Cytotoxic labdane-type diterpenes from Hedychium longipetalum inhibiting production of nitric oxide

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4572–4575

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Cytotoxic labdane-type diterpenes from Hedychium longipetalum inhibiting production of nitric oxide Hongmei Zhao a,b,y, Guangzhi Zeng c,y, Simeng Zhao c, Junju Xu a,⇑, Lingmei Kong c, Yan Li c, Ninghua Tan c, Shengchao Yang a,⇑ a b c

Yunnan Research Center on Good Agricultural Practice for Dominant Chinese Medicinal Materials, Yunnan Agricultural University, Kunming 650201, PR China School of Pharmacy, Yunnan University of Traditional Chinese Medicine, Kunming 650500, PR China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China

a r t i c l e

i n f o

Article history: Received 22 May 2015 Revised 5 August 2015 Accepted 21 August 2015 Available online 21 August 2015 Keywords: Zingiberaceae Hedychium longipetalum Labdane diterpenes Inhibition of NO production Cytotoxicity

a b s t r a c t Three new labdane diterpenes, hedylongnoids A (1), B (2) and C (3), were isolated from the rhizomes of Hedychium longipetalum, together with three known ones yunnancoronarin A (4), hedyforrestin C (5) and hedyforrestin B (6). Their structures were established by spectroscopic analysis, including 2D-NMR spectroscopic techniques. Compounds 1–6 exhibited inhibitory effects against nitric oxide (NO) production in LPS and IFN-c-induced RAW 264.7 murine macrophages with IC50 values ranging from 0.56 to 7.50 lg/ ml, and 3–6 showed cytotoxicities against cancer cell lines SGC-7901 and Hela with IC50 values ranging from 6.21 to 14.53 lg/ml and from 6.58 to 14.83 lg/ml, respectively. Ó 2015 Elsevier Ltd. All rights reserved.

The Hedychium (Zingibereae) genus comprises approximately 50 species, distributing in tropical areas of the world, and 21 of which grow in south of China.1,2 The species of Hedychium are known for their strong aromatic odor and used as hot natured drugs in traditional Chinese medicine.3 Some species, especially Hedychium spictatum and Hedychium coronarium, have been widely used as medicinal plants to treat liver diseases and stomach ailments such as pain, indigestion, swelling, diarrhea, hernia and so on.3,4 Previous chemical investigation on the constituents of them revealed the presence of diterpenes, sesquiterpenes, diarylheptanoids,5–7 some of which showed cytotoxic,3–5 anti-angiogenic,6 a-glucosidase inhibitory,7 antiinflammatory activities.8 However, Hedychium longipetalum was a new species found from Yunnan province of China in 2010,9 and no data were reported about its secondary metabolites and pharmacological activities. In order to reveal the constituents and their biological activities, we carried out a phytochemical study on this plant, and three new labdane diterpenoids, hedylongnoids A (1), B (2) and C (3), were isolated from the rhizomes of Hedychium longipetalum, along with three known diterpenes yunnancoronarin A (4),10 hedyforrestin C (5),11 hedyforrestin B (6) (Fig. 1).10 Moreover, all compounds were eval-

⇑ Corresponding authors. Tel./fax: +86 871 65227816. y

E-mail address: [email protected] (J. Xu). Contributed equally to this work.

http://dx.doi.org/10.1016/j.bmcl.2015.08.057 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

