Diterpenoids from Croton laui and Their Cytotoxic and Antimicrobial

Apr 15, 2014 - Giang , P. M.; Son , P. T.; Hamada , Y.; Otsuka , H. Chem. Pharm. .... Giang , P. T.; Jin , H. Z.; Son , P. T.; Lee , J. H.; Hong , Y. ...
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Diterpenoids from Croton laui and Their Cytotoxic and Antimicrobial Activities Cui-Ping Liu, Jin-Biao Xu, Jin-Xin Zhao, Cheng-Hui Xu, Lei Dong, Jian Ding, 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

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S Supporting Information *

ABSTRACT: Fourteen new diterpenoids including clerodane (1− 12), labdane (13), and norlabdane (14) types, as well as nine known analogues were isolated from the aerial parts of Croton laui. Their structures were established on the basis of spectroscopic analysis, and that of crotonolide H (11) was confirmed by singlecrystal X-ray crystallography. Crotonolide A (1) exhibited moderate cytotoxicity against two tumor cell lines, HL-60 (human premyelocytic leukemia, IC50 9.42 μM) and P-388 (murine leukemia, IC50 7.45 μM), and crotonolide G (10) displayed significant antibacterial activity against a panel of Gram-positive bacteria.

T

he genus Croton (Euphorbiaceae) comprises about 1300 species in tropical and subtropical regions worldwide.1 Many species in this genus have been used as a folk medicine to treat stomachache, abscesses, malaria, and inflammation.2−4 A large array of chemical constituents were identified from the genus Croton, of which especially terpenoids displayed a broad spectrum of biological activities, such as acetylcholinesterase inhibitory,2 antiplasmodial,5 cytotoxic,6,7 antiviral,8 and antiinflammatory activities.9,10 In continuing our efforts to identify structurally diverse and biologically important compounds from medicinal herbs, 14 new diterpenoids (1−14) and nine known compounds were isolated from the aerial parts of Croton laui Merr. et Metc. Cytotoxic and antimicrobial evaluations revealed that compound 1 exhibited moderate cytotoxic activity against HL-60 (human premyelocytic leukemia) and P-388 (murine leukemia) cell lines, and compound 10 displayed antibacterial activity against a panel of Gram-positive bacteria. We herein present the isolation, structural elucidation, and biological tests of these diterpenoids.

and 167.2), and a β-substituted furan ring (δC 144.2, 140.0, 124.6, and 108.5). These functionalities accounted for seven out of the 12 indices of hydrogen deficiency, and the remaining five thus required the existence of a pentacyclic system. The aforementioned data suggested that 1 was a clerodane diterpenoid.11 The assignment of the structure of 1 was facilitated by 2D NMR spectra. In the COSY spectrum (Figure 1A), the correlations of H-10/H2-1/H2-2/H-3 and H-6/H2-7 delineated two proton-bearing sequences of C(10)−C(1)−C(2)−C(3) and C(6)−C(7). In the HMBC spectrum (Figure 1A), the correlations from H-12 to C-13, C-14, and C-16 attached the furan ring to C-12. The chemical shift of C-20 (δC 112.5) and the multiple HMBC correlations from H-20 to C-9, C-12, and

■

RESULTS AND DISCUSSION Compound 1 possessed a molecular formula of C20H18O6 based on the 13C NMR data and the HRESIMS ion at m/z 731.2102 [2 M + Na]+ (calcd 731.2104), requiring 12 indices of hydrogen deficiency. The IR absorption bands at 1757 and 1738 cm−1 were indicative of the presence of carbonyl groups. The 1H NMR data (Table 1) displayed the diagnostic resonances (δH 6.46, 7.46, and 7.49) of a β-substituted furan ring. The 13C NMR spectrum (Table 4) showed 20 carbon resonances, which were classified by HSQC and DEPT experiments as four methylenes (δC 41.6, 36.5, 26.1, and 20.7), four methines (δC 112.5, 75.8, 75.5, and 38.8), two sp3 quaternary carbons (δC 50.5 and 48.2), two double bonds (δC 144.4, 139.8, 128.2, and 115.3), two ester carbonyls (δC 168.9 © 2014 American Chemical Society and American Society of Pharmacognosy

Received: January 16, 2014 Published: April 15, 2014 1013

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Table 1. 1H NMR Spectroscopic Data of Compounds 1−5 proton position 1α 1β 2α 2β 3 6α 6β 7α 7β 10 11 12 14 15 16 17 pro-Z 17 pro-E 19 20 HO-19 CH3O-18 Ac-6 a

1a

2a

3a

4a

5a

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

1.96, 2.16, 2.73, 2.54, 7.01,

m m ddd (20.8, 7.8, 2.5) m dd (4.5, 2.8)

4.68, 2.60, 3.01, 2.14, 2.51, 5.30, 6.46, 7.46, 7.49, 5.33, 5.35,

dd (12.3, 5.6) m dd (14.6, 5.6) m m dd (10.0, 6.0) br s br s s d (2.5) d (2.5)

1.96, 2.14, 2.23, 2.41, 7.00, 1.12, 2.70, 2.30, 3.03, 1.69, 2.35, 5.14, 6.40, 7.40, 7.42, 4.68, 4.77, 5.52, 5.33, 5.48, 3.76,

