MS m/z 687.3878 [2 M + Na]+ (calcd for C40H56O8Na, 687.3873). Eurifoloid D (4): white, amorphous powder; [α]27D −158 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 215 (4.47) nm; IR (KBr) νmax 3431, 3361, 2952, 2924, 2850, 1761, 1722, 1657, 1456, 1385, 1338, 1263, 1151, 1066, 760 cm−1; 1H NMR (CDCl3) see Table 1 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 371.1 [M + K]+, 687.4 [2 M + Na]+; ESI(−)MS m/z 331.6 [M − H]−, 377.2 [M + HCO2]−; HRESI(+)MS m/z 687.3881 [2 M + Na]+ (calcd for C40H56O8Na, 687.3873). Eurifoloid E (5): white, amorphous powder; [α]24D −199 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 222 (4.31) nm; IR (KBr) νmax 3477, 2931, 1749, 1693, 1371, 1226, 1061, 1014, 962 cm−1; 1H NMR (CDCl3) see Table 1 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 771.5 [2 M + Na]+; ESI(−)MS m/z 373.1 [M − H]−; HRESI(+)MS m/z 375.2177 [M + H]+ (calcd for C22H31O5, 375.2171). Eurifoloid F (6): white, amorphous powder; [α]27D +40 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 221 (4.05) nm; IR (KBr) νmax 3473, 2927, 2850, 1732, 1689, 1460, 1367, 1103, 1026, 872, 773 cm−1; 1 H NMR (CDCl3) see Table 1 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 355.2 [M + Na]+, 687.3 [2 M + Na]+; HRESI(+)MS m/z 687.3878 [2 M + Na]+ (calcd for C40H56O8Na, 687.3873). Eurifoloid G (7): white, amorphous powder; [α]27D +34 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 225 (4.42) nm; IR (KBr) νmax 2952, 2923, 2852, 1753, 1741, 1375, 1240, 1018, 972 cm−1; 1H NMR (CDCl3) see Table 2 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 397.2 [M + Na]+, 771.2 [2 M + Na]+; HRESI(+)MS m/z 397.1998 [M + Na]+ (calcd for C22H30O5Na, 397.1991). Eurifoloid H (8): white, amorphous powder; [α]27D −4 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 207 (4.50) nm; IR (KBr) νmax 3413, 2929, 2871, 2856, 1720, 1381, 1248, 1038, 999 cm−1; 1H NMR (CDCl3) see Table 2 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 385.3 [M + Na]+, 747.4 [2 M + Na]+; HRESI(+)MS m/z 747.4846 [2 M + Na]+ (calcd for C44H68O8Na, 747.4812). Eurifoloid I (9): white, amorphous powder; [α]27D +14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (4.22) nm; IR (KBr) νmax 3381, 2931, 2873, 2854, 1724, 1666, 1452, 1383, 1288, 1065, 1036, 933 cm−1; 1H NMR (CDCl3) see Table 2 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 343.3 [M + Na]+; ESI(−)MS m/z 365.6 [M + HCO2]−, 639.7 [2 M − H]−; HRESI(−)MS m/z 365.2330 [M + HCO2]− (calcd for C21H33O5, 365.2328). Eurifoloid J (10): white, amorphous powder; [α]27D 0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 210 (4.29) nm; IR (KBr) νmax 3361, 2956, 2922, 2852, 1720, 1658, 1633, 1246, 1082, 1030, 939 cm−1; 1H NMR (CDCl3) see Table 2 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 383.2 [M + Na]+; HRESI(+)MS m/z 743.4510 [2 M + Na]+ (calcd for C44H64O8Na, 743.4499). Eurifoloid K (11): white, amorphous powder; [α]27D −5 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 208 (4.13) nm; IR (KBr) νmax 3464, 3376, 2937, 2873, 1734, 1711, 1375, 1240, 1065, 1030, 1003, 920 cm−1; 1H NMR (CDCl3) see Table 2 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 775.5 [2 M + Na]+; ESI(−)MS m/z 774.0 [2 M − H]−; HRESI(+)MS m/z 775.4388 [2 M + Na]+ (calcd for C44H64O10Na, 775.4397). Eurifoloid L (12): white, amorphous powder; [α]27D −4 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 209 (4.25) nm; IR (KBr) νmax 3473, 2952, 2846, 1707, 1375, 1265, 1090, 1032, 904 cm−1; 1H NMR (CDCl3) see Table 2 and 13C NMR (CDCl3) see Table 4; ESI(+)MS

