Cytotoxic Clerodane Diterpenoids from the Leaves and Twigs of

Sep 13, 2013 - The minor component 2 had the same gross structure as 1. However, the splitting pattern ..... epicaseabalansin C. As has been reported,...
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Cytotoxic Clerodane Diterpenoids from the Leaves and Twigs of Casearia balansae Bo Wang,† Xiao-Ling Wang,‡ Shu-Qi Wang,† Tao Shen,† Yong-Qing Liu,§ Huiqing Yuan,§ Hong-Xiang Lou,† and Xiao-Ning Wang*,† †

Department of Natural Product Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, Jinan 250012, People’s Republic of China ‡ The Second Hospital of Shandong University, 247 Bei-Yuan Street, Jinan 250033, People’s Republic of China § Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, 44 West Wenhua Road, Jinan 250012, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new clerodane diterpenoids (1−10), caseabalansins A−G, 18-epicaseabalansin A, 2-epicaseabalansin B, and 2-epicaseabalansin C, one new triterpenoid, balansinone (11), and seven known diterpenoids (12−18) were obtained from the leaves and twigs of Casearia balansae. Compounds 1 and 2 are the first examples of clerodane diterpenoids with an oxygen bridge between C-2 and C-19, and compounds 5−7 are three new diterpenoid artifacts presumably formed during the extraction process. The structures of the new compounds were established on the basis of extensive spectroscopic data, and that of 11 was verified by single-crystal X-ray crystallographic analysis. Compound 15 showed cytotoxic activity against the tumor cell lines PC3, DU145, SKOV3, and A549 with IC50 values of 4.5, 4.3, 5.1, and 5.7 μM, respectively. Compounds 1a, 2a, and 4 showed selective activity against PC3 tumor cells.

T

he genus Casearia contains about 180 species distributed throughout tropical Africa, Asia, northwest Australia, and South America. Casearia species are rich sources of clerodanetype1−4 and some ent-kaurane-type glycosides5,6 and dolabellane-type diterpenes.7 Plants of this genus are known to contain compounds reported to have cytotoxic,8−10 immunomodulatory,11 trypanocidal,12,13 antibothropic, 14 anti-inflammatory,15,16 antimicrobial,17,18 and antioxidant activities.19−21 In the course of our ongoing efforts to discover potential anticancer agents from Chinese herbs,22 extracts of twigs and leaves of Casearia balansae Gagnep. (Flacourtiaceae) showed moderate cytotoxicity against PC3M human metastatic prostate adenocarcinoma, NCI-H460 human non-small cell lung cancer, SF-268 human central nervous system cancer, and MCF-7 human breast cancer cells. The extracts yielded 10 new clerodane diterpenes (1−10), a new 9,10-seco-9,19-cyclolanostane triterpenoid (11), and seven known diterpenoids (12−18; see Supporting Information). Compounds 1−7 and 9−17 were evaluated for their cytotoxic activity against PC3, DU145 human prostate cancer, SKOV3 human ovarian carcinoma, A549 human lung carcinoma, and WI-38 normal human fibroblast cells. Herein we report the isolation, structure elucidation, and cytotoxicity evaluation of these compounds.

and 2 by chromatographic methods (normal- and reversedphase TLC and HPLC), the structures of these two compounds were elucidated as a mixture. The HRESIMS displayed a pseudomolecular ion [M + Na]+ peak at m/z 341.2084 that suggested the molecular formula of C20H30O3 for one or both of them (calcd for C20H30O3Na, 341.2093). The NMR spectra of the major compound 1 were similar to that of rel(2R,4S,5R,8S,9S,10R,18S,19R)-2,18,19-triacetoxycleroda-13-

■

RESULTS AND DISCUSSION Compounds 1 and 2 were obtained as an inseparable mixture of two isomers in a ratio of approximately 1.3:1, as determined by 1 H NMR spectroscopy. Owing to difficulties in separating 1 © 2013 American Chemical Society and American Society of Pharmacognosy

Received: March 13, 2013 Published: September 13, 2013 1573

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Figure 1. Key NOESY (H↔H) correlations of 1 (left) and 2 (right).