5.55, s

m m m m dd (5.6, 2.3) td (13.2, 6.3) dd (13.2, 5.3) m m dd (13.0, 2.7) m m br s br s br s s s d (3.0) s d (3.0) s

1.91, 2.61, 2.23, 2.41, 7.06, 1.28, 2.54, 2.36, 2.83, 1.59, 2.44, 5.14, 6.38, 7.40, 7.41, 4.66, 4.75, 5.75, 5.15,

m m m m dd (5.6, 2.3) td (13.2, 6..2) dd (13.2, 6.0) m td (13.6, 6.7) m m m br s br s br s s s d (2.8) s

3.72, s

1.98, 2.16, 2.23, 2.38, 6.97,

m m m m dd (5.8, 1.9)

2.04, 2.65, 2.16, 2.18, 7.10,

m m m m dd (5.3, 2.1)

5.68, 2.50, 3.24, 2.16, 2.36, 5.20, 6.42, 7.40, 7.44, 4.79, 4.82, 5.57, 5.40, 5.02, 3.72, 1.94,

m dd (15.5, 2.0) br d (15.5) m m m br s br s s br s br s d (2.7) s d (2.7) s s

5.64, 2.43, 3.05, 2.12, 2.43, 5.18, 6.38, 7.40, 7.42, 4.76, 4.78, 5.70, 5.17,

m m br d (15.7) m m m br s br s s s s d (3.4) s

3.69, s 1.95, s

Data were measured in CDCl3 at 500 MHz.

Table 2. 1H NMR Spectroscopic Data of Compounds 6−10 6a proton position 1α 1β 2α 2β 3 4 6α 6β 7α 7β 8 10 11α 11β 12 14 15 16 17 18 19 20 HO-19 CH3O-18 a

7a

(mult., J in Hz) 1.51, 1.95, 2.20, 2.39, 6.94,

8a

(mult., J in Hz)

m m m m dd (5.7, 2.2)

2.20, 2.36, 1.95, 2.19, 7.06,

m m m m dd (5.2, 2.1)

1.03, 2.38, 1.66, 1.90, 1.68, 1.51, 2.16,

td (13.5, 5.5) m m m m t (7.8) m

1.23, 2.40, 1.25, 1.69, 2.35, 1.40, 1.92,

m m m m m m m

5.11, 6.43, 7.40, 7.42, 1.00,

dd (10.2, 5.5) br s br s s d (6.4)

5.15, 6.40, 7.40, 7.41, 1.00,

m br s br s s m

(mult., J in Hz) 1.78, 2.00, 2.32, 2.48,

m m td (14.3,7.3) ddd (14.3, 5.0, 1.6)

2.24, 1.85, 1.27, 1.60, 2.14, 2.23, 1.65, 2.38, 1.77, 5.51, 6.43, 7.43, 7.46,

m dt (13.5, 3.3) td (13.5, 3.6) m ddd (14.5, 6.8, 3.4) m dd (12.8, 2.9) dd (13.4, 5.6) m dd (11.5, 5.6) br s br s s

0.92, d (6.7) 5.40, 5.40, 4.44, 3.75,

d (2.3) s d (2.0) s

5.46, d (3.5) 5.26, s

0.82, s 1.10, s

9b (mult., J in Hz) 1.87, 1.50, 2.06, 1.58, 4.35,

m m m m br s

1.89, m 1.47, m 1.73, m 2.13, m 2.11, m 1.04, dd (12.4, 2.1) 2.34, dd (13.4, 5.7) 1.60 m 5.49, dd (11.4, 5.7) 6.40, br s 7.40, br s 7.42, s a 4.81, br s b 4.88, br s 1.32, s 1.11, s

10b (mult., J in Hz) 1.36, m 1.42, m 2.01, m 5.17, br s 1.17, m 1.69, dt (12.8, 3.0) 1.39, m 1.47,m 1.36, m 1.38, m 1.95, 5.57, 4.14, 4.11, 0.79, 1.57,

m t (7.0) d (7.0) s d (6.3) br s

0.98, s 0.71, s

3.73, s b

Data were measured in CDCl3 at 500 MHz. Data were measured in CDCl3 at 400 MHz.

C-19 suggested the presence of a C-20 acetal group and the formation of a five-membered lactone between C-19 and C-20, which were identical to those of croverin.12 The Δ8,17 exocyclic double bond was assigned by HMBC correlations from H2-17

to C-7, C-8, and C-9. HMBC correlations from H-3 and H-6 to C-18 permitted the location of a C-3 double bond and the formation of an α,β-unsaturated five-membered lactone between C-4 and C-6 via C-18. The relative configuration of 1014

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Table 3. 1H NMR Spectroscopic Data of Compounds 11−14 proton position 1α 1β 2α 2β 3α 3β 5 6α 6β 7

a

11b

12b

13a

14a

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

(mult., J in Hz)

1.57−1.62, m 1.67, m 2.05, m 3.58, br s

1.40, 1.60, 2.01, 1.69, 3.58,

m m tdd (14.0, 4.4, 3.7) m br s

1.38, m 1.58, m 1.34−1.44, m

1.40, m 1.58, m 1.38−1.47, m

0.98, 1.46, 1.43, 1.63, 1.14, 1.41, 1.05, 1.90, 1.31, 3.69,

td (12.9, 3.7) m m m m m dd (13.0, 2.7) ddd (13.4, 4.6, 2.7) ddd (13.4, 13.0, 11.0) dd (11.0, 4.6)

