Synthesis and evaluation of cytotoxic effects of novel α-methylenelactone tetracyclic diterpenoids

Synthesis and evaluation of cytotoxic effects of novel a-methylenelactone tetracyclic diterpenoids

Yao-fu Zeng^a, Jia-qiang Wu^a, Lian-yong Shi^a, Ke Wang^a, Bin Zhou^a, Yong Tang^a, Da-yong Zhang^a,⇑, Yang-chang Wu^b, Wei-yi Hua^a, Xiao-ming Wu^a

aCenter of Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Road Tongjia Xiang, Nanjing 210009, China

bGraduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan

a r t i c l e i n f o

Article history:

Received 8 November 2011 Revised 22 December 2011 Accepted 16 January 2012 Available online 26 January 2012

Keywords:

Tetracyclic diterpenoids Steviol

Isosteviol a-Methylenelactone Anti-tumor activity

a b s t r a c t

A series of tetracyclic diterpenoids bearing thea-methylenelactone group have been synthesized and screened for their in vitro anti-tumor activities against six human cancer cell lines. The results showed that compounds 1c, 2a and 2b exhibited significant cytotoxicity superior to the positive control doxorubicin hydrochloride against MDA-MB-231, K562 and HepG2 cell lines. In particular, compound 2b was identified as the most promising anticancer agent against HepG2 cells with IC50value of 0.09l^M.

Ó 2012 Elsevier Ltd. All rights reserved.

Cancer is one of the leading causes of death worldwide and causes serious problems in human life. Therefore, various catego- ries of anti-tumor agents have been developed. However, some side effects could happen simultaneously and the resistance to available chemo-therapeutic agents was rising. Hence, it is urgent to develop novel compounds as anticancer agents with higher bioactivities and lower toxicities.^1,2

Natural products have always been interesting sources for developing novel leading compounds. Stevioside (Fig. 1) is the pri- mary sweet component in the leaves of Stevia rebaudiana Bertoni which is a plant native to South America.^3,4Stevioside consists of three molecules of glucose and steviol as its aglycone. A large num- ber of researches have suggested that stevioside tastes 300 times sweeter than sucrose and can be used as a non-caloric sweetener in South America, Japan, and China. Moreover, stevioside along with its metabolic components steviol⁵ and isosteviol⁶possesses multiple pharmacological activities including anti-hyperglycemic, anti-inflammatory, anti-tumor and anti-diarrheal. It has been shown that these three compounds strongly inhibited the cancer formation induced by TPA (12-o-tetra-decanoylphorbol-13-ace- tate) and DMBA (7,12-dimethylbenz[a]anthracene) in a two-stage carcinogenesis test in mouse. In addition, isosteviol inhibited both mammalian DNA polymerases and human DNA topoisomerase II.

Taken together, these compounds could be served as promising

chemopreventive agents against chemical carcinogenesis.^7,8In or- der to develop potential anticancer agents of higher cytotoxicity, some structural modifications have been done. In our previous work, we built up a crucial fragment of exo-methylene cyclopenta- none in the ring D of steviol and isosteviol and obtained some com- pounds with significantly improved cytotoxicity.⁹ Tao and co- workers. synthesized various 15- and 16-substituted isosteviol derivatives by means of functional interconversions, then obtained some compounds with promising activities against B16-F10 mela- noma cells.¹⁰

Plenty of investigations have reported thata-methylenelactone is a crucial building block of many natural products and exhibits wide-ranging biological activities such as anti-tumor, anti-inflam- matory, antimicrobial and so on.¹¹Therefore, the synthesis of this structural moiety has received much attention,^12,13and the rela- tionship between its activities and structure has also been studied.

It appears thata-methylenelactone may be regarded as alkylating agents by virtue of Michael addition with biological nucleophiles such asL-cysteine or thiol-containing enzymes (Enz-SH).¹⁴ Many sesquiterpene lactones isolated from different kinds of natural plants have been reported to display interesting biological activi- ties. For instance, costunolide (Fig. 1) isolated from the root of Sau- ssurea lappa exhibited potent cytotoxicity against HepG2, OVCAR-3 and HeLa cell lines with CD₅₀values of 1.6, 2.0, 2.0lg/mL, respec- tively.¹⁵Kupchan et al. discovered that vernolepin (Fig. 1) bearing two a-methylenelactone groups showed significant cytotoxicity activity against Walker intramuscular carcinosarcoma in vitro

0960-894X/$ - see front matterÓ 2012 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bmcl.2012.01.051

⇑Corresponding author. Tel./fax: +86 25 83271307.

