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received: 23 February 2016 accepted: 18 May 2016 Published: 08 June 2016

Penicimenolides A-F, Resorcylic Acid Lactones from Penicillium sp., isolated from the Rhizosphere Soil of Panax notoginseng Ya-Nan An1,*, Xue Zhang1,*, Tian-Yuan Zhang1, Meng-Yue Zhang1,  Qian-Zhang2, Xiao-Yu Deng1, Feng Zhao2, Ling-Juan Zhu1, Guan Wang1, Jie Zhang1, Yi-Xuan Zhang1, Bo Liu3 & Xin-Sheng Yao1,4 Five new 12-membered resorcylic acid lactone derivatives, penicimenolides A-E (1–5), one new ring-opened resorcylic acid lactone derivative penicimenolide F (6), and six known biogenetically related derivatives (7–12) were isolated from the culture broth of a strain of Penicillium sp. (NO. SYP-F-7919), a fungus obtained from the rhizosphere soil of Panax notoginseng collected from the Yunnan province of China. Their structures were elucidated by extensive NMR analyses, a modified Mosher’s method, chemical derivatization and single crystal X-ray diffraction analysis. Compounds 2–4 exhibited potent cytotoxicity against the U937 and MCF-7 tumour cell lines and showed moderate cytotoxic activity against the SH-SY5Y and SW480 tumour cell lines. The substitution of an acetyloxy or 2-hydroxypropionyloxy group at C-7 significantly increased the cytotoxic activity of the resorcylic acid lactone derivatives. Subsequently, the possible mechanism of compound 2 against MCF-7 cells was preliminarily investigated by in silico analysis and experimental validation, indicating compound 2 may act as a potential MEK/ERK inhibitor. Moreover, proteomics analysis was performed to explore compound 2-regulated concrete mechanism underlying MEK/ERK pathway, which is still need further study in the future. In addition, compounds 2–4 and 7 exhibited a significant inhibitory effect on NO production induced by LPS. Panax notoginseng is widely distributed in Yunnan, Guangxi and Jiangxi province of China and its roots have been used as a traditional Chinese medicine for the treatment of hemorrhages, blood stasis and improvement of blood circulation and remission pain1. In recent years, the investigations regarding the bioactive secondary metabolites from endophytic or rhizospheric fungi of Panax notoginseng have been receiving increasing attention, leading to the various secondary metabolites with antimicrobial2,3, antifungal4, and cytotoxic activities5. During our studies for new natural bioactive constituents from rhizospheric fungus of Panax notoginseng, a strain of Penicillium sp. (SYP-F-7919) has drawn our interest because the EtOAc extract of the culture broth exhibited typical resorcylic acid lactones (RALs) UV absorptions (λmax) at 215, 264 and 297 nm6. RALs are a class of fungal polyketide derivatives that are produced by a variety of fungal strains, such as Lasiodiplodia theobromae 7,8, Penicillium sp. 9, Syncephalastrum racemosum 10, Pyrenophora teres 11 and Acremonium zeae12. Since the first RAL12, lasiodiplodin, was isolated in 197113, approximately 30 natural RAL12 compounds had been obtained6–26, some of which exhibit antibacterial15,16, antifungal12,14, antitumour10,17,18, anti-inflammatory19,20,23, antidiabetic21, and antihypertensive activities22. In our study, five new RAL12 derivatives, 1

Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China. 2School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, People’s Republic of China. 3State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, People’s Republic of China. 4Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.-X.Z. (email: [email protected]) or B. L. (email: [email protected]) or X.-S.Y. (email: [email protected]) Scientific Reports | 6:27396 | DOI: 10.1038/srep27396

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Figure 1.  Chemical structures of compounds 1–12.

penicimenolides A-E (1–5), a new ring-opened resorcylic acid lactone derivative penicimenolide F (6), and six known biogenetically related derivatives (7–12) (Fig. 1) were isolated from the culture broth of the fungus. In the cytotoxicity assay, compounds 2–4 showed potent cytotoxic activity against the U937 and MCF-7 tumour cell lines and displayed moderate cytotoxicity against the SH-SY5Y and SW480 tumour cell lines. Subsequently, the possible mechanism of compound 2 against MCF-7 cells was preliminarily investigated by in silico analysis and experimental validation, indicating compound 2 may act as a potential MEK/ERK inhibitor. Moreover, proteomics analysis was performed to explore compound 2-regulated concrete mechanism underlying MEK/ERK pathway, which is still need further study in the future. In addition, compounds 2–4 and 7 exhibited a significant inhibitory effect on the production of nitric oxide (NO) in murine macrophages (RAW 264.7) activated by lipopolysaccharide (LPS). Herein, we report the isolation, structure elucidation, absolute configuration, bioactivities and preliminary mechanism of the compounds obtained from the Penicillium sp. SYP-F-7919.

