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Three new diterpenoid alkaloids from the roots of Aconitum duclouxii a

a

b

a

Tian-Peng Yin , Le Cai , Jian-Min He , Jian-Wei Dong , Hai-Xian a

a

Fang , Hao Zhou & Zhong-Tao Ding

a

a

Key Laboratory of Medicinal Chemistry for Nature Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China b

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School of Resource and Environment, Baoshan University, Baoshan 678000, China Published online: 05 Feb 2014.

To cite this article: Tian-Peng Yin, Le Cai, Jian-Min He, Jian-Wei Dong, Hai-Xian Fang, Hao Zhou & Zhong-Tao Ding (2014) Three new diterpenoid alkaloids from the roots of Aconitum duclouxii, Journal of Asian Natural Products Research, 16:4, 345-350, DOI: 10.1080/10286020.2014.881802 To link to this article: http://dx.doi.org/10.1080/10286020.2014.881802

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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 4, 345–350, http://dx.doi.org/10.1080/10286020.2014.881802

Three new diterpenoid alkaloids from the roots of Aconitum duclouxii Tian-Peng Yina1, Le Caia1, Jian-Min Heb, Jian-Wei Donga, Hai-Xian Fanga, Hao Zhoua and Zhong-Tao Dinga* a

Key Laboratory of Medicinal Chemistry for Nature Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China; bSchool of Resource and Environment, Baoshan University, Baoshan 678000, China

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(Received 11 December 2013; final version received 7 January 2014) Three new C19-diterpenoid alkaloids, ducloudines C (1), D (2), and E (3), were isolated from the roots of Aconitum duclouxii. Their structures were established on the basis of extensive spectroscopic analyses. Ducloudine C (1) is the first aconitine-type C19-diterpenoid alkaloid with a CvO group at C-3 and a CvC bond between C-1 and C-2. All compounds were tested for their biological activities against one pathogenic fungi and two pathogenic bacteria. Keywords: Aconitum duclouxii; diterpenoid alkaloids; biological activity; ducloudines CZE

1. Introduction Aconitum L. is a large genus comprising 400 species and distributed in the temperate region of the northern hemisphere. About 76 Aconitum species in China have been used as folk medicine, which are mainly used for treatment of rheumatoid arthritis and various types of pains [1]. Aconitum duclouxii Levl. (Ranunculaceae), a perennial herb distributed in Dali Bai Autonomous Prefecture, Yunnan Province of China, has long been used as a folk medicine to treat rheumatism and pains [2]. However, phytochemical research on it is quite limited so far [3]. Our previous study had led to the isolation of two new diterpenoid alkaloids and two known alkaloids [4]. During our further phytochemical investigation on this plant, three new C19-diterpenoid alkaloids, ducloudines C (1), D (2), and E (3), were obtained from its roots (Figure 1). Herein, the isolation, structural elucidation, and biological activities of three compounds are described. *Corresponding author. Email: [email protected] q 2014 Taylor & Francis

2. Results and discussion Compound 1 was isolated as a white amorphous powder and its molecular formula was deduced to be C24H35NO6 by HR-ESI-MS at m/z 434.2545 [M þ H]þ. The IR spectrum indicated the presence of an OH group (3438 cm21), a keto-carbonyl (1716 cm21), and a CvC bond (1670 cm21). Inspection of the NMR data indicated an aconitine-type C19diterpenoid alkaloid [5]. The NMR spectra displayed signals of an N-ethyl group (dH 0.99, t, J ¼ 7.2 Hz; 2.47 –2.49, 2.49– 2.51, each 1H, m; dC 13.0, q; 48.7, t), three OMe groups (dH 3.28, 3.34, 3.38, each 3H, s; dC 59.1, q; 56.4, q; 58.0, q), a keto-carbonyl (dC 200.9, s), and a characteristic disubstituted CvC bond (dH 6.22, 1H, d, J ¼ 10.0 Hz; 6.47, 1H, d, J ¼ 10.0 Hz; dC 131.6, d; 148.3, d). The 13C NMR spectrum suggested the existence of other five oxygenated carbons (dC 72.2, t; 74.3, s; 75.8, d; 81.8, d; 82.0, d), indicating the presence of two hydroxyl groups and three OMe groups. Three OMe groups were

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1

22 2 21

O

3

N 4

H3CO

6

18

OH

OH

OCH3 O O CH3

OH

OH

15

N

8

5

19

16

9

11

OCH3

OCH3

13 12 14 17 10

7

OH

N OH

HO

OCH3

OCH3

H3CO

OH

HO

2

1

OCH3

H3CO 3

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Figure 1. Structures of compounds 1 –3.

