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New steroidal alkaloids from the bulbs of Fritillaria puqiensis
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
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New steroidal alkaloids from the bulbs of Fritillaria puqiensis Yan Jiang, Ping Li ∗ , Hui-Jun Li ∗ , Hua Yu MOE Key Laboratory of Modern Chinese Medicines, Department of Pharmacognosy, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing 210009, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Six new steroidal alkaloids, namely puqienines C–E (1–3), puqiedine (4), 3␣-puqiedin-7-ol
Received 7 February 2006
(5), and puqietinedione (6), along with two known steroidal alkaloids puqiedinone (7) and
Received in revised form 26 May
peimisine (8), were isolated from the bulbs of Fritillaria puqiensis G.D. Yu et G.Y. Chen (Lili-
2006
aceae). Their structures were elucidated on the basis of spectroscopic analysis, including 1D
Accepted 29 May 2006
and 2D NMR experiments. Among these alkaloids, 1–3 had a veratramine-type skeleton, 4, 5,
Published on line 10 July 2006
7 a cevanine-type skeleton, 6 a secosolanidine-type skeleton, and 8 a jervine-type skeleton. The existence of multiple types of steroidal skeletons, especially of relatively large amount
Keywords:
of veratramine-type alkaloids in one species is rare in the genus Fritillaria, and the results
Fritillaria puqiensis
might be of chemotaxonomic significance for this species. © 2006 Elsevier Inc. All rights reserved.
Steroidal alkaloids Puqienines C–E Puqiedine 3␣-Puqiedin-7-ol Puqietinedione
1.
Introduction
The genus Fritillaria, containing about 130 species, is one of the largest genera in the plant family of Liliaceae [1]. Many species of Fritillaria were traditionally used as herbal remedies in Japanese, Turkish, Pakistani and south-east Asian folk medicines [2–4], while the bulbs of several Fritillaria species growing in China, have been used as an antitussive, antiasthmatic and expectorant using the Chinese name “Beimu” in traditional Chinese medicine (TCM) for centuries [5]. Previous investigations on pharmacology and phytochemistry had suggested that steroidal alkaloids were responsible for the effects of this herbal drug [6,7]. The Fritillaria alkaloids were structurally divided into five main types, cevanines, jervines, veratramines, solanidines, and secosolanidines skeleton, with cevanine-type alkaloids occupying dominantly [8–11]. However, during the course of our investigation concerning the isolation and characterization of chemical constituents of Fritillaria puqiensis G.D. Yu et G.Y. Chen, endemic to Hubei Province ∗
of China, relatively copious veratramine- and secosolanidinetype steroidal alkaloids were obtained [12–14]. In continuation of our studies on F. puqiensis, six new steroidal alkaloids, namely, puqienines C–E (1–3), puqiedine (4), 3␣-puqiedin-7ol (5), puqietinedione (6) (Fig. 1), together with two known steroidal alkaloids puqiedinone (7) and peimisine (8), were isolated and identified from the dried ground bulbs of this plant. In this paper, we describe the extraction, isolation and structure elucidation of the new alkaloids.
2.
Experimental
2.1.
General procedures
The melting points were measured on an X4 micro-melting point apparatus and were uncorrected. Optical rotations were determined in MeOH on a PE-241 MC digital polarimeter at 20 ◦ C. IR spectra were recorded in KBr disks on a Nicolet
Corresponding authors. Tel.: +86 25 8324 2299; fax: +86 25 8324 2299. E-mail addresses: [email protected] (P. Li), [email protected] (H.-J. Li). 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.05.016
844
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
Fig. 1 – Structures of 1–10.
Impact 410 spectrophotometer. UV spectra were performed on a Shimadzu UV-2501 spectrophotometer. NMR spectra were obtained in CDCl3 or pyridine-d5 containing TMS as an internal standard on a Bruker Am-500 NMR spectrometer. The f1 and f2 resolution for heteronuclear 2D experiments was about 0.2 and 0.01 ppm, respectively. HRESIMS spectra were recorded on an Agilent 1100 LC/MSD TOF instrument in positive ion mode. Column chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Factory, China). TLC was conducted on pre-coated silica gel G plates (0.25 mm thick, Qingdao Marine Chemical Factory, China).
2.2.
Extraction and isolation procedure
The bulbs of F. puqiensis were collected from Puqi County, Hubei Province, People’s Republic of China, in May 2000, and
authenticated by one of us (Dr. Ping Li). A voucher specimen was deposited at the Herbarium of Department of Pharmacognosy, China Pharmaceutical University. Dried ground bulbs (10 kg) were extracted with 70% EtOH by soaking repeatedly (3× 20L). The concentrated extract was dissolved in 2% HCl (pH 3.5) and partitioned with ether. The pH of the aqueous solution was re-adjusted with NH4 OH to 9.2 and extracted with CHCl3 . The CHCl3 extract (crude alkaloid, 50 g) was chromatographed over silica gel using CHCl3 /CH3 OH of increasing polarity as eluent to obtain five fractions A–E. Fractions B–E were further separated by repeated column chromatography, and eluted with petroleum ether/Me2 CO/diethylamine (88:12:2, 80:20:2 and 75:25:2) and CHCl3 /CH3 OH/diethylamine (100:7:5), respectively. Fraction B afforded 4 (19 mg) and 8 (50 mg), fraction C yielded 3 (27 mg), 6 (10 mg) and 7 (12 mg), fraction D gave 5 (20 mg), and fraction E offered 1 (23 mg) and 2 (18 mg).
845
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
2.2.1.
Puqienine C (1) 291–293 ◦ C,
C28 H45 NO4 , colorless needles (MeOH), mp [˛]20 D + 10.9◦ (c = 0.1, MeOH); UV (MeOH) max 250 nm; IR (KBr)max 3446, 2952, 1660, 1124 cm−1 ; HRESIMS m/z 460.3430 [M + H]+ (calcd. for C28 H46 NO4 , 460.3421); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.2.
Puqienine D (2)
C28 H45 NO4 , colorless needles (MeOH), mp 275–277 ◦ C, [˛]20 D −73.1◦ (c = 0.1, MeOH); IR (KBr)max 3446, 2949, 1707, 1057 cm−1 ; HRESIMS m/z 460.3422 [M + H]+ (calcd. for C28 H46 NO4 , 460.3421); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.3.
Puqienine E (3)
C28 H43 NO3 , colorless squares (MeOH), mp 235–237 ◦ C, [˛]20 D −141.1◦ (c = 0.1, MeOH); UV (MeOH) max 305 nm; IR (KBr)max 3448, 2921, 1612, 1234 cm−1 ; HRESIMS m/z 442.3346 [M + H]+ (calcd for C28 H44 NO3 , 442.3315); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.4.
Puqiedine (4)
C27 H45 NO2 , colorless squares (Me2 CO), mp 139–141 ◦ C, −38.4◦ (c = 0.1, MeOH); IR (KBr)max 3396, 2925, [˛]20 D 2767 cm−1 ; HRESIMS m/z 416.3539 [M + H]+ (calcd. for
C27 H46 NO2 , 416.3734); 1 H and Tables 1 and 2.
2.2.5.
13 C
NMR data are shown in
3˛-Puqiedin-7-ol (5)
C27 H45 NO3 , colorless squares (Me2 CO), mp 267–269 ◦ C, [˛]20 D −47.7◦ (c = 0.1, MeOH); IR (KBr)max 3396, 2917, 2750 cm−1 ; HRESIMS m/z 432.3485 [M + H]+ (calcd for C27 H46 NO3 , 432.3472); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.6.
