Bioorganic & Medicinal Chemistry Letters 24 (2014) 3952–3955

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Lactones from Ficus auriculata and their effects on the proliferationfunction of primary mouse osteoblasts in vitro

http://dx.doi.org/10.1016/j.bmcl.2014.06.0350960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel./fax: +86 898 65889422.E-mail addresses: hchr116@hainnu.edu.cn (C.-R. Han), chgying123@163.com

(G.-Y. Chen).� Tai-Ming Shao and Cai-Juan Zheng are co-first authors.

Tai-Ming Shao a,c,�, Cai-Juan Zheng a,�, Chang-Ri Han a,⇑, Guang-Ying Chen a,⇑, Chun-Yan Dai a,b,Xiao-Ping Song a, Jin-Chao Zhang b, Wen-Hao Chen a

a Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, PR Chinab College of Chemistry & Environmental Science, Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding 071002, PR Chinac School of Biochemical Engineering, Hainan Institute of Science and Technology, Haikou 571126, PR China

a r t i c l e i n f o

Article history:Received 8 April 2014Revised 10 June 2014Accepted 12 June 2014Available online 21 June 2014

Keywords:Ficus auriculataLactonesChemical constituentsPrimary osteoblasts

a b s t r a c t

Bioassay-guided fractionation of the petroleum ether, chloroform and EtOAc extracts of the stems of Ficusauriculata led to the isolation of five new 12-membered lactones (3R,4R)-4-hydroxy-de-O-methyllasio-diplodin (1), 6-oxolasiodiplodin (2) and ficusines A�C (3–5), together with three known related ana-logues (6–8). The structures of the new compounds were elucidated by comprehensive spectroscopicdata. The absolute configurations of 3 and 8 were established by single crystal X-ray diffraction analysis.Compounds 3–5 represent the first 12-membered lactones with a quinone ring unit. Compounds 6 and 7exhibited significant proliferation function of primary osteoblasts (OBs) in vitro. Especially, the promo-tion rate of 6 reached 151.55 ± 1.34% (P < 0.001) at the concentration of 100 lM.

� 2014 Elsevier Ltd. All rights reserved.

The genus Ficus (Moraceae) comprising about 1000 speciesgrows mainly in tropical and subtropical.1 Ficus auriculata has beenused as a Chinese folk medicine for the treatment of hyperactivitycough and nocturnal emission.2 The leaves of F. auriculata exhib-ited antioxidant, antiinflammatory, antidiabetic and hepatoprotec-tive activities, and the fruits of F. auriculata exhibited significantantibacterial activity.3–5 Previous chemical studies on this genushave led to the isolation of an array of compounds including trit-erpenoids,6–10 flavonoids,11–14 alkaloids15–17 and so on. Preliminaryexperimental results showed that the petroleum ether, chloroformand EtOAc extracts of the stems of F. auriculata exhibited signifi-cant proliferation function of primary osteoblasts (OBs) in vitro[The promotion rate reached 147.56 ± 5.48% (P < 0.001),154.46 ± 6.15% (P < 0.001) and 212.3 ± 10.61% (P < 0.001), respec-tively, at the concentration of 100 lM]. A chemical investigationon the F. auriculata has led to the isolation and characterizationof five new 12-membered lactones18,19 (3R,4R)-4-hydroxy-de-O-methyllasiodiplodin (1), 6-oxolasiodiplodin (2), and ficusinesA�C (3–5), together with three known benzoic acid lactones(R)-(+)-lasiodiplodin (6), (+)-(R)-de-O-methyllasiodiplodin (7) and(3R,6S)-6-hydroxylasiodiplodin (8) (Fig. 1). The absolute configurations

of 3 and 8 were determined by single crystal X-ray diffraction analysis.Herein we report the isolation, structure elucidation, and the prolifer-ation function of primary osteoblasts (OBs) in vitro of the isolatedcompounds.