located at C-6, C-16, and C-18, respectively, on the basis of the HMBC correlations from OCH3-6 (dH 3.38, s) to C-6 (dC 81.8, d), OCH3-16 (dH 3.34, s) to C-16 (dC 82.0, d), and OCH3-18 (dH 3.28, s) to C-18 (dC 72.2, t) (Figure 2). A triplet signal at dH 4.30 (t, J ¼ 4.8 Hz) was attributed to H-14b, suggesting the presence of an OH14a [6]. The HMBC correlations observed from H-16, H-6, H-14, and H-9 to C-8 (dC 74.3, s) suggested that a hydroxyl group was attached to C-8. The rare a,bunsaturated ketone was determined according to the two characteristic H-atoms ( dH 6.22, d, J ¼ 10.0 Hz; 6.47, d, J ¼ 10.0 Hz) together with the C-atom signals (dC 131.6, d; 148.3, d; 200.9, s). The HMBC correlations between H-1 to C-3, H-1 to C-10, H-1 to C-5, H-2 to C-11, H-2 to C-4, H-18 to C-3, and H-19 to C-3 demonstrated that the CvO group is located at C-3, and the CvC bond between C-1 and C-2 [7]. All signals in the 1H (13C) spectra of compound 1 were assigned based on its 2D NMR spectra (Table 1). Therefore, the structure of ducloudine C

12

O

N

4 19 H3CO

OCH3

13 16

16 OH

1 2

(1) was determined, which is the first aconitine-type C19-diterpenoid alkaloid with a CvO group at C-3 and a CvC bond between C-1 and C-2. Compound 2 was isolated as a white amorphous powder, whose molecular formula was deduced to be C24H39NO7 by HR-ESI-MS at m/z 454.2799 [M þ H]þ. The NMR spectra of compound 2 displayed signals of an N-ethyl group (dH 1.07, 3H, t, J ¼ 7.2 Hz; 2.39 –2.41, 2.43– 2.45, each 1H, m; dC 13.0, q and 48.1, t) and three MeO groups (dH 3.24, 3.27, 3.28, each 3H, s; dC 59.1, q; 56.3, q; 57.8, q). The 13C NMR spectrum suggested the existence of seven oxygenated carbons (dC 71.9, d; 72.4, d; 74.0, s; 75.6, d; 79.7, t; 82.4, d; 82.9, d), indicating the presence of four hydroxyl groups and three OMe groups. The NMR spectra strongly suggested an aconitine-type C19-diterpenoid alkaloid for 2. The three OMe groups were located at C-6, C-16, and C-18, respectively, on the basis of the HMBC correlations between OCH3-6 (dH 3.28, s) and C-6 (dC 82.9, d), OCH3-16 (dH 3.27, s) and C-16 (dC 82.4, d),

14 9 5

Figure 2. Key 1HZ1H COSY ( compound 1.

15

8 OH 7

6 OCH3

17 2

1 N

O

), HMBC (

OCH3 OH

14 11

98

15 OH

6 OCH3 18 H3CO

), and NOESY (

) correlations of

Journal of Asian Natural Products Research

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Table 1. 1H and compound 1.

347

13

C NMR spectral data (400 MHz for 1H and 100 MHz for

13

C, CDCl3) for

No.

dH, J (Hz)

dC

No.

dH, J (Hz)

dC

1 2 3 4 5 6 7

6.47 d (10.0) 6.22 d (10.0)

148.3 d 131.6 d 200.9 s 51.1 s 49.0 d 81.8 d 53.2 d

13 14 15 16 17 18 19

40.2 d 75.8 d 42.3 t 82.0 d 61.2 d 72.2 t 51.5 t

74.3 s 47.9 d 41.7 d 49.5 s 30.8 t

21 22 OCH3-6 OCH3-16 OCH3-18

2.28 – 2.30 m 4.30 t (4.8) 2.39 – 2.41 m, 2.13 – 2.16 m 3.27 – 3.29 m 2.18 – 2.20 m 3.86 – 3.88 m 2.64 ABq (10.8), 2.31 ABq (10.8) 2.49 – 2.51 m, 2.47 – 2.49 m 0.99 t (7.2) 3.38 s 3.34 s 3.28 s