8 9 10 11α 11β 12 14 15 16 17

1.48, m

1.51, m

1.97, 1.63, 1.94, 4.83, 6.37, 7.36, 7.36, 0.74,

1.81, 1.51, 1.64, 2.30, 6.26, 7.34, 7.20, 0.81,

18

1.23, s

1.25, s

0.87, s

19 20

1.13, s 0.73, s

1.13, s 0.75, s

0.80, s 0.80, s

m m m dd (8.0, 2.7) br s br s br s d (6.5)

dd (12.3, 2.1) m m m br s br s s d (6.3)

1.27, m 1.80, dt (12.9, 3.6) 1.67−1.73, m 3.72, m 1.31, dd (12.4, 2.8) 1.58, m 1.46, ddd (13.0, 12.4, 4.3) α 2.03, td (13.0, 5.0) β 2.41, m

1.48 m

1.90, d (11.0)

1.95, 1.60, 4.29, 5.83, 4.18, 1.61, 1.12,

2.39, 2.52, 6.41, 9.34,

m m t (8.0) t (6.7) d (6.7) s s

m m t (5.9) s

1.76, br s pro-Z: 4.40, s pro-E: 4.86, s a 3.44, d (10.3) b 3.72, m 0.89, s 0.81, s

Data were measured in CDCl3 at 500 MHz. bData were measured in CDCl3 at 400 MHz.

Table 4. 13C NMR Spectroscopic Data of Compounds 1−14a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ac-6 CH3O-18 a

1

2

3

4

5

6

7

8

9

10

11

12

13

14

20.7 26.1 139.8 128.2 48.2 75.8 36.5 144.4 50.5 38.8 41.6 75.5 124.6 108.5 144.2 140.0 115.3 167.2 168.9 112.5

21.0 26. 8 142.0 135.3 41.3 31.4 32.1 151. 8 50.2 48.6 38.6 71.3 127.5 108.8 143.7 139.3 106.0 169.4 95.5 103.1

21.7 27.0 141.7 134.3 40.7 35.6 31.3 151.4 48.7 45.0 38.5 69.8 128.5 108.7 143.8 139.1 105.9 167.4 95.6 102.0

21.8 27.1 141.2 137.5 41.6 35.0 29.8 38.2 47.2 47.4 38.3 71.5 127.4 108.8 143.7 139.3 16.8 168.1 95.7 99.9

22.7 41.1 211.4 58.0 40.9 38.0 18.6 51.6 37.3 55.1 44.6 71.9 125.9 108.6 144.0 139.5 172.1 6.9 14.5 15.1

16.9 34.6 74.4 159.6 39.3 37.1 18.3 51.6 37.4 55.0 44.4 72.1 126.0 108.6 143.9 139.5 172.6 110.0 23.1 14.9

18.4 27.0 120.5 144.6 38.3 36.9 27.6 36.3 38.8 46.5 37.1 29.1 144.9 126.0 58.5 60.8 16.1 18.1 20.0 18.5

17.2 30.5 76.6 77.0 41.6 32.5 26.7 36.7 39.5 41.5 46.1 63.2 131.5 108.7 143.5 138.5 16.3 21.3 17.5 18.1

16.5 30.6 76.5 76.5 41.4 32.6 26.7 36.3 38.8 41.0 38.8 18.5 125.9 111.2 142.8 138.5 16.2 21.6 17.4 18.4

39.8 18.5 42.3 33.2 55.7 29.1 78.8 85.7 59.1 35.9 28.6 82.0 139.0 122.1 59.2 13.6 19.3 33.6 21.1 15.9

37.0 27.6 76.2 42.4 48.9 23.9 37.6 147.4 56.2 39.3 24.6 155.9 139.2 195.3

51.7

21.5 26.3 143.2 131.1 45.4 72.0 36.9 146.7 48.7 38.7 38.3 70.1 128.2 108.6 143.9 139.1 108.7 167.2 93.7 102.0 170.0 21.1 51.9

21.5 27.0 140.3 137.5 41.5 31.8 30.9 38.4 49.4 49.4 38.7 73.0 126.2 108.9 143. 6 139.6 17.0 168.9 97.3 99.5

52.5

21.4 26.3 142.1 133.6 45.9 70.0 38.0 147.0 51.2 42.4 38.8 72.4 126.5 108.8 143.7 139.5 108.8 169.0 95.6 102.1 170.0 21.1 52.5

52.2

51.8

9.6 108.4 71.5 11.6 15.0

Data were measured in CDCl3 at 125 MHz.

orientations. Consequently, H-12 was fixed in an α-orientation by the ROESY correlations of H-12/H-1α and H-1β. The ROESY correlation between H-7α and H-10 indicated that H-

1 was established on the basis of a ROESY experiment (Figure 1B), in which the correlation between H-20 and H-6 indicated that they were spatially close, and arbitrarily assigned in β1015

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Figure 1. Selected COSY (bold bond), HMBC (A), and ROESY (B) correlations of 1.

