Euphorbesulins AâP, Structurally Diverse ... - ACS Publications

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Euphorbesulins A−P, Structurally Diverse Diterpenoids from Euphorbia esula Bin Zhou,† Yan Wu,† Seema Dalal,‡ Maria B. Cassera,‡ and Jian-Min Yue*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China ‡ Department of Biochemistry and the Virginia Tech Center for Drug Discovery, MC 0308, Virginia Tech, Blacksburg, Virginia 24061, United States S Supporting Information *

ABSTRACT: Aqueous ethanol extracts of powdered twigs of Euphorbia esula afforded 16 new diterpenoids, named euphorbesulins A−P. These euphorbesulins included presegetane (1−3), jatrophane (4−14), paraliane (15), and isopimarane (16) diterpenoids as well as six known analogues. Compounds 1−3 represent a rare type of presegetane diterpenoid. Their structures were determined by analysis of the spectroscopic data, and the absolute configuration of 1 was established by X-ray crystallography. Diterpenoid 7 showed low nanomolar antimalarial activity, while the remaining compounds showed only moderate or no antimalarial activity.

T

he Euphorbia genus (Euphorbiaceae) comprises about 2000 species worldwide, of which about 80 species grow in China.1 The genus is well known for producing macrocyclic diterpenoid polyesters such as the ingenane,2−4 jatrophane,5−8 paraliane,3,6,9 and segetane2,3,6,10 diterpenoids. Euphorbia esula L. plants are distributed throughout China and are used in traditional Chinese medicine to treat cancer, swelling, and warts.11,12 A number of diterpenoids with a wide spectrum of bioactivities, including antiproliferative, cytotoxic, skin irritating, tumor promoting, antiviral, and antibacterial effects8,11−15 have previously been isolated from this species. In a continuing search for novel bioactive compounds from the genus Euphorbia,16,17 16 new diterpenoids (1−16) and six known analogues were identified from extracts of the twigs of E. esula. The structures of these compounds were characterized by physical data and X-ray diffraction data.

■

RESULTS AND DISCUSSION

Pre-Segetane-Type Diterpenoids. Compound 1, colorless crystals, had the molecular formula C33H44O9 as deduced by the (+)-HRESIMS ion at m/z 585.3071 [M + H]+ (calcd 585.3058) and 13C NMR data. IR absorption bands at 3410, 1750, and 1718 cm−1 revealed the presence of hydroxy and carbonyl functionalities. Two ester residues were readily identified as benzoate and 2,3-dimethylbutanoate groups using diagnostic 1H and 13C NMR data (Table 1). Two ketocarbonyls (δC 220.6 and 222.0) and one exocyclic double bond (δH 5.64 and 5.04 s; δC 142.6 and 122.0) were also distinguished by 1H and 13C NMR data. These groups accounted for nine out of the 12 indices of hydrogen deficiency, and the remaining indices required the presence of three additional rings. The above-mentioned data suggested that © XXXX American Chemical Society and American Society of Pharmacognosy

compound 1 is a rare presegetane-type diterpenoid.10 Its 2D structure was delineated using 2D NMR spectra. Four spin systems were established by 1H−1H COSY correlations (Figure 1A). The HMBC correlations (Figure 1A) of H-1 and H-2 with C-15 (δC 86.7), H-11 and H-12 with C-8 (δC 83.0), and H-20 with C-13 (δC 82.5) located three hydroxy groups at C-15, C-8, and C-13. The carbonyl groups were located at C-9 and C-14 by the HMBCs of H-18/C-9 (δC 220.6) and H-20/C-14 (δC Received: March 7, 2016

A

DOI: 10.1021/acs.jnatprod.6b00205 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compounds 1−3 in Pyridine-d5 1 δH

positon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3-OBz CO 1 2, 6 3, 5 4 5-OAcylsb CO 1 2 3 4 5 6 a

α 2.71, dd β 2.31, dda 2.71, m 6.08, brs 3.71, dd (11.1, 4.2) 6.37, d (11.1) a

2.62, da 3.53, da

α 2.12, dd (12.3, 6.7) β 2.74, dd (12.3, 12.4) 3.92, dd (12.4, 6.6)

1.03, 5.04, 5.64, 1.03, 1.37, 2.03,

d (6.3) brs brs s s s

2 δC 52.8 39.7 80.2 58.2 76.0 142.6 33.5 83.0 220.6 42.9 35.7 42.0 82.5 222.0 86.7 14.5 122.0 25.9 27.3 24.1

8.26, d (8.2) 7.27, t (7.8) 7.45, t (7.4)

166.9 131.6 130.6 129.2 133.6

ma m d (6.8) d (6.8) d (7.0)

175.2 46.4 32.1 19.8 20.9 14.0

2.34, 1.86, 0.80, 0.84, 1.15,

δH α 2.75, dd β 2.38, dda 2.75, m 6.16, brs 3.93, dd (10.9, 4.1) 6.64, d (10.5) a

2.77, da 3.61, da

α 2.15, dd (12.3, 6.5) β 2.78, dd (12.3, 12.6) 3.98, dd (12.6, 6.7)

1.02, 5.12, 5.79, 1.05, 1.37, 2.06,

3 δC


52.7 39.8 80.2 58.2 77.4 142.4 32.9 83.4 220.6 42.8 35.7 41.8 82.5 222.4 86.8 14.4 122.5 25.8 27.3 24.0

8.23, d (8.5) 7.26, t (7.7) 7.47, ta

166.8 131.5 130.5 129.2 133.6

8.12, 7.38, 7.47, 7.38, 8.12,

165.9 132.0 130.4 129.2 133.6 129.2 130.4

d (7.4) t (7.7) ta t (7.7) d (7.4)

δH

δC

α 2.71, dd β 2.32, dda 2.68, m 6.04, brs 3.68, dd (11.0, 3.9) 6.34, d (11.0) a

2.64, da 3.54, da

α 2.11, dd (12.3, 6.8) β 2.75, dd (12.3, 12.3) 3.89, dd (12.3, 6.8)

1.02, 5.08, 5.68, 1.02, 1.37, 2.03,


52.7 39.6 80.1 58,2 76.3 142.5 33.3 83.4 220.7 42.8 35.7 41.7 82.4 222.2 86.6 14.2 122.3 25.8 27.2 24.0

8.25, d (8.3) 7.25, t (7.8) 7.45, t (7.5)

167.0 131.5 130.5 129.1 133.6

2.03, s

170.0 21.9

Overlapping signals. b5-OAcyls: diMeBuO for 1, OBz for 2, and OAc for 3.