8 9 10 11 12

3.03 d (6.8) 4.26– 4.28 m 1.29 d (4.8) 2.29– 3.31 m 2.07– 2.09 m 2.09– 2.11 m, 1.29– 1.31 m

and OCH3-18 (dH 3.24, s) and C-18 (dC 79.7, t) (Figure 3). The triplet signal at dH 4.12 (t, J ¼ 4.5 Hz) was attributed to H-14b, suggesting the presence of an OH-14a. Meanwhile, a signal at dH 3.66– 3.69 (m) was attributed to H-1b, suggesting the presence of an OH-1a [8,9]. A oneproton doublet signal at d 4.02 (d, J ¼ 4.0 Hz) was assigned to H-3b based on the correlations between this signal and C-1 (dC 72.4, d) as well as C-18 (dC 79.7, t) in the HMBC spectrum. The a-orientation of OH-3 was established according to the correlation between H-3 and H-18 in its NOESY spectrum. All signals in the 1H (13C) NMR spectra of this compound were assigned based on its 2D NMR spectra (Table 2). All the evidence given above led to the assignment of the structure of ducloudine D as shown in Figure 1.

OH

N HO

4 19 H3CO

12

13 16

Compound 3 was obtained as a white amorphous powder. Its molecular formula was deduced to be C26H41NO8 by HRESI-MS at m/z 496.2904 [M þ H]þ. The NMR spectrum of 3 exhibited characteristic features of the aconitine-type C19diterpenoid alkaloid, bearing an N-ethyl group (dH 1.15, 3H, t, J ¼ 7.2 Hz; 2.50 – 2.52, 2.54 – 2.56, each 1H, m; dC 12.9, q; 48.2, t), three OMe groups (dH 3.28, 3.33 and 3.36, each 3H, s; dC 56.1, q; 59.1, q; 57.9, q), and one acetyl group (dH 2.07, 3H, s; dC 21.3, q; 170.3, s). Comparison of the MS and NMR spectra of 3 with those of 2 showed it had an additional acetoxyl group instead of a hydroxyl group. The 13C NMR spectra of 3 and 2 are very similar except for C-9, C-13, and C-14 due to the substituted effect (OH ! OAc) [10]. This extra acetyl group could be located at C-14

OCH3 16 OH

8 OH 7

6 OCH3

Figure 3. Key 1HZ1H COSY ( compound 2.

2

15

9 5

48.7 t 13.0 q 58.0 q 56.4 q 59.1 q

OH17 1 N

HO

), HMBC (

11

OCH3 OH

9 8

15 OH

6 OCH3 18 H3CO

), and NOESY (

) correlations of

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Table 2. 1H and 13C NMR spectral data (400 MHz for 1H and 100 MHz for compounds 2 and 3.

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Compound 2

13

C, CDCl3) for

Compound 3

No.

dH, J (Hz)

dC

dH, J (Hz)

dC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

3.66 – 3.69 m 1.67 – 1.69 m, 1.82 – 1.84 m 4.02 d (4.0)

72.4 d 37.9 t 71.9 d 44.2 s 46.3 d 82.9 d 52.2 d 74.0 s 48.0 d 43.7 d 49.5 s 29.7 t 40.3 d 75.6 d 42.2 t 82.4 d 63.1 d 79.7 t

3.78 t (3.2) 1.78 – 1.80 m, 1.95 – 1.97 m 4.13 d (4.0)

72.4 d 37.9 t 72.1 d 44.2 s 46.0 d 83.1 d 52.8 d 74.5 s 46.0 d 43.0 d 49.7 s 29.7 t 36.6 d 77.0 d 42.6 t 81.9 d 62.9 d 79.8 t

19 21 22 OCH3-6 OCH3-16 OCH3-18 OAc-14

2.00 d (6.4) 4.10 d (6.4) 1.97 – 1.99 m 2.41 – 2.43 m 1.72 – 1.74 m 1.59 – 1.61 m, 1.93 – 1.95 m 2.10 t (6.6) 4.12 t (4.5) 2.00 – 2.02 m, 2.25 – 2.27 m 3.25 – 3.27 m 2.73 br s 3.46 ABq (8.0), 3.51 ABq (8.0) 2.05 ABq (11.2), 3.04 ABq (11.2) 2.39 – 2.41 m, 2.43 – 2.45 m 1.07 t (7.2) 3.28 s 3.27 s 3.24 s

2.11 – 2.13 m 4.13 d (6.4) 2.08 – 2.10 m 2.25 – 2.27 m 1.88 – 1.90 m 1.17 – 1.19 m, 1.73 – 1.75 m 2.64 t (5.6) 4.86 t (4.8) 2.30 – 2.32 m, 2.35 – 2.38 m 3.29 – 3.31 m 2.78 – 2.80 m 3.53 ABq (8.0), 3.57 ABq (8.0) 2.48 ABq (11.2), 2.79 ABq (11.2) 2.50 – 2.52 m, 2.54 – 2.56 m 1.15 t (7.2) 3.36 s 3.28 s 3.33 s