Results and Discussion

Structural elucidation of resorcylic acid lactone derivatives.  The ethyl acetate extract of the culture broth of the fungus Penicillium sp. was isolated by a combination of column chromatography, including silica gel, ODS, Sephadex LH-20, and reversed phase high performance liquid chromatography (HPLC) to yield twelve resorcylic acid lactone derivatives (1–12). Penicimenolide A (1) was isolated as colourless needles, [α]25 D  + 68.1 (c 0.5, MeOH). Its molecular formula was determined to be C16H18O5 by HRESIMS at m/z 291.1231 [M + H]+ (calcd. for C16H19O5, 291.1232). The IR spectrum of 1 revealed the presence of hydroxyl group(s) at 3384 cm−1, carbonyl group(s) at 1708 and 1642 cm−1 and an aromatic ring at 1605 and 1449 cm−1. A comparison of the 1H and 13C NMR spectroscopic data (Table 1) for 1 with those of 8 showed that the two compounds possessed a similar structure, except for the loss of two methylenes and the appearance of a pair of olefinic signals in 1. The coupling constant (J6,7 = 15.5 Hz) indicated an E configuration for the double bond. The position of the double bond was confirmed by the 1H-1H COSY correlations of H-6/H-5 and H-7/H-8 (Fig. 2). Because the absolute configuration at C-3 in 10 was identified to be R based on the X-ray diffraction analysis (Cu Ka) (Fig. 3), the asymmetric carbon atom C-3 in the isolated compounds (except for 6) was proposed to be an R configuration because of a shared biogenesis. For compound 1, this conclusion was further confirmed by comparing the optical rotation value with 8 ([α]25 D  + 40.7). Based on the above evidence, the structure of 1 was identified to be (3R), (6E)-etheno-9-oxo-de-O-methyllasiodiplodin. Penicimenolide B (2) was obtained as colourless needles, [α]25 D  + 39.8 (c 0.5, MeOH). The molecular formula C18H22O7 was confirmed by HRESIMS at m/z 351.1435 [M + H]+ (calcd. for C18H23O7, 351.1444). Except for an additional acetyl group, the 1H and 13C NMR data (Table 1) for 2 were similar to those of 12. The HMBC correlation between H-7 and the carbonyl carbon of the acetyl group at δC 172.2 suggested that 7-OH was acetylated (Fig. 2). To confirm the absolute configuration of C-7 in 2, both compounds 2 and 12 were reacted with acetic anhydride and pyridine. Because of the same retention time in the HPLC analysis and the similar 1H NMR data of the diacetylated derivative 2a and the triacetylated derivative 12a (see Supplementary Fig. S1), the absolute configuration of C-7 was confirmed to be R. Therefore, compound 2 was established to be (3R,7R)-7-acetoxyl9-oxo-de-O-methyllasiodiplodin. Penicimenolide C (3) was obtained as yellow needles, [α]25 D +15.4 (c 0.5, MeOH) and was determined to have a molecular formula of C19H24O8 on the basis of HRESIMS analysis (m/z 381.1549 [M + H]+, calcd. for C19H25O8, 381.1549). The 1H and 13C NMR data for 3 (Table 1) were similar to those of 2, except for an additional oxygenated methine (δH 4.21, δC 68.1). The HMBC correlations from H-7 (δH 5.51), H-2′ (δH 4.21) and H3-3′ (δH 1.35) to C-1′ Scientific Reports | 6:27396 | DOI: 10.1038/srep27396

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2

δC

δH

172.3 (C)

δC

3 δH

172.4 (C)

δC

δH

172.4 (C)

3

74.8 (CH)

5.37 m

74.1 (CH)

5.16 m

74.1 (CH)

5.16 m

4

34.7 (CH2)

1.96 m;

34.0 (CH2)

1.65 m;

