A new steroidal alkaloid from Beaumontia grandiflora

Li Ma a, Jian Guang Luo a, Yu Cheng Gu b, Ling Yi Kong a,*a Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,

Nanjing 210009, People’s Republic of Chinab Syngenta Jealott’s Hill International Research Centre Bracknell, Berkshire RG42 6EY, United Kingdom

Received 4 January 2009

Abstract

A new steroidal alkaloid, beaumontamine (1), was isolated from the stems of Beaumontia grandiflora. The structure was

elucidated on the basis of spectral analysis.

# 2009 L.Y. Kong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Beaumontia grandiflora; Beaumontamine; Alkaloid

Beaumontia grandiflora (Apocynaceae) is distributed in Yunnan, Guangxi, Guangdong and Fujian provinces in

China. It is used in folk medicine to treat waist ache, leg pain of rheumatism, bone fracture and wound [1]. In our

research, a new steroidal alkaloid, beaumontamine (1), was obtained and its structure was elucidated on the basis of a

comprehensive analysis of the 1H NMR, 13C NMR and 2D NMR spectra. Herein, we report the isolation and structure

elucidation of beaumontamine.

The stems of B. grandiflora were collected from Xishuangbanna Botanical Garden in Yunnan Province, China, in

October 2006, and authenticated by Prof. Mian Zhang in China Pharmaceutical University. A voucher specimen (no.

20061001) was deposited in our Department of Natural Medicinal Chemistry. The dried stems (15 kg) were extracted

with 70% EtOH (1500 L � 3) under reflux. The combined solution was concentrated to dryness under vacuum. The

crude extract (800 g) was suspended in water and successively extracted with petroleum ether, ethyl acetate and n-

butanol. After concentration in vacuum, the ethyl acetate extract (140.0 g) was subjected to silica gel column eluted

with a gradient of CHCl3–MeOH (100:0–1:1) to afford ten fractions. Fraction 4 (8.6 g) [CHCl3–MeOH (10:1)] was

further purified by using silica gel column chromatography repeatedly to provide compound 1 (5 mg). Compound 1

was obtained as colorless needle crystals (MeOH), with mp 156–158 8C, and optical rotation [a]D25 18.0 (c 0.06,

MeOH). It showed blue color when sprayed with vanillin–H2SO4 reagent in co-TLC and positive Liebermann–

Burchard reaction. Its molecular formula was established as C21H35NO2 from the quasimolecular ion peak at m/z

334.2740 [M + H]+ (calcd. 334.2741) in HR-ESI-MS. The IR spectrum (KBr) revealed the presences of –NH2 (3389,

3387, 1632 cm�1) [2], C–N (1275 cm�1), –OH (3290, 3048 cm�1), –CH3 (2971, 2901 cm�1) and –CH2– groups

(2934, 2845 cm�1). In the NMR spectra, three methyl [dH 1.24 (s, H3-18), 1.04 (s, H3-19), 1.58 (d, J = 6.6, H3-21); dC

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Chinese Chemical Letters 20 (2009) 1094–1096

* Corresponding author.

E-mail address: cpu_lykong@126.com (L.Y. Kong).

1001-8417/$ – see front matter # 2009 L.Y. Kong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2009.03.044

15.2 (C-18), 19.6 (C-19), 19.6 (C-21)] and an olefinic methine groups [dH 5.46 (br s, H-6); dC 140.8 (C-5), 121.3 (C-6)]

were observed. On the basis of the above information, compound 1 was determined to be steroidal alkaloid [3,4].

All the proton and carbon signals were assigned as Table 1 by HMQC and HMBC spectra. The position of the –OH

and –NH2 groups was determined by using the HMBC correlations. Two hydroxyl groups were located to C-3 [dH 3.91

(br s, H-3a); dC 71.1 (C-3)] and C-14 [dC 85.3 (C-14)], respectively. The –NH2 group [dH 2.0 (br s, 2H)] was linked to

C-20 [dC 55.0] (Fig. 1). Comparing the NMR spectral data of H-17, C-3, C-14 and C-17 in compound 1 with those in

digitoxigenin b-gentiobiosyl-b-D-cymaroside [dH 2.79 (dd, J = 9, 5, H-17a); dC 73.2 (C-3), 84.6 (C-14), 51.5 (C-17)]

and 17a-digitoxigenin b-gentiobiosyl-a-L-cymaroside [dH 3.40 (dd, J = 9, 5, H-17b); dC 72.8 (C-3), 85.2 (C-14), 48.9

(C-17)] [5], 3b-,14b- and 17a-OH were determined, which was confirmed by the NOESY experiment (Fig. 2).

Therefore, the structure of compound 1 was elucidated as 3b, 14b-dihydroxyl-20-amino-17a-pregne-5-ne, named

beaumontamine (Fig. 1).

L. Ma et al. / Chinese Chemical Letters 20 (2009) 1094–1096 1095

Table 1

NMR spectral data of compound 1 in C5D5N (1H: 500 MHz; 13C: 125 MHz; d (ppm), J (Hz)).

Position dH dC

1 1.85 (m, 1H, Ha), 2.09 (m, 1H, Hb) 37.4

2 2.68 (m, 1H, Ha), 2.09 (m, 1H, Hb) 27.5

3 3.91 (m, 1H, Ha) 71.1

4 2.68 (m, 2H) 43.2

5 140.8

6 5.46 (m, 1H) 121.3

7 2.09 (m, 2H) 32.7

8 1.85 (m, 1H) 37.7

9 1.19 (m, 1H) 46.6

10 37.3

11 1.42 (m, 2H) 21.0

12 1.34 (m, 2H) 38.9

13 48.3

14 85.3

15 1.85 (m, 1H, Ha), 2.15 (m, 1H, Hb) 32.4

16 2.28 (m, 1H, Ha), 2.09 (m, 1H, Hb) 19.6

17 3.91 (m, 1H) 46.7

18 1.24 (s, 3H) 15.2

19 1.04 (s, 3H) 19.6

20 1.85 (m, 1H) 55.0

21 1.58 (d, 3H, 6.6) 19.6

NH2 2.01 (2H, m)

Fig. 1. Key HMBC correlations of compound 1.

Acknowledgments

The research work was financially supported by the National Natural Science Foundation of China for Outstanding

Young Scientists (No. 30525032) and Syngenta Postgraduate Fellowship (No. SPF-008).

References

[1] Zhongguozhiwuzhi, 63, Science Press, Beijing, 1995, p. 129.

[2] A.U. Rahman, D. Shahwar, M.I. Choudhary, B. Sener, G. Toker, K.H.C. Baser, Phytochemistry 50 (1999) 333.

[3] M.H. Qiu, R.L. Nie, X. Wang, J. Zhou, Acta Botanica Sinica 31 (1989) 535.

[4] I. Naeem, N. Khan, M.I. Choudhary, A.U. Rahman, Phytochemistry 43 (1996) 903.

[5] T. Yamauchi, F. Abe, T. Santisuk, Phytochemistry 29 (1990) (1961).

L. Ma et al. / Chinese Chemical Letters 20 (2009) 1094–10961096

Fig. 2. Key NOESY correlations of compound 1.