(3R,4R)-4-hydroxy-de-O-methyllasiodiplodin (1)20 was obtainedas a white powder. The molecular formula of 1 was determinedto be C16H22O5 by HRESIMS, with 6 degrees of unsaturation. TheIR spectrum of 1 revealed presence of hydroxyl at 3425 cm�1 andcarbonyl at 1706 cm�1. The 1H NMR spectrum (Table 1) of 1 dis-played a hydroxyl group bonding to an ester carbonyl at dH 11.83(1H, s), two aromatic protons at dH 6.28 (1H, d, J = 2.6 Hz) and6.23 (1H, d, J = 2.6 Hz), two methine protons at dH 5.02 (1H, m)and 3.90 (1H, m), 12 methylene protons at dH 3.57–1.54, and amethyl protons with doublet at dH 1.46 (3H, d, J = 6.2 Hz). The13C NMR spectrum (Table 2) of 1 revealed 16 signals, which weresorted by DEPT and HSQC techniques into one methyl (dC 17.6),six methylenes (dC 22.5, 24.3, 28.3, 32.2, 34.3 and 34.4), fourmethines including two O-methine carbons (dC 72.3 and 76.4),two aromatic carbons (dC 101.7 and 111.2), and five quaternarycarbons including an ester carbonyl carbon (dC 171.6). Thesespectroscopic data indicated that the basic skeleton of 1 should be12-membered lactone. The 1H NMR spectrum of 1 was similar tothat of 7.21 The only significant difference in the 1H NMR spectrumof 1 in comparison with 7 was the presence of an O-methine protonsignal for H-4 that was shifted downfield to dH 3.90 (instead of onemethylene signal at dH 1.92 and 1.79 in 7). The downfield shifts

O

O

HO

O

O

2 3 4 5

O

OR1

HO

R2O

1 3

5

79

11

15

17

13

O

OO

O

OH

OO

OO

O

OHO

OO

O

OH

O1 R1 = H R2 = α -OH R3 = H6 R1 = CH3 R2 = H R3 = H7 R1 = H R2 = H R3 = H8 R1 = CH3 R2 = H R3 = β -OH

R3

Figure 1. The structures for compounds 1–8.

Table 11H NMR data (400 MHZ, CDCl3) for compounds 1–5 (dH, J in Hz)

Position 1 2 3 4 5

3 5.02, m 5.21, m 4.92, m 4.60, m 4.66, m4 3.90, m 2.14, m 1.98, m 1.93, m 1.93, m

1.96, m 1.60, m 1.53, m5 1.97, m 2.54, m 2.86, m 1.90, m 1.24, m

1.65, m 2.50, m 2.42, m6 1.66, m — — 2.50, m 1.26, m

2.20, m7 1.57, m 2.68, m 2.55, m — 1.25, m

2.34, m 2.25, m8 1.54, m 1.52, m 1.88, m 2.80, m 1.26, m

1.57, m 2.16, m9 1.60, m 1.57, m 1.53, m 2.21, m 1.55, m

1.10, m 1.75, m10 3.57, m 2.60, m 2.48, m 2.47, m 2.32, m

2.28, m 2.45, m 2.11, m 2.07, m12 6.23, d (2.6) 6.17, br s 6.05, br s 6.08, m 6.07, d (1.4)14 6.28, d (2.6) 6.17, br s 5.51, d (1.3) 5.58, d (1.4) 5.55, d (1.4)17 1.46, d (6.2) 1.34, d (6.2) 1.32, d (6.2) 1.44, d (6.6) 1.40, d (6.5)OH-15 11.83, s — — — —OH-16 — — 4.03, br s 4.04, br s 4.13, br s–OCH3 — 3.69, s 3.72, s 3.75, s 3.73, s

Table 213C NMR data (100 MHZ, CDCl3) for compounds 1–5 (dC, mult.)

