Bioorganic & Medicinal Chemistry Letters 25 (2015) 1823–1826

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New cytochalasins from medicinal macrofungus Crodyceps taii and their inhibitory activities against human cancer cells Xiao-Gang Li a,b, , Wei-Dong Pan a,b, , Hua-Yong Lou b, Ru-Ming Liu a, Jian-Hui Xiao a,⇑, Jian-Jiang Zhong c,⇑ a

Division of Microbial Resources & Drug Development, Center for Translational Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, China The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, China c State Key Laboratory of Microbial Metabolism, and School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China b

a r t i c l e

i n f o

Article history: Received 18 November 2014 Revised 3 March 2015 Accepted 20 March 2015 Available online 27 March 2015 Keywords: Cytochalasin Cordyceps taii Secondary metabolite Isolation Antitumor activity

a b s t r a c t Three new cytochalasins (1–3) together with two known cytochalasin analogues (4 and 5) were isolated from the chloroform fraction of ethanolic extract of a medicinal macrofungus Cordyceps taii. The structures of the new compounds were elucidated on the basis of spectroscopic analysis, including HRESIMS, 1D and 2D NMR experiments. The cytotoxicities of Compounds 1–5 were investigated by the sulforhodamine B (SRB) method in vitro against human highly metastatic lung cancer cell 95-D, human lung cancer cell line A-549 and normal hepatocyte HL-7702. The results revealed that Compounds 4 and 5 showed potent antitumor activities against human lung cancer cell 95-D with IC50 value of 3.67 and 4.04 lM, respectively. In comparison with cisplatin, the first-line chemotherapy drug, they had little or no cytotoxicity on normal cells, but showed stronger cytotoxic effects on cancer cells 95-D. Ó 2015 Elsevier Ltd. All rights reserved.

The fungi of Cordyceps, mainly parasitizing on the larvae of insects or/and arthropods, have been used as famous traditional Chinese medicines or tonics for hundreds of years and have received increasing interest around the world in recent years.1–4 Interestingly, various potential anticancer compounds have been found in different Cordyceps species over the past two decades.2,5,6 Cordyceps taii, a folk medicinal macrofungus, is a new species of the genus Cordyceps discovered in Guizhou province of China in the 1990s.7 Our previous studies showed that C. taii possessed various important pharmacological activities such as immunomodulatory, antiaging, antioxidant and antimicrobial effects.8–10 During an ongoing program for screening antitumor secondary metabolites from Cordyceps fungi, it was found that the chloroform fraction of the ethanolic extract of C. taii showed potent anticancer activities in vitro and in vivo.11 As a further study for the antitumor active compounds from C. taii, three new cytochalasins (Compounds 1–3) and two known analogues (Compounds 4 and 5) were isolated from the chloroform fraction of C. taii. 12 The structures of the known Compounds 4 (Deacetylcytochalasin C) and 5 (Zygosporin D) (Fig. 1) were identified by comparing their spectroscopic data with reported values.13 Details of the isolation,

⇑ Corresponding authors. Tel.: +86 851 28609278 (J-.H.X.). E-mail addresses: [email protected], [email protected] [email protected] (J.-J. Zhong).   Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2015.03.059 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

(J.-H.

Xiao),

structural elucidation of all these compounds, and their in vitro antitumor activities against tumor cells are reported herein. Compound 114 was obtained as colorless needle crystals (CHCl3/ MeOH). The molecular formula C28H33NO4 was fixed by HR-ESI-MS (neg.) at m/z 446.2332 [MH], with 13° of unsaturation. The 1H NMR spectrum of 1 (Table 1) exhibited a signal of a single-substituted benzene at dH 7.28 (5H, m). Moreover, the signals of two methyls at dH 1.55 (3H, s) and dH 1.75 (3H, s) suggested both of them were connected to a quaternary carbon, respectively. The 13 C NMR and DEPT spectra of 1 revealed 28 carbon signals, including four methyls, two methylenes, fifteen methines and seven quaternary carbons. Among them, signals at dC 199.8 and dC 210.4 could be assigned to typical carbonylic carbons, and signal at dC 176.2 could be assigned to a typical acylamide carbon. In addition, signals at dC 125.1, 138.3, 148.7, 131.6, 129.4 and 133.6 suggested that there were three double bonds. The structure of 1 was further determined by the interpretation of the 2D NMR data, including 1 H–1H COSY, HMQC, HMBC, and NOESY spectra. In the COSY spectrum, correlations of H-3/H-4/H-10, H-8/H-13/H-14/H-15/H16/H-22, and H-18/H-19/H-20/21/H-23 suggested the presence of spin-systems of C-3, 4 to C-10 and C-8, 13, 14, 15, 16 to C-22 and C-18, 19, 20, 21 to C-23. In the HMBC spectrum, correlations from H-3 (dH 3.77, m), H-10 (dH 3.13) to C-10 revealed the connection of the phenyl to C-3 via C-10, correlations also displayed H-10 (dH 3.13), H-4 (dH 3.82) to C-3, and H-4 (dH 3.82), H-8 (dH 4.22), H-21 (dH 5.14) to C-9. Moreover, other correlations between H-12 (dH 1.75), H-8 (dH 4.22), and a carbonyl group (dC 199.8)

