Article pubs.acs.org/jnp
Nematicidal Stemona Alkaloids from Stemona parviflora Sheng-Zhuo Huang, Fan-Dong Kong, Qing-Yun Ma, Zhi-Kai Guo, Li-Man Zhou, Qi Wang, Hao-Fu Dai,* and You-Xing Zhao* Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, People’s Republic of China S Supporting Information *
ABSTRACT: Eight new alkaloids, 3β-n-butylstemonamine (1), 8-oxo-3β-n-butylstemonamine (2), 3-n-butylneostemonine (3), 10-epi-3-n-butylneostemonine (4), 8-oxo-oxymaistemonine (5) protostemonine N4-oxide (6), (19S)-hydroxy-21methoxystemofoline (7), and parvistemonine A (8), were isolated from the roots of Stemona parviflora, together with 17 known alkaloids. The structures of the new alkaloids were elucidated based on a comprehensive spectroscopic data analysis. The absolute configurations of 1−4 were determined by the ECD exciton chirality method and quantum ECD calculations. Protostemonine (10) and stemofoline (12) showed strong nematicidal activity against Panagrellus redivevus, with IC50 values of 0.10 and 0.46 μM, respectively.
T
thoxystemofoline,5 isoprotostemonine,16 didehydroprotostemonine,16 (−)-stemonine,2 and (12R)-dihydroprotostemonine.17 The known compounds were determined by comparing their NMR and MS data with those reported in the literature. The present report describes the compound isolation process and structural elucidation details, as well as the results of testing the alkaloids obtained in an antinematode assay.
o date, the Stemona alkaloids have been isolated only from plants in the relatively small monocotyledonous family, Stermonaceae. Secondary metabolites from species in this family are well-known for their interesting structures and biological activities and for the total synthesis methodology applied for these compounds.1,2 Due to their antitussive and insecticide activities, the roots of Stemona species have been used in folk medicine in East Asia for thousands of years.3 Currently, there are three species of this genus [S. tuberosa Lour., S. japonica Franch. & Sav., and S. sessilifolia Franch. & Sav.] listed in the Chinese Pharmacopoeia as cough remedies and as pesticides. In addition, the species Stemona parviflora C.H. Wright is also used as a medicinal plant herb and has similar fleshy tuberous roots and occurs in Hainan Island.4 Stemona parviflora, a herb used by the Li ethnic group, was investigated previously, leading to the isolation of several alkaloids such as stemofoline, oxystemofoline, and parvineostemonine.5,6 However, there is no prior systematic alkaloid analysis report on S. parviflora for its chemical relationship to other species in the genus or on its bioactivity evaluation. In the present study, the constituents of S. parviflora were investigated. As a result, eight new Stemona alkaloids, 3β-nbutylstemonamine (1), 8-oxo-3β-n-butylstemonamine (2), 3-nbuctylneostemonine (3), 10-epi-3-n-butylneostemonine (4), 8oxo-oxymaistemonine (5) protostemonine N4-oxide (6), and (19S)-hydroxy-21-methoxystemofoline (7), and parvistemonine A (8), inclusive of some rare alkaloids with a n-butyl substituent, were isolated and characterized structurally. Also obtained were 17 known analogues, protostemonamide (9),7 protostemonine (10),8 (+)-oxystemofoline (11),9 stemofoline (12),10 