Position 1 2 3 4 5

1 171.6, C 169.5, C 168.4, C 169.0, C 169.2, C3 76.4, CH 73.1, CH2 74.7, CH 79.1, CH 79.0, CH4 72.3, CH 31.8, CH2 28.7, CH2 32.4, CH2 32.4, CH2

5 34.4, CH2 40.7, CH2 38.6, CH2 21.0, CH2 23.0, CH2

6 24.3, CH2 213.3, C 211.4, C 41.0, CH2 25.1, CH2

7 22.5, CH2 41.5, CH2 40.4, CH2 211.0, C 23.2, CH2

8 28.3, CH2 20.9, CH2 24.1, CH2 38.2, CH2 24.3, CH2

9 32.2, CH2 28.6, CH2 27.2, CH2 19.5, CH2 24.0, CH2

10 34.3, CH2 30.3, CH2 29.5, CH2 27.3, CH2 27.4, CH2

11 149.7, C 141.5, C 154.0, C 152.8, C 154.3, C12 111.2, CH 108.0, CH 128.0, CH 126.1, CH 126.6, CH13 160.6, C 158.2, C 187.0, C 187.0, C 187.3, C14 101.7, CH 96.9, CH 101.5 CH 102.0, CH 102.0, CH15 165.9, C 158.1, C 172.1, C 171.7, C 171.8, C16 105.2, C 116.8, C 75.2, C 75.8, C 75.8, C17 17.6, CH3 20.4, CH3 20.5, CH3 20.7, CH3 20.7, CH3

–OCH3 — 55.6, CH3 56.5, CH3 56.5, CH3 56.4, CH3

T.-M. Shao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3952–3955 3953

observed for C-4 [dC 72.3 (CH) for 1 vs dC 31.2 (CH2) for 7] in the 13CNMR spectrum also reflected the presence of a hydroxy group atC-4 in 1 rather than a methylene group at C-4 in 7. The planarstructure of 1 was confirmed 1H–1H COSY, HMQC, and HMBCexperiments (Fig. 2). The relative configurations at C-3 and C-4 of1 were determined by the ROESY correlation from H-3 to H-4indicated that they were on the same side. Hence, the structureof 1 was determined to be 4-hydroxy-de-O-methyllasiodiplodin.

6-Oxolasiodiplodin (2)22 was obtained as a pale yellow powder.The molecular formula of 2 was determined to be C17H22O5 by

HRESIMS, with 7 degrees of unsaturation. The IR spectrum of 2revealed presence of hydroxyl at 3420 cm�1 and carbonyl at1711 cm�1. The 1H and 13C NMR spectra of 2 (Tables 1 and 2) weresimilar to those of 8,23 the obvious differences were the absence ofa methine proton signal at dH 3.58 (1H, m) in 2 and the 13C signal ofC-6 [(dC 213.3 (C) for 2 vs dC 68.6 (CH) for 8] indicated that themethine group for C-6 in 8 was replaced by a carbonyl group in2. The location of the ketone carbonyl at C-6 was confirmed bythe HMBC correlations from H-4, H-7, and H-8 to C-6 (Fig. 2).Accordingly, compound 2 was identified as 6-oxolasiodiplodin.

Ficusine A (3)24 was obtained as pale yellow crystals. Themolecular formula of 3 was determined to be C17H22O6 by HRE-SIMS, with 7 degrees of unsaturation. The IR spectrum of 3 revealedpresence of hydroxyl at 3424 cm�1 and carbonyl at 1726 cm�1. The1H NMR spectrum of 3 (Table 1) displayed two olefinic protons atdH 6.05 (1H, br s) and 5.51 (1H, d, J = 1.3 Hz), an O-methine protonat dH 4.92 (1H, m), a methoxy proton at dH 3.72 (3H, s), 12 methy-lene protons at dH 1.10–2.86, and a methyl proton at dH 1.32 (3H, d,J = 6.2 Hz). The 13C NMR spectrum of 3 (Table 2) revealed 17 signalsclassified as a methyl (dC 20.5), a methoxy (dC 56.5), six methylenes(dC 24.1, 27.2, 28.7, 29.5, 38.6 and 40.4), three methines (dC 74.7,101.5 and 128.0), and six quaternary carbons (dC 75.2, 154.0,168.4, 172.1, 187.0 and 211.4), including an ester carbonyl carbon(dC 168.4), two ketone carbonyl carbons (dC 187.0 and 211.4). Thesespectroscopic data indicated that the basic skeleton of 3 should be12-membered lactones. The structure of 3 was further demon-strated by analysis of HMBC spectrum (Fig. 2). The HMBC spectrumshowed correlations from H-14 to C-13, C-15 and C-16 indicatedthe presence of an a,b-unsaturation ketone, suggesting that there