33.9 (CH2)

1.68 m;

5

29.7 (CH2)

2.42 m;

19.2 (CH2)

1.56 m;

19.2 (CH2)

1.57 m;

6

138.5 (CH)

5.60 dt (15.5, 6.9)

33.5 (CH2)

1.88 m;

33.4 (CH2)

1.89 m;

7

122.5 (CH)

5.52 dt (15.5, 6.7)

71.6 (CH)

5.45 m

72.1 (CH)

5.51 m

8

46.2 (CH2)

2.99 m;

47.9 ( CH2)

3.09 dd (15.9, 10.4);

47.7 ( CH2)

3.14 dd (15.9, 10.4);

1.85 m

1.71 m

2.21 m

1.71 m

1.58 m

1.59 m

1.56 m

2.99 m 9

208.2 (C)

10

47.4 (CH2)

2.68 dd (15.9, 1.4) 206.7 (C)

4.37 d (17.0);

52.9 (CH2)

3.97 d (17.0) 11

139.1 (C)

12

113.7 (CH)

13

163.2 (C)

14

103.1 (CH)

15

165.5 (C)

16

107.9 (C)

17

19.3 (CH3)

4.55 d (18.7);

113.9 (CH) 103.2 (CH)

6.11 d (2.5)

1′

172.2 (C)

2′

21.3 (CH3)

3′

3.85 d (18.8) 113.9 (CH)

6.11 d (2.4)

164.1 (C) 6.24 d (2.5)

103.2 (CH)

6.25 d (2.4)

167.0 (C)

106.5 (C) 18.9 (CH3)

4.56 d (18.8);

139.9 (C)

167.0 (C) 1.34 d (6.5)

53.0 (CH2)

3.85 d (18.7)

164.1 (C) 6.23 d (2.5)

2.70 dd (15.9, 1.3) 206.6 (C)

139.9 (C) 6.12 d (2.5)

1.61 m

106.5 (C) 1.28 d (6.4)

18.9 (CH3)

1.29 d (6.4)

175.1 (C) 2.01 s

68.1 (CH)

4.21 dd (13.8, 6.9)

20.7 (CH3)

1.35 d (6.9)

Table 1.  1H (600 MHz) and 13C NMR (150 MHz) data of 1–3 in CD3OD (J in Hz).

Figure 2.  Key 1H-1H COSY (bold lines) and HMBC (arrows) correlations of 1–3, 5 and 6.

(δC 175.1), in combination with the 1H-1H COSY correlation between H3-3′ and H-2′, revealed the presence of a 2′-hydroxypropionyloxy fragment attached to C-7 (Fig. 2). Accordingly, a planar structure was proposed for 3. The absolute configuration of C-2′ in 3 was established using the modified Mosher method27. Compound 3 was treated separately with (R)- and (S)-MTPA-Cl to obtained the respective (S)- and (R)-MTPA esters (3a and 3b). The R configuration of C-2′ was determined by the positive ΔδH(S-R) value of H3-3´ and the negative ΔδH(S-R) value of H-7 (see Fig. 4 and Supplementary Table S2). In addition, the absolute configuration of C-7 was established by alkaline hydrolysis and HPLC analysis. Because of the same retention time of the alkaline hydrolysis product 3c 25 ([α]25 D  − 58.2) and the known compound 12 ([α]D  − 78.6), the configuration of C-7 was confirmed to be R. Based Scientific Reports | 6:27396 | DOI: 10.1038/srep27396

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Figure 3.  X-ray crystallographic structure of compound 10.