E-mail address:[email protected](D. Zhang).

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1922–1925

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j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m c l

and in vivo in rats.¹⁶However, ent-kaurane diterpenoids possess- inga-methylenelactone group are rare in the natural products dis- covered recently. Therefore, we tried to introduce this critical moiety into steviol and isosteviol and obtained three scaffolds of ent-kaurene diterpenoids. Some derivatives were also synthesized and screened for their anticancer activities against six cancer cell lines in vitro by MTT method.

The synthetic route towards the target compounds was de- scribed as follows: first, treatment of steviol with chloromethyl methyl ether and N,N-diisopropylethylamine afforded 4 in 1 h,¹⁷

then reaction of 4 with selenium oxide and tert-butyl hydroperox- ide led to 5 (Scheme 1).¹⁸Oxidation of 5 with PDC provided com- pound 6.¹⁹We next tried a phenylthio group as a stable protecting group of thea-methylene unit. Conjugate addition of p-thiocresol to enone 6 produced b-thioketone 7²⁰ which was successfully transformed into sulfone lactone 8 by Baeyer–Villiger oxidation with excessive mCPBA.^21,22Finally, desulfonylation of 8 with DBU in THF under mild condition gave the desired compound 1a.²³ Compound 1b was prepared from 1a by deprotection of methoxy- methyl group with 10% HCl in THF.²⁴Esterification of 1b with dif- ferent kinds of halohyrocarbons afforded 1c–f. Compound 1g could be obtained by acylation of 1f with acetic anhydride in the pres- ence of DMAP.²⁵

The synthetic approach employed to prepare 2a was outlined in Scheme 2. First, we attempted to reduce 4 with LiAlH4in anhy- drous THF under refluxing condition. Although the reaction could proceed smoothly, the yield was very low due to the poor liposol- ubility of the product. Therefore, we had to protect the 13-hydroxy of steviol with excessive MOM ether as well. By doing this, com- pound 10 could be obtained in a good yield (86%). Acylation of

O OH

COOMOM O

H H

OH

COOH

H H

OH

COOMOM

H H

OH

COOMOM

H H

OH

OH

COOMOM

H H

O

OH

COOMOM

H H

O S

O OH

COOMOM

H H O

O OH

COOH O

H H

1cR¹=CH3, R²=H 1dR¹=CH₂CH₂CH₃, R²=H 1eR¹=CH₂CH=CH₂, R²=H 1f R¹=Bn, R²=H 1gR¹=Bn, R²=Ac O

OR²

COOR¹ O

H H

1a

1b

a b c d

e f g

h

i

4 5 6

7 8

steviol

SO₂

Scheme 1. Reagents and conditions: (a) MOMCl, DIPEA, DMF (90.0%); (b) SeO2, t-BuOOH, THF (85.0%); (c) PDC, DMF (75.0%); (d) p-thiocresol, Et3N, THF (65.6%); (e) 85%

mCPBA, NaHCO3, CH2Cl2(53.8%); (f) DBU, THF (69.9%); (g) 10% HCl, THF, H2O (84.0%); (h) R¹R (for 1c, R = I; for 1d and 1f, R = Br; for 1e, R = Cl), K2CO3, DMF, KI (64.0–81.4%); (i) Ac2O, Et3N, THF, DMAP (65.2%).

O O costunolide

O OH

O H

O O

vernolepin O-β−glu-β−glu(²→1)

COO-β−glu

H H

stevioside

Figure 1. Chemical structures of stevioside, costunolide and vernolepin.