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12 11

1'

10

H 3 2

HN

5 4 1

6 7 8

9

21

O H 13 19

20

O OH

H

14

18

22

17

23

HN

HN

O

HN

O OH

O

HN OH O

HN

OH

O

OH H

O OH

OH O

Table 1 The NMR (400 MHz) data for Compounds 1–3a 2

3

dC

dH (multi, J in Hz)

dC

dH (multi, J in Hz)

dC

1 2 3 4 5 6 7

– 1.97 (m) 3.77 (m) 3.82 (br s) – – –

176.2 – 59.9 51.7 148.7 131.6 199.8

– 1.91 (m) 3.35 (m) 3.05 (br s) –

175.8 – 60.0 49.8 126.7 131.5 68.1

– 2.18 3.21 2.52 2.50

5.28 (br s)

176.0 – 55.4 53.5 35.3 137.9 127.3

8

4.22 (d, 9.6)

51.8

48.9

3.10 (d, 9.6)

41.4

9 10

– 3.13 (dd, 13.2, 6.8) 3.20 (dd, 13.2, 8.0) 1.55 (s) 1.75 (s) 5.44 (m) 6.85 (dd, 16.0, 10.0) 2.02 (m) 2.68 (m) 2.69 (m) – 3.43 (m) – 5.72 (m)

55.1 45.9

20 21 21-OH 22 23 10 20 , 60 40 30 , 50 a

1 dH (multi, J in Hz)

16 17 18 18-OH 19

23

16

22

18 17

3

HN

O

7

4

8 21 20

14 15

13 19

O OH

16

22

18 23

O

2

3

HN O OH

Figure 1. Chemical structures of Compounds 1–5 from the medicinal macrofungus C. taii.

15

O OH

19

15

10

5 (Zygosporin D)

11 12 13 14

21 20

10

12

6 7 14

4

9

8

21 20 OOH

13

15 16

22

19

OH

O

3 Figure 2. COSY (bold) and key HMBC (arrows) correlations for Compounds 1–3.

H

Position

8

OH 14

5

4 (Deacetylcytochalasin C)

3

9

13

11

OH H

H

H

4

1

2

O OH

7 3

1'

1

H

10

15 16

12

O

OH H

6.50 (br d, 16.0) 5.14 (br s) 5.08 (br s) 1.08 (d, 6.0) 1.24 (d, 7.2) 7.20–7.30 (m)

18.6 12.1 129.4 133.6 38.6 42.8 210.4 51.7 – 125.1 138.3 73.9 – 19.7 16.2 138.7 130.0 127.4 129.1

3.79 (br d, 10.0) 2.34 (dd, 10.4, 10.0) 2.83 (dd, 13.2, 9.2) 2.98 (dd, 13.2, 5.6) 1.54 (s) 1.70 (s) 5.21 (m) 5.92 (dd, 15.6, 10.0) 1.92 (m) 2.45 (m) 2.60 (m) 3.27 (m) – 5.12 (m) 6.04 (br d, 16.4) 4.44 (br s) 5.63 (br s) 1.14 (d, 7.2) 1.31 (d, 7.2) 7.20–7.30 (m)

53.6 45.3

13.9 16.2 127.1 131.0 37.2 43.1 210.2 50.7 – 124.4 137.3 74.7 – 17.2 19.7 138.6 128.9 134.7 129.1

(m) (m) (br s) (m)

2.54 (dd, 13.6, 8.8) 2.94 (dd, 13.6, 3.6) 1.22 (d, 7.2) 1.75 (s) 5.09 (m) 5.78 (dd, 16.0, 10.0) 1.96 (m) 2.42 (m) 2.71 (m) – 4.74 (s) 5.41 (dd, 16.0, 2.0) 6.22 (dd, 16.0, 2.4) 4.11 (br s) 5.52 (br s) 1.17 (d, 6.8) 1.53 (s) 7.10–7.30 (m)

1 was measured in Pyridine-d5, 2 and 3 were measured in CDCl3.