Puqietinedione (6)
C28 H45 NO2 , colorless needles (MeOH), mp 281–283 ◦ C, ◦ −1 [˛]20 D + 30.1 (c = 0.1, MeOH); IR (KBr)max 2950, 1719, 1698 cm ; HRESIMS m/z 428.3527 [M + H]+ (calcd. for C28 H46 NO2 , 428.3523); 1 H and 13 C NMR data are shown in Tables 1 and 2.
3.
Results and discussion
Air-dried ground bulbs were extracted with 70% EtOH, then the concentrate was dissolved in 2% HCl and extracted with ether. The acidic aqueous solution was alkalized with NH4 OH and extracted with CHCl3 . The CHCl3 -soluble portion was repeatedly chromatographed over silica gel to afford eight steroidal alkaloids (1–8). The known compounds (7 and 8) were iden-
Table 1 – 1 H NMR data of compounds 1–6a (ı) 1b
2b
3b
4c
5b
6c
1.36 m, 1.58 m 1.65 m, 1.99 m 3.86 br, m, 24.4d 1.76 m, 2.80 m 2.17 dd, 12.3, 3.1
1.27 m, 1.57 m 1.70 m, 1.97 m 3.78 br, m, 25.0d 1.80 m, 2.31 m 2.21 dd, 12.5, 2.4
1.37 m, 1.58 m 1.65 m, 2.02 m 3.87 br, m, 24.8d 1.71 m, 2.87 m 2.28 dd, 14.8, 3.2 5.96 m
2.56 m 1.32 m, 2.15 m
2.40 m 2.75 m 2.42 m 1.70 m 1.25 m, 2.03 m
1.34 m, 2.04 m 1.89 m, 2.04 m 4.46 m, 12.4d 1.90 m, 2.45 m 2.89 m 4.18 dd, 2.8, 2.1 4.42 dd, 2.8, 2.4
1.63 m, 2.08 m 2.38 m, 2.44 m
6.04 t, 2.5
1.03 m, 1.45 m 1.46 m, 1.79 m 3.64 br, m, 24.8d 1.70 m 1.10 m 3.86 br, m 1.09 m 1.98 m 1.78 m 1.00 m 1.08 m, 1.78 m
1.90 m
2.95 br, m
1.55 m 1.19 qd, 10.6, 2.8 1.46 m 1.51 m, 1.72 m 0.96 m, 1.69 m 0.61 qd, 10.5, 3.5 1.60 m, 2.79 m 0.99 s 0.99 m 0.86 d, 6.5 1.32 m 1.11 m, 1.94 m 0.86 m, 1.76 m 1.72 m 1.58 m, 2.77 m 0.84 d, 6.3
1.63 m 1.38 qd, 10.8, 3.2 2.43 m 1.60 m, 1.99 m 0.96 m, 1.64 m 0.62 qd, 10.5, 2.8 1.66 m, 2.92 m 1.32 s 0.97 m 0.78 d, 6.5 1.29 m 1.11 m, 1.86 m 0.82 m, 1.67 m 1.67 m 1.55 m, 2.73 m 0.76 d, 6.3
Position 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 a b c d
2.80 m 2.19 dd, 12.9, 3.2 2.44 dd, 12.9, 2.6 2.51 m
2.05 m 1.26 m, 2.03 m 1.40 m, 2.09 m 1.94 m 1.30 s 0.80 s 3.06 qd, 7.3, 1.9 1.24 d, 7.3 2.06 m 4.21 br, m 1.09 td, 12.9, 2.4 2.04 m 2.32 br, m 1.91 m, 2.85 m 0.76 d, 6.6 2.59 s
5.30 m 2.13 m, 2.45 m 2.29 m 1.22 s 0.73 s 3.15 qd, 7.1, 2.2 1.23 d, 7.1 2.15 m 4.19 br, m 1.08 td, 12.9, 2.5 2.02 m 2.30 br, m 1.94 m, 2.84 m 0.73 d, 6.5 2.59 s
1.94 m, 2.18 m 1.28 m, 1.86 m 1.96 m 1.01 d, 7.0 0.68 s 1.97 m 1.25 d, 6.1 1.91 m 4.12 br, m 1.10 td, 12.9, 2.5 1.97 m 2.27 m 1.77 m, 2.84 m 0.77 d, 6.6 2.23 s
Assignments were made using H–H COSY, HMQC and HMBC techniques. Measured in pyridine-d5. Measured in CDCl3. Multiplet width at half height.
2.45 m 2.30 m 1.31 m, 1.91 m
2.33 m, 2.59 m 2.60 m 1.99 t, 12.7 2.39 m 1.87 m 1.35 m 1.45 m, 1.68 m 1.27 m, 2.09 m 1.28 m 1.13 m, 1.55 m 1.31 m, 1.77 m 1.30 m 0.72 s 0.96 s 1.92 m 0.94 d, 6.6 1.76 m 1.31 m, 1.55 m 0.88 m, 1.77 m 1.73 m 1.76 m, 2.99 m 0.85 d, 6.0 2.21 s
846
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
Table 2 – 13 C NMR data of compounds 1–6a (ı) Position 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 27 28 a
b c
1b
2b
3b
4c
5b
6c
37.8 t 31.7 t 70.5 d 31.6 t 54.9 d 199.2 s 120.7 d 172.9 s 57.4 d 40.4 s 25.4 t 57.7 d 74.5 s 48.3 d 28.8 t 26.7 t 50.4 d 16.8 q 12.4 q 29.7 d 13.2 q 70.6 d 66.8 d 43.2 t 23.3 d 65.8 t 19.6 q 41.3 q
37.4 t 31.3 t 70.0 d 30.9 t 57.0 d 209.6 s 45.5 t 43.8 d 55.7 d 37.9 s 25.1 t 57.2 d 72.9 s 142.5 s 116.7 d 28.7 t 46.1 d 16.8 q 12.5 q 29.4 d 13.8 q 69.6 d 66.6 d 42.9 t 22.7 d 65.3 t 19.3 q 40.8 q
37.2 t 31.3 t 70.3 d 31.7 t 54.7 d 198.9 s 113.4 d 169.6 s 53.5 d 39.8 s 33.6 t 162.1 s 33.9 d 135.2 s 22.0 t 20.1 t 40.0 d 13.4 q 11.9 q 33.0 d 13.9 q 66.6 d 66.0 d 42.4 t 24.7 d 66.1 t 19.3 q 42.1 q
39.1 t 31.3 t 71.9 d 34.9 t 48.3 d 72.8 d 38.6 t 34.8 d 57.7 d 35.5 s 29.3 t 40.2 d 44.5 d 44.0 d 24.7 t 25.6 t 46.5 d 61.5 t 15.1 q 41.3 d 14.9 q 68.5 d 30.2 t 33.5 t 30.7 d 64.7 t 19.7 q
35.6 t 29.6 t 66.3 d 33.7 t 37.7 d 77.6 d 70.7 d 39.7 d 49.0 d 36.9 s 28.8 t 40.3 d 44.5 d 37.5 d 24.7 t 25.7 t 46.5 d 61.7 t 13.7 q 41.7 d 14.8 q 68.3 d 30.6 t 33.6 t 31.0 d 64.8 t 19.5 q
38.1 t 37.3 t 211.0 s 37.0 t 57.5 d 208.8 s 46.6 t 38.0 d 53.5 d 41.2 s 21.7 t 39.5 t 43.4 s 56.3 d 24.0 t 27.4 t 52.9 d 12.0 q 12.6 q 35.1 d 12.6 q 66.7 d 23.9 t 33.4 t 31.2 d 66.1 t 19.5 q 42.8 q
Assignments were made using H–H COSY, HMQC and HMBC techniques. Measured in pyridine-d5 . Measured in CDCl3 .