10 was α-oriented in the B-ring possessing a boat conformation. Thus, the structure of compound 1 was elucidated as shown and named crotonolide A. Compounds 2 and 3 were obtained as a 3:1 interconverting mixture as measured by 1H NMR peak integration and shared a molecular formula of C21H24O6 as determined by 13C NMR data and their HRESIMS ion at m/z 767.3046 [2 M + Na]+ (calcd 767.3043). Two sets of proton resonances in the 1H NMR data (Table 1) were distinguished by an HSQC experiment, including an olefinic proton resonance pair at δH 7.00/7.06 (H-3, dd, J = 5.6, 2.3 Hz), two pairs of oxygenated methine resonances at δH 5.52/5.75 (H-19) and 5.33/5.15 (H20), two pairs of proton resonances for terminal double bonds at δH 4.68/4.66 (Hpro‑Z-17) and δH 4.77/4.75 (Hpro‑E-17), a pair of methoxy groups at δH 3.76/3.72 (CH3O-18), and a set of proton resonances for a β-substituted furan ring. Analysis of the NMR data of 2/3 (Tables 1 and 4) indicated them to be structurally related to the coexisting diterpenoid croverin,12 the difference being the presence of a C-19 hemiacetal moiety in 2/ 3 replacing the C-19 lactone carbonyl (δC 166.5) of croverin. The hemiacetal is less stable and interconverts into a 3:1 mixture of compounds 2 and 3. Taking 2 as the example, the presence of the hemiacetal moiety was confirmed by HMBC correlations (Figure 2A) from H-19 to C-4, C-5, C-6, C-10, and C-20 and from OH-19 to C-19 and C-5. The relative configurations of 2 and 3 were established on the basis of their ROESY spectra (Figure 2B1 for 2 and B2 for 3). The ROESY correlation of H-6α/H-10 revealed that they were cofacial and arbitrarily assigned as α-oriented. The ROESY correlations of H-12/H2-1 and H-20/H-7β indicated that the protons within each correlation pair were spatially close, which allowed the assignment of H-12 and H-20 to αand β-orientations, respectively. The above-mentioned information indicated that 2 and 3 shared the same relative configurations with croverin at C-5, C-9, C-10, C-12, and C-20. Specifically, the strong ROESY correlations of OH-19/H-6β and H-19/H-1β of 2 indicated the relative configuration of C19 as depicted. Consequently, the ROESY correlation between H-19 and H-6β of 3 revealed that the orientation of H-19 in 3 was opposite that of 2. The structures of compounds 2 and 3 were thus established and named crotonolide B and isocrotonolide B, respectively. Compounds 4 and 5, named crotonolide C and isocrotonolide C, were also isolated as a 3:1 mixture and shared a molecular formula C23H26O8 as assigned by 13C NMR data and the HRESIMS molecular ion at m/z 883.3151 [2 M + Na]+ (calcd 883.3153). The NMR data (Tables 1 and 4) revealed the presence of an additional acetoxy group in 4/5 as compared

Figure 2. Selected HMBC (A) and ROESY (B) correlations of 2/3.

with 2/3. Its location was deduced from the HMBC correlations (Supporting Information, Figure S21) from H-6 (δH 5.68, 4; 5.64, 5) to the acetyl carbonyl (δC 170.0, 4; 170.0, 5), respectively. The structures of 4 and 5 were further confirmed by 2D NMR spectra, and in particular the ROESY correlations (Supporting Information, Figure S22) of H-6β/ OH-19 in 4 and H-6β/H-19 in 5 revealed that the C-6 acetoxy group was α-oriented. Compounds 6 and 7, a 3:1 mixture as measured by 1H NMR peak integration, shared a common molecular formula of C21H26O6 as assigned by 13C NMR data and the HRESIMS molecular ion at m/z 771.3352 [2 M + Na]+ (calcd 771.3356) with nine indices of hydrogen deficiency. The NMR data (Tables 2 and 4) of 6/7 showed a resemblance to those of 2/3. Major differences including the presence of an additional methyl group and the absence of the exocyclic methylene group of 2/3 indicated that the Δ8,17 double bond of 2/3 was reduced in 6/7. This assignment was corroborated by HMBC correlations (Supporting Information, Figure S29). The ROESY correlation (Supporting Information, Figure S30) between H3-17 and H-20 indicated a β-oriented C-17 methyl group. Compounds 6 and 7 were named crotonolide D and isocrotonolide D, respectively. The molecular formula of crotonolide E (8) was determined to be C20H26O4 by 13C NMR data and the HRESIMS ion at m/ z 683.3568 [2 M + Na]+ (calcd 683.3560) with eight indices of hydrogen deficiency. The 1H NMR data (Table 2) of 8 displayed three methyl resonances at δH 0.82 (s), 1.10 (s), and 0.92 (d, J = 6.7 Hz), along with the diagnostic resonances at δH 6.43, 7.43, and 7.46 for a β-substituted furan ring. All 20 carbons in the molecule were observed in the 13C NMR spectrum (Table 4), including three methyls (δC 6.9, 14.5, and 15.1), four olefinic carbons (δC 108.6, 125.9, 139.5, and 144.0), one ester carbonyl (δC 172.1), and one keto carbonyl (δC 211.4). The furan ring and two carbonyls accounted for five indices of hydrogen deficiency, and the remaining three implied 1016