protostemotinine,11 isooxymaistemonine,12 oxymaistemonine,13 isostemonamide,14 stemonamide,14 stemonamine,7 (2′R)-hydroxystemofoline,15 (2′S)-hydroxystemofoline,15 me© 2016 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Compound 1 was obtained as an orange amorphous solid, and its molecular formula was assigned as C22H31NO4, with eight degrees of unsaturation, from its HREIMS (m/z 396.2150 [M + Na]+, calcd for C22H31NO4Na 396.2152) and NMR data (Table 1). The IR spectrum displayed bands for the presence of carbonyl (1760, 1709 cm−1) and double bond (1661 and 1564 cm−1) absorptions. Analysis of its 13C NMR and DEPT spectra Received: June 8, 2016 Published: September 29, 2016 2599
DOI: 10.1021/acs.jnatprod.6b00528 J. Nat. Prod. 2016, 79, 2599−2605
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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of 1−4 (in CDCl3) 1 position
δH mult. (J in Hz)
1β α 2α β 3 5β α 6α β 7β α 8α
1.73 2.11 1.89 1.50 3.23 3.14 2.93 1.78 1.40 2.00 1.28 2.90
β
2.20 ddd (2.4, 10.5, 12.8)
9 9a 10 11 12 13 14 15 16 17 18 19 20 21 OMe
2.02 1.08 1.58 1.09 1.16 1.29 0.90 3.99
m m m m m m m m m m m m
s s m m m m t (7.5) s
2 δC
3
δH mult. (J in Hz)
36.1 t 30.4 t 59.5 d 45.4 t 27.5 t 27.1 t
2.09 2.02 1.98 1.60 3.19 3.24
m m m m m m
2.13 m 1.65 m 2.81 ddd (2.6, 7.0, 12.9) 2.56 ddd (5.7, 6.1, 12.9)
δC
δH mult. (J in Hz)
102.7 d
5.84 d (3.3)
103.1 d
30.1 t
5.82 d (3.1)
104.1 d
5.91 d (3.3)
104.2 d
58.9 d 42.4 t 21.2 t 41.4 t
4.18 m 3.63 m 2.13 m 1.56 m 2.58 m 1.82 m 3.78 ddd (3.5, 10.2, 11.0)
174.6 s 78.4 s 135.6 s 198.9 s 91.5 s 172.5 s 97.2 s 175.1 s 8.9 q 8.3 q 33.2 t
161.8 s 75.4 s 142.7 s 199.2 s 93.3 s 171.2 s 97.4 s 174.3 s 10.2 q 8.8 q 33.7 t
2.95 dd (9.8, 10.2)
27.2 23.1 14.2 59.1
26.5 23.2 14.3 58.6
t t q q
δC
5.91 d (3.1)
202.1 s
s s m m m m t (8.0) s
δH mult. (J in Hz)
36.9 t
28.3 t
2.05 2.02 1.58 1.09 1.18 1.29 0.90 4.01
4 δC
t t q q
134.3 s 44.3 t 26.0 t 34.2 t 86.2 d
4.20 m 3.64 m 2.54 m 2.12 m 2.57 m 1.84 m 3.87 ddd (3.7, 10.1, 10.8)
134.1 s 44.2 t 26.0 t 34.5 t 87.0 d
2.95 dd (9.7, 10.8)
2.11 s 1.53 d (6.5) 2.54 m
52.1 d 128.0 s 39.6 d 149.1 s 125.3 s 163.3 s 97.4 s 170.2 s 9.2 q 19.2 q 26.4 t
2.08 s 1.63 d (6.8) 2.55 m
50.4 d 127.7 s 41.7 d 150.9 s 126.2 s 163.6 s 97.9 s 170.8 s 8.8 q 16.9 q 26.4 t
1.58 1.42 0.96 4.20
31.5 22.6 13.9 58.9
1.59 1.41 0.96 4.18
31.5 22.6 14.0 59.5
3.52 dq (6.5, 9.8)
m m t (7.3) s
t t q q
3.58 dq (6.5, 9.8)
m m t (7.3) s
t t q q
Figure 1. Key HMBC (→) and 1H−1H COSY () correlations of 1−8.
26.5 (t, C-19), 23.2 (t, C-20), and 14.3 (q, C-21) and the absence of any signal at δC 51.4 (t, C-3),18 indicating that C-3 is substituted with a n-butyl group in compound 1. This inference was confirmed by the 1H−1H COSY cross-peaks and HMBC (Figure 1) correlations observed for 1. The sequential 1H−1H
(Table 1) showed 22 carbon resonances, including four methyls (one methoxy), nine methylenes, one methine, and eight quaternary carbons (two carbonyls and four olefins). The 13C NMR data of 1 were similar to those of stemonamine, except for the additional signals at δC 59.5 (d, C-3), 33.2 (t, C-18), 2600
DOI: 10.1021/acs.jnatprod.6b00528 J. Nat. Prod. 2016, 79, 2599−2605
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Figure 2. Key ROESY (↔) correlations of 1−8.