O

OH

HO

O

O

HO

OO

1H-1H COSY HMBC

O

O O

O

OHO

O O

O

O

OHO

O O

O

OH

O

4 521 3

O1 3

5

79

11

15

17

13

OH

Figure 2. 1H–1H-COSY( ) and key HMBC(H ? C) correlations of compounds 1–5.

3954 T.-M. Shao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3952–3955

was a quinone ring rather than a benzene ring. The correlationsfrom H-10 to C-9, C-11, C-12 and C-16 indicated the connectionpoints of the aliphatic ring and the quinone ring were at C-11.The location of the methoxy was confirmed by the HMBC correla-tions from –OCH3 to C-14 and C-15 (Fig. 2). The location of theketone carbonyl was confirmed by the HMBC correlations fromH-4, H-7, and H-8 to C-6 (Fig. 2). Therefore, ficusine A (3) repre-sents the first 12-membered lactones with the skeleton of quinonering. Finally, by slow crystallization from MeOH–CHCl3, singlecrystals of 3 suitable for X-ray diffraction analysis using Cu Karadiation were obtained, allowing the structure of 3 (CCDC No.952019) to be fully established (Fig. 3). The absolute configurationsof 3 were determined as 3R,16R.

Ficusine B (4)25 was obtained as a pale yellow powder. Themolecular formula of 4 was determined to be C17H22O6 by HRE-SIMS, with 7 degrees of unsaturation. The IR spectrum of 4 revealedpresence of hydroxyl at 3424 cm�1 and carbonyl at 1726 cm�1.Detailed analysis of the 1H and 13C NMR spectra (Tables 1 and 2)revealed that 4 is also a resorcylic acid lactone, which is similarto those of 3. The significant differences were the chemical shiftsof C-6 [dC 41.0 (CH2) for 4 vs dC 211.4 (C) for 3], and C-7 [dC

211.0 (C) for 4 vs dC 40.4 (CH2) for 3], indicating that ketone wasat the C-7 position in 4 rather than at C-6 in 3. The structure wasfurther confirmed by 1H–1H COSY and HMBC spectra (Fig. 2).Therefore, the structure of 4 was considered to be ficusine B.

Ficusine C (5)26 was obtained as a pale yellow powder. Themolecular formula of 5 was determined to be C17H24O5 by HRE-SIMS, with 6 degrees of unsaturation. The IR spectrum of 5 revealedpresence of hydroxyl at 3424 cm�1, carbonyl at 1726 cm�1. The 1Hand 13C NMR spectra of 5 (Tables 1 and 2) were closely resembledthose of 3, the significant differences were the 13C signals of C-6 [dC

25.1 (CH2) for 5 vs dC 211.4 (C) for 3], indicated that the carbonylfor C-6 in 3 was replaced by the methylene in 5. The structurewas also confirmed by HMBC and 1H–1H COSY analysis (Fig. 2).Therefore, the structure of 4 was considered to be ficusine C.

Figure 3. X-ray crystallographic structure of compound 3.

The absolute configurations of 4 and 5 were tentativelyassigned as 3R,16R on the basis of the absolute configurations of3, according to a shared biogenesis for these compounds.