tified as puqiedinone [15] and peimisine [16], respectively, by detailed spectral analysis and compared with reference data. Puqienine C (1) was isolated as colorless needles from MeOH, giving a positive Dragendorff test. The HRESIMS exhibited a quasimolecular ion [M + H]+ at m/z 460.3430 corresponding to the molecular formula C28 H45 NO4 , possessing seven degrees of unsaturation. The UV spectrum showed absorption maximum at 250 nm, which was diagnostic of an ␣, unsaturated ketone. The IR spectrum indicated the presence
of hydroxyl (3446 cm−1 ) and carbonyl (1660 cm−1 ) groups. The NMR spectrum of 1 displayed two methyl singlet signals at ı 0.80 (3H, s, H-19) and 1.30 (3H, s, H-18), two methyl doublet signals at ı 0.76 (3H, d, J = 6.6 Hz, H-27) and 1.24 (3H, d, J = 7.3 Hz, H-21), as well as an N-methyl signal at ı 2.59 (3H, s, H-28), which suggested that 1 was a veratramine-type alkaloid [17]. The 13 C NMR and DEPT spectra disclosed the presence of a carbonyl carbon [ı 199.2 (C-6)], an oxygenated quaternary carbon [ı 74.5 (C-13)], and two oxy-methines [ı 70.5 (C-3) and 66.8 (C-23)]. The 13 C NMR feature of 1 was very similar to that of puqienine F (9) [14], with the major differences being the presence of characteristic signals of a conjugated double bond (ı 172.9 and 120.7), and the absence of a 12,16-epoxy ring in 1. The position of double bond was confirmed to locate between C-7 and C-8 through the HMBC correlations between olefinic proton (ı 6.04) and C-5 (ı 54.9), C-9 (ı 57.4), and C-14 (ı 48.3). In the NOESY spectrum of 1, H3 -18 showed clear cross peak with H3 -19, confirming the configuration of 13-OH to be ␣. Then, the key NOEs were observed between H-1␣/H-3, H-3/H-5, H-5/H-9, H-1/H-19, H-9/H-12, H-14/H-17, H-22/H-27, H-23/H-26␣ and H-26␣/H-27, which proved A/B trans, 3-OH, 5␣-H, 12␣-H, 17side chain, 22␣-H, 23-OH and 25␣-CH3 (Fig. 2). The relative stereochemistries of C-20 and C-22 were tentatively established to be R* from biogenesis. Thus, 1 was elucidated as (20R* , 22R* )-N-methyl-5,6,12,13-tetrahydro-3, 13␣, 23-trihydroxy5␣12␣, 17, 25␣-veratraman-7-en-6-one, named puqienine C. Puqienine D (2) was crystallized from MeOH as colorless needles, and gave a positive reaction to Dragendorff reagent. The molecular formula, C28 H45 NO4 , was assigned on the basis of the quasimolecular ion [M + H]+ at m/z 460.3422 in its HRESIMS. The IR spectrum showed absorption bands for hydroxyl (3446 cm−1 ) and carbonyl (1707 cm−1 ) groups. The 1 H NMR spectrum of 2 indicated the presence of two methyl singlet signals [ı 0.73 (3H, s, H-19) and 1.22 (3H, s, H-18)], two methyl doublet signals [ı 0.73 (3H, d, J = 6.5 Hz, H-27) and 1.23 (3H, d, J = 7.1 Hz, H-21)], a methyl group located at the nitrogen atom [ı 2.59 (3H, s, H-28)], two oxy methine protons [ı 3.78 (1H, br, m, H-3␣) and 4.19 (1H, br, m, H-23␣)], and an olefinic proton [ı 5.30 (1H, m)]. The 13 C NMR spectrum of 2 gave almost the same chemical shifts as those of 1 except for the signals due to different position of double bond. The signals resonating at ı 142.5 and 116.7 implied the presence of a nonconju1H
Fig. 2 – Key NOESY correlations of 1.
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
gated double bond in 2, and the HMBC correlations between olefinic proton (ı 5.30) and C-17 (ı 46.1), C-12 (ı 57.2) assigned the position of the double bond between C-14 and C-15. In the NOESY spectrum, the correlations between methyl protons H3 -19/H-8 and H-1␣/H-9 revealed B/C trans. The remaining cross peaks suggested the same configurations of A/B ring fusion and piperidine moiety as those of 1. Therefore, 2 was established as (20R* , 22R* )-N-methyl-5,6,12,13-tetrahydro3, 13␣, 23-trihydroxy-5␣, 12␣, 17, 25␣-veratraman-14-en-6one, named puqienine D. Puqienine E (3) was obtained as colorless squares from MeOH, and gave a positive reaction to Dragendorff reagent. The HRESIMS showed a quasimolecular ion [M + H]+ at m/z 442.3346, consistent with the molecular formular C28 H43 NO3 . The UV spectrum exhibited absorption maximum at 305 nm, which revealed the presence of a long conjugated system. The IR spectrum displayed absorption bands for hydroxyl (3448 cm−1 ) and carbonyl (1612 cm−1 ) groups. Analysis of its 1 H and 13 C NMR data suggested that 3 was a veratraminetype steroidal alkaloid closely related in structure to 1, with the exception of signals occurring for additional conjugated double bond, with signals for a hydroxyl group being absent. The position of another conjugated double bond was confirmed to locate between C-12 and C-14 through the HMBC correlations between olefinic proton [ı 5.96 (1H, m, H-7)] and C-14 (ı 135.2), C-18 methyl protons [ı 1.01 (3H, d, J = 7.0 Hz, H-18)] and C-12 (ı 162.1). As the same time, H3 -18 resonating as a doublet disclosed C-13 to be a methine carbon. In the NOESY spectrum, the correlations between methyl protons H3 -19 and methyl protons H3 -18 demonstrated methyl group at C-13 was in -orientation. Similarly, the configurations of A/B ring fusion together with piperidine part were established by corresponding NOEs. Accordingly, 3 was determined as (20R* , 22R* )-N-methyl-5,6,12,13-tetrahydro-3, 23dihydroxy-5␣, 13, 17, 25␣-veratraman-7,12(14)-dien-6-one, named puqienine E. Puqiedine (4) was crystallized from Me2 CO as colorless squares, and gave a positive Dragendorff test. The molecular formula, C27 H45 NO2 , was confirmed from a quasimolecular ion [M + H]+ at m/z 416.3539 by HRESIMS. The IR spectrum exhibited the diagnostic absorption of hydroxyl (3396 cm−1 ) and trans-quinolizidine (2767 cm−1 ) groups. The 1 H NMR spectrum of 4 only showed three methyl signals, of which one methyl singlet [ı 0.99 (3H, s, H-19)] revealed the existence of OH at C-6 [18], and two methyl doublet [ı 0.84 (3H, d, J = 6.3 Hz, H-27) and 0.86 (3H, d, J = 6.5 Hz, H-21)] indicated the presence of 25␣-CH3 and the absence of 20-OH, respectively [15]. The above data suggested that 4 was a cevanine-type alkaloid. Furthermore, two oxy methine protons appeared at ı 3.64 (br, m, H-3␣) and 3.86 (br, m, H-6␣). The 13 C NMR spectrum of 4 also displayed the signals of three methyl carbons at ı 14.9 (C-21), 15.1 (C-19) and 19.7 (C-27), three nitrogen-linked carbons at ı 61.5 (C-18), 64.7 (C-26) and 68.5 (C-22), and two oxygenated carbons at ı 71.9 (C-3) and 72.8 (C-6). The overall 1 H and 13 C NMR features were quite similar to those of puqiedinone (7) with the only difference being the replacement of the carbonyl group at C-6 by a hydroxyl group in 4. Thus, 4 was characterized as (20R, 22S, 25R)-5␣-cevane-3, 6-diol, named puqiedine. 3␣-Puqiedin-7-ol (5) was obtained as colorless squares from Me2 CO, gave a positive reaction to Dragendorff reagent. The