uated for their inhibitory effects against nitric oxide (NO) production in LPS and IFN-c-induced RAW 264.7 murine macrophages, and activities against human gastric (SGC-7901) and human cervical (Hela) cancer cell lines. Hedylongnoid A (1)12 was obtained as a colorless oil with the formula C20H22O7, in agreement with the HR-ESI-MS at m/z 329.1744 ([M+H]+ (calcd for C20H23O+7; calcd: 329.1753). The 1H NMR spectrum showed signals corresponding to three tertiary methyls at dH 1.16 (3H, s, H-18), 1.15 (3H, s, H-19) and 1.02 (s, H-20), four olefinic protons at dH 5.88 (s, 1H, H-7), 8.32 (s, 1H, H-16), 7.47 (br s, 1H, H-15) and 6.86 (s, 1H, H-14), three methine protons including an oxygenated one at dH 4.58 (d, 1H, J = 7.7 Hz, H-11), four methylene groups including an oxygenated one at dH 4.60 (br d, 1H, J = 14.9 Hz,H-17a) and 4.50 (br d, 1H, J = 14.9 Hz, H-17b) (Table 1). Analysis of the 13C NMR, DEPT, and HSQC data revealed signals due to 20 carbons, which were classified into six quaternary carbons including two olefinic and two carbonyl ones at dC 193.4 (s, C-12) and 199.2 (s, C-6), seven methines including four olefinic ones, four methylenes including an oxygenated one at dC 70.2 (t, C-17), and three methyls. These data were consistent with the HR-ESI-MS empirical formula and suggested that 1 was a labdane diterpene, which was further supported by 1H–1H COSY correlations and HMBC correlations (Fig. 2). In the 1H–1H COSY spectrum, the correlation peaks between H-1 and H-2, H-2 and H-3, H-9 and H-11, H-14 and H-15, which enabled us to identify

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O

O

14

16

12 13 11 O 20 O 1 9 2 17 8 10 4 5 6 H O 19 18

1

O

O

O

OCH 3

O

H

2

OH

H

OH

OH

3

H

4

O

O

O

O

OCH3

O

O

OH

OH

OH

O

1 H-H COSY HMBC

O

HO

O

O

O

O

OH

2

3

Figure 2. 1H–1H COSY and key HMBC correlations for compounds 1–3.

H

OH

OH

H

5

6

Figure 1. Structures of compounds 1–6 isolated from H. longipetalum.

three spin systems due to the units of CH@CH, CHACH, and CH2ACH2ACH2. The HMBC spectrum showed key correlations between H-11 with C-12, C-13 and C-17, H-9 with C-12, H-5 with C-6, H-7 with C-17, H-15 with C-16. Particularly, C-11 and C-17, C-15 and C-16 were linked by oxygen atom due to the correlations of H-11 with C-17, H-15 with C-16, respectively (Fig. 2). Since the key NOE interactions between the proton signals of H-9 and H-5, H-11 and H-20 were observed in ROESY spectrum (Fig. 3), the relative configuration of H-9 and H-11 should be a and b-oriented, respectively. Thus, compound 1 was identified as 13b-furanolabda7-en-11,17-epoxy-6,12-dione (Fig. 1), and named hedylongnoid A. Hedylongnoid B (2)13 ([a]19.3 6.26°), was obtained as a colorless D gum, and its molecular formula was determined as C20H28O4 by HRESI-MS data at m/z 355.1878 [M+Na]+ (calcd for C20H28O4Na+; 355.1885). The 1H NMR spectrum showed signals assignable to three methyls at dH 1.03 (3H, s), 1.24 (3H, s) and 1.16 (3H, s), seven methine protons including two oxygenated ones at dH 4.36 (1H, br s, H-6) and 4.03 (1H, br s,H-7) and a pair of trans olefinic ones at dH 6.97 (1H, dd,