Compound 827 was identified to be (3R,6S)-6-hydroxylasiodip-lodin by X-ray diffraction analysis using Cu Ka radiation (CCDCNo. 933371) (Fig. 4). Therefore, the absolute configurations of 1,2, 6 and 7 were proposed to determined as 3R configuration dueto the biosynthesis pathway. Known compounds 628 and 728 wereidentified as (R)-(+)-lasiodiploidin and (+)-(R)-de-O-methyllasio-diplodin by comparing their spectroscopic data with the reporteddata.

Previous chemical research suggested that most of lactoneswere isolated from fungus such as Lasiodiplodia theobromae,29

Botryosphaeria rhodina30 and Neocosmospora sp.,31 only few lactoneswere isolated from plants (Durio zibethinu32 and Osbeckiaopipara33).

Compounds 2–8 were evaluated for the proliferation function ofprimary osteoblasts (OBs) in vitro by the MTT method.34 Theresults indicated that these compounds could promote prolifera-tion of OBs at different concentration (Table 3). Compounds 3, 4,6 and 7 had slight inhibitory effect at a concentration of 1 lM. Only3, 4 and 6 promote proliferation of OBs at a concentration of10 lM. Compounds 3, 6 and 7 could promote proliferation ofOBs, the promotion rate reached 119.62 ± 0.76% (P < 0.001),151.55 ± 1.34% (P < 0.001) and 119.48 ± 1.09% (P < 0.01), respec-tively, at the concentration of 100 lM. In summary, the effect ofthese compounds on the proliferation of OBs depended on concen-tration. The exact relationships need further studies by structuremodification of the compounds.

In summary, eight 12-membered lactones including five newones were isolated from the stems of the Ficus auriculata. Previouschemical research suggested that most of lactones were isolatedfrom fungus rather than plants. The quinone ring skeleton of 3–5was reported for the first time for 12-membered lactones. All ofthe isolated compounds including absolute configurationswere established. The single crystal X-ray diffraction analysis of 8was reported for the first time. Compounds 6 and 7 exhibited

Figure 4. X-ray crystallographic structure of compound 8.

Table 3The effect of compounds 2–8 on the proliferation of OBs

Compounds Proliferation rate (%)

1 (lM) 10 (lM) 100 (lM)

2 2.87 ± 0.34 -4.29 ± 0.56 3.28 ± 0.093 12.54 ± 0.97** 10.92 ± 0.87** 19.62 ± 0.76***

4 10.00 ± 0.13** 8.94 ± 0.98** 2.61 ± 0.765 �7.91 ± 1.07* �43.92 ± 2.76*** �68.32 ± 1.76***

6 3.45 ± 0.98* 14.19 ± 0.43*** 51.55 ± 1.34***

7 1.67 ± 0.12 �4.54 ± 0.65 19.48 ± 1.09**

8 3.97 ± 0.23 �6.67 ± 0.12 2.1 ± 0.14NaF 12.15 ± 1.34 18.76 ± 1.23 22.54 ± 1.76

* P < 0.05.** P < 0.01.

*** P < 0.001 versus control group, n = 6.

T.-M. Shao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3952–3955 3955

significant proliferation function of primary osteoblasts (OBs)in vitro. This is the first report of proliferation function of primaryosteoblasts (OBs) in vitro for this class of metabolites.

Acknowledgments

This project was supported by the Ministry of Science andTechnology of China (973 Program, 2011CB512010), the NationalNatural Sciences Foundation of China (Nos. 21166009, 81160391and 81360478) and the Major Technology Project of Hainan(ZDZX2013008-4), the Natural Science Foundation of HainanProvince (Nos. 213021, 213017), the Pharmaceutical Joint ResearchFundation of the Natural Science Foundation of Hebei Province andChina Shiyao Pharmaceutical Group Co., Ltd (No. B2012201044),Youth Foundation of Hainan Institute of Science and Technology(HKYZQJ2014-07).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06.035.