847
molecular formular, C27 H45 NO3 , was determined from a quasimolecular ion [M + H]+ at m/z 432.3485 in HRESIMS. The IR spectrum displayed absorption bands for hydroxyl (3396 cm−1 ) and trans-quinolizidine (2750 cm−1 ) groups. The 1 H NMR spectrum of 5 indicated the presence of two methyl doublet signals [ı 0.76 (3H, d, J = 6.3 Hz, H-27) and 0.78 (3H, d, J = 6.5 Hz, H-21)] and one methyl singlet signals [ı 1.32 (3H, s, H-19)], consistent with a cevanine-type alkaloid. In addition, the NMR spectra showed three oxygenated methines (ıH 4.18, 4.42 and 4.46; ıC 66.3, 70.7 and 77.6, respectively), which showed that 5 possessed one more hydroxyl group than 4. The 13 C NMR spectrum of 5 gave almost the same chemical shifts as that of 4 except for the signals due to the A- and B-ring carbons. The locations of three hydroxyl groups were assigned through a HMBC experiment: correlations between proton ı 4.18 and C-10 (ı 36.9) and C-8 (ı 39.7), proton ı 4.42 and C-5 (ı 37.7), C-9 (ı 49.0) indicated the steroid nucleus possessing 6,7-diol group; correlations between protons [ı 1.34 (H-1␣), 1.89 (H-2), 2.04 (H-1) and 2.04 (H-2␣)] and the oxygenated carbon at ı 66.3 positioned the other hydroxyl group at C3. The configuration of hydroxyl group at C-3 was inferred to be axial, since its geminal proton appeared characteristically at ı 4.46 with W1/2 = 12.4 Hz, suggesting its equatorial orientation [4]. Based on a consideration of the 1 H-splitting pattern and coupling constants [ı 4.18 (dd, J = 2.1, 2.8 Hz, H-6) and 4.42 (dd, J = 2.4, 2.8 Hz, H-7)], the relative stereochemistries of 6,7-diol groups were proposed to be 6, 7␣ orientation, respectively [19]. Consequently, 5 was deduced as (20R, 22S, 25R)-5␣-cevane-3␣, 6, 7␣-triol, named 3␣-puqiedin7-ol. Puqietinedinone (6) was crystallized from MeOH as colorless needles, and gave a positive reaction to Dragendorff reagent. The HRESIMS displayed a quasimolecular ion [M + H]+ at m/z 428.3527, consistent with the molecular formular C28 H45 NO2 . The IR spectrum indicated the presence of two carbonyl (1719, 1698 cm−1 ) groups. The 1 H NMR spectrum of 6 showed two methyl singlet signals [ı 0.72 (3H, s, H-18) and 0.96 (3H, s, H-19)], two methyl doublet protons [ı 0.85 (3H, d, J = 6.0 Hz, H-27) and 0.94 (3H, d, J = 6.6 Hz, H-21)], and a methyl group located at nitrogen atom [ı 2.21 (3H, s, H-28)], which suggested that 6 belonged to a secosolanidane-type alkaloid. The 13 C NMR spectrum of 6 displayed the presence of two carboxyl carbons at ı 208.8 and 211.0, assigned to C-6 and C-3, respectively, based on the HMBC correlations between H-5, H2 -7 and C-6, H-5, H-2␣, H-1 and C-3. Comparing the NMR data of 6 with those of puqietinone (10) [13], the only difference was that the hydroxyl group at C-3 of puqietinone was replaced by a carbonyl group in 6. Hence, 6 was formulated as (22R, 25S)-N-methyl-22,26-epiminocholest-3, 6-dione, named puqietinedinone. Generally, the steroidal alkaloids occurring in the genus Fritillaria are characterized with 5␣-cevenine isosteroidal alkaloids [11,20]. Nevertheless, similar to our recent investigation [12,14], the presence of relatively large amount of veratramineand secosolanidine-type alkaloids from F. puqiensis makes this species distinct. Actually, the taxonomical state of this species still remains controversial between botanists: Yu et al. [21] published it as a specific rank, while Xiao et al. [22] treated it as a subspecies of F. thunbergii. From the point of chemtaxonomy, our findings are compatible with the taxonomical treatment
848
steroids
71
proposed by Yu et al. And further investigation will be required to explore this proposal in depth.
Acknowledgments We are grateful for the cultivation fund of the key scientific and technical innovation project, Ministry of Education of China (No.2004-295), and the National Natural Science Foundation of China (No. 30070886).
references
[1] Wang FZ, Tang J. Liliaceae: In: Delectis Florae Republicae Popularis Sinicae Agenda Academiae Sinicae Edita, (ed.). Flora republicae popularis sinicae, vol. 14. Beijing: Science Press; 1980:101–16. [2] Lin G, Li P, Li SL, Chan SW. Chromatographic analysis of Fritillaria isosteroidal alkaloids, the active ingredients of Beimu, the antitussive traditional Chinese medicinal herb. J Chromatogr A 2001;935:321–38. [3] Qian ZZ, Nohara ZT. Steroidal alkaloids of Fritillaria maximowiczii. Phytochemistry 1995;40:979–81. [4] Akhtar MN, Atta-Ur-Rahman, Choudhary MI, Sener B, Erdogan I, Tsuda Y. New class of steroidal alkaloids from Fritillaria imperialis. Phytochemistry 2003;63:115–22. [5] The Pharmacopoeia Commission of People’s Republic of China. Pharmacopoeia of the People’s Republic of China, vol. 1. Chemical Industry Press: Beijing; 2005, 25/6. [6] Li P, Ji H, Xu GJ, Xu LS. Studies on the antitussive and expectorant effects of Chinese drug Beimu. J Chin Pharm Univ 1993;24:360–2. [7] Zhou Y, Ji H, Li P, Jiang Y. Antimuscarinic function of five Fritillaria alkaloids on guinea pig tracheal strips. J Chin Pharm Univ 2003;34:58–60. [8] Atta-Ur-Rahman, Akhtar MN, Choudhary MI, Tsuda Y, Sener B, Khalid A, Parvez M. New steroidal alkaloids from Fritillaria imperialis and their cholinesterase inhibiting activities. Chem Pharm Bull 2002;50:1013–6.