6.37) to the corresponding ester carbonyls at δC 166.9 and 175.2, respectively. In the NOESY spectrum (Figure 1B), the correlations of H2/H-1α, H-2/H-4, H-2/H-3, H-3/H-4, and H-18/H-11α revealed that they were cofacial and were assigned randomly as α-oriented. Consequently, H-12 and H3-20 were assigned as β-oriented by NOESY interactions of H-20/H-12 and H-19/H12. The large 3J4,5 value of 11.1 Hz showed their antiperiplanar arrangement,2 suggesting that the 2,3-dimethylbutanoate group was α-oriented. Finally, the assignments of OH-8α and OH15β were confirmed by an X-ray diffraction study (Figure 2), which also determined the absolute configuration of 1 as (2S, 3S, 4R, 5R, 8R, 12S, 13R, 15R, 2′R) [absolute structure parameter: 0.04(13)] (Table S1, Supporting Information). The structure of compound 1, euphorbesulin A, was thus unambiguously assigned as shown. The molecular formula of compound 2 was assigned as C34H38O9 by the sodium adduct (+)-HRESIMS ion at m/z 613.2408 [M + Na]+ (calcd 613.2408) and 13C NMR data. The

Figure 1. (A) Key HMBC and COSY (bold bonds) and (B) NOESY correlations for 1.

222.0), respectively. The HMBC correlations of H-17/C-5, C6, and C-7 indicated a Δ6(17) exocyclic double bond. The benzoate and 2,3-dimethylbutanoate groups were located at C3 and C-5 by the HMBCs from H-3 (δH 6.08) and H-5 (δH B



Article

Compound 3 had the molecular formula C29H36O9 as established by the sodium adduct HRESIMS ion at m/z 551.2261 [M + Na]+ (calcd 551.2252) and 13C NMR data. The 1 H and 13C NMR data (Table 1) of 3 showed similarities to compound 2 except for the acyl group at C-5. An acetoxy group (δH 2.03; δC 170.0, 21.9) was located at C-5 by the HMBC cross-peak from H-5 (δH 6.34) to the carbonyl carbon of the acetate group (Figure S22, Supporting Information). The structure of compound 3, euphorbesulin C, was thus defined as depicted. Jatrophane-Type Diterpenoids. Compound 4 was a white, amorphous powder and had a molecular formula of C37H46O14 as assigned by the (+)-HRESIMS and 13C NMR data. Analysis of the 1H and 13C NMR data (Tables 2 and 4) revealed that compound 4 was a jatrophane-type diterpenoid polyester.18 Four structural fragments as drawn with bold bonds were delineated by interpretation of the 1H−1H COSY spectrum (Figure 3A). The HMBC correlation networks (Figure 3A) of H-1 (δH 5.30)/C-15; OH-15 (δH 4.00)/C-4, C-14 (δC 210.7), and C-15 (δC 87.2); and H-20/C-13 and C-14 allowed the connections of C-15 and C-13 via C-14 and of C-1 and C-4 via C-15, which also assigned a hydroxy and a carbonyl group at C-15 and C-14, respectively. The HMBCs of H-18 and H-19/C-9, C-10, and C-11 attached C-9, C-11, C-18, and C-19

Figure 2. ORTEP drawing of 1.

NMR data (Table 1) revealed that compound 2 was also a presegetane-type diterpenoid and structurally related to 1. The NMR data showed that a benzoate group was attached to C-5 of 2 in place of the 2,3-dimethylbutanoate group in 1. This was verified by HMBC cross-peaks (Figure S14, Supporting Information) from H-3 (δH 6.16) and H-5 (δH 6.64) to the corresponding carbonyl carbons of two benzoate groups at δC 166.8 and 165.9, respectively. The structure of compound 2, euphorbesulin B, was thus defined as depicted. Table 2. 1H NMR Data of Compounds 4−8 (500 MHz, CDCl3) 4

7

(mult., J in Hz)

(mult., J in Hz)

4.46, dd (10.9, 4.3)

2 3 4 5 7 8 9 11 12 13 16 17

2.86, m 5.68, dd (5.9, 4.7) 3.07, t (4.3) 5.57, da 5.54, s 5.11, s 4.95, s 5.93, d (15.9) 5.57, dda 3.79, m 0.97, d (7.4) 5.10, brs 5.10, brs 0.95, s 1.34, s 1.35, d (7.2) 2.02,b s 2.05,b s

2.55, 5.85, 3.44, 5.35, 5.68, 5.01, 4.92, 5.80, 5.51, 3.72, 1.13, 5.18, 4.97, 0.95, 1.21, 1.26,

m t (3.2) t (3.3) brs s s s d (16.1) dd (16.1, 9.2) dq (9.2, 6.5) d (6.5) brs brs s s d (6.5)

α 2.10, dda β 2.78, dd (15.7, 9.0) 2.41, m 5.53, t (3.6) 2.98, dd (5.1, 3.3) 5.50, d (5.1) 5.68, s 5.04, s 4.91, s 5.77, d (16.0) 5.58, dd (16.0, 9.3) 3.50, dq (9.3, 6.6) 0.99, d (6.6) 5.13, brs 5.12, brs 0.92, s 1.23, s 1.30, d (6.6)

α 2.17, dd (15.6, 10.7) β 2.85, dd (15.6, 9.0) 2.59, m 5.77, t (3.8) 3.31, dd (5.7, 3.6) 5.84, d (5.7) 6.03, s 5.22, s 4.96, s 5.90, d (15.9) 5.66, dd (15.9, 9.5) 3.61, dq (9.5, 6.7) 1.03, d (6.7) 5.33, brs 5.25, brs 0.92, s 1.31, s 1.34, d (6.7)