Table 1. 1H NMR Spectroscopic Data for 1−5a 1b

position 1α 1β 2 3α 3β 4 6α 6β 7α 7β 8 10 11 12 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 2″ 2‴ 6-OMe

2b

1.79, 1.26, 3.89, 1.40, 2.65, 2.54, 1.48, 1.42, 1.95, 1.35, 1.82, 1.85, 1.29, 1.46, 2.19, 6.39, 5.26, 5.05, 4.99,

m m t (4.8) m dd (13.8, 4.8) dd (9.8, 3.4) m m m m m m m m m dd (17.6, 10.9) d (17.6) d (10.9) br s

1.80, 1.21, 3.92, 1.82, 1.87, 2.47, 2.32, 1.38, 1.95, 1.34, 1.82, 1.95, 1.29, 1.46, 2.19, 6.39, 5.26, 5.05, 4.99,

m dd (13.1, 6.5) t (4.7) m m d (9.4) dt (13.6, 3.5) m m m m m m m m dd (17.6, 10.9) d (17.6) d (10.9) s

1.02, 5.59, 4.65, 1.13,

d (7.9) d (3.4) s s

1.04, 5.19, 4.86, 1.11,

d (7.9) s s s

3c

4c

1.26, 2.13, 4.31, 2.01, 1.38, 2.30, 1.58, 1.74, 1.41, 1.22, 1.55, 1.81, 1.21, 1.44, 2.04, 6.43, 5.23, 5.03, 5.03, 4.92, 0.86, 6.48, 6.13, 0.94,

m ddd (13.7, 8.7, 1.8) m m m ddd (14.9, 7.2, 3.6) m dt (14.0, 2.8) m m m d (13.7) m m m dd (17.6, 10.9) d (17.6) d (10.8) s s d (6.8) d (7.2) s s

1.80, 1.60, 4.14, 1.68, 1.43, 1.84, 1.60, 1.72, 1.42, 1.32, 1.53, 2.32, 1.25, 1.47, 2.11, 6.43, 5.25, 5.05, 5.04, 4.93, 0.86, 6.43, 6.15, 0.94,

m m q (7.7) m m m m m m m m d (13.1) m m t (8.5) dd (17.6, 9.9) d (17.6) d (9.9) s s d (6.7) d (7.1) s s

2.25, 1.62, 0.94, 1.90,

td (7.3, 1.4) sextet (7.3) t (7.3) s

2.25, 1.62, 0.93, 1.93,

t (7.4) sextet (7.4) t (7.4) s

5c 1.66, 2.18, 5.59, 5.95,

td (13.4, 9.4) ddd (13.4, 7.3, 2.7) dd (8.2, 8.2) br s

3.49, dd (12.1, 3.8) 1.87, 1.50, 1.76, 2.36, 1.25, 1.45, 2.06, 6.41, 5.19, 5.02, 5.03, 4.92, 0.93, 5.46, 6.34, 0.94, 3.76, 3.56, 1.18,

m q (12.9) m dd (13.5, 2.7) d (7.1) m m dd (17.3, 10.7) d (17.3) d (10.7) s s d (6.6) s s s dq (9.5, 7.3) dq (9.5, 7.3) t (7.3)

1.85, s 2.09, s 3.33, s

Recorded at 600 MHz, δ in ppm, and J in Hz. Assignments were made on the basis of HSQC and 1H−1H COSY data. bRecorded in methanol-d4. Recorded in CDCl3.

a c

and H-4 (3.4 Hz) in 1 was in agreement with a β-orientation of OH-18, in which case the dihedral angle of H-18 relative to H-4 was 29.9° in the MM2 energy minimized model and the vicinal coupling constant would be relatively small (Figure 1). Therefore, the structure of 1 was determined to be rel(2S,4S,5R,8S,9S,10R,18R,19S)-2,19:18,19-diepoxycleroda-13(16),14-dien-18-ol, named caseabalansin A. The minor component 2 had the same gross structure as 1. However, the splitting pattern of H-18 (δ 5.19, s) and chemical shift of C-18 (δ 108.1) indicated that 18-OH was α-oriented, in

(16),14-dien-18,19-oxolane,23 except for the absence of the acetoxy groups at C-2, C-18, and C-19 and the presence of an oxygen bridge between C-2 and C-19, which was supported by the HMBC correlations of H-2/C-10 and C-19 and H-19/C-2, C-5, and C-6 (Supporting Information). The NOESY correlations (Figure 1) of H-10/H2-11 and H-19, H-7β/H317, and H-19/H-6β disclosed that these protons were βoriented and the A/B rings cis-fused. The NOESY correlations of H-4/H-7α and H3-20 indicated that ring A adopted a boat conformation. The observed coupling constant between H-18 1574

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Table 2. 1H NMR Spectroscopic Data for 6−10a 6b

position 1α 1β 2 3 6 7α 7β 8 10 11 12 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 2″ 2‴ 3‴ 4‴ 5‴ 6-OMe

6ab

1.88, 1.98, 5.47, 6.01, 3.26, 1.84, 1.50, 1.73, 2.25, 1.28, 1.48, 2.09,

m m m d (3.7) dd (12.4, 3.6) dt (13.4, 3.6) q (13.0) m dd (13.5, 3.7) m m m

1.88, 2.11, 5.51, 6.88, 3.72,

m m m d (5.1) dd (13.7, 6.9)