58.7 45.5

13.8 19.8 132.5 130.7 37.9 42.3 210.8 77.2 – 126.1 137.8 75.9 – 19.3 24.2 137.9 129.2 127.1 128.8

indicated the presence of a carbonyl group at C-7. And the correlations from H-23 (dH 1.24) to C-18 (dC 51.7), C-17 (dC 210.4), C-19 (dC 125.1), and H-21 (dH 5.14) to C-9 (dC 55.1) indicated a methyl connected to C-18, and a hydroxy connected to C-21, respectively (Fig. 2). All the information implied that Compound 1 was a cytochalasin type compound similar to Deacetylcytochalasin C6 (Fig. 1). The NOE correlations of H-4/H-8, H-4/21-OH, H-16/23-CH3 in the NOESY experiment showed they were co-facial (Fig. 3), and the CD spectrum of 1 had a high degree of similarity with Deacetylcytochalasin C (4),17 which further established the absolute configuration of 1 as shown in Figure 1. To the best of our knowledge, this is a new cytochalasin compound. Compound 215 was obtained as colorless lump crystals (CHCl3/ MeOH). The molecular formula was established as C28H35NO4 by HR-ESI-MS at m/z 450.2635 [M+H]+, with 12° of unsaturation. Consistent with its molecular formula, 28 carbon resonances were observed in the 13C NMR spectrum and were categorized by DEPT experiment into four primary carbons, two secondary carbons, sixteen tertiary carbons and six quaternary carbons. The 1H (Table 1) and 13C NMR spectra data of 2 were similar to 1, except for carbonyl group signal for 1 signal at (dC 199.8), and hydroxy group for 2 signal at [(dH 3.79, CH, d, J = 9.1 Hz), dC 68.1)], respectively. The COSY spectrum showed correlations of H-3/H-4/H-10, H-7/H8/H-13/H-14/H-15/H-16/H-22, and H-18/H-19/H-20/21/H-23. The HMBC spectrum showed correlations from H-7 (dH 3.79) to C-12 (dC 16.2), C-8 (dC 48.9), C-13 (dC 127.1) suggesting a hydroxyl group linked to C-7 (Fig. 2). The relative structure of 2 was determined by NOE correlations of H-3/H-4, H-4/H-8, H-16/23-CH3, and H-3/21OH indicted they were syn oriented (Fig. 3). Moreover, the CD spectrum of 2 was recorded, which was very similar to that of Deacetylcytochalasin C (4).17 The absolute structure of 2, therefore, was determined as a new natural compound (Fig. 1). Compound 316 was obtained as amorphous flavescent powder. The molecular formula was determined as C28H35NO4 by HR-ESIMS (neg.) at m/z 448.2489 [MH], with 12° of unsaturation. The 1 H NMR spectrum of 3 (Table 1) exhibited four methyls (two singlets and two doublets) at dH 1.22 (3H, d, J = 7.2 Hz), dH 1.75 (3H, s), dH 1.17 (3H, d, J = 6.8 Hz), and dH 1.53 (3H, s). The 13C NMR and DEPT spectra of 3 revealed 28 carbon signals, including four methyls, two methylenes, sixteen methines and six quaternary carbons. Signals at dC 176.0 and dC 210.8 indicated there were only two carbonyl groups with six resonances assignable to olefinic carbons (dC 137.9, 127.3, 132.5, 130.7, 126.1, and 137.8) in the 13C NMR. The above results implied that this compound was an analogue of Compound 1. In the 13C NMR spectrum, signal at dC 77.2 was assigned to a quaternary carbon and chemical shift changed increase than 1 (dC 51.7) illustrated linkage with a carbonyl group to C-18. In the COSY spectrum, the correlations of H-10/H-3/H-4/

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H HN

8

4

O H

H

H

4

8

OH H

3 16

21

O OH

23

HN

H H O

1

H

4

H O

H

8

3

HN

23

2

11

H

16

21

O OH

16

21

O OH

18

H

OH O

3 Figure 3. Selected NOESY correlations (double arrows) for Compounds 1–3.

Table 2 Cytotoxicity of Compounds 1–5 after 48 h of treatment by the SRB method (mean ± s.d., n = 3) IC50(lM)

Cell lines

95-D A-549 HL-7702 MRC-5 * #

1

2

3

4

5

Cisplatin

23.67 ± 2.36 32.28 ± 3.02 29.84 ± 3.15 >200

26.03 ± 2.84 17.13 ± 2.23 35.68 ± 4.55 >200

20.69 ± 1.82 19.92 ± 2.50 30.85 ± 2.92 >200

3.67 ± 1.45*,# 13.62 ± 1.13 31.08 ± 3.68 >200

4.04 ± 1.98*,# 16.72 ± 1.35 30.18 ± 2.59 >200

8.27 ± 0.86* 2.96 ± 0.39* 4.94 ± 0.24* 22.54 ± 6.25

P