Figure 4.  Δδ Values (δa-δb in ppm) obtained for the MTPA esters of compounds 3, 5 and 6. on the above evidence, the structure of 3 was determined to be (3R,7R,2′R)-7-(2′-hydroxypropionyloxy)-9-oxode-O-methyllasiodiplodin. Penicimenolide D (4) was isolated as a yellowish oil, [α]25 D  + 31.6 (c 0.5, MeOH). The HRESIMS of 4 (m/z 381.1548 ([M + H]+, calcd. for C19H25O8, 381.1549) showed the same molecular formula as 3. The 1H and 13C NMR spectroscopic data for 4 (Table 2) were very similar to those for 3. The analyses of the 1H-1H COSY, HSQC, and HMBC spectra of 4 indicated that the compound had an identical planar structure to that of 3. A detailed comparison of the 13C NMR data for compounds 3 and 4 highlighted the differences in the chemical shifts at C-5, C-6, C-7 and C-8, suggesting that compound 4 is a C-7 epimer of 3. Thus, the structure of 4 was determined to be (3R,7S,2′R)-7-(2′-hydroxypropionyloxy)-9-oxo-de-O-methyllasiodiplodin. To compare computed electronic circular dichroism (ECD) with experimental results is a valid method to assign absolute configurations of natural products28–32. Thus, the absolute configurations of 3 and 4 were confirmed by quantum chemical TDDFT calculations of theirs ECD spectra. Conformational searches were performed using MMFF94S force field for 3 and 4. All geometries (33 lowest energy conformers for 3 and 98 for 4, respectively) with relative energy from 0-18 kcal/mol used in optimizations at the B3LYP/6-31G(d) level using Gaussian09 package33. The B3LYP/6-31G(d)-optimized conformers (32 lowest energy conformers for 3) with relative energy from 0 to 10 kcal/mol and conformers (28 lowest energy conformers for 4) with relative energy from 0 to 10 kcal/mol were then re-optimized at the B3LYP/6-311+G(d) level. ECD computations for all conformers were carried out at the B3LYP/6-311++G(2d,p) level in the gas phase. Boltzmann statistics were performed for ECD simulations with standard deviation of σ 0.22 eV. The computed ECD of 3 and 4 were looked similar to the experimental values of 3 and 4, respectively (Fig. 5). Thus, the absolute configurations of 3 and 4 were further confirmed. Penicimenolide E (5) was obtained as a yellowish oil, [α]25 D  − 57.8 (c 0.25, MeOH). Its molecular formula was confirmed to be C16H20O5, which was deduced by the HRESIMS (m/z 293.1385 [M + H]+ calcd. for C16H21O5, 293.1389). The detailed analysis of the 1H and 13C NMR (Table 2) spectroscopic data revealed that 5 possessed a similar structure to trans-resorcylide6, and the only difference was the replacement of the ketone carbonyl at C-9 (δC 201.7) in trans-resorcylide by an oxygenated methine (δC 74.9) in 5, which was further proven by the HMBC correlations from H-7, H-8, and H-10 to C-9 (Fig. 2). To confirm the absolute configuration of C-9 in 5, a modified Mosher’s method27 was carried out to produce (S)- and (R)-MTPA esters (5a and 5b). The negative ΔδH(S-R) value of H-10 and the positive ΔδH(S-R) value of H3-17, H-4, H-5 and H-6 unambiguously confirmed the S configuration of C-9 (see Fig. 4 and Supplementary Table S3). Based on the above evidence, the structure of compound 5 was confirmed to be (3R,9S)-(7E)-etheno-9-hydroxy-de-O-methyllasiodiplodin. Penicimenolide F (6) was isolated as a yellowish oil, [α]25 D  − 4.6 (c 0.25, MeOH). The HRESIMS exhibited a quasimolecular ion peak at m/z 285.0971 [M + H]+ (calcd. for C13H17O7, 285.0974), indicating a molecular Scientific Reports | 6:27396 | DOI: 10.1038/srep27396

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5

δC

δH

172.6 (C)

δC

6 δH

170.8 (C)

δC

2

105.9 (C)

3

76.1 (CH)

4.96 m

73.0 (CH)

5.08 m

166.4 (C)

4

33.8 (CH2)

1.85 m;

33.4 (CH2)

1.93 m;

101.9 (CH)

1.76 m 5

21.6 (CH2)

1.66 m;

34.0 (CH2)

1.74 m;

6.15 d (2.4)

1.66 m 22.5 (CH2)

1.42 m 6

δH

173.0 (C)

1.70 m;

164.1 (C)

1.60 m 31.4 (CH2)

1.69 m

2.15 m;

112.7 (CH)

6.19 d (2.4)

1.99 m

7

70.8 (CH)

5.52 m

129.8 (CH)

5.49 dt (15.7, 6.3)

144.6 (C)

8

50.1 (CH2)

2.99 dd (13.4, 3.0);

133.4 (CH)

5.35 dt (15.7, 5.2)

24.6 (CH3)

2.45 s

2.63 dd (13.4, 10.3) 9

207.3 (C)

10

51.0 (CH2)

11

139.6 (C)