6.42, 5.20, 5.02, 5.04, 4.94, 0.94, 5.48, 6.42, 0.93, 3.78, 3.60, 1.20,

dd (17.6, 10.9) d (17.6) d (10.9) s s d (6.6) s s s dq (9.8, 7.1) dq (9.8, 7.1) t (7.1)

6.34, dd (17.6, 10.9) 5.22, d (17.6) 5.04, d (10.9) 5.03, s 4.98, s 0.94, d (6.8) 10.50, s 9.40, s 0.97, s

2.43, dd (13.1, 3.9)

1.95, m

1.88, s 2.12, s

2.09, s

3.29, s

3.27, s

7b

8c

1.90, m 5.45, 6.06, 3.77, 1.70, 1.78,

br s d (3.9) m m m

2.33, 1.26, 1.54, 2.07,

dd (12.4, 4.6) m m m

6.44, 5.15, 5.02, 5.04, 4.96, 0.92, 5.58, 6.48, 0.90, 3.76, 3.61, 1.18,

dd (17.1, 10.8) d (17.1) d (10.8) s s d (6.7) t (1.5) s s dq (10.0, 7.1) dq (10.0, 7.1) t (7.1)

1.88, 2.47, 1.70, 1.54, 0.96, 1.16,

s m m m t (7.4) d (6.9)

1.86, 1.98, 5.40, 5.91, 3.34, 1.80, 1.40, 1.74, 2.37, 1.47,

d (14.5) m br s d (4.1) m dt (12.6, 3.1) q (12.6) m dd (14.5, 3.2) m

2.49, 1.98, 6.38, 5.20, 4.99, 5.03, 4.96, 0.92, 5.38, 5.49, 0.92, 3.41,

m m dd (17.6, 10.8) d (17.6) d (10.8) s s d (7.7) br s s s s

10b

2.50, d (12.7) 2.52, d (6.7)

2.50, d (12.6) 2.52, d (6.6)

6.14, 3.48, 1.96, 1.55, 1.82, 2.81, 1.24, 1.49, 2.07,

d (1.2) dd (12.8, 3.7) dt (12.8, 3.7) q (12.8) m dd (12.7, 6.7) m m m

6.12, 3.48, 1.96, 1.54, 1.83, 2.81, 1.23, 1.52, 2.09,

d (1.3) dd (12.7, 3.7) dt (12.7, 3.7) q (12.7) m dd (12.6, 6.6) m dt (12.0, 3.1) m

6.43, 5.20, 5.04, 5.05, 4.92, 0.99, 6.78, 6.53, 0.93,

dd (17.7, 10.9) d (17.7) d (10.9) s s d (6.8) d (1.5) s s

6.43, 5.20, 5.04, 5.05, 4.93, 0.99, 6.80, 6.53, 0.93,

dd (17.6, 11.2) d (17.6) d (11.2) s s d (6.8) d (1.6) s s

2.30, 1.65, 0.95, 1.88,

t (7.5) sextet (7.5) t (7.5) s

2.08, s

1.89, s 2.42, 1.66, 1.52, 0.95, 1.14, 3.27,

m m m t (7.4) d (6.9) s

3.32, s

3.32, s

Recorded at 600 MHz, δ in ppm, and J in Hz. Assignments were made on the basis of HSQC and H− H COSY data. Recorded in CDCl3. Recorded in methanol-d4.

a c

9b

1

1

b

ent-2β-hydroxy-3,4-dihydro-4α-isozuelanin on the basis of NOESY correlation (Supporting Information) and their identical coupling constants. Therefore, the structure of 3 was determined as rel-(2R,4S,5R,8S,9S,10R,18S,19R)-18-butanoyloxy-19-acetoxy-18,19-epoxycleroda-13(16),14-dien-2-ol, named caseabalansin B. Comparison of the NMR data of 4 with those of 3 indicated a different orientation of 2-OH and thus a different C-2 configuration. The differences in the chemical shift of some protons and carbons in ring A and lack of the NOESY correlation (Supporting Information) between H-2 and H-10 suggested that H-2 was α-oriented. Thus, 4 was the 2-epimer of 3 and was named 2-epicaseabalansin B. The HRESIMS of compound 5 did not give the pseudomolecular ion peak but revealed a fragment ion [M + H − 2(AcOH)]+ peak at m/z 357.2436 (calcd for C23H33O3, 357.2430), which suggested the molecular formula C27H40O7. The NMR data indicated that 5 was similar to casearlucin B,2 except for replacement of the acetoxy group at C-18 with an ethoxy group. The relative configuration of 5 was the same as that of casearlucin B on the basis of a NOESY experiment (Supporting Information) and their similar coupling constants and chemical shift. Thus, 5 was established as rel-

