Cassaine diterpene alkaloids from Erythrophleum fordii and their anti-angiogenic effect

Bioorganic & Medicinal Chemistry Letters 24 (2014) 168–172

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Cassaine diterpene alkaloids from Erythrophleum fordii and their anti-angiogenic effect Tran Manh Hung a, To Dao Cuong a, Jeong Ah Kim b, Nara Tae c, Jeong Hyung Lee c, Byung Sun Min a,⇑ a

College of Pharmacy, Catholic University of Daegu, Gyeongbuk 712-702, Republic of Korea College of Pharmacy, Kyungpook National University, Daegu 702-701, Republic of Korea c College of Natural Science, Kangwon National University, Kangwon 200-701, Republic of Korea b

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 3 September 2013 Revised 1 November 2013 Accepted 20 November 2013 Available online 27 November 2013

Angiogenesis plays a critical role in embryonic development and various physiological processes. However, excessive angiogenesis is associated with several pathological conditions including cancer. Angiogenesis is closely related to tumor growth, invasion and metastasis, and is considered a prime target for anticancer therapy. In this study, two new mono cassaine diterpenoid amides (1, 5) and four known compounds (2–4, 6) were isolated from the bark of Erythrophleum fordii (Leguminosae). Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. The effects of isolates on endothelial tube formation on Matrigel were investigated. Among them, compound 3 was found to have the most potent inhibitory effect on the capillary-like structure formation of human umbilical vein endothelial cells (HUVECs). Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

Keywords: Erythrophleum fordii Leguminosae Cassaine diterpenoid amide Anti-angiogenesis

During our continued search to find anti-angiogenic agents from natural plants, the methylene chloride fraction of the methanol extract of Erythrophleum fordii Oliver (Leguminosae) was found

to show potential inhibitory activity. E. fordii is widely distributed in China, Taiwan, and Vietnam, and is used in Chinese traditional medicine to invigorate and promote blood circulation.1 In prior

21

O 20

3

11

H

22

O

OH

13

HO O

19 18

O

2

N

H O

O OH

26

N

O

OH

H

O 25

3

O

OH

H H

O

H O

1

O

H

H

O O

H O

O OH

H O

H

O O

5

4

OH

H O O

O

H

N

H O

O

24

O

O

OH

H

7

6

N

H 17

H

4

HO O

H

23

N

16 15

Figure 1. Chemical structure of isolated compounds (1–6) from E. fordii.

⇑ Corresponding author. Tel.: +82 53 850 3613; fax: +82 53 850 3602. E-mail address: [email protected] (B.S. Min). 0960-894X/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.048

6

N

OH

169

T. M. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 168–172 Table 1 H and 13C NMR data of compounds 1 and 5

1

Position

1 (in CD3OD) dHa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 a b c

5 (in Pyridine)

(J in Hz)

dC c

1.80 1.25 2.30 1.80 3.42

(1H, (1H, (1H, (1H, (1H,

m) m) m) m)c dd, 11.5, 4.0)

1.72 1.48 1.80 2.19 1.78

(1H, (1H, (1H, (1H, (1H,

m) m), 1.29 (1H, m)c m),c 1.20 (1H, m) m) m)

72.1 46.8 41.4 25.5c 29.3 49.5 45.0 54.1 28.1 25.5c 167.7 39.1 112.2 171.7 13.4 20.6 179.4 14.2 40.6 54.0 60.5 51.9

1.08 (3H, d, 7.0) 1.26 (3H, s) s) s) m) t, 5.5) s)

d Cb

(J in Hz)

1.85 1.34 2.45 1.78 4.99

29.2

2.83 (1H, m) 5.76 (1H, s)

(3H, (3H, (2H, (2H, (3H,

dHa

38.3

2.03 (1H, m),c 1.29 (1H, m)c 3.74 (1H, m), 2.03 (1H, m)c

0.63 2.67 3.42 4.25 3.64

b

(1H, (1H, (1H, (1H, (1H,

m) m) m)c m) dd, 12.0, 4.4)

36.9 25.8 79.6 47.0 52.1 77.2 209.3 51.2 47.0 42.9 27.3 24.7 167.0 41.3 114.1 167.3 14.3 24.6 173.8 15.2 36.9 51.8 64.9 53.9 170.8 21.3

1.38 (1H, d, 12.5) 5.39 (1H, d, 12.5) 2.48 (1H, dd, 13.0, 3.5) 1.82 (1H, m) 1.88 (1H, m), 1.30 (1H, m) 3.21 (1H, m), 2.14 (1H, m) 2.87 (1H, m) 5.92 (1H, s) 1.30 (3H, d, 6.5) 1.40 (3H, s) 1.14 2.45 3.50 4.20 3.83

(3H, (3H, (2H, (2H, (3H,

s) s)c m) t, 5.6) s)

2.05 (3H, s)

Recorded at 400 MHz. Recorded at 100 MHz. Overlapped signals. All assignments were confirmed by DEPT-135, HMQC and HMBC experiments.