48.1 t 48.1 t 13.0 q 57.8 q 56.3 q 59.1 q

2.07 s

according to the HMBC correlation between H-14 (4.86, t, J ¼ 4.8 Hz) and OAc-14 (dC 170.3, s) (Figure 4). This was further confirmed by H-14 in compound 3 was shifted downfield from dH 4.12 (t, J ¼ 4.5 Hz) in compound 2 to dH 4.86 (t, J ¼ 4.8 Hz) [11]. All signals in the 1 H (13C) spectra of this compound were

OH 2 HO 19

N 4

H3CO

12

13 16

OCH3

16

O 15

5

8 OH 7

6 OCH3

Figure 4. Key 1HZ1H COSY ( compound 3.

48.2 t 12.9 q 57.9 q 56.1 q 59.1 q 170.3 s 21.3 q

assigned based on its 2D NMR spectra (Table 2). Therefore, the structure of ducloudine E (3) was determined. The results of bioassays (Table 3) showed that compound 1 exhibited antifungal activity against Candida albicans with an MIC of 128 mg/ml. Compounds 2 and 3 showed antifungal activity against

OH17 1

O 9

48.1 t

CH3 2 HO

), HMBC (

N

11

OCH3 O O

9

CH3

8 15 OH

6 OCH3 18 H3CO

), and NOESY (

) correlations of

Journal of Asian Natural Products Research Table 3. Antifungal and antibacterial activities of compounds 1 – 3. MIC (mg/ml) Compound

C. albicans

E. coli

B. subtilis

1 2 3 Nystatin Kanamycin

128 256 256 16

. 512 512 . 512

.512 256 256

1

1

Yunnan Province, China, in December 2012, and were identified by Prof. ShuGang Lu from School of Life Sciences, Yunnan University. A voucher specimen (2012-bcw-1) has been deposited in the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Kunming, China.

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3.3 C. albicans with MICs of 256 mg/ml and antibacterial activity against Bacillus subtilis with MICs of 256 mg/ml, respectively. 3.

Experimental

3.1 General experimental procedures Melting points were determined on an XRC-1 Melting Point Apparatus (Sichuan University Science Instrument Co., Chengdu, China) and were uncorrected. A Shimadzu UV –vis 2550 spectrometer (Shimadzu, Kyoto, Japan) was used for colorimetric measurements and UV spectra. Optical rotations were measured with a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Nicolet Magna-IR 550 spectrometer (Thermo Nicolet Co., Madison, WI, USA) was used for scanning IR spectroscopy with KBr pellets. NMR spectra were recorded on Bruker AM-400 spectrometers (Bruker, Karlsruhe, Germany) using TMS as the internal reference. ESI-MS analyses were carried out with Agilent G3250AA (Agilent, Santa Clara, CA, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, MA, USA). Silica gel (200 – 300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China) was used for column chromatography (CC). Fractions were monitored by TLC and visualized by spraying with modified Dragendorff’s reagent. 3.2 Plant material The roots of A. duclouxii were collected from Dali Bai Autonomous Prefecture in

349

Extraction and isolation

The air-dried and powdered roots (6.5 kg) of A. duclouxii were percolated with 0.5% HCl. The aqueous acidic solution was basified with ammonia (10%) to pH 9.0 and then extracted with EtOAc. Removal of the solvent under reduced pressure afforded the total crude alkaloids (51.5 g) as a yellowish amorphous powder. The total alkaloids were chromatographed over SiO2 column eluting with chloroform – methanol (100:1 – 1:1) gradient system to give 13 fractions (A – M). Fraction C (2.3 g) was further subjected to CC by petroleum ether – acetone – diethylamine (100:2:1 – 50:50:1) to give ducloudine C (105.5 mg). Further normal phased CC purification of Fraction F (3.6 g) was accomplished by elution with a chloroform – methanol (30:1 – 10:1) to afford ducloudine D (15.0 mg). Fraction H (1.1 g) was chromatographed over SiO2 column with chloroform –methanol (10:1 –5:1) to provide ducloudine E (35.5 mg). 3.3.1 Ducloudine C (1) White amorphous powder; m.p. 66– 688C, ½a20 D þ 58.36 (c ¼ 1.5, CHCl 3 ); UV (MeOH) lmax nm (log 1): 219 (1.24); IR (KBr, cm21): nmax 3438, 2926, 2811, 1716, 1670, 1455, 1108. For 1H and 13C NMR spectral data, see Table 1. HR-ESIMS m/z: 434.2545 [M þ H]þ (calcd for C24H36NO6, 434.2543). 3.3.2 Ducloudine D (2) White amorphous powder; m.p. 86– 888C, ½a20 D þ 24.54 (c ¼ 1.6, CHCl 3 ); IR

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(KBr, cm21): nmax 3428, 2931, 2814, 1634, 1462, 1100. For 1H and 13C NMR spectral data, see Table 2. HR-ESI-MS m/z: 454.2799 [M þ H] þ (calcd for C24H40NO7, 454.2805).