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the presence of three additional rings in 8. The aforementioned data and analysis of the NMR spectra suggested 8 to be a clerodane-type diterpenoid, with a structure closely related to furocrotinsulolide A13 except for the A-ring. In the HMBC spectrum (Supporting Information, Figure S37), the multiple correlations of H3-18/C-3, C-4, and C-5; H319/C-4, C-5, C-6, and C-10; and H-4/C-2, C-3, C-5, and C-10 permitted the assignment of the carbonyl group at C-3 and also verified the planar structure. The relative configuration of 8 was assigned by a ROESY experiment (Supporting Information, Figure S38), in which the correlations of H3-18/H3-19, H3-19/ H3-20, and H3-20/H-12 indicated that they were cofacial and randomly assigned α-orientations. Consequently, the ROESY correlations of H-4/H-6β, H-4/H-10, and H-8/H-6β showed them to be β-directed. Compound 9, named crotonolide F, had the molecular formula C20H26O4 as determined by 13C NMR data and the HRESIMS ion at m/z 683.3566 [2 M + Na]+ (calcd 683.3560). Its NMR data (Tables 2 and 4) showed many similarities to the data of furocrotinsulolide A.13 Its 13C NMR spectrum showed the presence of two additional olefinic carbon resonances at δC 159.6 and 110.0 in 9 instead of the C-18 methyl group at δC 21.8 and the oxygenated quaternary C-4 at δC 76.3 of furocrotinsulolide A, indicating the presence of a double bond between C-4 and C-18 in 9. The relative configuration of the remaining stereogenic centers in 9 was assigned to be identical to those of furocrotinsulolide A by the ROESY spectrum (Supporting Information, Figure S46), in which the correlations of H-3/H2-2, H3-19/H3-20, and H3-20/H-12 suggested that OH-3, H-12, CH3-19, and CH3-20 were αoriented, and the correlation of H-8/H-10 indicated their βorientations. Compound 10, named crotonolide G, was assigned the molecular formula C20H32O based on 13C NMR data and the HRESIMS ion at m/z 289.2536 [M + H]+ (calcd 289.2531). The NMR data (Tables 2 and 4) revealed that its structure was similar to that of 3-{2-[(4aR,5R,6R,8aR)-3,4,4a,5,6,7,8,8aoctahydro-l,5,6,8a-tetramethyl-5-naphthyl]ethy1}furan, except for the presence of a β-substituted 2,5-dihydrofuran ring in 10 replacing the furan ring at C-12 of the known analogue.14,15 This conclusion was confirmed by the chemical shifts of C-13 (δC 144.9), C-14 (δC 126.0), C-15 (δC 58.5), and C-16 (δC 60.8) and, in particular, by the HMBC correlations of H-14/C12, C-13, C-15, and C-16; H-15/C-14 and C-16; and H-16/C12, C-13, and C-14 (Supporting Information, Figure S53). In the ROESY spectrum (Supporting Information, Figure S54), the correlations of H3-19/H3-20 and H3-19/H-6α indicated that the two methyls were cofacial and arbitrarily assigned as αoriented. The ROESY correlation of H-6β/H-8 showed that CH3-17 was α-oriented. The molecular formula of crotonolide H (11) was defined as C20H32O4 based on 13C NMR data and the HRESIMS ion at m/z 381.2291 [M + HCOO]− (calcd 381.2277). The NMR data (Tables 3 and 4) of 11 closely resembled the data of 3α,4β-dihydroxy-15,16-epoxy-12-oxo-cleroda-13(16),14diene,16 except for the presence of the C-12 oxygenated methine in 11 instead of the C-12 carbonyl group (δC 195.4) in the latter. This difference indicated that 11 possessed a hydroxy group at C-12, which was confirmed by HMBC correlations (Figure 3A) from H-12 to C-9, C-11, C-13, C-14, and C-16. Because OH-12 was located in a conformationally mobile side chain, the relative configuration of C-12 could not be assigned by the ROESY spectrum. The absolute configuration of 11 was

Figure 3. Selected HMBC (A) correlations and X-ray structure (B) of 11.

defined by X-ray crystallographic analysis as 3R, 4R, 5R, 8R, 9S, 10R, and 12S (Figure 3B). Compound 12 possessed a molecular formula of C20H32O3 based on its 13C NMR data and the HREIMS ion at m/z 320.2350 [M]+ (calcd 320.2352), which showed one fewer oxygen atom than 11, suggesting it was a deoxygenated product of 11. Comparison of the NMR data (Tables 3 and 4) of 12 and 11 showed that a methylene group (δC 18.5, C-12) in 12 replaced the oxygenated methine group (δC 63.2, C-12) in 11. This was confirmed by the HMBC spectrum (Supporting Information, Figure S69). Thus, the structure of 12 was assigned and named 12-deoxycrotonolide H. Crotonolide I (13) was assigned a molecular formula of C20H34O3 based on its 13C NMR data and the HRESIMS ion at m/z 667.4937 [2 M + Na]+ (calcd 667.4914). The 1H NMR data of 13 (Table 3) were similar to those of carterochaetol,17 except for a deshielded proton resonance at δH 3.69 (H-7), which showed HMBC correlations (Figure 4A) to C-5, C-6, C-

Figure 4. Selected HMBC (A) and ROESY (B) correlations of 13.