α 2.18, dd (15.8, 10.8) β 2.78, dd (15.8, 9.2) 2.52, m 5.59, ta 3.28, dd (6.7, 3.5) 5.59, da 6.05, s 5.17, s 4.94, s 5.88, d (16.0) 5.58, dda 3.57, m 1.01, d (6.7) 5.18, brs 5.18, brs 0.91, s 1.28, s 1.30, d (6.6)

s s s s s

2.04,b s 1.99, s 2.05,b s 2.06,b s 2.11,b s 2.12,b s

1.92, s

1.57, 2.18, 2.09, 2.01, 2.23,

1.97, s 1.52, s

2.08, s 1.48, s 1.88, s

2.12, s 1.50, s

OBz 2, 6 3, 5 4 OH

2.06,b s 2.07,b s 2.08,b s

−5 8.06, 7.41, 7.54, 4.00,

d (7.8) t (7.7) t (7.5) s

−3 8.00, 7.44, 7.58, 3.35,

(mult., J in Hz)

8

5.30, d (9.8)

18 19 20 OAc-1 OAc-3 OAc-5 OAc-7 OAc-8 OAc-9 OAc-15 HOCH2CO2-15

a

6

1

position

(mult., J in Hz)

5

−5/7 7.83, d (7.7)/7.99, d (7.7) 7.35, t (7.7)/7.35, t (7.7) 7.38, t (7.5)/7.50, t (7.4)

d (8.3) t (7.3) t (7.5) s

(mult., J in Hz)

4.19, 4.29, −7 8.07, 8.30, 7.58,

d (17.0) d (17.0) d (8.3) dd (8.3, 7.2) t (7.5)

Overlapping signals. bExchangeable within each column. C



Article

Table 3. 1H NMR Data of Compounds 9−13 (500 MHz, CDCl3) position

10

11

12

13

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

1

4.29, d (11.1)

2 3 4 5 7 8 9 11 12 13 16 17

2.51, 5.63, 3.44, 5.69, 5.82, 5.10, 4.94, 5.84, 5.52, 3.69, 1.10, 5.13, 5.18, 0.96, 1.25, 1.23,

18 19 20 OAc-2 OAc-3 OAc-5 OAc-7 OAc-8 OAc-9 OAc-15 OBz 2, 6 3, 5 4 a

9 (mult., J in Hz)

m t (3.7) t (3.8) brs s s s d (16.0) dd (16.0, 9.4) dq (9.4, 6.3) d (6.3) brs brs s s d (6.3)

2.02,a s

α 2.13, dd (15.6, 10.9) β 2.83, dd (15.6, 8.9) 2.50, m 5.60, t (3.7) 3.15, dd (6.9, 3.5) 5.65, d (6.9) 5.76, s 3.97, s 4.69, s 5.82, d (15.9) 5.56, dd (15.9, 9.4) 3.52, dq (9.4, 6.6) 1.01, d (6.5) 5.36, brs 5.54, brs 1.01, s 1.21, s 1.33, d (6.6)

α 2.62, d (17.0) β 3.23, d (17.0)

α 2.73, d (17.1) β 3.28, d (17.1)

α 2.59, d (16.3) β 2.86, d (16.3)

d (3.8) dd (7.1, 3.8) d (7.1) s s s d (16.0) dd (16.0, 9.5) dq (9.5, 6.6) s brs brs s s d (6.6) s s s

6.00, t (3.7) 3.51, t (4.1) 5.49, brd (2.5) 5.76, s 5.10, s 4.94, s 5.82, d (15.9) 5.63, dd (15.9, 9.2) 3.68, dq (9.2, 6.7) 1.59, s 5.04, brs 5.12, brs 0.94, s 1.26, s 1.30, d (6.7) 1.61,a s

5.67, 3.98, 5.52, 5.93, 5.14, 4.96, 5.90, 5.64, 4.18, 1.41, 5.06, 5.21, 0.92, 1.29, 1.31,

1.98, s 1.49, s

5.83, 3.67, 5.66, 5.87, 4.03, 4.71, 5.80, 5.58, 3.69, 1.57, 5.35, 5.53, 1.02, 1.22, 1.31, 2.14, 2.00, 1.44,

1.41, s

1.56, 2.13, −7 8.05, 7.46, 7.57,

1.54, 2.32, −7 8.05, 7.46, 7.58,

s s

2.01,a s 2.14, s 2.07,a s 2.19,a s 2.24,a s −3 8.08, d (7.5) 7.46, t (7.7) 7.59, t (7.3)

a

2.02, s 2.08,as 2.08,a s 1.73, s −5 8.07, d (7.8) 7.44, t (7.6) 7.57, t (7.5)

s s d (7.5) t (7.6) t (7.4)

d (7.7) t (7.6) t (7.5)

d (3.2) dd (4.4, 3.2) d (4.4) s s s d (16.0) dd (16.0, 9.6) dq (9.6, 6.6) s brs brs s s d (6.6)

2.10, s 1.52, s −3/7 8.03, d (7.6)/8.11, d (7.5) 7.43, t (7.7)/7.49, t (7.7) 7.56, t (7.5)/7.62, t (7.5)

Exchangeable within each column.

assigned in an α-orientation by the 3J1,2 value of 10.9 Hz. Thus, the structure of euphorbesulin E (5) was defined. Euphorbesulin F (6) had the molecular formula C32H44O13 as deduced by the sodiated (+)-HRESIMS ion at m/z 659.2675 [M + Na]+ (calcd 659.2674) and 13C NMR data. The NMR data (Tables 2 and 4) indicated that 6 was a jatrophane-type diterpenoid possessing six acetate groups and was structurally similar to 17,13 except for the presence of an acetate rather than a benzoate group at C-7 as in 17. This was corroborated by the HMBC correlation from H-7 (δH 5.68) to the acetate carbonyl carbon (δC 167.8) (Figure S48, Supporting Information). Therefore, the structure of 6, euphorbesulin F, was assigned as shown. The molecular formula of 7 was assigned as C42H48O13 by the sodiated (+)-HRESIMS ion at m/z 783.2993 [M + Na]+ (calcd 783.2987) and 13C NMR data. Analysis of the 1H and 13 C NMR data (Tables 2 and 4) indicated that the structure of 7 was similar to that of 6. The NMR data also showed the presence of two benzoate and four acetate groups in 7. Differences between the two compounds indicated the presence of two benzoate groups at C-5 and C-7 in 7 instead of the two acetate groups in 6. This was confirmed by the HMBC cross-peaks from H-5 and H-7 to the corresponding carbonyl carbons of the benzoate groups (Figure S56, Supporting Information). Therefore, the structure of compound 7, euphorbesulin G, was established. The molecular formula (C37H46O14) of 8 was assigned using the sodium adduct (+)-HRESIMS ion at m/z 737.2776 [M +