O

N

O

OH

N

OH

O O O

HO O COSY

O

O

HMBC

1

O OH 5

Figure 2. Selected COSY and HMBC correlations for 1 and 5.

studies on the genus Erythrophleum, cassaine diterpenoid amines, amides and oleanane-type triterpene saponins were found as the main constituents of this genus;2 minor amounts of cassaine acids were also identified.3 Furthermore, cassaine alkaloids showed a digitalis-like action on the heart4 and cytotoxic activity against some tumor cell lines.2c,5 Many cassaine diterpenoid dimers recently reported in Erythrophleum succirubrum possess

O

O

O

HO

H

H H

O

N

H H

an unsymmetrical dimeric structure due to intramolecular ester bond formation of two diterpenoid units.6 Recently, a phytochemical investigation on the leaves of E. fordii resulted in the isolation of cassaine diterpenoid–diterpenoid amide dimers and cassaine diterpenoid amides.7 Furthermore, cytotoxic activities of the isolated compounds were examined in several human cancer cell lines.7 In the present study, we report the isolation and structural

O

OH

O

O

O

H HO H

1

H

H H

H

NOE Figure 3. Selected NOEs correlations for 1 and 5.

N

H

O

5

H

OH

170

T. M. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 168–172

Figure 4. Effect of compounds from E. fordii on HUVEC tube-like structure formation. Proliferating HUVECs were plated on the surface of Matrigel in complete media with indicated doses of the isolates (1–6) or solvent controls. The tube formation was evaluated 15 h later. (A) Tube number of HUVECs treated with compounds 1–6 (3 lg/mL), curcumin (3 lg/mL) was used as positive control; (B) Effects of compound 3 on LDH release from endothelial cells; (C) Morphological analysis and tube formation of cells treated with 3 (3–30 lg/mL); Bar = 100 lm. Quantitative data are presented as mean ± SD. The solvent-treated controls (n = 3) were normalized to 100%. % Controls (n = 3) from compound-treated groups. ⁄P <0.05, difference versus control group.