OH

COOH

H H

OMOM

COOMOM

H H

OMOM

CH₂OR

H H

OMOM

CH₂OAc

H H

OH

OR

CH₂OAc

H H

O

OH

CH₂OAc

H H

O S

O OH

CH2OAc

H H O O

OH

CH₂OR

H H O

steviol 10R=H

11R=Ac

9 12

13R=MOM 14R=H

15 16 2aR=Ac

2bR=H

a b

c

d e

f

g h i

j SO₂

Scheme 2. Reagents and conditions: (a) MOMCl, DIPEA, DMF (85.3%); (b) LiAlH4, THF, reflux (86.0%); (c) Ac2O, Et3N, THF, DMAP (77.6%); (d) SeO2, t-BuOOH, THF (75.6%); (e) PDC, DMF (72.0%); (f) 10% HCl, THF, H2O (82.3%); (g) p-thiocresol, Et3N, THF (87.6%); (h) 85% mCPBA, NaHCO3, CH2Cl2(55.2%); (i) DBU, THF (70.6%); (j) 10% KHCO3, CH3OH, reflux (71.0%).

Y. Zeng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1922–1925 1923

10 with acetic anhydride afforded 11. The following procedures for preparing 2a were exactly the same as for preparing 1a. Finally, 2a was converted to 2b with 10% KHCO3in refluxing CH3OH. It was noteworthy that conjugate addition of p-thiocresol to 13 could not get theb-thioketone, so we had to deprotect the MOM ether to reduce the steric hindrance.

Scheme 3illustrated the synthesis of compounds 3a–d starting from isosteviol. First of all, we attempted to construct a hydroxy- methyl in thea-position of 16-ketone with aqueous formaldehyde under base condition. Surprisingly, the 16-ketone was reduced at the same time. The mechanism of the reaction had been reported and was proposed as a one-spot ‘Aldol–Cannizzaro reaction’ pro- cess.²⁶After the diol 18 had been successfully achieved, we chose acetyl, a cleanly and conveniently deprotected group, to protect the primary alcohol selectively. Oxidation of 19 with PDC provided 20. However, the key intermediate lactone 22 couldn’t be prepared from 20 with excessive mCPBA until the deprotection of acetyl was completed. Treatment of 22 with p-toluenesulfonyl chloride in pyr- idine produced tosylate 23 which was heated under reflux in pyr- idine for 6 h to give the target compound 3a.²⁷Compound 3b was obtained via elimination of 24 which was accessed by removing the benzyl of 23 with 10% Pd-C. Eventually, two analogues (3c, d) of 3b have been synthesized.

In order to investigate whether thea-methylenelactone group is essential for the bioactivity of the compound; we changed the

a-methylene group into the epoxy. Oxidation of 1f and 3a with excessive mCPBA afforded compounds 1h²⁸ and 3e, respectively (Scheme 4).

The structures of the target compounds were elucidated using spectroscopic techniques (IR,¹H and¹³C NMR, ESI/MS, HRMS).

The cytotoxic activities²⁹ of compounds 1a-3e³⁰ were deter- mined in vitro against six cell lines: prostatic carcinoma (PC-3),

colotectal carcinoma (HCT-116), breast carcinoma (MDA-MB- 231), human erythroleukemic cell line (K562), hepatocellular car- cinoma (HepG2) and gastric carcinoma (MGC-803). Doxorubicin hydrochloride was selected as a positive control. The IC50values were used to determine the growth inhibition in the presence of tetracyclic diterpenoids 1a–3e against PC-3, HCT-116, MDA-MB- 231, K562, HepG2 and MGC-803 cancer cell lines. From the IC50

values summarized inTable 1, the compounds 1c, 2a and 2b have

COOH H H

O

COOR H

H O

O

COOR H H

OH

OH

17R=H 18R=Bn

COOBn H H

OH

OAc

COOBn H H

O

OR

20R=Ac 21R=H

O

COOBn H

H O

OH

O

COOR H

H O

OTs

23R=Bn

24R=H 3aR=Bn

3bR=H 3cR=CH₃ 3dR=CH2CH2CH3

isosteviol 19

b e

22

j

a c d

f g

h i

j

Scheme 3. Reagents and conditions: (a) HCHO (aq), NaOH, C2H5OH–H2O, 75°C, 3 h (56.1%); (b) BnBr, K2CO3, DMF, KI (70.4%); (c) Ac2O, Et3N, THF, DMAP, 45 min (85.2%); (d) PDC, DMF (83.1%); (e) 10% KOH, CH3OH, rt (89.4%); (f) 85% mCPBA, NaHCO3, CH2Cl2(56.1%); (g) TsCl, pyridine, DMAP (53.7%); (h) 10% Pd-C, H2, C2H5OH (92.3%); (i) pyridine, DMAP, reflux (66.2%); (j) RR¹(for 3c, R¹= I; for 3d, R¹= Br), K2CO3, DMF, KI (75.2%, 80.1%).