to C-10. The HMBCs of H-17/C-5, C-6, and C-7 placed C-5, C-7, and C-17 at C-6. The five acetate groups were attached to C-1, C-3, C-7, C-8, and C-9 by the HMBC correlations from H-1 (δH 5.30), H-3 (δH 5.68), H-7 (δH 5.54), H-8 (δH 5.11), and H-9 (δH 4.95) to the corresponding acetate carbonyl carbons (δC 170.7, 170.7, 170.3, 170.1, and 169.7), respectively. Finally, the benzoate group was placed at C-5 by the HMBC cross-peak from H-5 (δH 5.57) to its carbonyl carbon (δC 165.2). The NOESY correlations (Figure 3B) of H-1/H-13, H-1/H2, H-1/H-4, H-2/H-3, H-3/H-4, H-4/H-7, H-4/H-13, H-11/ H-18, and H-11/H-13 indicated that these protons were cofacial and were randomly assigned as α-oriented. Subsequently, the NOESY correlations of H-12/H-19, H-9/H-19, H-9/H-8, H-8/H-5, and OH-15/H-5 suggested that they were β-oriented. The small 3J4,5 value of 4.3 Hz indicated that the Δ6(17) double bond is parallel to the macrocyclic plane of the molecule.6 The structure of euphorbesulin D (4) was thus defined as shown. Compound 5 possessed a molecular formula of C37H46O14 as assigned by the sodium adduct (+)-HRESIMS ion at m/z 737.2779 (calcd 737.2780) and 13C NMR data. Comparison of NMR data (Tables 2 and 4) of 5 with those of 1818 suggested that they are structural analogues except for the presence of one more hydroxy moiety in compound 5, which was located at C-1 by the HMBC correlation from OH-1 (δH 3.35) to C-1 (δC 87.3) (Figure S40, Supporting Information). The H-1 was D


E

a

Exchangeable signals.

5165.2 130.3 130.1 128.4 133.2

170.3, 20.9a 170.1, 20.8a 169.7, 20.6a

170.7, 21.3

75.4 39.4 74.8 47.1 69.2 143.5 68.1 70.5 80.7 40.7 137.1 130.1 44.9 210.7 87.2 9.4 113.6 26.4 23.3 20.3 170.7, 21.3a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OAc 2-OAc 3-OAc 5-OAc 7-OAc 8-OAc 9-OAc 15-OAc/HOCH2CO2 OBz CO 1 2, 6 3, 5 4

a

4

position

168.3, 170.2, 169.8, 169.8, 171.9, 3165.1 130.3 129.7 128.5 133.4

87.3 44.7 75.6 48.2 68.3 141.9 68.3 68.3 80.3 40.4 136.1 131.1 47.4 204.4 97.2 12.1 115.1 25.7 24.0 19.2

5

20.6a 21.4a 20.8a 20.8a 21.6a

170.1, 168.5, 170.0, 169.8, 169.7, 170.2,

43.5 37.9 76.3 49.9 69.6 141.2 67.8 70.0 80.8 40.4 135.7 130.8 44.6 204.4 91.9 14.2 117.5 25.7 23.6 20.2

6

20.8 20.2 20.9a 21.1a 21.2a 21.6a

a

Table 4. 13C NMR Data of Compounds 4−13 (125 MHz, CDCl3)

170.2, 21.0 170.1, 20.4 170.0, 21.5 5/7164.8/165.6 129.6/130.0 129.8/130.0 128.0/128.7 133.0/133.6

170.0, 21.0

43.5 38.4 76.9 49.9 70.5 140.9 68.0 70.5 81.0 40.5 135.6 131.0 44.9 204.5 92.1 14.6 118.3 25.8 23.7 20.3

7

170.3, 21.1 170.1, 20.4 171.9, 60.9 7165.7 130.2 129.9 128.9 133.8

170.0, 21.0 169.0, 20.6

43.6 37.9 76.8 50.0 70.5 139.9 67.4 70.3 81.1 40.3 136.1 130.3 44.6 204.1 92.9 14.2 119.4 25.6 23.7 20.7

8

170.0, 169.8, 169.7, 172.4, 5164.4 130.2 130.0 128.5 133.4

a

21.4a 20.7a 20.8a 21.3

169.7, 21.2

86.8 44.7 68.8 47.8 68.3 141.8 75.4 69.9 80.4 40.8 136.0 131.4 47.6 204.1 97.4 12.1 115.0 25.7 23.8 19.0

9

172.8, 20.5 169.5, 21.4 7165.2 129.8 129.8 128.8 133.7

170.0, 20.8 168.9, 20.6

43.6 37.9 76.9 49.8 70.6 140.2 68.2 69.5 86.9 39.6 134.8 130.6 44.5 204.5 91.6 14.2 120.7 25.9 23.8 20.3

10

173.0, 20.6 169.9, 22.5 7165.1 130.1 129.9 128.9 133.7

168.3,a 19.9 169.8, 21.5 169.3, 21.0 169.1, 20.6

169.7,a 20.7a 169.5, 21.4 170.0, 20.9a 169.7, 21.5a 170.0,a 22.5a 3164.7 130.0 129.9 128.6 133.6

48.3 87.8 77.1 47.3 69.4 141.6 67.7 70.0 80.7 40.6 136.3 130.6 44.1 204.0 92.2 19.6 116.3 25.8 23.5 19.9

12

48.6 87.6 77.6 46.9 70.9 140.0 68.3 69.3 87.1 39.8 135.2 130.7 44.0 204.4 91.2 19.2 121.4 26.1 23.8 20.2

11

170.0, 20.9 170.1, 20.5 169.8, 21.6 3/7165.1/165.7 130.1/129.9 129.8/130.0 128.5/128.9 133.5/133.9

168.4, 23.4

50.0 79.1 80.5 47.0 69.1 141.6 68.8 70.3 80.9 40.5 135.5 131.5 43.5 204.7 92.4 23.4 115.8 26.0 23.6 19.9 −5/8/9/15