which case the dihedral angle of H-18 to H-4 was 87.9° (Figure 1), and a value close to zero would be expected and was found for JH‑18/H‑4.24 Moreover, the NOE correlation between H-18 (δ 5.19) and H-3β (δ 1.87) (Figure 1) also supported the above conclusion. Therefore, 2 was the 18-epimer of 1 and was named 18-epicaseabalansin A. Although in some cases natural products were obtained as inseparable isomeric mixtures,25 it was of interest to stabilize them by derivatization and characterize them as pure compounds. This mixture when acetylated with Ac2O−pyridine afforded the 18-acetate 1a and 2a, which were separated by HPLC in a ratio of 1:3, and their structures were elucidated by HRMS and NMR experiments (Supporting Information). It was interesting that although 2 was minor in the natural epimeric mixture, the 18-acetate (2a) was the major product owing to an anomeric-type effect that favored the formation of the α-anomer.26 Compound 3 was obtained as a colorless oil, and the molecular formula was suggested to be C26H 40 O6 by HRESIMS. The NMR data (Tables 1 and 3) of 3 were similar to those of ent-2β-hydroxy-3,4-dihydro-4α-isozuelanin,27 except that a butyryl group instead of the acetyl group was present at C-18. The relative configuration of 3 was the same as that of 1575

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Table 3. 13C NMR Spectroscopic Data for 1−10a position

1b

2b

3c

4c

5c

6c

6ac

7c

8b

9c

10c

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

29.5 64.2 24.9 38.0 43.3 24.2 24.9 34.6 36.8 38.0 35.9 23.7 147.4 138.9 112.0 114.4 13.3 98.1 106.5 18.9

29.3 65.2 32.7 35.1 41.0 24.5 25.0 34.6 36.8 41.2 37.9 23.7 147.5 138.9 112.0 114.4 13.1 108.1 107.5 18.8

30.1 67.4 30.4 46.3 50.2 23.9 28.0 37.9 37.1 35.7 33.3 27.3 145.4 140.4 112.3 115.4 15.6 98.2 100.2 26.7 172.2 36.4 18.2 13.7 169.8 21.5

28.7 67.1 33.9 48.5 50.0 23.8 28.0 36.7 37.8 33.0 31.4 27.4 145.5 140.3 112.6 115.3 15.7 98.1 100.1 26.7 172.5 36.3 18.2 13.7 170.1 21.5

26.8 71.3 122.9 146.8 52.9 83.4 31.5 37.0 38.2 41.2 27.5 23.8 145.3 140.3 112.5 115.3 15.9 103.2 97.7 25.7 64.3 15.5

27.3 66.4 120.6 147.3 52.8 82.4 31.2 36.8 37.5 36.5 28.1 23.9 145.4 140.4 112.3 115.4 16.0 103.9 98.2 25.6 64.6 15.5

25.8 65.2 146.6 149.4 54.8 81.6 32.8 35.8 37.9 39.8 31.7 23.2 140.3 138.9 112.9 116.2 15.6 202.4 191.2 25.7

27.1 66.3 121.6 145.2 53.7 73.3 36.4 37.4 37.5 37.3 28.0 23.7 146.5 140.5 112.1 115.4 15.7 103.7 97.5 25.5 64.7 15.4

28.2 68.4 121.7 149.5 55.3 84.0 32.5 37.9 38.7 37.3 31.6 25.7 148.9 141.1 112.7 115.6 16.1 105.0 100.1 26.2 54.8

35.8 198.4 124.2 165.8 54.4 82.5 31.0 36.5 38.0 41.3 27.4 23.9 144.9 140.2 112.6 115.5 15.8 95.4 98.3 24.6 169.7 21.1

170.2 21.8 170.7 21.3

170.2 21.9 170.9 21.5

169.5 21.6

35.8 198.6 124.2 166.0 54.5 82.5 31.0 36.5 38.0 41.3 27.4 23.9 144.9 140.2 112.6 115.5 15.8 95.2 98.3 24.6 172.4 36.3 18.3 13.6 169.4 21.5

170.4 21.1

57.5

57.5

57.2

57.6

57.6

170.2 21.5 176.2 41.0 27.0 11.6 16.5

178.0 42.4 27.9 12.1 16.9 57.8

Recorded at 150 MHz, and δ in ppm. Assignments were made on the basis of HSQC and HMBC data. bRecorded in methanol-d4. cRecorded in CDCl3. a