T. M. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 168–172

elucidation as well as anti-angiogenic activities of two new cassaine diterpenoid amines (1 and 5) and four known compounds (2–4, 6) from the bark of E. fordii. Repeated column chromatographies (silica gel, RP-18) of the CH2Cl2-soluble fraction resulted in the isolation of six compounds.8 Among them, four known compounds were identified by comparing the physicochemical and spectroscopic data (IR, UV, MS, 1D and 2D NMR) with literature2c,7 as cassaine-type diterpenoid amides, namely, nor-erythrophlamide (2), 3b-acetyl-nor-erythrophlamide (3), 6a-hydroxy-nor-cassamide (4) and nor-cassamide (6) (Fig. 1). Compound 1 was isolated as a white powder. A quasi-molecular ion at m/z 422.2909 (calcd 422.2906) [M+H]+ in the positive HR-FAB-MS indicated a molecular formula of C24H40NO5. Its IR spectrum showed bands at 3375 and 1720 cm1 indicative of the presence of hydroxyl and carbonyl groups in the molecule.9a The 13C NMR spectrum displayed resonances for two carbonyls at dC 171.7 (C-16) and 179.4 (C-19), a pair of olefinic carbons at dC 112.2 (C-15) and 167.7 (C-13) and one oxymethine at dC 72.1 (C-3). The 1H NMR spectrum displayed resonances of three methyls at dH 1.08 (3H, d, 7.0 Hz, H-17), 1.26 (3H, s, H-18) and 0.63 (3H, s, H-20), one methoxyl at dH 3.64 (3H, s, H-24), one exocyclic olefinic proton at dH 5.76 (1H, s, H-15), one oxymethine at dH 3.42 (1H, dt, 11.5, 4.0 Hz, H-3), and a methyl amide N–CH3 at dH 2.67 (3H, s, H-21) (Table 1). All signals in the above spectra closely resembled those of the cassaine diterpenoid amide skeleton,4 and this was further supported by analyses of HMQC and HMBC spectroscopic data. The HMBC spectrum confirmed correlations between protons H-3, H-5 and H-18 and C-4, between H-20 and C-1, C-9 and C-10. Diagnostic HMBC correlations were also observed between H-12 and H-15 and C-13, and between H-15 and H-22 and C-16 (Fig. 2). The correlation signals between H24 and C-19 and between H-18 and C-4 and C-19 indicated the presence of a methoxycarbonyl group at C-4. A comparison of the 1H and 13C NMR spectroscopic data of 1 with those of 2 (nor-erythrophlamide)2c suggested they share the same skeleton, except for the 7-methylene group in 1 and the 7-keto group in 2. This difference was supported by cross-peaks from H-5 to H-6 and H-7 in the COSY spectrum (Fig. 2). According to the biogenetic isoprene rule of the cassaine skeleton, the two methyls attached to the rings were in the 10b and 14a orientations, respectively.10 The J value of H-3 (dt, 11.5, 4.0 Hz) suggested a b-orientation for the hydroxyl group. The above-mentioned considerations were further substantiated by the significant NOE correlations between H-3, H-5 and H-18, between H-5 and H-9, and between H-9 and H-17 (a-orientation) (Fig. 3). The NOE correlations between H-8 and H-14, H-8 and H-20, and H-20 and H-24 indicated these protons were b-orientated (Fig. 3). Furthermore, the NOE correlation between H-14 and H-15 indicated double bond at C-13/C-15 had the E-conformation.2c,6,10 Thus, the chemical structure of 1 was assigned as in Figure 1 and named erythroformide. Compound 5 was isolated as a white powder. Its molecular formula was established as C26H40NO8 by positive-ion mode HR-FABMS, which showed a pseudo-molecular ion peak at m/z 494.2757 (calcd 494.2754) [M+H]+. IR absorptions at 3342 and 1720 cm1 showed the presence of hydroxyl and carbonyl groups, respectively.9b Based on 13C NMR and DEPT spectra, compound 5 possessed four carbonyls (dC 167.3, 170.8, 173.8 and 209.3), and a pair of olefinic carbons (dC 114.1 and 167.0). Analysis of its 1H NMR spectrum indicated the presence of four methyl groups at dH 1.30 (3H, d, 6.4 Hz, H-17), 1.40 (3H, s, H-18), 1.14 (3H, s, H20) and 2.05 (3H, s, H-26), a methoxyl group at dH 3.83 (3H, s, H24), an exocyclic vinyl proton at dH 5.92 (1H, s, H-15), and a methyl amide proton at dH 2.45 (3H, s, H-21) (Table 1). In addition, the two oxygenated methines at dH 4.99 (1H, dd, 12.0, 4.4 Hz, H-3) and 5.39 (1H, d, 12.5 Hz, H-6) indicated the H-3a and H-6b orientations,7