8, C-9, and C-17, indicating that 13 was the 7-hydroxy derivative of carterochaetol. This conclusion was supported by the deshielded carbon resonance at δC 78.8 (C-7) in its 13C NMR spectrum. The ROESY correlation (Figure 4B) of H-16/ H2-15 indicated the E-geometry for the C-13 double bond. The ROESY correlations of H-5/H-7, H-7/H-9, and H-9/H-12 revealed that they were cofacial and arbitrarily assigned an αorientation. Consequently, the ROESY correlations of H3-17/ H3-20 and H3-19/H3-20 indicated that they were β-oriented. The molecular formula of crotonolide J (14) was established as C19H30O3 by 13C NMR data and the HREIMS ion at m/z 306.2194 [M]+ (calcd 306.2195). Its NMR data (Tables 3 and 4) were similar to those of 3β-hydroxy-15-nor-14-oxo-8(17),12labdadien-14-al,18 and the major differences were the chemical 1017

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Shanghai Institute of Materia Medica, Chinese Academy of Sciences (accession number Crola-2010-HN-1Y). Extraction and Isolation. The air-dried, powdered twigs of C. laui (5 kg) were extracted with 95% EtOH, and the resulting residue (400 g) was suspended in H2O and partitioned with EtOAc. The EtOAcsoluble fraction (175 g) was subjected to CC (D101-macroporous absorption resin, eluted with 30, 50, 80, and 90% EtOH in H2O) to obtain fractions 1−4. Fractions 2 (57 g) and 3 (85 g) were fractionated using the same procedure (MCI gel CC, eluted with 50 to 100% MeOH in H2O) to obtain subfractions 2A−2C and 3A−3D, respectively. Fraction 2A (4.5 g) was subjected to silica gel CC and eluted with petroleum ether/acetone (15:1 to 1:3) to give subfractions 2A1 to 2A5. Similarly, fraction 2B (9.4 g) gave 2B1 to 2B6. Fraction 2A2 was separated on an RP-18 silica gel column (MeOH/H2O, 40 to 100%) to afford compounds 1 (3 mg, 0.000 06%) and 14 (2 mg, 0.000 04%). Fraction 2A4 was purified by semipreparative HPLC with a mobile phase of 40% MeCN in H2O to afford compound 11 (7 mg, 0.000 14%). Fraction 2B2 was chromatographed over an RP-18 silica gel column (MeOH/H2O, 50 to 100%) to obtain one major component, which was crystallized from MeOH to yield compound 13 (15 mg, 0.0003%). Similarly, fraction 2B3 yielded two major components, each of which was purified by semipreparative HPLC (60% MeCN/H2O), affording 7β,12α-dihydroxy-13-epi-manoyl oxide (9 mg, 0.000 18%) and (11E)-14,15-bisnor-8α-hydroxy-11-labden-13one (4 mg, 0.000 08%), respectively. Fraction 2B5 was purified by semipreparative HPLC (45% MeCN in H2O) to give labda-8(17),13Edien-3-one (1 mg, 0.000 02%). Fraction 2C (5.8 g) was passed over a column of silica gel (petroleum ether/EtOAc, 80:1 to 1:5) to obtain a major fraction, which was purified by semipreparative HPLC (60% MeCN/H2O) to yield compound 12 (2 mg, 0.000 24%). Fraction 3B (25 g) was separated by silica gel CC eluted with petroleum ether/ EtOAc (50:1 to 1:5) to yield virescenol B (712.0 mg, 0.014 24%) and fractions 3B1−3B5. Fraction 3B2 (2.9 g) was fractionated over an RP18 silica gel column eluted with MeOH/H2O (50 to 100%) to obtain the six subfractions 3B2a to 3B2f. Fraction 3B2a was subjected to silica gel CC and eluted with petroleum ether/acetone (20:1 to 2:1) to afford three major components, each of which was further purified on a column of Sephadex LH-20 gel eluted with EtOH to yield dihydrocroverin (94.0 mg, 0.001 88%), compound 8 (53.0 mg, 0.001 06%), and croverin (9 mg, 0.000 18%), respectively. Fractions 3B2c and 3B2d were purified by semipreparative HPLC (75% MeOH/ H2O) to afford compounds 2/3 (7 mg, 0.000 14%) and 6/7 (2 mg, 0.000 04%), respectively. Fraction 3B3 (3.7 g) was separated on RP-18 silica gel (MeOH/H2O, 30 to 100%) to afford four fractions, 3B3a to 3B3d. Fraction 3B3a was subjected to silica gel CC (CHCl3/MeOH, 500:1 to 100:1) to give three subfractions, 3B3a1−3B3a3. Fraction 3B3a1 (159.0 mg) was passed over a column of Sephadex LH-20 gel eluted with EtOH to afford compounds 4/5 (6 mg, 0.000 12%), 9 (17 mg, 0.000 34%), and gelomulide G (13 mg, 0.000 26%). Fractions 3B3a2 and 3B3a3 were purified by the same procedure (silica gel column, CHCl3/MeOH, 200:1 to 50:1) to give compound 10 (52 mg, 0.001 04%) and ent-3β,19-dihydroxylabda-12,14-diene (367 mg, 0.007 34%), respectively. Fraction 3B4 (98 mg) was purified on a column of RP-18 silica gel (MeOH/H2O, 50 to 100%) to afford furocrotinsulolide A (4 mg, 0.000 08%). Crotonolide A (1): colorless gum; [α]25D −101 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.78) nm; IR (KBr) νmax 2962, 2925, 2854, 1757, 1738, 1668, 1342, 1228, 1259, 1080, 1018, 802 cm−1; 1H NMR (CDCl3) see Table 1; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 731.2102 [2 M + Na]+ (calcd for C40H36O12Na, 731.2104). Crotonolide B (2) and isocrotonolide B (3): pale gum; [α]25D −58 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.25) nm; IR (KBr) νmax 3423, 2958, 2925, 2860, 1709, 1643, 1436, 1259, 1146, 1090, 1026, 874, 796 cm−1; 1H NMR (CDCl3) see Table 1; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 767.3046 [2 M + Na]+ (calcd for C42H48O12Na, 767.3043). Crotonolide C (4) and isocrotonolide C (5): pale gum; [α]25D −232 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 220 (3.81) nm; IR (KBr) νmax 3419, 2952, 2924, 1734, 1647, 1436, 1373, 1267, 1238, 1099, 1022, 953, 876 cm−1; 1H NMR (CDCl3) see Table 1; 13C NMR