Thus, 7 was determined as rel-(2S,5R,6R,8S,9S,10R,18S,19R)19-acetoxy-18-ethoxy-18,19-epoxy-2-(2ξ-methylbutanoyloxy)cleroda-3,13(16),14-trien-6-ol and has been accorded the trivial name caseabalansin D. The O-ethyl group at C-18 in 5−7 may be artifactual; hence they were analyzed by HPLC together with the acetone extract of the plant (Supporting Information). The absence of 5−7 in the acetone extract suggested that the ethoxy group was introduced during the extraction process involving EtOH. The molecular formula of compound 8 was deduced as C27H42O6 by HRESIMS. The NMR data (Tables 2 and 3) indicated that 8 was similar to caseamembrin B,31 except for the absence of the C-19 acetoxy moiety and the presence of a methoxy group at C-6. The relative configuration was the same as that of 7 by analysis of NOESY data and coupling constants. Thus, 8 was assigned as rel-(2S,5R,6R,8S,9S,10R,18S,19R)18,19-epoxy-6,18-dimethoxy-2-(2ξ- methylbutanoyloxy)cleroda-3,13(16),14-trien-19-ol, named caseabalansin E. HPLC analysis indicated the presence of 8 in the acetone extract of the plant (Supporting Information), excluding the possibility that 8 is an artifact. The molecular formula of compound 9 was assigned as C25H34O7 by HRESIMS ([M + NH4]+ at m/z 464.2647, calcd for C25H38O7N, 464.2648). The NMR data (Tables 2 and 3) indicated that 9 was similar to casearlucin B2 except for

(2R,5R,6R,8S,9S,10R,18S,19R)-2,19-diacetoxy-18-ethoxy-6-methoxycleroda-3,13(16),14-trien-18,19-oxolane, named caseabalansin C. Compound 6 was a C-2 stereoisomer of 5 by comparison of the HRESIMS and NMR data (Tables 2 and 3). As reported previously, with an α-oriented ester side chain, C-2 usually resonates near δ 71; otherwise it would resonate near δ 66.28 The chemical shift value of C-2 (δ 66.4) in 6 indicated a βorientation of the side chain. Thus, 6 was established as the 2epimer of 5 and was accorded the trivial name 2epicaseabalansin C. As has been reported,28,29 the casearintype clerodanes undergo opening of the diacetal ring in chloroform to form the 18,19-dialdehyde. The hydrolysis product 6a was observed in the 1H NMR spectrum when 6 was stored in CDCl3 for more than 24 h. Compound 6a has not previously been reported, and the 1H and 13C data (Tables 2 and 3) were assigned by 2D NMR spectra. In addition, the NMR data of 5 and 6 measured in methanol-d4 were also provided (Supporting Information). The molecular formula of compound 7 was deduced as C29H44O7 by HRESIMS. The NMR data (Tables 2 and 3) were similar to those of caseamembrin I,30 except for the presence of an ethoxy group at C-18 instead of the methoxy group. The configuration was evaluated by comparison with literature values and a NOESY experiment (Supporting Information).30 1576

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clohexa-2,5-dienone moiety. The HMBC correlations of H219/C-1, C-10, C-5, C-9, and C-8 and H2-6/C-4, C-5, C-7, C-8, and C-10 together with the spin system of CH2(6)−CH2(7)− CH(8) suggested a substituted cycloheptane B-ring.32 The structure of rings C and D and the side chain was established by 1 H−1H COSY and HMBC experiments (Figure 2). An

hydrolysis of the 2-acetoxy group and oxidation of C-2 to a carbonyl group. The HMBC correlations of H-1/C-2, C-5, and C-10 served to confirm this structure. The NOE correlations (Supporting Information) confirmed that it had the same stereostructure as casearlucin B. Thus, 9 was established as rel(5R,6R,8S,9S,10R,18S,19R)-18,19-diacetoxy-18,19-epoxy-6-methoxycleroda-3,13(16),14-trien-2-one, named caseabalansin F. Compound 10 was also a colorless oil. The molecular formula was deduced as C27H38O7 by HRESIMS ([M + NH4]+ at m/z 492.2969, calcd for C27H42O7N, 492.2961). The only difference between 10 and 9 was the C-18 butanoate moiety replacing the acetate group in 9. The structure of 10 was thus assigned as rel-(5R,6R,8S,9S,10R,18S,19R)-19-acetoxy-18-butanoyloxy-18,19-epoxy-6-methoxycleroda-3,13(16),14-trien-2one, named caseabalansin G. Compound 11 was obtained as colorless needles. A molecular formula of C31H46O3 was assigned on the basis of HRESIMS, showing a pseudomolecular ion [M + H]+ peak at m/z 467.3517 (calcd for C31H47O3, 467.3525), implying nine indices of hydrogen deficiency. The 1H NMR spectrum (Table 4) revealed five methyl singlets, two methyl doublets, a pair of Table 4. 1H and 13C NMR Spectroscopic Data for 11a position