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[9] Atta-Ur-Rahman, Choudhary MI. Diterpenoid and steroidal alkaloids. Nat Prod Rep 1997;14:191–203. [10] Atta-Ur-Rahman, Choudhary MI. Diterpenoid and steroidal alkaloids. Nat Prod Rep 1999;16:619–35. [11] Li SL, Li P, Lin G, Ren YJ, Que NN. Existence of 5␣-cevenine isosteroidal alkaloids in bulbs of Fritillaria L. Acta Pharm Sin 1999;34:842–7. [12] Jiang Y, Li HJ, Li P, Cai ZH, Ye WC. Four new steroidal alkaloids from the bulbs of Fritillaria puqiensis. J Nat Prod 2005;68:664–7. [13] Li P, Li XG, Xu GJ, Kaneko KJ. The structural elucidation of puqietinone from Fritillaria puqiensis. J Chin Pharm Univ 1990;24:198–200. [14] Li HJ, Jiang Y, Li P, Ye WC, Puqienine F. A novel veratramine alkaloid from the bulbs of Fritillaria puqiensis. Chem Pharm Bull 2006;54:722–4. [15] Lin G, Ho YP, Li P, Li XG. Puqiedinone, a novel 5␣-cevenine alkaloid from the bulbs of Fritillaria puqiensis. J Nat Prod 1995;58:1662–7. [16] Wang FP, Zhang R, Tang XY. Revision of structure of peimisine. Acta Pharm Sin 1992;27:273–8. [17] Li QH, Wu ZH, Zhang LL, Shao L. Isolation and identification of alkaloids from Fritillaria ningguoensis S C. Chen et S. F. Yin. Acta Pharm Sin 1988;23: 415–21. [18] Lee P, Kitmaura Y, Kaneko K, Shiro M, Xu GJ, Chen YP, Hsu HY. The structural elucidation of Fritillaria alkaloids from Fritillaria ebeiensis var. purpurea. I. The structures of ebeienine, ebeiedine and ebeiedinone. Chem Pharm Bull 1988;36:4316–29. [19] Kadota S, Chen SZ, Li JX, Xu GJ, Namba T. A steroidal alkaloids from Veratrum oblongum. Phytochemistry 1995;38:777–81. [20] Yu SC, Xiao PG. The existence of isosteroidal alkaloids in Fritillaria L (Liliaceae) and its taxonomical significance. Acta Phytotax Sin 1992;30:450–9. [21] Yu GD, Li P, Xu GJ. Studies on the Chinese drug Bei-mu IV. Medicinal plant resources of Fritillaria from Hubei Provice. J Chin Pharm Univ 1985;16:25–32. [22] Yu SC, Xiao PG. On the taxonomical state of Fritillaria sulcisquamosa and F. puqiensis. Acta Phytotax Sin 1992;30:277–8.
available at www.sciencedirect.com
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New steroidal alkaloids from the bulbs of Fritillaria puqiensis Yan Jiang, Ping Li ∗ , Hui-Jun Li ∗ , Hua Yu MOE Key Laboratory of Modern Chinese Medicines, Department of Pharmacognosy, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing 210009, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Six new steroidal alkaloids, namely puqienines C–E (1–3), puqiedine (4), 3␣-puqiedin-7-ol
Received 7 February 2006
(5), and puqietinedione (6), along with two known steroidal alkaloids puqiedinone (7) and
Received in revised form 26 May
peimisine (8), were isolated from the bulbs of Fritillaria puqiensis G.D. Yu et G.Y. Chen (Lili-
2006
aceae). Their structures were elucidated on the basis of spectroscopic analysis, including 1D
Accepted 29 May 2006
and 2D NMR experiments. Among these alkaloids, 1–3 had a veratramine-type skeleton, 4, 5,
Published on line 10 July 2006
7 a cevanine-type skeleton, 6 a secosolanidine-type skeleton, and 8 a jervine-type skeleton. The existence of multiple types of steroidal skeletons, especially of relatively large amount
Keywords:
of veratramine-type alkaloids in one species is rare in the genus Fritillaria, and the results
Fritillaria puqiensis
might be of chemotaxonomic significance for this species. © 2006 Elsevier Inc. All rights reserved.
Steroidal alkaloids Puqienines C–E Puqiedine 3␣-Puqiedin-7-ol Puqietinedione
1.
Introduction
The genus Fritillaria, containing about 130 species, is one of the largest genera in the plant family of Liliaceae [1]. Many species of Fritillaria were traditionally used as herbal remedies in Japanese, Turkish, Pakistani and south-east Asian folk medicines [2–4], while the bulbs of several Fritillaria species growing in China, have been used as an antitussive, antiasthmatic and expectorant using the Chinese name “Beimu” in traditional Chinese medicine (TCM) for centuries [5]. Previous investigations on pharmacology and phytochemistry had suggested that steroidal alkaloids were responsible for the effects of this herbal drug [6,7]. The Fritillaria alkaloids were structurally divided into five main types, cevanines, jervines, veratramines, solanidines, and secosolanidines skeleton, with cevanine-type alkaloids occupying dominantly [8–11]. However, during the course of our investigation concerning the isolation and characterization of chemical constituents of Fritillaria puqiensis G.D. Yu et G.Y. Chen, endemic to Hubei Province ∗
of China, relatively copious veratramine- and secosolanidinetype steroidal alkaloids were obtained [12–14]. In continuation of our studies on F. puqiensis, six new steroidal alkaloids, namely, puqienines C–E (1–3), puqiedine (4), 3␣-puqiedin-7ol (5), puqietinedione (6) (Fig. 1), together with two known steroidal alkaloids puqiedinone (7) and peimisine (8), were isolated and identified from the dried ground bulbs of this plant. In this paper, we describe the extraction, isolation and structure elucidation of the new alkaloids.
2.
Experimental
2.1.
General procedures
The melting points were measured on an X4 micro-melting point apparatus and were uncorrected. Optical rotations were determined in MeOH on a PE-241 MC digital polarimeter at 20 ◦ C. IR spectra were recorded in KBr disks on a Nicolet
Corresponding authors. Tel.: +86 25 8324 2299; fax: +86 25 8324 2299. E-mail addresses: [email protected] (P. Li), [email protected] (H.-J. Li). 0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2006.05.016
844
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
Fig. 1 – Structures of 1–10.
Impact 410 spectrophotometer. UV spectra were performed on a Shimadzu UV-2501 spectrophotometer. NMR spectra were obtained in CDCl3 or pyridine-d5 containing TMS as an internal standard on a Bruker Am-500 NMR spectrometer. The f1 and f2 resolution for heteronuclear 2D experiments was about 0.2 and 0.01 ppm, respectively. HRESIMS spectra were recorded on an Agilent 1100 LC/MSD TOF instrument in positive ion mode. Column chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Factory, China). TLC was conducted on pre-coated silica gel G plates (0.25 mm thick, Qingdao Marine Chemical Factory, China).
2.2.
Extraction and isolation procedure
The bulbs of F. puqiensis were collected from Puqi County, Hubei Province, People’s Republic of China, in May 2000, and
authenticated by one of us (Dr. Ping Li). A voucher specimen was deposited at the Herbarium of Department of Pharmacognosy, China Pharmaceutical University. Dried ground bulbs (10 kg) were extracted with 70% EtOH by soaking repeatedly (3× 20L). The concentrated extract was dissolved in 2% HCl (pH 3.5) and partitioned with ether. The pH of the aqueous solution was re-adjusted with NH4 OH to 9.2 and extracted with CHCl3 . The CHCl3 extract (crude alkaloid, 50 g) was chromatographed over silica gel using CHCl3 /CH3 OH of increasing polarity as eluent to obtain five fractions A–E. Fractions B–E were further separated by repeated column chromatography, and eluted with petroleum ether/Me2 CO/diethylamine (88:12:2, 80:20:2 and 75:25:2) and CHCl3 /CH3 OH/diethylamine (100:7:5), respectively. Fraction B afforded 4 (19 mg) and 8 (50 mg), fraction C yielded 3 (27 mg), 6 (10 mg) and 7 (12 mg), fraction D gave 5 (20 mg), and fraction E offered 1 (23 mg) and 2 (18 mg).