References and notes

1. Hou, K. Z. A Dictionary of the Families and Genera of Chinese Seed Plants; SciencesPress: Beijing, China, 1982. 2nd ed. pp 195.

2. Institute of Medicinal Plant Development Hainan Branch Species list of SouthChina Medicinal Plant Garden; China Agriculture Press: Beijing, 2007. pp 150.

3. Shi, Y. X.; Xu, Y. K.; Hu, H. B.; Na, Z.; Wang, W. H. Food Chem. 2011, 128, 889.4. Fishawy, A. E.; Zayed, R.; Afifi, S. J. Nat. Prod. (Gorakhpur, India) 2011, 4, 184.5. Saklani, S.; Chandra, S. Int. J. Pharm. Sci. Rev. Res. 2012, 12, 61.6. Tuyen, N. V.; Kim, D. S. H. L.; Fong, H. S.; Soejarto, D. D.; Khanh, T. C.; Tri, M. V.;

Xuan, L. T. Phytochemistry 1998, 50, 467.7. Chiang, Y. M.; Kuo, Y. H. J. Nat. Prod. 2000, 63, 898.8. Chiang, Y. M.; Kuo, Y. H. J. Nat. Prod. 2001, 64, 436.9. Chiang, Y. M.; Su, J. K.; Liu, Y. H.; Kuo, Y. H. Chem. Pharm. Bull. 2001, 49, 581.

10. Chiang, Y. M.; Chang, J. Y.; Kuo, C. C.; Chang, C. Y.; Kuo, Y. H. Phytochemistry2005, 66, 495.

11. Li, Y. C.; Kuo, Y. H. J. Nat. Prod. 1997, 60, 292.12. Kitajima, J.; Kimizuka, K.; Arai, M.; Tanaka, Y. Chem. Pharm. Bull. 1998, 46, 1647.13. Ya, J.; Zhang, X. Q.; Wang, Y.; Zhang, Q. W.; Chen, J. X.; Ye, W. C. Nat. Prod. Res.

2010, 24, 621.14. Bankeu, J. J. K.; Khayala, R.; Lenta, B. N.; Noungoue, D. T.; Ngouela, S. A.;

Mustafa, S. A.; Asaad, K.; Choudhary, M. I.; Prigge, S. T.; Hasanov, R. J. Nat. Prod.2011, 74, 1370.

15. Damu, A. G.; Kuo, P. C.; Shi, L. S.; Li, C. Y.; Kuoh, C. S.; Wu, P. L.; Wu, T. S. J. Nat.Prod. 2005, 68, 1071.

16. Subramaniam, G.; Ang, K. K. H.; Ng, S.; Buss, A. D.; Butler, M. S. Phytochem. Lett.2009, 2, 88.

17. Lin, H. Y.; Chiu, H. L.; Lu, T. L.; Tzeng, C. Y.; Lee, T. H.; Lee, C. K.; Shao, Y. Y.; Chen,C. R.; Chang, C. I.; Kuo, Y. H. Chem. Pharm. Bull. 2011, 59, 113.

18. The stems of F. auriculata were collected from Jianfengling National ForestPark, Hainan Province China, in June 2010, and identified by Prof. Qiong-XinZhong, College of Life Science, Hainan Normal University. A voucher specimenwas deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry ofMinistry of Education, Hainan Normal University.