δC

1 2 3

185.9 124.8 157.9

4 5 6

40.5 167.7 29.9

7

25.2

8

48.9

9 10 11 12

63.4 132.1 61.4 34.2

13 14 15

45.5 46.9 34.4

16

27.2

δH (mult, J in Hz) 6.15, d (9.8) 6.93, d (9.8)

2.82, dd (14.4, 7.7) 2.54, t (14.4) 1.80, m 1.40, m 2.22, dd (12.1, 2.7)

3.11, d (6.3) 1.99, dd (15.0, 6.4) 1.90, d (15.0)

1.30, 1.40, 1.80, 1.36,

m m m m

Figure 2. 1H−1H COSY (bold line) and key HMBC (H→C) correlations of 11.

δH (mult, J in Hz)

position

δC

17 18 19

40.5 14.9 30.6

20 21 22

42.6 11.2 70.6

23

34.3

24

37.9

1.16, m 1.39, m 2.48, m

25 26 27 28

148.8 110.1 20.0 17.2

4.74, s 1.62, s 0.80, s

29 30 31

23.4 23.8 16.9

1.51, 0.95, 3.04, 2.56, 1.65, 0.90, 3.56,

m s d (14.3) d (14.3) m d (6.6) d (10.3)

additional methyl was present at C-24, as verified by the HMBC correlations of CH3-31 to C-23, C-24, and C-25. A hydroxy group was attached to C-22 from the HMBC correlations of CH3-21 to C-22 and H-22 to C-21, C-23, and C-24, and a Δ25-double bond was evident from correlations of CH3-27 to C-24, C-25, and C-26. As eight of the nine indices of hydrogen deficiency were consumed by four rings, three double bonds, and one carbonyl, there must be an additional ring in 11. The two oxygenated carbons at δ 61.4 and 63.4 as well as the remaining one unassigned oxygen atom suggested that an epoxide is located between C-9 and C-11. Finally, a singlecrystal X-ray diffraction study confirmed the gross structure of 11 and allowed the determination of its relative configuration as depicted (Figure 3). Therefore, the structure of 11 was elucidated as rel-(8S,9S,11R,13R,14S,17R,20S,22R,24S)-22-hy-

1.34, s 1.25, s 1.03, d (7.0)

a1

H and 13C NMR data were recorded in methanol-d4 at 600 and 150 MHz, respectively. Assignments were made on the basis of HSQC and HMBC data.

protons of a cis-disubstituted double bond, a terminal double bond, and two oxymethines. The 13C NMR spectrum (Table 4) exhibited 31 carbon signals, which were divided into seven methyl, seven sp2 (including one carbonyl at δ 185.9), seven sp3 methylene, six sp3 methine (including two oxygen-bearing methines at δ 61.4 and 70.6), and four sp3 quaternary carbons as analyzed via HSQC data. The HMBC correlations of CH3-29 (CH3-30)/C-3, C-4, and C-5 and H-2/C-1, C-4, and C-10 indicated that ring A was a 1,2-disubstituted 4,4-dimethylcy-