171

respectively. Furthermore, these signals were similar to those of 3b-hydroxydinorcassamide isolated from the leaves of E. fordii,7 except for the presence of an acetate group. This was supported by an HMBC correlation from the proton at dH 4.99 (H-3) to the acetate carbonyl carbon at dC 170.8 (C-25) (Fig. 2). Further analysis of HMQC, HMBC and COSY spectra permitted the assignments of all proton and carbon resonances (Fig. 2). The relative stereochemistry of 5 was confirmed by its NOESY spectrum. In addition to similar correlation signals with 1, NOE correlations between H-6, H-8, and H-20 in 5 indicated these protons were in the same b-orientation, and therefore, the hydroxyl group at C-6 was a-orientated (Fig. 3). Thus, compound 5 is identified as 3b-acetoxynorcassamide. Since the effects of methylene chloride soluble fraction has been examined for its inhibitory effect on the capillary-like structure formation on Matrigel of HUVECs, we were interested in investigating the effects of the isolated compounds on the proliferation of endothelial cells. To investigate cytotoxic effect of isolated compounds on HUVECs, we performed MTT assay in various concentration of 1–6 (3–30 lg/mL) and a 0.1% DMSO as control.11 After 48 h incubation, all isolates did not significantly affect the viabilities of the HUVECs (data not shown). The formation of tube-like structures is an essential step in angiogenesis and involves matrix degradation, rearrangement, and the apoptosis of endothelial cells.12a Therefore, in the present study, we examined anti-angiogenic activities of all isolates using the capillary-like tube formation assay on Matrigel.12b Capillary tube structures were observed in the control group after HUVECs were placed in the wells, whereas the presence of the compounds 1–6 at 3 lg/mL significantly reduced the formation of tube-like structures (Fig. 4A). Curcumin (3 lg/mL), a non-toxic nature and was developed to treat chronic diseases associating with extensive neovascularisation,13 reduced 45% formation of tube-like structure vs. control (Fig. 4A). Since compound 3, 3b-acetyl-nor-erythrophlamide, effectively inhibited tube-like structures formation (up to 42% of the control), LDH cytotoxic assay was carried out to examine whether 3b-acetyl-nor-erythrophlamide would result in toxic effects of HUVEC.14 The results found that 3 caused minute toxicity on HUVECs (Fig. 4B), suggesting that inhibitory effect on HUVECs might be partially due to general cytotoxicity of 3. In addition, treatment of a noncytotoxic dose of 3 (3–30 lg/mL) also significantly reduced this tube-like structure formation. At a concentration 30 lg/mL, 3 effectively inhibited tube-like structure formation (4.5% vs control) (Fig. 4C). These results indicate cassaine diterpenoid amines isolated from E. fordii can suppress angiogenesis in vitro, and E. fordii might be a potent anti-angiogenic agent. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (KRF2012K2A4A1034671) and BK21 Plus program. The authors are grateful at the Korea Basic Science Institute for measuring the MASS spectrometry. References and notes 1. Chen, J. S.; Zhen, S. Chinese Virose Plant, 1st ed.; Science Press: Beijing, 1987; p 321. 2. (a) Culvenor, C. C. J.; Loder, J. W.; Nearn, R. Phytochemistry 1971, 10, 2793; (b) Cronlund, A.; Sandberg, F. Acta Pharm. Suec. 1976, 13, 35; (c) Qu, J.; Hu, Y. C.; Yu, S. S.; Chen, X. G.; Li, Y. Planta Med. 2006, 72, 442. 3. (a) Li, N.; Yu, F.; Yu, S. S. Acta Bot. Sin. 2004, 46, 371; (b) Yu, F.; Li, N.; Yu, S. S. J. Asian Nat. Prod. Res. 2005, 7, 19; (c) Tsao, C. C.; Shen, Y. C.; Su, C. R.; Li, C. Y.; Liou, M. J.; Dung, N. X. Wu. T.S Bioorg. Med. Chem. 2008, 16, 9867. 4. Verotta, L.; Aburjai, T.; Rogers, C. B.; Dorigo, P.; Maragno, I.; Fraccarollo, D.; Giovanni, S.; Gaion, R. M.; Floreani, M.; Carpenedo, F. Planta Med. 1995, 61, 271.

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5. Loder, W.; Culvenor, C. C. J.; Nearn, R. H.; Russell, G. B.; Stanton, D. W. Aust. J. Chem. 1974, 27, 179. 6. Miyagawa, T.; Ohtsuki, T.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. Tetrahedron Lett. 2009, 50, 4658. 7. Du, D.; Qu, J.; Wang, J. M.; Yu, S. S.; Chen, X. G.; Xu, S.; Ma, S. G.; Li, Y.; Ding, G. Z.; Fang, L. Phytochemistry 2010, 71, 1749. 8. The bark of Erythrophleum fordii Oliver (Leguminosae) was collected at Quang Nam province, Vietnam, in May 2012. Professor Tran Cong Luan at Hochiminh city University of Medicine and Pharmacy performed the botanical identification, and a voucher specimen (CUD-3177) was deposited at the Herbarium of the College of Pharmacy, Catholic University of Daegu, Korea. The bark (2.0 kg) was extracted with MeOH three times (3 h  3 L) under reflux. After solvent removal under reduced pressure, the residue (426.0 g) was suspended in H2O and partitioned sequentially versus CH2Cl2 and EtOAc. The CH2Cl2 soluble fraction (40.0 g) was separated on a silica gel column (50  10 cm) using a stepwise gradient of CHCl3/MeOH (40: 1 ? 1: 1, v/v) to yield 27 fractions (A1A27) according to their TLC profiles. Fraction A27 (3.7 g) was subjected to silica gel column chromatography (60  5 cm) using CHCl3–MeOH plus 0.1% of acetic acid (7:1) as eluent to yield seven subfractions (A27-1-27.7). Fraction A27-2 (106.0 mg) was chromatographed on a silica gel column (50  2.5 cm) using a CHCl3-MeOH (7:1) to give compound 1 (5.5 mg). Fraction A27.4 (56.0 mg) was further chromatographed on a silica gel column (50  2.5 cm) using a CHCl3–MeOH (6:1) to yield 2 (9.5 mg). Compound 3 (45.2 mg) was obtained by recrystallization of fraction A22 (250.0 mg) in cold MeOH. Fraction A25 (250.0 mg) was chromatographed on an ODS RP column (50  2.5 cm) using a MeOH–H2O (4:5 plus 0.1% acetic acid) to give 4 (6.5 mg). Fraction A27-5 (350.0 mg) was subjected to an ODS RP column (60  2.5 cm), and eluted MeOH–H2O (4:5 plus 0.1% acetic acid) to yield fraction A27-5-1 and 27-5-2. Compounds 5 (11.0 mg) and 6 (4.0 mg) were obtained by further purification of fraction A27-5-1 by silica gel column chromatography using a CHCl3–MeOH gradient (7:1  4:1). 9. (a) Erythroformide (1). White amorphous powder; ½a25 D 19.5 (c 0.05, MeOH); IRmmax (KBr): 3445, 2947, 1718, 1646, 1215, 1126 cm–1; UV (MeOH) kmax (log e): 232 (3.18) nm; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD) spectroscopic data, see Table 1; HR-FAB-MS m/z 422.2909 [M+H]+ (calcd for C24H40NO5, 422.2906); (b) 3b-Acetoxynorcassamide (5). White amorphous powder; ½a25 D 46.7 (c 0.10, MeOH); IRmmax (KBr): 3455, 2945, 1721, 1612, 1475, 1405, 1212 cm–1; UV (MeOH) kmax (log e): 236 (4.16) nm; 1H NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5) spectroscopic