O OH

COOBn O

H H

1f

O OH

COOBn O

H H

O

1h

O

COOBn H

H O

O

COOBn H

H O

O

3a 3e

a a

Scheme 4. Reagents and conditions: (a) 85% mCPBA, NaHCO3, CH2Cl2(51.3%, 55.7%).

Table 1

Cytotoxicities of compounds 1a–3e in vitro^a

Compound Anti-tumor activity in 48 h^b(IC50,l^M)

PC-3 HCT-

116

MDA-MB- 231

K562 HepG2 MGC803

1a 0.12 2.56 0.78 24.21 0.38 2.23

1b 8.32 2.31 21.10 18.4 5.68 3.23

1c 1.56 0.22 0.56 0.13 0.16 1.21

1d 88.67 67.56 36.43 24.43 67.21 35.14

1e 56.32 24.12 37.32 22.12 67.21 34.18

1f 15.52 45.24 28.23 6.23 78.15 2.12

1g 10.11 35.23 18.10 0.34 25.23 1.56

1h 56.24 45.46 38.69 102.10 48.34 29.97

2a 5.71 0.34 1.10 0.89 0.12 2.56

2b 2.10 3.83 0.22 0.16 0.09 1.26

3a 88.76 78.90 102.50 19.53 28.65 19.92

3b 56.21 35.23 78.43 56.24 46.54 98.78

3c 34.21 56.45 22.87 25.45 35.24 11.23

3d 65.35 23.45 56.76 35.23 12.39 19.76

3e 189.92 222.62 127.35 87.81 231.26 123.54

Dox^c 1.18 3.03 1.45 2.35 1.09 2.56

aInhibition of cell growth by the listed compounds was determined using MTT assay.

bData represent the mean value of three independent determinations.

c Dox = Doxorubicin hydrochloride.

1924 Y. Zeng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1922–1925

shown significant cytotoxities against all the six cell lines with the IC50values ranging from 0.09 to 5.71lM. Compounds 1a and 1c were found to be more effective than doxorubicin hydrochloride in HCT-116, MDA-MB-231, HepG2 and MGC803 cell lines. Esterifi- cation of 19-acid with MOM ether or methyl improved the cytotox- icity (1a vs 1b, 1b vs 1c), while esterification of 19-acid with propyl, allyl and benzyl group decreased the activity (1b vs 1d, 1b vs 1e, 1b vs 1f). Compound 1f showed selective inhibition against K562 (IC50= 6.23lM) and MGC803 (IC50= 2.12l^{M) cell}

lines. Compound 1g with the 13-hydrogen acylated exhibited slightly higher activity compared to compound 1f. On the contrary, removing the 19-acetyl of 2a afforded 2b with better cytotoxity against all the cell lines except HCT-116. Compared with 1h, com- pound 1f bearing thea-methylenelactone group displayed better activity, which indicated thata-methylenelactone group played an important role in their anticancer activities. However, the com- pounds (3a–e) with the isosteviol scaffold exhibited weak activities.

In summary, we have successfully synthesized three scaffolds of tetracyclic diterpenoids bearing the a-methylenelactone moiety and evaluated their anticancer activities against six cell lines. We also proved thata-methylenelactone group was essential for the bioactivity of the compound, which was consistent with the previ- ous literature.¹¹Compound 2b was found to be the most potent compound in HepG2 with IC50value of 0.09lM. Further researches on identifying their cellular targets are ongoing in our laboratory and the results will be reported in due course.

Acknowledgment

We thank the National Natural Science Foundation of China (No. 30973607 and No. 81172934) for financial support.

References and notes

1. Wang, T. T.; Liu, J.; Zhong, H. Y.; Chen, H.; Lv, Z. L.; Zhang, Y. K.; Zhang, M. F.;

Geng, D. P.; Niu, C. J.; Li, Y. M.; Li, K. Bioorg. Med. Chem. Lett. 2011, 21, 3381.