4.77 d (18.5);

74.9 (CH)

4.05 m

43.6 (CH2)

3.11 d (18.8);

3.77 d (18.5) 12

114.1 (CH)

13

163.9 (C)

14

103.1 (CH)

15

166.4 (C)

2.82 d (18.8) 140.8 (C)

6.13 d (2.5)

111.1 (CH)

6.25 d (2.4)

161.2 (C) 6.25 d (2.5)

102.3 (CH)

6.19 d (2.4)

NDa

16

106.9 (C)

17

21.2 (CH3)

115.1 (C)

1′

176.0 (C)

62.4 (CH2)

4.47 m

2′

68.1 (CH)

4.25 dd (13.8, 6.9)

34.4 (CH2)

2.24 m;

3′

20.6 (CH3)

1.37 d (6.9)

68.9 (CH)

4.32 m

1.32 d (6.1)

19.0 (CH3)

1.31 d (6.4)

2.06 m 4′

176.1 (C)

5′

52.7 (CH3)

3.68 s

Table 2.  1H (600 MHz) and 13C NMR (150 MHz) data of 4–6 in CD3OD (J in Hz). aDesignate signal not detected.

Figure 5.  Calculated ECD spectra and experimental CD spectra of 3 and 4.

formula of C13H16O7. The 1H NMR spectrum of 6 (Table 2) showed two meta-coupled aromatic protons at δH 6.15 ( d, J = 2.4 Hz, H-4) and 6.19 (d, J = 2.4 Hz, H-6), two methyl signals at δH 3.68 (s, H3-5′) and 2.45 (s, H3-8), four methylene protons at δH 4.47 (m, H2-1′) and 2.06/2.24 (m, H-2′), and one methine proton at δH 4.32 (m, H-3′). The 13C NMR and DEPT spectra revealed the presence of 13 carbon resonances, including two ester carbonyls (δC 173.0, 176.1), six aromatic carbons, two methyls (one oxygenated), two methylenes (one oxygenated) and one oxygenated methine. The HMBC correlations (Fig. 2) from H-4 to C-1, C-2, C-3, C-5, and C-6, from H-6 to C-1, C-2, C-4, C-5, and C-8, and from H-8 to C-1, C-2, C-3, C-6, and C-7, combined with the molecular formula and the 13C NMR data, revealed the presence of a benzoyloxy fragment with a methyl group located at C-7 and two hydroxyl groups at C-3 and C-5, respectively. The 1H-1H COSY correlations between H-1′, H-3′ and H-2′, together with HMBC correlations from H3-5′ to C-3′ and C-4′ and from H-3′ to C-1′, C-2′, and C-4′, suggested the presence of a 3′-hydroxy-4′-oxo-4′-methyloxy-butyl fragment, which was attached to the benzoyloxy fragment, according to the correlation between H-1′ and C-1 in the HMBC spectrum. Additionally, the absolute configuration of C-3′ was established by a modified Mosher’s method27. The differences in the 1H-NMR chemical shifts between (S)- and (R)-MTPA esters (ΔδH(S-R)) at H-1´, H-2´ and H3-5´ were analyzed to confirm the Scientific Reports | 6:27396 | DOI: 10.1038/srep27396

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MCF-7

U937

SH-SY5Y

HepG2

SW480

A549

9.89 ± 0.41

1.38 ± 0.18

44.41 ± 1.42

>100

22.66 ± 1.22

>100

3

11.59 ± 0.23

6.49 ± 0.41

47.62 ± 0.81

>100

24.82 ± 1.85

>100

4

10.47 ± 0.44

2.19 ± 0.074

73.47 ± 1.62

>100

41.18 ± 2.3

>100

0.0152

0.0553

0.454

1.562

1.845

1.512

Taxol

Table 3.  Cytotoxicity of compounds 2–4 against human tumour cell lines.

absolute configuration of C-3´ as R (see Fig. 4 and Supplementary Table S4). Thus, the structure of compound 6 was determined to be (3′R)-3′-hydroxy-4′-oxo-4′-methoxy-orsellinate. In addition, six known compounds were isolated and identified as cis-resorcylide (7)6, dihydroresorcylide (8)24, (13S, 14R)-13-hydroxydihydroresorcylide (9)25, (11S)- methoxyresorcylide (10)6, (11R)-methoxyresorcylide (11)6 and 7-hydroxydihydroresorcylide (12)6 by a comparison of the observed and published data.