J = 10.1, 15.8 Hz, H-11) and 6.14 (1H, d, J = 15.8 Hz, H-12), five methylenes including an exomethylene at dH 5.26 (1H, s), 4.94 (1H, s) (Table 1). Among them, the signals for three methyl groups and an exomethylene were characteristic of a labdane-type diterpene.14 The 13C NMR, DEPT and HSQC data confirmed the presence of 20 carbons, which contains five quaternary carbons including two olefinic ones at dC 147.5 (s) and 129.2 (s), and an ester carbonyl one at dC 172.3 (s), seven methines including three olefinic ones and two oxygenated ones at dC 72.4 (d), 74.9 (d), five methylenes including an oxygenated one at dC 69.6 (t) and a terminal olefinic one at dC 108.6 (t), and three methyls. These data also suggested that 2 was a labdane diterpene, which was further confirmed by 1H–1H COSY correlations and HMBC correlations (Fig. 2). In the 1H–1H COSY spectrum, the correlation peaks between H-1 and H-2, H-2 and H-3, H-5 and H-6, H-6 and H7, H-9 and H-11, H-11 and H-12, H-14 and H-15, led us to identify four spin systems of CH2ACH2ACH2, CHACHACH, CHACH@CH, CHACH2. The HMBC spectrum showed key correlations between H-17 with C-7, C-8, and C-9, H-3, H-5, H-18 and H-19 with C-4, H-1, H-9 and H-20 with C-10, H-11, H-12, and H-15 with C-13, H-12, H-14 and H-15 with C-16. Especially, the location of a ester carbonyl carbon was determined by HMBC correlation of H-12 and C-16. In addition, the key NOE interactions between H-9 with H-7 and H-5, H-6 with H-5, H-11 with H-20 were observed in the ROESY spectrum (Fig. 3). Thus, the configuration of H-5, H-6, H-7 and H-9 should be

Table 1 NMR spectroscopic data of 1–3 C

1 d Ca

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

39.9 17.8 43.2 32.4 63.6 199.2 121.5 159.6 57.9 42.8 82.7 193.4 124.9 109.3 143.9 149.3 70.2 33.3 21.4 15.0

2

dHb 1.48 1.38 1.56 1.43

dCa (m), (m) (m), 1.41 (m) (m), 1.21 (m)

2.16 (s) 5.88 (s) 3.39 (br d, 7.7) 4.58 (d, 7.7)

6.86 7.47 8.32 4.60 4.50 1.16 1.15 1.02

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

The spectra were taken in CDCl3 at 150 MHz. The spectra were taken in CDCl3 at 600 MHz.

s) s) d, 14.9), d, 14.9)

dHb

3 d Ca

dHb

43.3

1.56 (m), 1.00 (m)

42.2

1.70 (m), 1.10 (m)

19.1 43.7 34.4 55.5 72.4 74.9 147.5 60.6 40.0 134.8 121.3 129.2 143.2 69.6 172.3 108.6

1.56 (m), 1.43 (m) 1.54 (m), 1.20 (m)

19.4 43.8 34.4 57.2 69.1 47.3 143.5 56.7 40.6 25.4 142.4 124.4 32.8 102.2 169.6 111.5

1.63 (m), 1.50 (m) 1.39 (m), 1.19 (m)

33.5 24.1 17.8

1.09 (s) 4.36 (br s) 4.03 (br s) 2.35 (d, 10.1) 6.97 (dd, 10.1, 15.8) 6.14 (d, 15.8) 7.20 (s) 4.83 (s) 5.26 (s), 4.94 (s) 1.03 (s) 1.24 (s) 1.16 (s)

33.6 23.6 17.1 56.7

1.09 (1H, s) 4.39 (br s) 2.34 (m) 1.90 (br d, 13.0) 2.35 (m), 2.27 (m) 6.74 (m) 2.99 (m), 2.71 (dd, 17.1, 1.9) 5.46 (dd, 6.4, 1.9) 5.01 (s), 4.68 (s) 1.01 1.21 1.03 3.52

(s) (s) (s) (s)

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1

2

3

Figure 3. Key ROESY correlations for compounds 1–3.