2. Sangthong, S.; Krusong, K.; Ngamrojanavanich, N.; Vilaivan, T.; Puthong, S.;

Chandchawan, S.; Muangsin, N. Bioorg. Med. Chem. Lett. 2011, 21, 4813.

3. Brusick, D. J. Food Chem. Toxicol. 2008, 46, S83.

4. Geuns, J. M. C. Phytochemistry 2003, 64, 913.

5. Ogawa, T.; Nozaki, M.; Matsui, M. Tetrahedron 1980, 36, 2641.

6. Mosettig, E.; Beglinger, U.; Dolder, F.; Lichti, H.; Quitt, P.; Waters, J. A. J. Am.

Chem. Soc. 1963, 85, 2305.

7. Chatsudthipong, V.; Muanprasat, C. Pharmacol. Therapeut. 2009, 121, 41.

8. Takasaki, M.; Konoshima, T.; Kozuka, M.; Tokuda, H.; Takayasu, J.; Nishino, H.;

Miyakoshi, M.; Mizutani, K.; Lee, K. H. Bioorg. Med. Chem. Lett. 2009, 17, 600.

9. Li, J.; Zhang, D. Y.; Wu, X. M. Bioorg. Med. Chem. Lett. 2011, 21, 130.

10. Wu, Y.; Dai, G. F.; Yang, J. H.; Zhang, Y. X.; Zhu, Y.; Tao, J. C. Bioorg. Med. Chem.

Lett. 1818, 2009, 19.

11. Hoffmann, H. M. R.; Babe, J. Angew. Chem., Int. Ed. Engl. 1985, 24, 94.

12. Grieco, P. A. Synthesis 1975, 67.

13. Petragnani, N.; Ferraz, H. M. C.; Silva, G. V. J. Synthesis 1986, 157.

14. Kupchan, S. M.; Fessler, D. C.; Eakin, M. A.; Giacobbe, T. J. Science 1970, 168, 376.

15. Sun, C. M.; Syu, W. J.; Don, M. J.; Lu, J. J.; Lee, G. H. J. Nat. Prod. 2003, 66, 1175.

16. Kupchan, S. M.; Hemingway, R. J.; Werner, D.; Karim, A. J. Org. Chem. 1969, 34, 3903.

17. Cui, Y. M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Katsutoshi, I.; Sawada, K.;

Ohwada, T. Bioorg. Med. Chem. Lett. 2008, 18, 5197.

18. Blay, G.; Garcia, B.; Molina, E.; Pedro, J. R. Tetrahedron 2007, 39, 9621.

19. Corey, E. J.; Schmidt, G. Tetrahedron Lett. 1979, 5, 399.

20. Tamura, R.; Watabe, K.; Kamimura, A.; Hori, K.; Yokomori, Y. J. Org. Chem. 1992, 57, 4903.

21. Adam, W.; Carballeira, N.; Peters, E. M.; Peters, K.; Schnering, H. G. J. Am. Chem.

Soc. 1983, 105, 5132.

22. Whitesell, J. K.; Matthews, R. S.; Minton, M. A.; Helbling, A. M. J. Am. Chem. Soc.

1981, 103, 3468.

23. Pohmakotr, M.; Komutkul, T.; Tuchinada, P.; Prabpai, S.; Kongsaeree, P.;

Reutrakul, V. Tetrahedron 2005, 61, 5311.

24. Song, L.; Chen, Q. H.; She, X. K.; Chen, X. G.; Wang, F. P. J. Asian. Nat. Prod. Res.

2011, 13, 787.

25. White, J. D.; Amedio, J. C.; Gut, J. S.; Ohira, S.; Jayasinghe, L. R. J. Org. Chem. 1992, 57, 2271.

26. Tao, J. C.; Tian, G. Q.; Zhang, Y. B.; Fu, Y. Q.; Dai, G. F.; Wu, Y. Chin. Chem. Lett.

2005, 11, 1441.