Figure 3. Absolute configurations of compounds 1 and 2 as determined by the ECD exciton chirality method and calculated ECD spectra for 1.
COSY cross-peaks of H-2 [δH 1.89 (1H, m) and 1.50 (1H, m]/ H-3 [δH 3.23 (1H, m)], H-3/H-18 [δH 1.58 (1H, m) and 1.09
(1H, m)], H-18/H-19[δH 1.16 (2H, m)], H-19/H-20 [δH 1.29 (2H, m)], and H-20/H-21 [δH 0.90 (1H, d, J = 7.5 Hz)] 2601
DOI: 10.1021/acs.jnatprod.6b00528 J. Nat. Prod. 2016, 79, 2599−2605
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Figure 4. Experimental and calculated ECD curves of 3 and 4.
Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data of 5−8 (in CDCl3) 5 position 1β α 2α β 3 5β α 6α β 7β α 8 9 9a 10 11 12 13 14 15 16 17 18 19 20 21 22/OMe OMe
δH mult. (J in Hz)
6 δC
2.16 m
36.5 t
1.98 1.91 3.35 3.70 3.26 2.26 1.72 2.77 2.61
26.1 t
2.06 2.10 3.85 2.41 1.50 2.63
m m m m m m m m m
s s m m m m
1.29 d (7.0) 4.05 s
63.4 d 44.6 t 21.3 t 40.9 t 201.2 s 159.8 s 76.5 s 144.0 s 198.6 s 93.3 s 171.4 s 97.2 s 174.2 s 10.4 q 8.8 q 85.3 d 34.5 t 34.8 t 179.5 s 14.9 q 59.2 q
δH mult. (J in Hz)
7 δC
δH mult. (J in Hz)
2.30 1.89 2.09 2.06 3.95 3.92 3.71 1.99
m m m m m m m m
24.4 t
2.53 1.79 5.13 1.83 4.17 3.01
m m m dd (4.0, 9.1, 11.0) m dd (6.6, 9.1)
34.1 t
2.07 1.47 4.31 2.39 1.74 2.81
s d (6.6) ddd (4.1, 11.0, 11.1) m m m
1.26 d (7.0) 4.17 s
23.7 t 76.6 d 68.0 t 19.5 t
74.1 d 50.6 d 85.5 d 39.8 d 148.6 s 124.9 s 163.9 s 96.6 s 171.4 s 18.3 q 7.5 q 81.5 t 32.6 d 34.8 t 179.3 s 13.4 q 58.9 q
2.07 m 1.88 m 4.42 m
3.26 3.04 2.04 1.92 2.68
m m m m d (6.1)
1.83 dd (3.4, 10.1) 3.55 m 3.13 m
1.40 2.10 1.71 3.89
d (6.5) s m m
1.75 3.55 3.49 4.17 3.36
m m m s s
8 δC
δH mult. (J in Hz)
δC
33.7 t
5.88 d (3.4)
103.0 d
78.6 d
5.84 d (3.4)
104.3 d
82.9 s 47.1 t 27.0 t 51.7 d 112.0 s 47.5 d 60.9 d 41.7 d 147.8 s 128.1 s 162.7 s 98.7 s 169.6 s 18.3 q 9.2 q 37.8 t 67.0 d
4.21 3.65 2.13 1.57 2.54 1.79 3.91 3.02
dd (14.7, 5.6) dd (14.7, 11.6) m m m m dd (9.5, 3.6) m
2.98 m 1.43 2.55 1.58 1.41 0.96
d (6.6) t (7.1) m m t (7.3)
134.3 s 44.2 t 26.0 t 34.4 t 81.8 d 49.4 d 128.1 s 39.4 d 178.5 s 13.9 q 26.3 t 31.6 t 22.6 t 13.9 q
36.7 t 69.1 q 58.9 q 58.8 q
comparison of their NMR data. The α-orientation of H-3 was proposed by a NOE interaction between H-1α and H-3. The ECD Cotton effects observed for 1 were attributed to exciton coupling between the transition dipoles of the α,β-unsaturated ketone and α,β-unsaturated ester moieties and could be used to predict the absolute configuration according to the Harada− Nakanishi nonempirical rule.19−21 The negative long-wave Cotton effect around λmax = 300 nm and positive short-wave Cotton effect around λmax = 230 nm (Figure 3) indicated that the front chromophore (α,β-unsaturated ketone) existed in a clockwise arrangement relative to the rear chromophore (α,βunsaturated ester) (Figure 3) and implied that compound 1 has