10. 11.

12.

13. 14.

data, see Table 1; HR-FAB-MS m/z 494.2757 [M+H]+ (calcd for C26H40NO8, 494.2754). Maurya, R.; Ravi, M.; Singh, S.; Yadav, P. P. Fitoterapia 2012, 83, 272. (a) Cell culture. HUVECs were cultured in M199 medium supplemented with 20% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 0.01% heparin (Sigma–Aldrich, St. Louis, MO, USA) and 30 lg/ml endothelial cell growth supplement (ECGS) (Sigma– Aldrich), and maintained at 37 °C in a humidified 5% CO2 atmosphere. Cells were seeded on plates coated with 0.2% gelatin (Sigma–Aldrich) and allowed to grow. Cell media were changed every other day. (b) Cell viability assay. The colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was modified and performed to quantitate the effect of isolated on cell viability. Briefly, HUVECs were seeded in a 96-well microtiter plate (Falcon, Franklin Lakes, NJ, USA), and allowed to reach 80% confluency after which various doses of isolates were treated for 48 h. After completion of the treatment, MTT stock solution (0.25%) (Sigma, St. Louis, MO, USA) was added to the cells to a final concentration of 0.05% and the cells were incubated for 3 h at 37 °C. Then, the MTT solution was removed and replaced by 50 ll DMSO, and the plates were shaken for 3 min. The optical density of each condition was determined using a microplate reader at a wavelength of 570 nm with a reference wavelength of 630 nm. The percentage of cell viability was calculated against untreated cells. (a) Yeh, J. C.; Cindrova-Davies, T.; Belleri, M.; Morbidelli, L.; Miller, N.; Cho, C. W.; Chan, K.; Wang, Y. T.; Luo, G. A.; Ziche, M.; Presta, M.; Charnock-Jones, D. S.; Fan, T. P. Angiogenesis 2011, 14, 187 (b) In vitro capillary tube formation assay. The modification of the Matrigel assay was used to evaluate in vitro angiogenesis activity by quantitating the capillary tube formation of HUVECs as described in the protocol of CHEMICON’s In Vitro Angiogenesis Assay Kit (ECM625). HUVECs (1  104 cells) were suspended in 50 ll of media containing various concentrations of compound and then added on the polymerized Matrigel. After incubation at 37 °C for 2–10 h, each culture was photographed at a magnification of 100 with a microscope video system (Carl Zeiss, Chester, VA). Gururaj, A. E.; Belakavadi, M.; Venkatesh, D. A.; Marmé, D.; Salimath, B. P. Biochem. Biophys. Res. Commun. 2002, 297, 934. (a) Lactate dehydrogenase (LDH) toxicity assay. The LDH release assay was performed using a cytotoxicity detection kit plus (LDH) (Roche Diagnostics) according to the manufacturer’s instructions.; (b) Saraswati, S.; Agrawal, S. S. Cancer Lett. 2013, 332, 83.