845
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
2.2.1.
Puqienine C (1) 291–293 ◦ C,
C28 H45 NO4 , colorless needles (MeOH), mp [˛]20 D + 10.9◦ (c = 0.1, MeOH); UV (MeOH) max 250 nm; IR (KBr)max 3446, 2952, 1660, 1124 cm−1 ; HRESIMS m/z 460.3430 [M + H]+ (calcd. for C28 H46 NO4 , 460.3421); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.2.
Puqienine D (2)
C28 H45 NO4 , colorless needles (MeOH), mp 275–277 ◦ C, [˛]20 D −73.1◦ (c = 0.1, MeOH); IR (KBr)max 3446, 2949, 1707, 1057 cm−1 ; HRESIMS m/z 460.3422 [M + H]+ (calcd. for C28 H46 NO4 , 460.3421); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.3.
Puqienine E (3)
C28 H43 NO3 , colorless squares (MeOH), mp 235–237 ◦ C, [˛]20 D −141.1◦ (c = 0.1, MeOH); UV (MeOH) max 305 nm; IR (KBr)max 3448, 2921, 1612, 1234 cm−1 ; HRESIMS m/z 442.3346 [M + H]+ (calcd for C28 H44 NO3 , 442.3315); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.4.
Puqiedine (4)
C27 H45 NO2 , colorless squares (Me2 CO), mp 139–141 ◦ C, −38.4◦ (c = 0.1, MeOH); IR (KBr)max 3396, 2925, [˛]20 D 2767 cm−1 ; HRESIMS m/z 416.3539 [M + H]+ (calcd. for
C27 H46 NO2 , 416.3734); 1 H and Tables 1 and 2.
2.2.5.
13 C
NMR data are shown in
3˛-Puqiedin-7-ol (5)
C27 H45 NO3 , colorless squares (Me2 CO), mp 267–269 ◦ C, [˛]20 D −47.7◦ (c = 0.1, MeOH); IR (KBr)max 3396, 2917, 2750 cm−1 ; HRESIMS m/z 432.3485 [M + H]+ (calcd for C27 H46 NO3 , 432.3472); 1 H and 13 C NMR data are shown in Tables 1 and 2.
2.2.6.
Puqietinedione (6)
C28 H45 NO2 , colorless needles (MeOH), mp 281–283 ◦ C, ◦ −1 [˛]20 D + 30.1 (c = 0.1, MeOH); IR (KBr)max 2950, 1719, 1698 cm ; HRESIMS m/z 428.3527 [M + H]+ (calcd. for C28 H46 NO2 , 428.3523); 1 H and 13 C NMR data are shown in Tables 1 and 2.
3.
Results and discussion
Air-dried ground bulbs were extracted with 70% EtOH, then the concentrate was dissolved in 2% HCl and extracted with ether. The acidic aqueous solution was alkalized with NH4 OH and extracted with CHCl3 . The CHCl3 -soluble portion was repeatedly chromatographed over silica gel to afford eight steroidal alkaloids (1–8). The known compounds (7 and 8) were iden-
Table 1 – 1 H NMR data of compounds 1–6a (ı) 1b
2b
3b
4c
5b
6c
1.36 m, 1.58 m 1.65 m, 1.99 m 3.86 br, m, 24.4d 1.76 m, 2.80 m 2.17 dd, 12.3, 3.1
1.27 m, 1.57 m 1.70 m, 1.97 m 3.78 br, m, 25.0d 1.80 m, 2.31 m 2.21 dd, 12.5, 2.4
1.37 m, 1.58 m 1.65 m, 2.02 m 3.87 br, m, 24.8d 1.71 m, 2.87 m 2.28 dd, 14.8, 3.2 5.96 m
2.56 m 1.32 m, 2.15 m
2.40 m 2.75 m 2.42 m 1.70 m 1.25 m, 2.03 m
1.34 m, 2.04 m 1.89 m, 2.04 m 4.46 m, 12.4d 1.90 m, 2.45 m 2.89 m 4.18 dd, 2.8, 2.1 4.42 dd, 2.8, 2.4
1.63 m, 2.08 m 2.38 m, 2.44 m
6.04 t, 2.5
1.03 m, 1.45 m 1.46 m, 1.79 m 3.64 br, m, 24.8d 1.70 m 1.10 m 3.86 br, m 1.09 m 1.98 m 1.78 m 1.00 m 1.08 m, 1.78 m
1.90 m
2.95 br, m
1.55 m 1.19 qd, 10.6, 2.8 1.46 m 1.51 m, 1.72 m 0.96 m, 1.69 m 0.61 qd, 10.5, 3.5 1.60 m, 2.79 m 0.99 s 0.99 m 0.86 d, 6.5 1.32 m 1.11 m, 1.94 m 0.86 m, 1.76 m 1.72 m 1.58 m, 2.77 m 0.84 d, 6.3
1.63 m 1.38 qd, 10.8, 3.2 2.43 m 1.60 m, 1.99 m 0.96 m, 1.64 m 0.62 qd, 10.5, 2.8 1.66 m, 2.92 m 1.32 s 0.97 m 0.78 d, 6.5 1.29 m 1.11 m, 1.86 m 0.82 m, 1.67 m 1.67 m 1.55 m, 2.73 m 0.76 d, 6.3
Position 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 a b c d
2.80 m 2.19 dd, 12.9, 3.2 2.44 dd, 12.9, 2.6 2.51 m
2.05 m 1.26 m, 2.03 m 1.40 m, 2.09 m 1.94 m 1.30 s 0.80 s 3.06 qd, 7.3, 1.9 1.24 d, 7.3 2.06 m 4.21 br, m 1.09 td, 12.9, 2.4 2.04 m 2.32 br, m 1.91 m, 2.85 m 0.76 d, 6.6 2.59 s
5.30 m 2.13 m, 2.45 m 2.29 m 1.22 s 0.73 s 3.15 qd, 7.1, 2.2 1.23 d, 7.1 2.15 m 4.19 br, m 1.08 td, 12.9, 2.5 2.02 m 2.30 br, m 1.94 m, 2.84 m 0.73 d, 6.5 2.59 s
1.94 m, 2.18 m 1.28 m, 1.86 m 1.96 m 1.01 d, 7.0 0.68 s 1.97 m 1.25 d, 6.1 1.91 m 4.12 br, m 1.10 td, 12.9, 2.5 1.97 m 2.27 m 1.77 m, 2.84 m 0.77 d, 6.6 2.23 s
Assignments were made using H–H COSY, HMQC and HMBC techniques. Measured in pyridine-d5. Measured in CDCl3. Multiplet width at half height.