a-oriented, as shown in Figure 1. Moreover, orientations of H-5 and H-6 were further determined by comparison of 1H NMR spectra of compound 2 and known compounds yunnancoronarin E, hedyforrestin D, and the upfield shift and singlet peak of H-5 and H-6 supported the above conclusion.3,15 Thus, compound 2 was elucidated to be (E)-labda-8(17),11,13-triene-6b,7b-dihydroxy-16-oic lactone (Fig. 1), and named hedylongnoid B. Hedylongnoid C (3),16 a colorless gum, had the molecular formula C21H32O4 as determined by positive HR-ESI-MS at m/z 371.2190 [M+Na]+ (calcd for C21H32O4Na+; 371.2198). The 13C NMR spectrum (Table 1) of compound 3 revealed 21 resonances including a methoxy signal and 1H NMR showed two oxygenated methine proton signals at dH 4.39 (1H, br s, H-6) and 5.46 (1H, dd, J = 6.4, 1.9 Hz, H-15), a trisubstituted olefinic proton signal at dH 6.74 (1H, m, H-12), and five characteristic proton signals of a labdane diterpene due to three tertiary methyl groups at dH 1.01 (s), 1.21 (s), 1.03 (s), and an exocyclic methylene group at dH 5.01 (1H, s), 4.68 (1H, s).14 Inspection of the 1H,1H-COSY spectrum of 3 enabled us to identify four spin systems corresponding to the fragments of CH2ACH2ACH2, CHACHACH2, CHACH2ACH, and CH2ACH. Assignment of the former fragments was corroborated by HMBC correlations between H-1 and C-10, H-18 and C-3, H-19 and C-5, H-17 and C-7, C-9, H-11 and C-13, H-12 and C-16, H-14 and C-13, C-16 (Fig. 2). The location of ester carbonyl on C-16, a methoxy group on C-15, and a hydroxy group on C-6, were determined by the HMBC correlations from H-12 to C-16, the proton signal of methoxy to C-15, H-6 to C-10, C-4 and C-8, respectively (Fig. 2). The relative configuration of 3 was established by the key NOE interactions from H-5 to H-9 and H-6, H-11 to H-20 in a ROESY experiment, which revealed that H-5, H-6 and H-9 were a-oriented (Fig. 3). The above conclusion was also supported by the similarity of NMR data of H-5 and H-6 of 2 and 3. Therefore, compound 3 was identified to be 6b-hydroxy-15n-methoxylabda-8(17),12-dien-16oic lactone (Fig. 1), and named hedylongnoid C. In previous studies, some labdane diterpenes from the species of Hedychium were found to show inhibitory activity against NO production and cytotoxicity.8,17,18 Therefore, compounds 1–6 were also evaluated for inhibitory effects against NO production in LPS and IFN-c-induced RAW 264.7 murine macrophages, and cytotoxic activities against two cancer cell lines SGC-7901 and Hela according to the reported methods.19,20 Results revealed that compounds 1–6 exhibited inhibitory effects against NO production in LPS and IFN-c-induced RAW 264.7 murine macrophages with the IC50 values ranging from 0.56 to 7.5 lg/ml, and 3–6 showed cytotoxicities against cancer cell lines SGC-7901 and Hela with the IC50 values ranging from 6.21 to 14.53 lg/ml and from 6.58 to 14.83 lg/ml, respectively. However, compounds 1 and 2 showed no cytotoxicities (Table 2).

Table 2 Cytotoxicity against two human cancer cell lines and inhibitory activity against NO production in LPS and IFN-c-induced RAW 264.7 macrophages for compounds 1–6 (IC50, lg/mL) Compounds

1 2 3 4 5 6 Taxola MG132b a,b

Cell lines

NO

SGC-7901

Hela

>25 >25 8.74 6.21 7.29 14.53 0.2

>25 >25 11.31 6.58 9.21 14.83 0.1

7.50 5.58 6.10 0.56 2.77 6.52 0.08

Positive control.