shifts of H2-18 (δH 3.44, 3.72) and C-18 (δC 71.5), suggesting that a hydroxy group was attached to C-18. This was verified by the HMBC spectrum (Supporting Information, Figure S84). In the ROESY spectrum (Supporting Information, Figure S85), the correlations of H-3/H2-1, H-5/H-9, and H-5/H2-18 indicated that they were cofacial, and the OH-3, H-5, H-9, and H2-18 were assigned as α-oriented randomly. Thus, the ROESY correlations of H3-20/H3-19 and H3-20/H2-11 indicated that they were β-oriented. Additionally, the ROESY correlation of H-12/H-14 revealed that the C-12 double bond adopted an E-geometry. Nine known diterpenoids, dihydrocroverin,12 croverin,12 furocrotinsulolide A,13 virescenol B,19 gelomulide G,20 ent3β,19-dihydroxylabda-12,14-diene,21 7β,12α-dihydroxy-13-epimanoyl oxide,22 (11E)-14,15-bisnor-8α-hydroxy-11-labden-13one,23 and labda-8(17),13E-diene-3β,15-diol,24 were also isolated. Their structures were identified by NMR and MS analyses as well as by comparison with reported data. Compounds 1−14 were evaluated for cytotoxic activity against the HL-60 (human premyelocytic leukemia) and P-388 (murine leukemia) cell lines. Only crotonolide A (1) showed moderate activity against the HL-60 and P-388 cell lines, with IC50 values of 9.42 and 7.45 μM, respectively, while the other compounds were inactive (IC50 ≥ 10 μM). Additionally, the antimicrobial activities of these compounds were tested against a panel of microbes including fungi and Gram-positive and Gram-negative bacteria. The results showed that crotonolide G (10) exhibited significant antibacterial activity with an MIC value of 43.4 μM against four strains of Gram-positive bacteria, including Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, and Bacillus subtilis CMCC 63501, and the other compounds were inactive (MIC > 150 μM) against all tested microbes.

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General Experimental Procedures. Melting points were measured on an SGM X-4 apparatus (Shanghai Precision & Scientific Instrument Co., Ltd.). Optical rotations were recorded on a PerkinElmer 341 polarimeter (Wellesley, MA, USA). UV spectra were obtained using a Shimadzu UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were obtained using a PerkinElmer 577 spectrometer with KBr disks (Wellesley, MA, USA). NMR spectra were acquired using Bruker AM-400 or AM500 NMR spectrometers (Bruker Biospin Rheinstetten, Germany). EIMS (70 eV) and HREIMS were measured on a Finnigan MAT-95 mass spectrometer in m/z (rel %) (Thermo Fisher Scientific, ZuidHolland, The Netherlands), and ESIMS and HRESIMS were obtained on a Bruker Daltonics Esquire 3000 Plus (Bruker Daltonics, Bremen, Germany) and a Waters-Micromass Q-TOF Ultima Global mass spectrometer (Milford, MA, USA), respectively. X-ray crystallographic analysis was performed on a Bruker APEX-II CCD detector (Bruker Biospin Rheinstetten, Germany) employing graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Semipreparative HPLC chromatography was performed on a Waters 1525 binary pump system with a Waters 2489 UV detector (210 nm) and a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, Japan). Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd.), C18 reversed-phase (RP18) silica gel (150−200 mesh, Merck), CHP20P MCI gel (75−150 μm, Mitsubishi Chemical Industries, Ltd.), and Sephadex LH-20 gel (Amersham Biosciences) were used for CC. All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co. Ltd.), and all solvents used for HPLC were of HPLC grade. Plant Material. Twigs of Croton laui were collected from Hainan Province, People’s Republic of China, and were authenticated by Prof. S.-M. Huang, Department of Biology, Hainan University, People’s Republic of China. A voucher specimen has been deposited in 1018