27. Kretchmer, R. A.; Thompson, W. J. J. Am. Chem. Soc. 1976, 98, 3379.

28. Yang, L. M.; Hsu, F. L.; Chang, S. F.; Cheng, J. T.; Hsu, J. Y.; Hsu, C. Y.; Liu, P. C.;

Lin, S. J. Phytochemistry 2007, 68, 562.

29. Cytotoxicity assay in vitro: The human cancer cell lines were provided by Mr.

Yangchang Wu research group (China Medical University) and maintained in a humidified atmosphere at 37°C in 5% CO2. The cells were cultured in RPMI- 1640 media containing 10% FBS, 100 IU/mL penicillin and 100l^g/mL streptomycin. Cell cytotoxity was determined by MTT assay. Briefly, cells were seeded in 96-well-plate (1 10⁴cells/well) and incubated for 48 h. Then, the tested compounds with various concentrations were added to the wells and 48 h later the MTT solution (0.5 mg/mL) was added and incubated for 4 h.

Two hundred microliters of DMSO was added to each well to dissolve the reduced MTT crystals. The absorbance of each well was measured at 570 nm with a microplate reader.

30. Selected spectral data for compounds 1a–2b: Compound 1a: white solid, mp 172–174°C; IR (KBr, cm¹) 3415, 2952, 2928, 2879, 1731, 1717, 1696, 1629, 1466, 1384, 1370, 1311, 1170, 1136, 1073, 1035, 999, 942;¹H NMR (300 MHz, CDCl3) d: 6.56(s, 1H), 6.07(s, 1H), 5.29(d, J=6 Hz, 1H), 5.16(d, J = 6 Hz, 1H), 3.49(s, 3H), 2.35(d, J = 13.2 Hz, 1H), 2.26(d, J = 13.41 Hz, 1H), 1.98–2.04(m, 3H), 1.86–1.94(m, 4H), 1.43–1.83(m, 6H), 1.26(s, 3H), 0.91–1.21(m, 3H), 0.88(s, 3H);

13C NMR (75 MHz, CDCl3) d: 176.6, 165.4, 143.3, 125.8, 90.5, 84.9, 70.3, 57.9, 56.1, 50.2, 43.9, 42.6, 41.7, 40.9, 39.7, 38.1, 37.4, 28.8, 20.6, 19.1, 18.8, 16.4; ESI/

MS: 393 [M+H]⁺; HRMS: calcd for C22H32NaO6 [M+Na]⁺415.2091, found 415.2102. Compound 1b: white solid, mp 218–220°C; IR (KBr, cm¹) 3539, 3423, 3221, 2957, 2868, 1704, 1630, 1467, 1385, 1370, 1353, 1292, 1264, 1234, 1202, 1164, 1095, 999, 970, 956, 812, 499;¹H NMR (300 MHz, DMSO-d6) d:

12.1(s, 1H), 6.26(d, J = 1.77 Hz, 1H), 5.94(d, J = 1.1 Hz, 1H), 2.20(d, J = 13.38 Hz, 1H), 2.04(d, J = 12.93 Hz, 1H), 1.77–1.90(m, 5H), 1.61–1.75(m, 4H), 1.41–

1.45(m, 1H), 1.33(m, 1H), 1.18–1.27(m, 2H), 1.14(s, 3H), 0.93–1.05(m, 2H), 0.88(s, 3H), 0.74–0.85(m, 1H);¹³C NMR (75 MHz, DMSO-d6) d: 178.5, 164.7, 144.1, 124.6, 84.4, 68.6, 54.9, 50.1, 42.6, 41.6, 41.2, 40.4, 39.2, 38.3, 37.1, 28.4, 20.4, 18.9, 18.5, 15.9; ESI/MS: 331 [M+1–H2O]⁺. Compound 1c: white solid, mp 200202 °C; IR (KBr, cm ¹) 3437, 2954, 2928, 2868, 1721, 1697, 1625, 1456, 1437, 1371, 1307, 1291, 1237, 1202, 1167, 1149, 1118, 1083, 1039, 995, 971;

1H NMR (300 MHz, CDCl3) d: 6.55(s, 1H), 6.07(s, 1H), 3.65(s, 3H), 2.35(d, J = 13.2 Hz, 1H), 2.23(d, J = 13.32 Hz, 1H), 1.96–2.0(m, 2H), 1.85–1.91(m, 4H), 1.76–1.82(m, 2H), 1.62–1.74(m, 2H), 1.25–1.58(m, 4H), 1.21(s, 3H), 1.03–