2.45 m 2.30 m 1.31 m, 1.91 m
2.33 m, 2.59 m 2.60 m 1.99 t, 12.7 2.39 m 1.87 m 1.35 m 1.45 m, 1.68 m 1.27 m, 2.09 m 1.28 m 1.13 m, 1.55 m 1.31 m, 1.77 m 1.30 m 0.72 s 0.96 s 1.92 m 0.94 d, 6.6 1.76 m 1.31 m, 1.55 m 0.88 m, 1.77 m 1.73 m 1.76 m, 2.99 m 0.85 d, 6.0 2.21 s
846
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
Table 2 – 13 C NMR data of compounds 1–6a (ı) Position 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 27 28 a
b c
1b
2b
3b
4c
5b
6c
37.8 t 31.7 t 70.5 d 31.6 t 54.9 d 199.2 s 120.7 d 172.9 s 57.4 d 40.4 s 25.4 t 57.7 d 74.5 s 48.3 d 28.8 t 26.7 t 50.4 d 16.8 q 12.4 q 29.7 d 13.2 q 70.6 d 66.8 d 43.2 t 23.3 d 65.8 t 19.6 q 41.3 q
37.4 t 31.3 t 70.0 d 30.9 t 57.0 d 209.6 s 45.5 t 43.8 d 55.7 d 37.9 s 25.1 t 57.2 d 72.9 s 142.5 s 116.7 d 28.7 t 46.1 d 16.8 q 12.5 q 29.4 d 13.8 q 69.6 d 66.6 d 42.9 t 22.7 d 65.3 t 19.3 q 40.8 q
37.2 t 31.3 t 70.3 d 31.7 t 54.7 d 198.9 s 113.4 d 169.6 s 53.5 d 39.8 s 33.6 t 162.1 s 33.9 d 135.2 s 22.0 t 20.1 t 40.0 d 13.4 q 11.9 q 33.0 d 13.9 q 66.6 d 66.0 d 42.4 t 24.7 d 66.1 t 19.3 q 42.1 q
39.1 t 31.3 t 71.9 d 34.9 t 48.3 d 72.8 d 38.6 t 34.8 d 57.7 d 35.5 s 29.3 t 40.2 d 44.5 d 44.0 d 24.7 t 25.6 t 46.5 d 61.5 t 15.1 q 41.3 d 14.9 q 68.5 d 30.2 t 33.5 t 30.7 d 64.7 t 19.7 q
35.6 t 29.6 t 66.3 d 33.7 t 37.7 d 77.6 d 70.7 d 39.7 d 49.0 d 36.9 s 28.8 t 40.3 d 44.5 d 37.5 d 24.7 t 25.7 t 46.5 d 61.7 t 13.7 q 41.7 d 14.8 q 68.3 d 30.6 t 33.6 t 31.0 d 64.8 t 19.5 q
38.1 t 37.3 t 211.0 s 37.0 t 57.5 d 208.8 s 46.6 t 38.0 d 53.5 d 41.2 s 21.7 t 39.5 t 43.4 s 56.3 d 24.0 t 27.4 t 52.9 d 12.0 q 12.6 q 35.1 d 12.6 q 66.7 d 23.9 t 33.4 t 31.2 d 66.1 t 19.5 q 42.8 q
Assignments were made using H–H COSY, HMQC and HMBC techniques. Measured in pyridine-d5 . Measured in CDCl3 .
tified as puqiedinone [15] and peimisine [16], respectively, by detailed spectral analysis and compared with reference data. Puqienine C (1) was isolated as colorless needles from MeOH, giving a positive Dragendorff test. The HRESIMS exhibited a quasimolecular ion [M + H]+ at m/z 460.3430 corresponding to the molecular formula C28 H45 NO4 , possessing seven degrees of unsaturation. The UV spectrum showed absorption maximum at 250 nm, which was diagnostic of an ␣, unsaturated ketone. The IR spectrum indicated the presence
of hydroxyl (3446 cm−1 ) and carbonyl (1660 cm−1 ) groups. The NMR spectrum of 1 displayed two methyl singlet signals at ı 0.80 (3H, s, H-19) and 1.30 (3H, s, H-18), two methyl doublet signals at ı 0.76 (3H, d, J = 6.6 Hz, H-27) and 1.24 (3H, d, J = 7.3 Hz, H-21), as well as an N-methyl signal at ı 2.59 (3H, s, H-28), which suggested that 1 was a veratramine-type alkaloid [17]. The 13 C NMR and DEPT spectra disclosed the presence of a carbonyl carbon [ı 199.2 (C-6)], an oxygenated quaternary carbon [ı 74.5 (C-13)], and two oxy-methines [ı 70.5 (C-3) and 66.8 (C-23)]. The 13 C NMR feature of 1 was very similar to that of puqienine F (9) [14], with the major differences being the presence of characteristic signals of a conjugated double bond (ı 172.9 and 120.7), and the absence of a 12,16-epoxy ring in 1. The position of double bond was confirmed to locate between C-7 and C-8 through the HMBC correlations between olefinic proton (ı 6.04) and C-5 (ı 54.9), C-9 (ı 57.4), and C-14 (ı 48.3). In the NOESY spectrum of 1, H3 -18 showed clear cross peak with H3 -19, confirming the configuration of 13-OH to be ␣. Then, the key NOEs were observed between H-1␣/H-3, H-3/H-5, H-5/H-9, H-1/H-19, H-9/H-12, H-14/H-17, H-22/H-27, H-23/H-26␣ and H-26␣/H-27, which proved A/B trans, 3-OH, 5␣-H, 12␣-H, 17side chain, 22␣-H, 23-OH and 25␣-CH3 (Fig. 2). The relative stereochemistries of C-20 and C-22 were tentatively established to be R* from biogenesis. Thus, 1 was elucidated as (20R* , 22R* )-N-methyl-5,6,12,13-tetrahydro-3, 13␣, 23-trihydroxy5␣12␣, 17, 25␣-veratraman-7-en-6-one, named puqienine C. Puqienine D (2) was crystallized from MeOH as colorless needles, and gave a positive reaction to Dragendorff reagent. The molecular formula, C28 H45 NO4 , was assigned on the basis of the quasimolecular ion [M + H]+ at m/z 460.3422 in its HRESIMS. The IR spectrum showed absorption bands for hydroxyl (3446 cm−1 ) and carbonyl (1707 cm−1 ) groups. The 1 H NMR spectrum of 2 indicated the presence of two methyl singlet signals [ı 0.73 (3H, s, H-19) and 1.22 (3H, s, H-18)], two methyl doublet signals [ı 0.73 (3H, d, J = 6.5 Hz, H-27) and 1.23 (3H, d, J = 7.1 Hz, H-21)], a methyl group located at the nitrogen atom [ı 2.59 (3H, s, H-28)], two oxy methine protons [ı 3.78 (1H, br, m, H-3␣) and 4.19 (1H, br, m, H-23␣)], and an olefinic proton [ı 5.30 (1H, m)]. The 13 C NMR spectrum of 2 gave almost the same chemical shifts as those of 1 except for the signals due to different position of double bond. The signals resonating at ı 142.5 and 116.7 implied the presence of a nonconju1H
Fig. 2 – Key NOESY correlations of 1.