13

Journal of Natural Products Article



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which replaced the acetate group in 17. This was verified by the HMBC cross-peaks (Figure S64, Supporting Information) from HOCH2− (δH 4.19 and 4.29, d) to the carbonyl carbon (δC 171.9) of the 2-hydroxyacetate group. Compound 8, euphorbesulin H, was thereby structurally characterized. Compound 9 shared a molecular formula of C37H46O14 with 4. The NMR data (Tables 3 and 4) revealed that these two compounds possessed the same number of acetate and benzoate groups. Compared to 4, the C-15 carbon resonance was deshielded (ΔδC +10.2) and the H-1 proton resonance was shielded (ΔδH −1.01), suggesting the presence of OAc-15 and OH-1 in 9. The structure of compound 9, euphorbesulin I, was subsequently confirmed by its HMBC data (Figure S72, Supporting Information). Compound 10, euphorbesulin J, possessed the molecular formula C35H44O12 as deduced from the (+)-HRESIMS sodium


Na]+ (calcd 737.2780) and 13C NMR data. Comparison of its 1 H and 13C NMR data (Tables 2 and 4) with those of 1713 showed that the only difference between compounds 8 and 17 was the presence of a 2-hydroxyacetate moiety at C-15 in 8,

Table 5. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compounds 14−16 in CDCl3 14 δH

position

15 δC

1

4.40, s

2 3 4 5 6

5.82, d (3.8) 4.15, t (4.0) 5.73, brs

7

5.97, s

68.8

8 9 10 11

5.21, s 4.99, s

70.3 80.7 40.7 136.0

12 13 14

5.53, dd (15.9, 9.7) 4.39, dq (9.7, 6.5)

15 16 17 18 19 20 OAc-3 OAc-5 OAc-8 OAc-9 OAc-15 OAc-17 OBz CO 1 2, 6 3, 5 4 4-OH 15-OH

5.96, d (15.9)

1.39, s 5.12, 5.22, 0.91, 1.30, 1.21, 2.06,

s s s s d (6.5) s

2.09, s 1.52, s 1.39, s

86.1 78.8 78.6 45.3 69.0 141.8

131.7 45.6 204.7 97.2 20.8 114.7 26.0 23.6 19.1 169.2, 21.5

α 3.04, dd (14.9, 11.2) β 1.50, dd (14.9, 5.1) 2.84, m 5.51, d (6.0) 6.12, s

α 1.56, dd (13.7, 12.0) β 2.28, dd (13.6, 8.5) 3.38, m

α 1.70, dd (12.9, 11.4) β 1.81, dd (12.9, 7.6) 3.22, m

1.03, d (7.5) 4.10, 4.23, 1.00, 1.10, 1.28,

d (12.2) d (12.2) s s s

16 δC 41.0 34.5 80.5 85.3 72.7 57.2 37.7 50.0 221.8 48.1 38.8 49.6 60.7 208.4

δH

δC

α 1.40, dd (14.8, 3.5) β 2.18, dd (14.8, 3.2) 4.11, q (3.3) 3.23, d (3.4)

43.3

1.27, 2.01, 2.05, 5.45,

m m m dd (5.6, 2.5)

α 1.76, m β 1.63, m 3.64, brs

66.5 24.7 24.4 17.6

1.01, s 1.11, s 0.89, s

1.97, s

169.7, 20.8

2.07, s 3-

169.9, 21.1

122.0 134.5 46.5 34.2 26.0

2.06, m

α 2.55, d (14.0) β 1.79, d (14.0) 5.82, dd (17.6, 10.9) 5.19, dd (10.9, 1.3) 5.11, dd (17.6, 1.3) 1.08, s

86.1 15.9

71.4 78.5 38.0 50.1 23.2

72.9 41.8 37.4 146.1 114.1 16.7 30.2 17.6 23.2

169.9, 20.8 170.2, 20.5 172.6, 21.3

5/7-

8.01, d (7.7)/8.10, d (7.7) 7.35, t (7.7)/7.43, t (7.7) 7.49, t (7.5)/7.56, t (7.5)

δH

164.4/165.2 130.1/129.8 129.9/130.0 128.7/128.7 133.2/133.7

7.95, 7.49, 7.60, 3.97, 2.81,

d (8.3) t (7.6) t (7.4) s s F

165.2 129.5 129.7 128.8 133.6



Article

adduct ion and 13C NMR data. Its NMR data (Tables 3 and 4) were similar to those of 17,13 suggesting that these two compounds are structural analogues. Analysis of its MS and NMR data showed that the only difference was one acetate group less in compound 10 than in 17. The H-8 (δH 3.97) of 10 was significantly shielded (ΔδH −1.2) as compared with that of the known compound, indicating the presence of a hydroxy group at C-8 of 10. This assignment was confirmed by the HMBC spectrum (Figure S80, Supporting Information). Euphorbesulin K (11) possessed a molecular formula of C37H46O14 as established by the sodium adduct (+)-HRESIMS ion and 13C NMR data. Comparison of its NMR data (Tables 3 and 4) with those of 10 indicated that their structures were closely related, differing only by an additional acetate group at C-2 of 11. This was supported by the observation of an oxygenated tertiary carbon resonance (δC 87.6) for 11 in place of the C-2 methine signal (δC 37.9) of 10. Compound 11 was thereby structurally characterized. Compound 12 had the molecular formula C39H48O15 as determined by the sodium adduct (+)-HRESIMS ion and 13C NMR data. The NMR data (Tables 3 and 5) analysis showed that its structure was closely related to compound 19,13 differing only in their acylation patterns. As opposed to 19, compound 12 had a benzoate and an acetate group located at C-3 and C-7, respectively, based on the HMBC cross-peaks (Figure S96, Supporting Information) from H-3 (δH 6.00) and H-7 (δH 5.76) to the respective acyl carbonyl carbons at δC 164.7 and 169.5. Thus, the structure of compound 12, euphorbesulin L, was characterized as shown. Comparison of the NMR data (Tables 3 and 4) of compound 13 (C42H48O14) with those of sieboldianine A (22)19 revealed that their structures were closely related, differing only in their acylation patterns at C-5 and C-7. An acetate and a benzoate group were located at C-5 and C-7 for 13, respectively, based on the HMBC cross-peaks (Figure S105, Supporting Information) from H-5 (δH 5.52) to the acetate carbonyl carbon (δC 168.4) and from H-7 (δH 5.93) to the benzoate carbonyl carbon (δC 165.7). This is opposite the assignments in 22. The remaining benzoate and four acetate moieties were placed at C-3, C-5, C-8, C-9, and C-15, respectively, by the HMBC data (Figure S105, Supporting Information), which is the same oxygenation pattern as in 22. Thus, the structure of euphorbesulin M (13) was defined as shown. The molecular formula (C42H48O15) of compound 14 showed 16 mass units more than that of sieboldianine C.19 Analysis of the NMR data (Table 5) revealed that it was a structural analogue of sieboldianine C, and the only difference was the presence of an extra hydroxy group at C-1 of 14, which caused deshielding of its H-1 (δH 4.40, s) resonance as compared to the H2-1 resonances (δH 2.49 and 2.81) of sieboldianine C. The OH-1 group was established to be βoriented by the NOESY correlations of H-1/H-3 and H-1/H-5. The remaining stereocenters of 14 were assigned as the same as those of sieboldianine C. Therefore, the structure of 14, euphorbesulin N, was defined as shown and verified by 2D NMR data (Figures S112 and S113, Supporting Information). A Paraliane-Type Diterpenoid. Compound 15, euphorbesulin O, possessed a molecular formula of C31H38O10. Analysis of the NMR data (Table 5) showed the presence of a benzoate, two acetate, and two ketocarbonyl groups. These functional groups accounted for nine indices of hydrogen deficiency, and the remaining four indices suggested the