Figure 3. ORTEP drawing of 11. 1577

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hybrid FTMS (Fourier transform mass spectrometer). All solvents used were of analytical grade (Laiyang Chemical Reagent Co., Ltd., Shandong, P. R. China). HPLC was performed on an Agilent 1100 G1310A isopump equipped with an Agilent 1100 G1322A degasser, an Agilent 1100 G1314A VWD detector (210 nm), and an Agilent ZORBAX SB-C18 column (9.4 mm × 250 mm, 5 μm). Silica gel (200− 300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China), C18 reversed-phase silica gel (YMC ODS-A gel, YMC Co., Ltd.), MCI-gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.), and Sephadex LH-20 (Pharmacia Biotek, Denmark) were used for CC. TLC was carried out with high-performance TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd.). TLC spots were visualized within iodine vapor or by spraying with H2SO4−EtOH (1:9) followed by heating. Implementation of the MM2 force field in ChemBio3D Ultra software from CambridgeSoft Corporation (Cambridge, MA, USA, ver. 11.0) was used to calculate molecular models. Plant Material. The twigs and leaves of Casearia balansae were collected from Xishuangbanna County, Yunnan Province, P. R. China, in October 2008. The plant material was identified by one of the authors (T.S.). A voucher specimen (CB01-2008-10) was deposited at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, P. R. China. Extraction and Isolation. The air-dried and powdered plant material (7.5 kg) was extracted with 90% aqueous EtOH (4 × 20 L, 5 days each) at room temperature. The combined extracts were concentrated under reduced pressure to afford a dark gum (430 g), which was suspended in 80% aqueous MeOH and partitioned with petroleum ether (5 × 1 L). The combined petroleum ether fraction was evaporated under reduced pressure to afford a brown gum (84 g). The 80% aqueous MeOH fraction was diluted to 50% MeOH by addition of H2O and then extracted with CH2Cl2. Evaporation of CH2Cl2 under reduced pressure yielded a brown semisolid (105 g). The petroleum ether-soluble fraction showed similar TLC to that of the CH2Cl2-soluble fraction; hence they were combined and subjected to silica gel CC with a gradient of petroleum ether−EtOAc (100:1 → 1:1) to yield eight fractions (Fr. 1−Fr. 8). Fr. 5 (10.7 g) was separated by CC on silica gel and then reversed-phase C18 to give 18 (10.0 mg). Fr. 6 (25.5 g) was first separated by silica gel CC eluted with petroleum ether−EtOAc (40:1) to give four fractions (Fr. 6.1− Fr.6.4). Fr. 6.3 (18.6 g) was further purified by CC on MCI-gel, C18 reversed-phase silica gel, silica gel, and Sephadex LH-20 and by preparative HPLC to give 6 (44.6 mg), 9 (13.3 mg), 17 (26.8 mg), 14 (16.2 mg), 13 (6.0 mg), 8 (1.6 mg), 7 (18.9 mg), 16 (13.7 mg), 5 (15.0 mg), and 10 (7.0 mg). Fr. 6.4 (9.1 g) was separated by CC on MCI-gel and reversed-phase silica gel and by preparative HPLC to give 3 (57.0 mg), 4 (4.8 mg), and 11 (75.0 mg). Fr. 7 (15.7 g) was first separated by MCI-gel (0 → 90% EtOH(aq)) to give seven fractions (Fr. 7.1−Fr. 7.7). Fr. 7.2 (7.0 g) was purified by CC on Sephadex LH20 and silica gel to give 12 (50.7 mg). Fr. 7.3 (3.1 g) was separated by silica gel CC and HPLC to give 1 and 2 as a mixture (19.5 mg) and 15 (1.9 mg) (for detailed procedures for extraction and isolation, see the Supporting Information).

droxy-9,11-epoxy-24-methyl-9(10→19)abeo-lanost-2,5(10),25trien-1-one, named balansinone. The known diterpenoids were identified as rel(2R,4R,5S,8R,9R,10S,18R,19R)-18,19-diacetoxy-18,19-epoxycleroda-3(16),14-dien-2-ol (12),27 rel(2S,5R,6R,8S,9S,10R,18S,19S)-18,19-diacetoxy-6-methoxy-2(2ξ-methylbutanoyloxy)cleroda-3,13(16),14-trien-18,19-oxolane (13),2 rel-(2R,4S,5R,8S,9S,10R,18S,19R)-2,18,19-triacetoxycleroda-13(16),14-dien-18,19-oxolane (14), 2 3 rel(2S,5R,6R,8S,9S,10R,18S,19R)-18,19-diacetoxy-18,19-epoxy-2(2ξ-methylbutanoyloxy)cleroda-3,13(16),14-trien-6-ol (15),2 caseamembrin A (16),31 casearlucin B (17),2 and trans-phytol (18)33 by comparing spectroscopic data with literature values. Among the compounds encountered in this investigation, 1 and 2 possess an oxygen bridge between C-2 and C-19, which are the first examples in clerodane-type diterpenoids. Although 5− 7 were artifacts formed during isolation, they provide structural diversity for our bioassays. Compounds 1a, 2a, 3−7, and 9−17 were evaluated for their antiproliferative activity against cancer cell lines PC3, DU145, SKOV3, and A549 and normal human fibroblast (WI-38) cells. As shown in Table 5, compound 15 Table 5. Cytotoxicity of Compounds 1a, 2a, 3−7, and 9−17 against Human Tumor Cells and Normal Human Primary Fibroblast Cellsa compound

PC3

DU145

SKOV3

A549

WI-38

1a 2a 3 4 5 6 7 9 10 11 12 13 14 15 16 17 paclitaxel

6.2 5.6 6.3 7.2 34.2 46.4 46.9 6.5 4.6 22.5 8.2 5.6 36.1 4.5 10.5 4.7 0.02

13.3 14.1 5.8 67.2 47.2 51.7 14.8 5.6 7.2 26.1 6.6 4.8 20.0 4.3 5.0 4.3 0.04

30.6 36.2 10.2 25.3 35.9 99.8 15.3 17.6 7.2 73.8 17.9 7.2 38.2 5.1 8.7 6.2

19.7 24.0 16.2 13.9 53.3 45.2 49.8 13.3 11.3 37.7 15.2 7.9 66.2 5.7 15.7 7.1

41.0 51.0 21.1 58.1 55.7 57.6 60.2 20.3 14.8 78.4 26.3 13.3 56.9 9.8 19.6 13.3 0.05

Results are expressed as mean IC50 values in μM from duplicate measurements. Paclitaxel and DMSO were used as positive and negative controls. a

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was the most active of the compounds tested and showed stronger activity against all tumor cell lines (IC50 < 5.7 μM) compared to normal WI-38 cells (IC50 = 9.8 μM). Compounds 5, 6, 11, and 14 were nearly inactive. Moreover, compounds 1a, 2a, and 4 showed selective activity against PC3 tumor cells.