Acknowledgments This work was supported by the National Natural Science Foundation of China (31360080), Young Academic and Technical Leader Raising Foundation of Yunnan Province to J.-J. Xu (2014HB013). The authors are grateful to the staff of analytical group at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences for the spectral data. References and notes 1. Delectis Florae Reipularis Agendae Academiae Sinicae Edita. ‘Flora Reipublicae Popularis Sinicae’, Science Press, Beijing, 1976, 16, 26. 2. Insitute Botanicum Kunmingense Academiae Sinicae Edita, ‘Flora Yunnancia’, Science Press, Beijing, 1997, 8, 96. 3. Zhao, Q.; Qing, C.; Hao, X. J.; Han, J.; Zuo, G. Y.; Zou, C.; Xu, G. L. Chem. Pharm. Bull. 2008, 56, 210. 4. Jiangsu New Medical College. ‘Chinese Materia Medica’, Shanghai People’s Press, Shanghai, 1977; 89. 5. Zhao, Q.; Hao, X. J.; Chen, Y. Z.; Zou, C. Acta Bot. Sin. 1995, 17, 201. 6. Zhan, Z. J.; Wen, Y. T.; Ren, F. Y.; Rao, G. W.; Shan, W. G.; Li, C. P. Chem. Biodivers. 2012, 9, 2754. 7. Reddy, P. P.; Tiwari, A. K.; Rao, R. R.; Madhusudhana, K.; Rao, V. R. S.; Ali, A. Z.; Babu, K. S.; Rao, J. M. Bioorg. Med. Chem. Lett. 2009, 19, 2562. 8. Matsuda, H.; Morikawa, T.; Sakamoto, Y.; Toguchida, I.; Yoshikawa, M. Bioorg. Med. Chem. 2002, 10, 2527. 9. Hu, X.; Liu, N. Ann. Bot. Fenn. 2010, 47, 237. 10. Kumrit, I.; Suksamrarn, A.; Meepawpan, P.; Songsri, S.; Nuntawong, N. Phytother. Res. 2010, 24, 1009. 11. Zhao, Q.; Hong, X.; Wang, Y. S.; Zou, C.; Hao, X. J. Chin. Chem. Lett. 2003, 14, 1141. 12. Hedylongnoids A (1): colorless oil; [a]19.93 +7.14° (c 0.1, CDCl3); UV (CDCl3): 141 D (3.63), 203 (3.14); IR (KBr): 3439, 2927, 2870, 2953, 1722, 1673, 1641; 1H and 13 C NMR: See Table 1; HR-ESI-MS m/z: 329.1744 ([M+H]+, C20H23O+7; calcd: 329.1753). 13. Hedylongnoids B (2): colorless gum; [a]19.3 6.26° (c 0.1, CDCl3); UV (CDCl3): D 251 (3.97), 327 (2.83); IR (KBr): 3442, 2925, 2864, 2852, 1752, 1645, 1447; 1H 13 and C NMR: See Table 1; HR-ESI-MS m/z: 355.1878 ([M+Na]+, C20H28O4Na+; calcd: 355.1885).

H. Zhao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4572–4575 14. Chokchaisiri, R.; Chaneiam, N.; Svasti, S.; Fucharoen, S.; Vadolas, J.; Suksamrarn, A. J. Nat. Prod. 2010, 73, 724. 15. Zhao, Q.; Zou, C.; Hao, X. J.; Cheng, Y. Z. Acta Bot. Yunnanica 2000, 22, 116. 16. Hedylongnoids C (3): colorless gum; [a]19.3 +34.29° (c 0.2, CDCl3); UV (CDCl3): D 240 (3.33), 226 (2.59), 203 (2.49); IR (KBr): 3443, 2926, 2851, 1760, 1675, 1 13 1643, 1448; H and C NMR: See Table 1; HR-ESI-MS m/z: 371.2190 ([M+Na]+, C21H32O4Na+; calcd: 371.2198). 17. Reddy, P.; Rao, R.; Shashidhar, J.; Sastry, B. S.; Rao, M.; Babu, S. Bioorg. Med. Chem. Lett. 2009, 19, 6078.

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18. Reddy, P.; Rao, R.; Rekha, K.; Babu, S.; Shashidhar, J.; Shashikiran, G.; Lakshmi, V.; Rao, M. Bioorg. Med. Chem. Lett. 2009, 19, 192. 19. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107. 20. Fan, J. T.; Su, J.; Peng, Y. M.; Li, Y.; Li, J.; Zhou, Y. B.; Zeng, G. Z.; Yan, H.; Tan, N. H. Bioorg. Med. Chem. 2010, 18, 8226.