19. The air-dried stem of F. auriculata (13.5 kg) were extracted three times with95% EtOH (20 L � 3) at room temperature and concentrated in vacuo to yield aextract (520 g), which was suspended in 2.0 L of water and then partitionedwith petroleum ether, CHCl3, EtOAc (2 L � 3) successively, to yield petroleumether extract (240 g), CHCl3 extract (60 g), EtOAc extract (75 g). The EtOAcextract (75 g) was subjected to CC on silica gel using gradient elution withpetroleum ether/EtOAc (100:1 to 1:100, v/v) to yield eight fractions A–H.Fraction A (4.60 g) was subjected to CC on silica gel using petroleum ether/EtOAc (4:1, v/v) to give five subfractions A1–A5. Subfraction A2 (670 mg) wasfurther chromatographed over Sephadex LH-20 gel column eluted with CHCl3/CH3OH (2:3, v/v), followed by PTLC to give 6 (7.5 mg). Subfraction A3 (710 mg)was further chromatographed over Sephadex LH-20 gel column eluted withCHCl3/CH3OH (2:3, v/v), followed by PTLC to give 7 (6.1 mg) and 8 (7.7 mg).Fraction B (3.4 g) was subjected to CC on silica gel using petroleum ether/EtOAc (4:1, v/v) to give four subfractions B1–B4. Subfraction B2 (270 mg) wasfurther chromatographed over Sephadex LH-20 gel column eluted with CHCl3/CH3OH (2:3, v/v), followed by PTLC to give 1 (1.5 mg). Subfraction B3 (310 mg)was further chromatographed over Sephadex LH-20 gel column eluted withCHCl3/CH3OH (2:3, v/v), followed by PTLC to give 2 (8.6 mg). Fraction C(240 mg) was further chromatographed over Sephadex LH-20 gel columneluted with CHCl3/CH3OH (2:3, v/v), followed by PTLC to give 5 (12.6 mg).Fraction D (6.8 g) was subjected to CC on silica gel using petroleum ether/EtOAc (3:1, v/v) to give five subfractions D1–D5. Subfraction D2 (670 mg) wasfurther chromatographed over Sephadex LH-20 gel column eluted with CHCl3/CH3OH (2:3, v/v), followed by PTLC to give 4 (10.4 mg), Subfraction D4(530 mg) was further chromatographed over Sephadex LH-20 gel columneluted with CHCl3/CH3OH (2:3, v/v), followed by PTLC to give 3 (11.9 mg).

20. (3R,4S)-4-Hydroxy-de-O-methyllasiodiplodin (1): White powder; [a]D25 +2.0 (c

1.0, MeOH); UV (MeOH) kmax (loge) 208 (4.24), 231 (3.18), 254 (4.84) nm; IR(KBr) mmax 3425, 2926, 1706, 1609, 1267 cm�1; 1H and 13C NMR data, seeTables 1 and 2; HRESIMS m/z 295.1541 [M+H]+ (calcd for C16H23O5, 295.1540).

21. Yang, R. Y.; Li, C. Y.; Lin, Y. C.; Peng, G. T.; She, Z. G.; Zhou, S. N. Bioorg. Med.Chem. Lett. 2006, 16, 4205.

22. 6-Oxolasiodiplodin (2): Pale yellow powder; [a]D25 +1.9 (c 1.0, MeOH); UV

(MeOH) kmax (loge) 209 (4.39), 235 (3.71), 260 (4.56) nm; IR (KBr) mmax 3382,1603, 1462, 1435, 1248, 1211, 1067 cm�1; 1H and 13C NMR data, see Tables 1and 2; HRESIMS m/z 307.1536 [M+H]+ (calcd for C17H23O5, 307.1540).

23. Li, P.; Takahashi, K.; Matsuura, H.; Yoshihara, T. Biosci., Biotechnol., Biochem.2005, 69, 1610.

24. Ficusine A (3): Pale yellow crystals; mp 154�156 �C; [a]D25 +2.2 (c 1.0, MeOH);

UV (MeOH) kmax (loge) 203 (5.21), 237 (4.20), 258 (4.74) nm; IR (KBr) mmax

3382, 1603, 1462, 1435, 1248, 1211, 1067 cm�1, C17H22O6, M = 322.35, spacegroup P212121 with a = 6.6767 (2) Å, b = 7.3343 (2) Å, c = 34.2153 (11) Å,a = b = c = 90�, V = 1675.49 (9) Å3, Z = 4, T = 293 (2) K, Dc = 1.278 g/cm3,l = 0.804 mm�1, F(000) = 688. Independent reflections: 2922 withRint = 0.0220. The final agreement factors are R1 = 0.0345 and wR2 = 0.0882[I > 2r(I)]. Flack parameter = 0.0 (2), CCDC number: 952019; 1H and 13C NMRdata, see Tables 1 and 2; HRESIMS m/z 345.1304 [M+Na]+ (calcd forC17H23O6Na, 345.1309).