CX21BIM-set5 microscope (Olympus Corp., Tokyo, Japan). MICs were determined as the lowest concentrations that produce complete growth inhibition of the tested microorganisms.

3.3.3

Acknowledgments

Ducloudine E (3)

White amorphous powder; m.p. 83 – 858C, ½a20 D þ 24.33 (c ¼ 1.0, CHCl3); IR (KBr, cm21): nmax 3432, 2925, 2858, 1731, 1099. For 1H and 13C NMR spectral data, see Table 2. HR-ESI-MS m/z: 496.2904 [M þ H]þ (calcd for C 26 H42NO8 , 496.2910).

This work was financially supported by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13095), a grant from the Natural Science Foundation of China (No. 21002084) and a grant from the Natural Science Foundation of the Yunnan Province (No. 2010CD017).

Note 3.4 Antifungal and antibacterial activity One pathogenic fungi (C. albicans) and two pathogenic bacteria (Escherichia coli ATCC 25922 and B. subtilis), collected from Yunnan Institute of Microbiology, were selected for antifungal and antibacterial assays, respectively. Antifungal and antibacterial assays were performed in 96well sterilized microplates using a microdilution method described previously [12]. The 4-day-old spores from C. albicans were transferred to sterile PDB broth medium to reach 1 £ 103 spores/ml. The 18-h-old bacterial cultures from E. coli and B. subtilis were added to sterile LB broth medium to reach 1 £ 105 colony-forming units/ml. The test compounds were dissolved in DMSO and their final concentrations ranged from 512 to 0.5 mg/ml by using a twofold serial dilution method. The wells containing test strains and diluted compounds were incubated at 288C (4 days) for fungi and 378C (24 h) for bacteria. The wells containing a culture suspension and DMSO were run as negative controls. Nystatin and kanamycin were introduced in the experiments as positive controls for antifungal and antibacterial assays, respectively. All experiments were repeated three times. The growth of test strains was observed with a

1.

These authors contributed equally to this study.

References [1] P.G. Xiao, F.P. Wang, F. Gao, L.P. Yan, D.L. Chen, and Y. Liu, Acta Phytotax. Sin. 44, 1 (2006). [2] People’s Government of Dali Bai Autonomous Prefecture, The Natural Resources of Chinese Medicinal Materials of Dali (Yunnan Ethnic Publishing House, Kunming, 1991), p. 77. [3] C.Y. Wang, J.B. Chen, and R.H. Zhu, Acta Pharm. Sin. 19, 445 (1984). [4] T.P. Yin, L. Cai, G. Lei, J.W. Dong, Y.X. Liu, and Z.T. Ding, Chin. J. Org. Chem. 33, 2528 (2013). [5] S.W. Pelletie, N.V. Mody, B.S. Joshi, and L.C. Schram, Alkaloids: Chemical and Biological Perspectives (Wiley, New York, 1984), pp. 206– 462. [6] G.B. Xie and F.P. Wang, Heterocycles 3, 631 (1997). [7] Z.T. Zhang, X.Y. Liu, D.L. Chen, and F.P. Wang, Helv. Chim. Acta 93, 811 (2005). [8] C.S. Peng, D.L. Chen, Q.H. Chen, and F.P. Wang, Chin. J. Org. Chem. 25, 1235 (2005). [9] S.W. Pelletier and Z. Djarmati, J. Am. Chem. Soc. 98, 2626 (1976). [10] F.P. Wang, Chin. J. Org. Chem. 3, 161 (1982). [11] L.S. Ding and W.X. Chen, Nat. Prod. Res. Dev. 1, 6 (1989). [12] M.C. Carpinella, C.G. Ferrayoli, and S.M. Palacios, J. Agric. Food Chem. 53, 2922 (2005).

Three new diterpenoid alkaloids from the roots of Aconitum duclouxii.

Three new C₁₉-diterpenoid alkaloids, ducloudines C (1), D (2), and E (3), were isolated from the roots of Aconitum duclouxii. Their structures were es...
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