m/z 369.2 [M + Na]+, 715.4 [2 M + Na]+; HRESI(+)MS m/z 715.4910 [2 M + Na]+ (calcd for C44H68O6Na, 715.4914). Eurifoloid M (13): white, amorphous powder; [α]27D −46 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 248 (4.20) nm; IR (KBr) νmax 3390, 2924, 2852, 1724, 1662, 1442, 1288, 1132, 1074 cm−1; 1H NMR (CDCl3) see Table 3 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 301.2 [M + H]+, 323.2 [M + Na]+, 623.3 [2 M + Na]+; HRESI(+)MS m/z 623.3339 [2 M + Na]+ (calcd for C38H48O6Na, 623.3349). Eurifoloid N (14): white, amorphous powder; [α]27D −41 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 207 (4.18) nm; IR (KBr) νmax 3346, 2927, 2870, 1703, 1684, 1454, 1387, 1282, 1070, 920 cm−1; 1H NMR (CDCl3) see Table 3 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 341.2 [M + Na]+; ESI(−)MS m/z 363.3 [M + HCO2]−, 635.6 [2 M − H]−; HRESI(−)MS m/z 363.2169 [M + HCO2]− (calcd for C21H31O5, 363.2171). Eurifoloid O (15): white, amorphous powder; [α]27D −43 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 206 (4.24) nm; IR (KBr) νmax 3460, 2951, 2922, 2856, 1691, 1450, 1389, 1290, 1084, 887 cm−1; 1H NMR (CDCl3) see Table 3 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 303.2 [M + H]+, 325.2 [M + Na]+; EIMS m/z 302 [M]+ (2), 285 (22), 284 (100), 269 (43), 204 (30), 131 (23), 118 (26), 105 (30), 91 (30); HREIMS m/z 302.2241 [M]+ (calcd for C20H30O2, 302.2246). Eurifoloid P (16): white, amorphous powder; [α]27D −31 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 211 (3.68) nm; IR (KBr) νmax 2964, 2935, 2866, 1722, 1701, 1462, 1444, 1383, 1184, 1097, 1011, 889 cm−1; 1H NMR (CDCl3) see Table 3 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 289.2 [M + H]+, 311.3 [M + Na]+, 599.3 [2 M + Na]+; EIMS m/z 288 [M]+ (100), 273 (8), 233 (36), 203 (77), 189 (31), 177 (28), 159 (41), 147 (24); HREIMS m/z 288.2086 [M]+ (calcd for C19H28O2, 288.2089). Eurifoloid Q (17): white, amorphous powder; [α]23D −17 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.37) nm; IR (KBr) νmax 3402, 2931, 2509, 1691, 1446, 1034 cm−1; 1H NMR (CD3OD) see Table 3 and 13C NMR (CD3OD) see Table 4; ESI(+)MS m/z 337.3 [M + H]+, 695.4 [2 M + Na]+; ESI(−)MS m/z 381.2 [M + HCO2]−; HRESI(−)MS m/z 381.2272 [M + HCO2]− (calcd for C21H33O6, 381.2277). Eurifoloid R (18): white, amorphous powder; [α]27D −21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 215 (4.20) nm; IR (KBr) νmax 3359, 2924, 2852, 1718, 1658, 1468, 1273, 1254, 1065 cm−1; 1H NMR (CDCl3) see Table 3 and 13C NMR (CDCl3) see Table 4; ESI(+)MS m/z 419.2 [M + H]+, 441.2 [M + Na]+, 859.5 [2 M + Na]+; HRESI(+)MS m/z 419.2807 [M + H]+ (calcd for C25H39O5, 419.2797). Bioassays. Anti-HIV and Cytotoxicity Assays. The anti-HIV activity and compound-induced cytotoxicity in MT-4 cell culture were measured as described previously26 with minor modifications. The concentration of the compound achieving 50% protection on the MT4 cells against the HIV-induced cytopathic effect, which is defined as the 50% effective concentration (EC50), was determined using Prism 5.0 software (GraphPad, San Diego, CA, USA). Similarly, the concentration of the compound that killed 50% of the MT-4 cells was determined as the 50% cytotoxic concentration (CC50). The selectivity index (SI) is defined as the ratio of the concentration of the compound that killed 50% of the MT-4 cells (CC50) to the concentration of the compound achieving 50% protection on the MT-4 cells against the HIV-induced cytopathic effect (EC50). XBP1 mRNA Splicing Inhibitory Activity Assays. XBP1 mRNA splicing inhibitory activity was tested via the reported protocol.11 NF-κB Signaling Pathway Inhibitory Activity Assays. NF-κB signaling pathway inhibitory activity was evaluated according to the previously reported protocol.27 PTP1B Inhibitory Activity Assay. The PTP1B inhibitory activity was measured as reported previously.28 I

dx.doi.org/10.1021/np5004752 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Article

(23) Lal, A. R.; Cambie, R. C.; Rutledge, P. S.; Woodgate, P. D. Phytochemistry 1990, 29, 1925−1935. (24) He, F.; Pu, J. X.; Huang, S. X.; Xiao, W. L.; Yang, L. B.; Li, X. N.; Zhao, Y.; Ding, J.; Xu, C. H.; Sun, H. D. Helv. Chim. Acta 2008, 91, 2139−2147. (25) Garg, R.; Gupta, S. P.; Gao, H.; Babu, M. S.; Debnath, A. K.; Hansch, C. Chem. Rev. 1999, 99, 3525−3601. (26) Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protoc. 2008, 3, 427−434. (27) Peng, Y. M.; Zheng, J. B.; Zhou, Y. B.; Li, J. Acta Pharm. Sin. 2013, 34, 939−950. (28) Zhang, W.; Hong, D.; Zhou, Y.; Zhang, Y.; Shen, Q.; Li, J.; Hu, L.; Li, J. Biochim. Biophys. Acta 2006, 1760, 1505−1512.

ASSOCIATED CONTENT

S Supporting Information *

IR, ESIMS/EIMS, HRESIMS/HREIMS, 1D and 2D NMR spectra of compounds 1−18. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-50806718. Fax: +86-21-50806718. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the National Natural Science Foundation (Nos. U1302222; 81273398) and the Foundation from the Ministry of Science and Technology (No. 2012CB721105) of P. R. China is gratefully acknowledged. We thank Prof. Y.-K. Xu of Xishuangbanna Topical Botanical Garden for the identification of the plant material.

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