presence of four rings in compound 15. The aforementioned data and the compound classes identified from this plant genus suggested that 15 is a paraliane-type diterpenoid.6,9 Analysis of the 1H−1H COSY spectrum (Figure 4A) indicated the


presence of three structural fragments as drawn with bold bonds. The scaffold of 15 was delineated by interpretation of the HMBC spectrum (Figure 4A), in which the correlation networks from OH-15 (δH 2.81) to C-1 (δC 41.0), C-4 (δC 85.3), C-14 (δC 208.4), and C-15 (δC 86.1); from OH-4 (δH 3.97) to C-3 (δC 80.5), C-4 (δC 85.3), and C-5 (δC 72.7); from H-7 to C-9 (δC 221.8); from H-17 to C-5, C-6, and C-7; from H-18 and H-19 to C-9, C-10, and C-11; and from H-20 to C12, C-13, and C-14 were observed. These HMBC correlations not only constituted the diterpenoid backbone but also placed hydroxy groups at C-4 and C-15 and ketocarbonyl groups at C9 and C-14. Furthermore, one benzoate and two acetate moieties were located at C-3, C-5, and C-17 by the HMBC cross-peaks from H-3 (δH 5.51), H-5 (δH 6.12), and H-17 (δH 4.10 and 4.23) to the corresponding carbonyl carbons (δC 165.2, 169.7, and 169.9) of the acyl groups, respectively. The relative configuration of 15 was established by the NOESY data (Figure 4B). The NOESY cross-peaks of H3-16/ H-1β, H-1β/OH-15, OH-15/H-5, H-5/H-7β, H-5/H-8, H-8/ H-12, H-11β/H-12, and H-12/H3-19 suggested that these protons were spatially close and were randomly assigned a βorientation. Subsequently, the NOESY interactions of H-2/H3, H-3/OH-4, OH-4/H3-20, and H3-20/H-17 indicated that they were α-oriented. Thus, the structure of 15 was deduced as shown. An Isopimarane-Type Diterpenoid. Compound 16 (C20H32O3) was obtained as a pale yellow powder. Analysis of its NMR data (Table 5) revealed that it was an isopimaranetype diterpenoid structurally similar to 3β,12α-dihydroxypimara-7,15-dien-2-one.20 The only structural variation was a hydroxy group at C-2 in 16 in place of a ketocarbonyl group of the latter. This assignment was verified by 2D NMR data (Figure 5). In particular, the NOESY (Figure 5B) correlations

Figure 5. (A) Key HMBC and COSY (bold bonds) and (B) NOESY correlations for 16. G