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(CDCl3) see Table 4; HRESIMS m/z 883.3151 [2 M + Na]+ (calcd for C46H52O16Na, 883.3153). Crotonolide D (6) and isocrotonolide D (7): pale gum; [α]25D −109 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 241 (4.15) nm; IR (KBr) νmax 3477, 3145, 2952, 2924, 2870, 1712, 1689, 1639,1502, 1460, 1437, 1255, 1022, 937, 756, 602 cm−1; 1H NMR (CDCl3) see Table 2; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 771.3352 [2 M + Na]+ (calcd for C42H52O12Na, 771.3356). Crotonolide E (8): white powder; [α]25D +95 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 207 (3.90) nm; IR (KBr) νmax 2966, 1709, 1510, 1379, 1225, 1144, 1024, 874, 789 cm−1; 1H NMR (CDCl3) see Table 2; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 683.3568 [2 M + Na]+ (calcd for C40H52O8Na, 683.3560). Crotonolide F (9): pale gum; [α]25D +56 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (3.93) nm; IR (KBr) νmax 3419, 2945, 2875, 1726, 1633, 1382, 1227, 1138, 1026, 875, 787 cm−1; 1H NMR (CDCl3) see Table 2; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 683.3566 [2 M + Na]+ (calcd for C40H52O8Na, 683.3560). Crotonolide G (10): pale gum; [α]25D −51 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 226 (2.78) nm; IR (KBr) νmax 3323, 2954, 2937, 2875, 1664, 1450, 1437, 1383, 999, 796 cm−1; 1H NMR (CDCl3) see Table 2; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 289.2536 [M + H]+ (calcd for C20H33O, 289.2531). Crotonolide H (11): colorless crystals (MeCN/H2O, 2:3); mp 87− 88 °C; [α]25D −12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.05) nm; IR (KBr) νmax 3425, 2951, 2927, 2868, 1500, 1460, 1387, 1322, 1161, 1093, 1051, 1024, 978, 874, 768 cm−1; 1H NMR (CDCl3) see Table 3; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 381.2291 [M + HCO2]− (calcd for C21H33O6, 381.2277). 12-Deoxycrotonolide H (12): pale gum; [α]25D −37 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 213 (4.25) nm; IR (KBr) νmax 3444, 2937, 2870, 1700, 1456, 1385, 1101, 1051, 1020, 978, 874, 777 cm−1; 1H NMR (CDCl3) see Table 3; 13C NMR (CDCl3) see Table 4; HREIMS m/z 320.2350 [M]+ (calcd for C20H32O3, 320.2352). Crotonolide I (13): white powder; [α]25D −24 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (4.10) nm; IR (KBr) νmax 3421, 3309, 2999, 2958, 2919, 2866, 1645, 1456, 1441, 1389, 1041, 1012, 978, 721 cm−1; 1 H NMR (CDCl3) see Table 3; 13C NMR (CDCl3) see Table 4; HRESIMS m/z 667.4937 [2 M + Na]+ (calcd for C40H68O6Na, 667.4914). Crotonolide J (14): pale gum; [α]25D +5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 232 (4.04) nm; IR (KBr) νmax 3400, 2929, 2854, 1684, 1643, 1444, 1385, 1261, 1086, 1034, 800 cm−1; 1H NMR (CDCl3) see Table 3; 13C NMR (CDCl3) see Table 4; HREIMS m/z 306.2194 [M]+ (calcd for C19H30O3, 306.2195). X-ray Crystallographic Analysis. Colorless crystals of 11 were obtained by crystallization from a MeCN/H2O (2:3) solvent. The crystal data were collected on a Bruker APEX-II CCD detector employing graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). The structure was solved by the direct method using the SHELXS97 program and refined with full-matrix least-squares difference Fourier techniques. Crystallographic data for 11 have been deposited at the Cambridge Crystallographic Data Center with the deposition number of CCDC 988339. A copy of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [tel: (+44) 1223-336-408; fax: (+44) 1223-336-033; e-mail: [email protected]]. X-ray crystal data of 11: C20H32O4, MW = 336.23, orthorhombic, space group P212121, a = 10.4173(2) Å, b = 12.2196(2) Å, c = 29.6743(6) Å, α = 90°, β = 90°, γ = 90°, V = 3777.40(12) Å3, Z = 8, Dcalc =1.215 Mg/m3, μ = (Cu Kα) 0.675 mm−1, F(000) = 1512; crystal size 0.320 × 0.260 × 0.100 mm3; independent reflections 6755 [R(int) = 0.0415]; final indices R1 = 0.0395, wR2 = 0.1065 [I > 2σ(I)]. Cytotoxicity Assay. The cytotoxicities of compounds 1−14 against the HL-60 (human premyelocytic leukemia) and P-388 (murine leukemia) cells were tested by using the MTT method25,26 with adriamycin as the positive control (Supporting Information, S1.1). Antimicrobial Activity Assay. The in vitro antibacterial activities against Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis

ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Shigella flexneri ATCC 12022, and Pseudomonas aeruginosa ATCC 14502 were conducted using the microdilution method27 with ofloxacin as the positive control (Supporting Information, S1.2). The in vitro antifungal activities against Candida albicans ATCC 1600, Saccharomyces sake ATCC 26421, Microsporum gypseum ATCC 14683, and Trichophyton rubrum ATCC 10218 were assessed by using the agar dilution method28 with amphotericin B as the positive control (Supporting Information, S1.2).

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ASSOCIATED CONTENT

S Supporting Information *

Bioassay, X-ray crystallographic data of compound 11, and 1D and 2D NMR, ESIMS/EIMS, HRESIMS/HREIMS, and IR spectra of compounds 1−14. 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 This work was financially supported by a grant (U1302222) from the National Natural Science Foundation of the People’s Republic of China. We thank Prof. S.-M. Huang of Hainan University for the identification of the plant material.

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REFERENCES

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