s t e r o i d s 7 1 ( 2 0 0 6 ) 843–848
gated double bond in 2, and the HMBC correlations between olefinic proton (ı 5.30) and C-17 (ı 46.1), C-12 (ı 57.2) assigned the position of the double bond between C-14 and C-15. In the NOESY spectrum, the correlations between methyl protons H3 -19/H-8 and H-1␣/H-9 revealed B/C trans. The remaining cross peaks suggested the same configurations of A/B ring fusion and piperidine moiety as those of 1. Therefore, 2 was established as (20R* , 22R* )-N-methyl-5,6,12,13-tetrahydro3, 13␣, 23-trihydroxy-5␣, 12␣, 17, 25␣-veratraman-14-en-6one, named puqienine D. Puqienine E (3) was obtained as colorless squares from MeOH, and gave a positive reaction to Dragendorff reagent. The HRESIMS showed a quasimolecular ion [M + H]+ at m/z 442.3346, consistent with the molecular formular C28 H43 NO3 . The UV spectrum exhibited absorption maximum at 305 nm, which revealed the presence of a long conjugated system. The IR spectrum displayed absorption bands for hydroxyl (3448 cm−1 ) and carbonyl (1612 cm−1 ) groups. Analysis of its 1 H and 13 C NMR data suggested that 3 was a veratraminetype steroidal alkaloid closely related in structure to 1, with the exception of signals occurring for additional conjugated double bond, with signals for a hydroxyl group being absent. The position of another conjugated double bond was confirmed to locate between C-12 and C-14 through the HMBC correlations between olefinic proton [ı 5.96 (1H, m, H-7)] and C-14 (ı 135.2), C-18 methyl protons [ı 1.01 (3H, d, J = 7.0 Hz, H-18)] and C-12 (ı 162.1). As the same time, H3 -18 resonating as a doublet disclosed C-13 to be a methine carbon. In the NOESY spectrum, the correlations between methyl protons H3 -19 and methyl protons H3 -18 demonstrated methyl group at C-13 was in -orientation. Similarly, the configurations of A/B ring fusion together with piperidine part were established by corresponding NOEs. Accordingly, 3 was determined as (20R* , 22R* )-N-methyl-5,6,12,13-tetrahydro-3, 23dihydroxy-5␣, 13, 17, 25␣-veratraman-7,12(14)-dien-6-one, named puqienine E. Puqiedine (4) was crystallized from Me2 CO as colorless squares, and gave a positive Dragendorff test. The molecular formula, C27 H45 NO2 , was confirmed from a quasimolecular ion [M + H]+ at m/z 416.3539 by HRESIMS. The IR spectrum exhibited the diagnostic absorption of hydroxyl (3396 cm−1 ) and trans-quinolizidine (2767 cm−1 ) groups. The 1 H NMR spectrum of 4 only showed three methyl signals, of which one methyl singlet [ı 0.99 (3H, s, H-19)] revealed the existence of OH at C-6 [18], and two methyl doublet [ı 0.84 (3H, d, J = 6.3 Hz, H-27) and 0.86 (3H, d, J = 6.5 Hz, H-21)] indicated the presence of 25␣-CH3 and the absence of 20-OH, respectively [15]. The above data suggested that 4 was a cevanine-type alkaloid. Furthermore, two oxy methine protons appeared at ı 3.64 (br, m, H-3␣) and 3.86 (br, m, H-6␣). The 13 C NMR spectrum of 4 also displayed the signals of three methyl carbons at ı 14.9 (C-21), 15.1 (C-19) and 19.7 (C-27), three nitrogen-linked carbons at ı 61.5 (C-18), 64.7 (C-26) and 68.5 (C-22), and two oxygenated carbons at ı 71.9 (C-3) and 72.8 (C-6). The overall 1 H and 13 C NMR features were quite similar to those of puqiedinone (7) with the only difference being the replacement of the carbonyl group at C-6 by a hydroxyl group in 4. Thus, 4 was characterized as (20R, 22S, 25R)-5␣-cevane-3, 6-diol, named puqiedine. 3␣-Puqiedin-7-ol (5) was obtained as colorless squares from Me2 CO, gave a positive reaction to Dragendorff reagent. The
847
molecular formular, C27 H45 NO3 , was determined from a quasimolecular ion [M + H]+ at m/z 432.3485 in HRESIMS. The IR spectrum displayed absorption bands for hydroxyl (3396 cm−1 ) and trans-quinolizidine (2750 cm−1 ) groups. The 1 H NMR spectrum of 5 indicated the presence of two methyl doublet signals [ı 0.76 (3H, d, J = 6.3 Hz, H-27) and 0.78 (3H, d, J = 6.5 Hz, H-21)] and one methyl singlet signals [ı 1.32 (3H, s, H-19)], consistent with a cevanine-type alkaloid. In addition, the NMR spectra showed three oxygenated methines (ıH 4.18, 4.42 and 4.46; ıC 66.3, 70.7 and 77.6, respectively), which showed that 5 possessed one more hydroxyl group than 4. The 13 C NMR spectrum of 5 gave almost the same chemical shifts as that of 4 except for the signals due to the A- and B-ring carbons. The locations of three hydroxyl groups were assigned through a HMBC experiment: correlations between proton ı 4.18 and C-10 (ı 36.9) and C-8 (ı 39.7), proton ı 4.42 and C-5 (ı 37.7), C-9 (ı 49.0) indicated the steroid nucleus possessing 6,7-diol group; correlations between protons [ı 1.34 (H-1␣), 1.89 (H-2), 2.04 (H-1) and 2.04 (H-2␣)] and the oxygenated carbon at ı 66.3 positioned the other hydroxyl group at C3. The configuration of hydroxyl group at C-3 was inferred to be axial, since its geminal proton appeared characteristically at ı 4.46 with W1/2 = 12.4 Hz, suggesting its equatorial orientation [4]. Based on a consideration of the 1 H-splitting pattern and coupling constants [ı 4.18 (dd, J = 2.1, 2.8 Hz, H-6) and 4.42 (dd, J = 2.4, 2.8 Hz, H-7)], the relative stereochemistries of 6,7-diol groups were proposed to be 6, 7␣ orientation, respectively [19]. Consequently, 5 was deduced as (20R, 22S, 25R)-5␣-cevane-3␣, 6, 7␣-triol, named 3␣-puqiedin7-ol. Puqietinedinone (6) was crystallized from MeOH as colorless needles, and gave a positive reaction to Dragendorff reagent. The HRESIMS displayed a quasimolecular ion [M + H]+ at m/z 428.3527, consistent with the molecular formular C28 H45 NO2 . The IR spectrum indicated the presence of two carbonyl (1719, 1698 cm−1 ) groups. The 1 H NMR spectrum of 6 showed two methyl singlet signals [ı 0.72 (3H, s, H-18) and 0.96 (3H, s, H-19)], two methyl doublet protons [ı 0.85 (3H, d, J = 6.0 Hz, H-27) and 0.94 (3H, d, J = 6.6 Hz, H-21)], and a methyl group located at nitrogen atom [ı 2.21 (3H, s, H-28)], which suggested that 6 belonged to a secosolanidane-type alkaloid. The 13 C NMR spectrum of 6 displayed the presence of two carboxyl carbons at ı 208.8 and 211.0, assigned to C-6 and C-3, respectively, based on the HMBC correlations between H-5, H2 -7 and C-6, H-5, H-2␣, H-1 and C-3. Comparing the NMR data of 6 with those of puqietinone (10) [13], the only difference was that the hydroxyl group at C-3 of puqietinone was replaced by a carbonyl group in 6. Hence, 6 was formulated as (22R, 25S)-N-methyl-22,26-epiminocholest-3, 6-dione, named puqietinedinone. Generally, the steroidal alkaloids occurring in the genus Fritillaria are characterized with 5␣-cevenine isosteroidal alkaloids [11,20]. Nevertheless, similar to our recent investigation [12,14], the presence of relatively large amount of veratramineand secosolanidine-type alkaloids from F. puqiensis makes this species distinct. Actually, the taxonomical state of this species still remains controversial between botanists: Yu et al. [21] published it as a specific rank, while Xiao et al. [22] treated it as a subspecies of F. thunbergii. From the point of chemtaxonomy, our findings are compatible with the taxonomical treatment
848
steroids
71
proposed by Yu et al. And further investigation will be required to explore this proposal in depth.
Acknowledgments We are grateful for the cultivation fund of the key scientific and technical innovation project, Ministry of Education of China (No.2004-295), and the National Natural Science Foundation of China (No. 30070886).
references
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