Article

column (petroleum ether/acetone, 50:1 to 1:5) to give four parts (C1−C4). Part C1 (6.8 g) was chromatographed on a silica gel column (petroleum ether/CHCl3, 5:1 to 1:3) to yield compounds 1 (30 mg) and 7 (23 mg). Fraction C2 (4.1 g) was subjected to an RP18 silica gel column (MeOH/H2O, 30−80%) and yielded three major fractions (C2a−C2c). Fraction C2a (2.1 g) was subjected to Sephadex LH-20 CC and afforded compounds 8 (28 mg), 14 (12 mg), and 18 (5 mg). Fraction C3 (5.3 g) was separated on an RP-18 column (MeOH/H2O, 30−80%) to yield three major subfractions (C3a− C3c). Fraction C3c (2.2 g) was fractionated on a column of silica gel (CHCl3/MeOH, 500:1 to 50:1) to obtain three major fractions, C3c1 to C3c3. After purification by HPLC (68% CH3CN in H2O, 3 mL/ min), subfraction C3c1 yielded compounds 2 (20 mg), 9 (19 mg), and 10 (6.5 mg); fraction C3c2 gave compounds 11 (6.5 mg) and 12 (21 mg); and fraction C3c2 afforded compounds 13 (14 mg) and 22 (2.2 mg). Euphorbesulin A (1): colorless crystals (MeOH); mp 172−175 °C; [α]22D +49 (c 0.6, MeOH); UV(MeOH) λmax (log ε) 228 (4.11) nm; IR (KBr) νmax 3413, 2965,1751, 1719, 1453, 1383, 1314, 1275, 1180, 1140, 1070, 921, 711 cm−1; 1H and 13C NMR (pyridine-d5), see Table 1; (+)-ESIMS m/z 585.1 [M + H]+, 445.2 [2 M + Na]+; (−)-ESIMS m/z 583.4 [M − H]−; (+)-HRESIMS m/z 585.3071 [M + H]+ (calcd for C33H44O9, 585.3058). Euphorbesulin B (2): white, amorphous powder; [α]22D +78 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 229 (4.30) nm; IR (KBr) νmax 3560, 3366, 2968, 1732, 1720, 1602, 1453, 1371, 1324, 1270, 1170, 1068, 926, 708 cm−1; 1H and 13C NMR (pyridine-d5), see Table 1; (+)-ESIMS m/z 613.0 [M + Na]+; (−)-ESIMS m/z 589.2 [M − H]−; (+)-HRESIMS m/z 613.2408 [M + Na]+ (calcd for C34H38O9Na, 613.2408). Euphorbesulin C (3): white, amorphous powder; [α]22D +54 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 228 (4.13) nm; IR (KBr) νmax 3431, 2966, 1744, 1723, 1452, 1373, 1274, 1229, 1070, 1026, 929, 713 cm−1; 1H and 13C NMR (pyridine-d5), see Table 1; (+)-ESIMS m/z 1079.1 [2 M + Na]+; (−)-ESIMS m/z 527.6 [M − H] −; (+)-HRESIMS m/z 551.2261 [M + Na]+ (calcd for C29H36O9Na, 551.2252). Euphorbesulin D (4): white, amorphous powder; [α]22D +63 (c 0.3, MeOH); UV(MeOH) λmax (log ε) 229 (4.15) nm; IR (KBr) νmax 3451, 2977, 1742, 1452, 1375, 1277, 1250, 1226, 1109, 1070, 1036, 713 cm−1; 1H NMR (CDCl3), see Table 2 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 697.1 [M − H2O + H]+; (+)-HRESIMS m/z 737.2771 [M + Na]+ (calcd for C37H46O14Na, 737.2780). Euphorbesulin E (5): white, amorphous powder; [α]22D +97 (c 0.3, MeOH); IR (KBr) νmax 3445, 2975, 1747, 1453, 1375, 1278, 1227, 1073, 1042, 963, 713 cm−1; 1H NMR (CDCl3), see Table 2 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 736.9 [M + Na]+; (−)-ESIMS m/z 759.6 [M + HCO2]−; (+)-HRESIMS m/z 737.2779 [M + Na]+ (calcd for C37H46O14Na, 737.2780). Euphorbesulin F (6): white, amorphous powder; [α]22D +20 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 231 (4.11) nm; IR (KBr) νmax 3432, 2924, 1742, 1630, 1456, 1375, 1260, 1233, 1104, 1040, 1020, 958 cm−1; 1H NMR (CDCl3), see Table 2 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 1295.1 [2 M + Na]; (+)-HRESIMS m/z 659.2675 [M + Na]+ (calcd for C32H44O13Na, 659.2674). Euphorbesulin G (7): white, amorphous powder; [α]22D +24 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 230 (4.58) nm; IR (KBr) νmax 3434, 2979, 1745, 1724, 1453, 1373, 1320, 1281, 1226, 1109, 1063, 944, 715 cm−1; 1H NMR (CDCl3), see Table 2 and 13C NMR (CDCl 3), see Table 4; (+)-ESIMS m/z 783.1 [M + Na]+; (+)-HRESIMS m/z 783.2993 [M + Na]+ (calcd for C42H48O13Na, 783.2987). Euphorbesulin H (8): white, amorphous powder; [α]22D +31 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 232 (3.98) nm; IR (KBr) νmax 3469, 2975, 1747, 1718, 1452, 1377, 1233, 1119, 1043, 952, 713 cm−1; 1 H NMR (CDCl3), see Table 2 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 737.3 [M + Na], 1451.2 [2 M + Na]+; (+)-HRESIMS m/z 737.2776 [M + Na]+ (calcd for C37H46O14Na, 737.2780). Euphorbesulin I (9): white, amorphous powder; [α]22D +60 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 228 (3.95) nm; IR (KBr) νmax

of H3-19/H-1β, H-1β/H3-20, H3-20/H-6β, H3-20/H-11β, H11β/H3-17, H3-17/H-12, and H3-17/H-14β indicated that these protons were spatially close and were arbitrarily assigned as β-oriented. Accordingly, the NOESY interactions of H-2/H3, H-3/H3-18, H3-18/H-5, and H-5/H-9 showed that these protons were α-directed. The structure of euphorbesulin P (16) was thus defined as shown. The six known diterpenoids 3β,5α,8α,15β-tetraacetoxy-7βbenzoyloxyjatropha-6 (17),11E-dien-9,11-dione (17), 13 5α,7β,8α,9α,15β-pentaacetoxy-3β-benzoyloxyjatropha6(17),11E-dien-14-one (18),18 2α,3β,5α,8α,9α,15β-hexaacetoxy-7β-benzoyloxyjatropha-6(17)-11E-dien-14-one (19),13 sieboldianine B (20),19 3β,5α,7β,8α,15β 8α,15β-pentaacetoxy-2αbenzoyloxyjatropha-6(17),11E-diene-9,14-dione (21),13 and sieboldianine A (22)19 were identified by physical data and/ or by comparing the MS and NMR data with reported data. Thirteen of the new compounds were tested for antimalarial effects against chloroquine-resistant Plasmodium falciparum strain Dd2 using a SYBR-Green assay with artemisinin as the positive control.21 Compound 7 showed low nanomolar antimalarial activity, while the rest of the diterpenoids showed only moderate to no antimalarial activity (Table 6). Table 6. Antimalarial Activity of 1−5, 7−12, 14, and 15 no.

IC50 (μM)

no.

IC50 (μM)

1 2 3 4 5 7 8

2.41 ± 0.44 >5 >5 >5 >5 0.12 ± 0.04 >5

9 10 11 12 14 15

>5 >5 >10 >5 >5 N.A.a

Positive control: artemisinin 7 ± 0.1 nM > 10 μM was considered inactive. a

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EXPERIMENTAL SECTION

General Experimental Procedures. The general experiments were completed according to the reported procedures with minor modification (General Experimental Procedures, Supporting Information).22 Plant Material. Twigs of E. esula were collected in June 2012 in Baise County, Guangxi Province, China. The plant species was identified by Professor Shao-Qing Tang of Guangxi Normal University. A voucher specimen (accession no. Eue-2012-1Y) has been deposited in the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. Dried powder of E. esula (7 kg) was percolated with 95% EtOH (3 × 25 L, rt), and the crude extract (550 g) was partitioned with EtOAc/H2O. The EtOAc phase sample was fractionated using an MCI gel column eluted with MeOH/H2O (from 3:7 to 9:1) to afford four major fractions (A−D). Fraction B (45 g) was chromatographed over silica gel column chromatography (CC) with petroleum ether/acetone (from 50:1 to 1:5) as the eluant to give four subfractions (B1−B4). Fraction B1 (500 mg) was purified by semipreparative HPLC (75% CH3CN in H2O, 3 mL/min) to obtain 17 (20 mg) and 19 (2.3 mg). Fraction B2 (5.4 g) was separated over a column of silica gel (petroleum ether/EtOAc, 8:1 to 1:3) to give three fractions (B2a−B2c). Fraction B2a (1.3 g) afforded compounds 4 (3.1 mg), 5 (2.5 mg), 6 (3.9 mg), and 20 (3.1 mg) by HPLC preparation (60% CH3CN in H2O, 3 mL/min). Fraction B2b was further fractionated over a column (Sephadex LH-20, eluted with EtOH) to give compounds 3 (7.8 mg) and 15 (4.6 mg). By using the same conditions of fraction B2b, fraction B2c afforded compounds 16 (2.1 mg) and 21 (2.3 mg). Fraction C (40 g) was subjected to a silica gel H