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ASSOCIATED CONTENT

S Supporting Information *

Details for extraction and isolation, preparation of 1a and 2a, Xray crystallographic study of 11, cytotoxicity assay, and analysis of the acetone extract of the plant and 5−8 by HPLC; selected 1 H−1H COSY and HMBC correlations of 1−10; selected NOESY correlations of 3−10; copies of 1D and 2D NMR, HRESIMS, and IR spectra of 1−11, 1a, and 2a. This material is available free of charge via the Internet at http://pubs.acs.org.

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an X-6 melting-point apparatus (Beijing TECH Instrument Co. Ltd.) and were uncorrected. Optical rotations were measured on a Perkin-Elmer 241 MC polarimeter. UV spectra were obtained on a Shimadzu UV-2550 spectrophotometer. IR spectra were recorded on a Thermo-Nicolet 670 spectrophotometer using KBr disks. NMR spectra were measured on a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz with TMS as internal standard. HRESIMS was carried out on an LTQ-Orbitrap XL

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-531-88382012. Fax: 86-531-88382548. E-mail: [email protected]. 1578

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Notes

(22) Wang, B.; Wang, X. N.; Shen, T.; Wang, S. Q.; Guo, D. X.; Lou, H. X. Phytochem. Lett. 2012, 5, 271−275. (23) Mosaddik, M. A.; Forster, P. I.; Booth, R.; Waterman, P. G. A. Biochem. Syst. Ecol. 2007, 35, 631−633. (24) Kuo, Y. H.; Chaiang, Y. M. Chem. Pharm. Bull. 1999, 47, 498− 500. (25) (a) Kwon, Y. J.; Sohn, M. J.; Kim, C. J.; Koshino, H.; Kim, W. G. J. Nat. Prod. 2012, 75, 271−274. (b) Li, J.; Fronczek, F. R.; Ferreira, D.; Burandt, C. L., Jr.; Setola, V.; Roth, B. L.; Zjawiony, J. K. J. Nat. Prod. 2012, 75, 728−734. (26) Wiberg, K. B.; Wilson, S. M.; Wang, Y. G.; Vaccaro, P. H.; Cheeseman, J. R.; Luderer, M. R. J. Org. Chem. 2007, 72, 6206−6214. (27) Khan, M. R.; Gray, A. I.; Sadler, I. H.; Waterman, P. G. Phytochemistry 1990, 29, 3591−3595. (28) Williams, R. B.; Norris, A.; Miller, J. S.; Birkinshaw, C.; Ratovoson, F.; Andriantsiferana, R.; Rasamison, V. E.; Kingston, D. G. I. J. Nat. Prod. 2007, 70, 206−209. (29) Santos, A. G.; Ferreira, P. M. P.; Vieira-Júnior, G. M.; Perez, C. C.; Gomes Tininis, A.; Silva, G. H.; Bolzani, V. S.; Costa-Lotufo, L. V.; Pessoa, C. Ó .; Cavalheiro, A. J. Chem. Biodiversity 2010, 7, 205−215. (30) Shen, Y. C.; Lee, C. L.; Khalil, A. T.; Cheng, Y. B.; Chien, C. T.; Kuo, Y. H. Helv. Chim. Acta 2005, 88, 68−77. (31) Shen, Y. C.; Wang, C. H.; Cheng, Y. B.; Wang, L. T.; Guh, J. H.; Chien, C. T.; Khalil, A. T. J. Nat. Prod. 2004, 67, 316−321. (32) Ali, Z.; Khan, S. I.; Fronczek, F. R.; Khan, I. A. Phytochemistry 2007, 68, 373−382. (33) Brown, G. D. Phytochemistry 1994, 36, 1553−1554.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from National Natural Science Foundation of China (No. 21272139) and Scientific Research Foundation for the Excellent Middle-Aged and Youth Scientists of Shandong Province (No. BS2009YY001) is gratefully acknowledged. We thank Prof. L. Gunatilaka from the University of Arizona (USA) for bioassay of the plant extracts.

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