1.18(m, 1H), 0.93–1.00(m, 1H), 0.85(s, 3H);¹³C NMR (75 MHz, CDCl3) d: 177.6, 165.4, 143.3, 125.8, 85.0, 70.3, 56.0, 51.3, 50.2, 43.6, 42.5, 41.7, 40.9, 39.6, 38.1, 37.5, 28.7, 20.6, 19.1, 18.8, 16.2; ESI/MS: 380 [M+NH4]⁺; HRMS: calcd for C21H30NaO5[M+Na]⁺385.1985, found 385.1996. Compound 1f: colorless oil, IR (KBr, cm ¹) 3427, 2956, 2932, 2871, 1718, 1627, 1455, 1370, 1351, 1275, 1259, 1231, 1207, 1163, 1141, 1092, 999, 996, 806, 755, 745, 699;¹H NMR (300 MHz, CDCl3) d: 7.34(m, 5H), 6.54(s, 1H), 6.05(s, 1H), 5.15(d, J = 12.34 Hz, 1H), 5.04(d, J = 12.33 Hz, 1H), 2.25(d, J = 13.17 Hz, 1H), 1.68–2.00(m, 9H), 1.57–1.63(m, 2H), 1.45–1.55(m, 3H), 1.24(s, 3H), 0.92–1.22(m, 3H), 0.76(s, 3H); ¹³C NMR (75 MHz, CDCl3) d: 176.8, 165.3, 143.3, 135.8, 128.5, 128.3(2), 128.2(2), 125.8, 84.9, 70.4, 66.1, 56.2, 50.1, 43.76, 42.5, 41.7, 40.9, 39.6, 38.1, 37.5, 28.8, 20.6, 19.1, 18.8, 16.3; ESI/MS: 461 [M+Na]⁺. Compound 2a: white solid, mp 152–154°C; IR (KBr, cm ¹) 3433, 2935, 2866, 1717, 1654, 1629, 1458, 1384, 1374, 1307, 1275, 1260, 1241, 1159, 1090, 1036, 1000; ¹H NMR (300 MHz, CDCl3) d: 6.48(s, 1H), 6.00(s, 1H), 4.11(d, J = 11.1 Hz, 1H), 3.83(d, J = 11 Hz, 1H), 2.26(d, J = 13.2 Hz, 1H), 1.98(s, 3H), 1.82–1.94(m, 5H), 1.71–

1.81(m, 2H), 1.58–1.67(m, 2H), 1.36–1.53(m, 4H), 1.26–1.32(m, 1H), 1.15–

1.23(m, 1H), 1.03–1.09(m, 1H), 0.99(s, 3H), 0.93(s, 3H), 0.78–0.90(m, 1H); ESI/

MS: 375 [M H] . 2b: white solid, 92–94°C; IR (KBr, cm¹) 3424, 2961, 2930, 2870, 1701, 1626, 1475, 1446, 1384, 1369, 1348, 1307, 1267, 1174, 1164, 1029, 1000;¹H NMR (300 MHz, DMSO-d6) d: 6.26(s, 1H), 5.94(s, 1H), 3.45–3.51(m, 1H), 3.16–3.22(m, 1H), 2.22(d, J = 13.4 Hz, 1H), 1.80–1.98(m, 4H), 1.67–1.76(m, 4H), 1.42–1.54(m, 3H), 1.23–1.36(m, 3H), 1.18(m, 1H), 1.03–1.15(m, 1H), 0.99(s, 3H), 0.89(s, 3H), 0.83(m, 1H);¹³C NMR (75 MHz, DMSO-d6) d: 164.7, 144.3, 124.5, 84.6, 68.6, 63.0, 55.5, 51.1, 43.5, 41.8, 41.7, 40.1, 39.2, 38.3, 35.1, 27.7, 18.8, 18.4, 18.0, 17.7; ESI/MS: 333 [M H] ; HRMS: calcd for C40H60NaO8

[2M+Na]⁺691.4180, found 691.4207.

Y. Zeng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1922–1925 1925