25. Ficusine B (4): Pale yellow powder; [a]D25 +2.4 (c 1.0, MeOH); UV (MeOH) kmax

(loge) 203 (5.18), 235 (4.51), 255 (4.65) nm; IR (KBr) mmax 3420, 2924, 1712,1671, 1230 cm�1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z345.1307 [M+Na]+ (calcd for C17H23O6Na, 345.1309).

26. Ficusine C (5): Pale yellow powder; [a]D25 +3.3 (c 1.0, MeOH); UV (MeOH) kmax

(loge) 203 (5.10), 234 (4.13), 254 (4.58) nm; IR (KBr) mmax 3424, 2932, 1726,1665, 1227 cm�1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z309.1699 [M+H]+ (calcd for C17H25O5, 309.1697).

27. (3R,6S)-6-Hydroxylasiodiplodin (8): C17H24O5, M = 308.36, space group P212121

with a = 7.95092 (5) Å, b = 12.06551 (7) Å, c = 16.92045 (9) Å, a = b = c = 90�,V = 1623.211 (16) Å3, Z = 4, T = 100 (2) K, Dc = 1.262 g/cm3, l = 0.755 mm�1,F(000) = 664. Independent reflections: 2906 with Rint = 0.0159. The finalagreement factors are R1 = 0.0233 and wR2 = 0.0594 [I > 2r(I)]. Flackparameter = 0.03 (12), CCDC number: 933371.

28. Matsuura, H.; Nakamori, K.; Omer, E. A.; Hatakeyama, C.; Yoshihara, T.;Ichihara, A. Phytochemistry 1998, 49, 579.

29. Rukachaisirikul, V.; Arunpanichlert, J.; Sukpondma, Y.; Phongpaichit, S.;Sakayaroj, J. Tetrahedron 2009, 65, 10590.

30. Yang, Q.; Asai, M.; Matsuura, H.; Yoshihara, T. Phytochemistry 2000, 54, 489.31. Gao, J. T.; Radwan, M. M.; Leon, F.; Dale, O. R.; Husni, A. S.; Wu, Y. S.; Lupien, S.;

Wang, X. N.; Manly, S. P.; Hill, R. A.; Dugan, F. M.; Cutler, H. G.; Cutler, S. J. J. Nat.Prod. 2013, 76, 824.

32. Rudiyansyah, J.; Garson, M. J. J. Nat. Prod. 2006, 69, 1218.33. Wang, H. S.; Wang, Y. H.; Shi, Y. N.; Li, X. Y.; Long, C. L. J. Chin. Mater. Med. 2009,

34, 414.34. Cell proliferation assay: The cell viability was analyzed by MTT assay Briefly,

OBs (2 � 103 cells per well) were plated in 96-well plates and cultured for 24 h.Then, Compounds 2–8 was added at final concentrations of 1, 10, and 100 lM.Wells containing cells without compounds treatment were used as control.Wells containing complete medium without cells were used as blank. After48 h treatment, 10 ll MTT (5.0 mg/ml) was added to each well. After incubatedfor additional 4 h at 37 �C, the supernatant was removed and 100 ll/welldimethyl sulfoxide (DMSO) was added. Optical density (OD) was measured at570 nm using microplate spectrophotometer (MDVersaMax, USA). The cellviability (%) was expressed as a percentage of [ODsample � ODblank]/[ODcontrol � ODblank] � 100.