Cytotoxicity of compounds against human tumour cell lines.  Most of the isolated compounds (2–5,

7–12) were evaluated for their cytotoxic activity against six human tumour cell lines, including U937, MCF7, A549, SH-SY5Y, HepG2 and SW480 (Table 3). Taxol, a well-known anticancer drug, was used as a positive control. Compounds 2-4 exhibited potent cytotoxicity against the U937 and MCF-7 cell lines, with IC50 values ranging from 1.4 to 11.6 μM and showed moderate cytotoxic activity against the SH-SY5Y and SW480 tumour cell lines. In contrast, compounds 5 and 7–12 were inactive against the six tested cell lines. The above data indicated that the substitution of an acetyloxy or a 2-hydroxypropionyloxy group at C-7 significantly increased the cytotoxic activity of the resorcylic acid lactone derivatives.

Molecular docking of compound 2 with MEK1 and ERK1.  Molecular docking is a widely used com-

putational approach to screen potential active structures based on their interactions with the binding site34. The predicted results obtained from SEA showed that our candidate compounds potentially chose MEK1 or ERK1 as a target. In this study, compound 2 was selected as a potential MEK1-ERK1 inhibitor, based upon its favorable “-CDOCKER ENERGY” scores. In order to understand the conformation of compound 2 in the ATP binding site of MEK1 and ERK1, molecular docking analysis and MD simulations were conducted to evaluate the binding affinity of compound 2 and MEK1, as well as ERK1. After 3ns MD simulations, one frame (time of 3000ps) of the equalized trajectory was extracted, we found that compound 2 could bind to ATP binding domain of MEK1 and ERK1 stably. Compound 2 could form hydrogen bonds with residues Ala76, Asn78, Lys97, Gly210 and Val211 of the MEK1 (Fig. 6A). For ERK1, the hydrogen bonds were found between compound 2 and residue Lys71 and Asp123 of the ERK1, respectively (Fig. 6B). Moreover, we found compound 2 can form hydrophobic interactions in the hydrophobic pocket of MEK1 with lipophilic residue Val82. For ERK1 protein, the hydrophobic interactions were also formed with lipophilic residues Val56, Ala69, Met125 and Leu173. Altogether, these results suggest that compound 2 exhibits efficient binding to ATP binding domain of MEK1 and ERK1, which may support its inhibitory effect against MEK1 and ERK1. Root mean square deviation (RMSD) fluctuation was an important criterion to gauge whether the protein-ligand system was stable. For MEK1-compound 2 system, RMSD of the complex were process about 2ns equilibrium during the whole simulation (Fig. 6C). During the process of MD simulation, RMSD of ERK1-compound 2 complex were limited to 0.3 nm, indicating a well-balanced system (Fig. 6D). Notably, we found RMSD of compound 2 in MEK1 protein remains stable after 800ps simulation, and then the RMSD fluctuations were narrow, indicating a stable binding in the ATP binding domain of MEK1 protein (Fig. 6C). The similar situation was also found in ERK1-compound 2 system, in which RMSD of compound 2 reached its dynamic equilibrium after 800ps simulation (Fig. 6D).

Compound 2 induces apoptosis through MEK/ERK pathway in MCF-7 cells.  In vitro experiments were employed with MCF-7 cells to elucidate the possible mechanism of compound 2, as ERK are often excessively activated in breast cancer35. Interestingly, we found that compound 2 demonstrated potent cytotoxic effect against MCF-7 cells in a time- and concentration-dependent manner, with an IC50 value of 9.893 μM (24 h) (Fig. 7A). In order to determine features of cell death induced by compound 2, the morphologic changes of cells were examined by phase-contrast microscope. Compared with control group, compound 2 caused remarkable morphologic changes such as membrane blebbing and apoptotic bodies (Fig. 7B). These changes were further confirmed by Hoechst 33258 staining. The cells in control group showed uniform dispersion of low-density fluorescence, but compound 2 treated cells showed condensed, bright fluorescence and nuclear fragmentation (Fig. 7C). In addition, apoptotic ratio was further evaluated by Annexin-V/PI double staining. We found the early apoptotic cells ratio was increased from 1.68 ± 0.25% to 10.81 ± 2.47% (p