Article

3478, 2976, 1744, 1374, 1277, 1225, 1123, 1069, 1018, 960, 715 cm−1; 1 H NMR (CDCl3), see Table 3 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 737.0 [M + Na]; (−)-ESIMS m/z 759.4 [M + HCO2]−; (+)-HRESIMS m/z 737.2772 [M + Na]+ (calcd for C37H46O14Na, 737.2780). Euphorbesulin J (10): white, amorphous powder; [α]22D +72 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 231 (4.13) nm; IR (KBr) νmax 3440, 2968, 1730, 1452, 1374, 1325, 1240, 1107, 1058, 927, 721 cm−1; 1 H NMR (CDCl3), see Table 3 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 679.0 [M + Na]; (−)-ESIMS m/z 701.4 [M + HCO2]−; (+)-HRESIMS m/z 679.2718 [M + Na]+ (calcd for C35H44O12Na, 679.2725). Euphorbesulin K (11): white, amorphous powder; [α]22D +31 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 229 (3.97) nm; IR (KBr) νmax 3479, 2974, 1748, 1723, 1374, 1240, 1113, 1070, 1028, 945, 715 cm−1; 1 H NMR (CDCl3), see Table 3 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 736.9 [M + Na]; (−)-ESIMS m/z 759.4 [M + HCO2]−; (+)-HRESIMS m/z 737.2780 [M + Na]+ (calcd for C37H46O14Na, 737.2780). Euphorbesulin L (12): white, amorphous powder; [α]22D +10 (c 0.9, MeOH); UV (MeOH) λmax (log ε) 228 (3.95) nm; IR (KBr) νmax 3437, 2977, 1752, 1453, 1373, 1278, 1240, 1123, 1033, 957, 714 cm−1; 1 H NMR (CDCl3), see Table 3 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 779.1 [M + Na]; (+)-HRESIMS m/z 779.2872 [M + Na]+ (calcd for C39H48O15Na, 779.2885). Euphorbesulin M (13): white, amorphous powder; [α]22D +35 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 230 (4.35) nm; IR (KBr) νmax 3465, 2974, 1747, 1722, 1452, 1375, 1273, 1237, 1114, 1071, 1026, 711 cm−1; 1H NMR (CDCl3), see Table 3 and 13C NMR (CDCl3), see Table 4; (+)-ESIMS m/z 759.1 [M − H2O + H]+; (−)-ESIMS m/z 821.4 [M + HCO2]−; (+)-HRESIMS m/z 799.2946 [M + Na]+ (calcd for C42H48O14Na, 799.2936). Euphorbesulin N (14): white, amorphous powder; [α]22D +39 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 231 (4.13) nm; IR (KBr) νmax 3484, 2979, 1747, 1452, 1374, 1269, 1230, 1103, 1046, 998, 952, 718 cm−1; 1H and 13C NMR (CDCl3), see Table 5; (−)-ESIMS m/z 837.5 [M + HCO2]−; (+)-HRESIMS m/z 815.2891 [M + Na]+ (calcd for C42H48O15Na, 815.2885). Euphorbesulin O (15): white, amorphous powder; [α]22D +174 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 230 (4.18) nm; IR (KBr) νmax 3455, 2967, 1719, 1453, 1376, 1277, 1227, 1123, 1028, 965, 715 cm−1; 1 H and 13C NMR (CDCl3), see Table 5; (+)-ESIMS m/z 1163.1 [2 M + Na]+; (−)-ESIMS m/z 1139.3 [2 M − H]−; (+)-HRESIMS m/z 593.2355 [M + Na]+ (calcd for C31H38O10Na, 593.2357). Euphorbesulin P (16): pale yellow powder; [α]22D −40 (c 0.2, MeOH); IR (KBr) νmax 3425, 2965, 2918, 1746, 1640, 1364, 1271, 1068, 1036, 996, 921 cm−1; 1H and 13C NMR (CDCl3), see Table 5; (+)-ESIMS m/z 343.2 [M + Na]+; (+)-HRESIMS m/z 343.2241 [M + Na]+ (calcd for C20H32O3Na, 343.2244). Crystal Structure Analysis. Colorless crystals of 1 were obtained by recrystallization in MeOH at room temperature. X-ray crystal data were acquired on a Bruker APEX-II CCD detector with graphitemonochromated Cu Kα radiation (λ = 1.541 78 Å). The structure of 1 was directly elucidated using SHELXL-97 (Sheldrick 2008) and refined by the full-matrix least-squares difference Fourier method. The X-ray data of 1 have been deposited at the Cambridge Crystallographic Data Center (CCDC 1437352) and are available free of charge via the Internet at www.ccdc.cam.uk/conts/retrieving.html. Antimalarial Activity Assay. Antimalarial activity against the P. falciparum strain Dd2 (chloroquine-resistant) was measured in a 72 h growth assay with minor modifications21,23 and with artemisinin as the positive control.

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IR, ESIMS, HRESIMS, 1D and 2D NMR data of 1−16 (PDF) X-ray crystallographic data of 1 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-50806718. Fax: +86-21-50806718. E-mail: [email protected] (J.-M. Yue). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The National Natural Science Foundation (21532007) and the Foundation (2012CB721105) of the MOST of P. R. China are greatly acknowledged. We thank Prof. S.-Q. Tang of Guangxi Normal University for authentication of the plant samples. We would like to thank Dr. J. Webster and Dr. D. G. I. Kingston for comments and corrections.

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REFERENCES

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(23) Liu, J.; He, X. F.; Wang, G. H.; Merino, E. F.; Yang, S. P.; Zhu, R. X.; Gan, L. S.; Zhang, H.; Cassera, M. B.; Wang, H. Y.; Kingston, D. G. I.; Yue, J. M. J. Org. Chem. 2014, 79, 599−607.

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