confirmed the linkage of C-2−C-3−C-18−C-19−C-20−C-21. The HMBC (Figure 1) correlations from H-1 [δH 2.11 (1H, m) and 1.73 (1H, m)], H-5 [δH 3.14 (1H, m) and 2.93 (1H, m)], and H-19 [δH 1.16 (2H, m)] to C-3 supported the linkage of the five-membered ring system, as shown in Figure 1. The other correlations that occurred in the 1H−1H COSY and HMBC spectra confirmed the atom connectivity of a stemonamine moiety in compound 1. Compound 1 was found to have the same relative configuration of the stemonamine skeleton based on its ROESY data (Figure 2), H-1α [δH 2.11 (1H, m)]/H-8α [δH 2.90 (1H, m)], H-1α/H-3, H-3/H-5α [δH 2.93 (1H, m)], and H-5β [δH 3.14 (1H, m)]/H-18 [δH 1.58 (1H, m)], and 2602
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maistemonine,13 compound 5 could be proposed as being derived from this latter alkaloid via the oxygenation of C-8 to a carbonyl group. A key HMBC correlation from H-7 [δH 2.77 (1H, m) and 2.61 (1H, m)] to C-8 [δC 201.2 (s)] and 1H−1H COSY correlations between H-7/H-6 [δH 2.26 (1H, m) and 1.72 (1H, m)] and H-6/H-5 [δH 3.70 (1H, m) and 3.26 (1H, m)] in compound 5 (Figure 1) supported this proposal. Compound 5 was assigned the same configuration as oxymaistemonine based on its NOESY NMR spectrum (Figure 2). Thus, the structure of compound 5 (8-oxo-oxymaistemonine) was determined as shown. Compound 6, was isolated as an orange amorphous substance, and its molecular formula was determined to be C23H31NO7, having nine degrees of unsaturation, from its HRESIMS (m/z 434.2178 [M + H]+, calcd for C23H32NO7 434.2179). Compound 6 exhibited 13C NMR data (Table 1) similar to those of protostemonine (10).8 However, differences were observed for the signals at δC 76.6 (d, C-3), 68.0 (t, C-5), and 85.5 (s, C-9a) in 6 that replaced those at δC 64.2 (d, C-3), 46.5 (t, C-5), and 58.7 (s, C-9a) in alkaloid 10. This indicated the presence of an N-oxide functionality at N-4 in compound 6, which was supported by its molecular formula of C23H32NO7, by having one more oxygen atom than compound 10. The HMBC and 1H−1H COSY correlations of compound 6 (Figure 1) were used to verify its planar structure. Compound 6 was assigned the same configuration as protostemonine (10) based on the analysis of NOE experiments (Figure 2). The N→O bond was assigned as β-oriented for the NOE correlation from H-3 [δH 3.95 (1H, m)] to H-5α [δH 3.71 (1H, m)]. Thus, the structure of compound 6 was assigned as protostemonine N4oxide. The molecular formula of compound 7 was determined as C23H31NO7, with nine degrees of unsaturation, from its HREIMS (m/z 434.2184 [M + H]+, calcd for C23H32NO7 434.2179). Compound 7 exhibited 13C NMR data (Table 1) similar to those of (2′S)-hydroxystemofoline,24 with the differences observed due to an additional resonance at δC 58.9 (q, OMe-21) and with the resonance at δC 69.1 (t, C-21) in 7 replacing that at δC 9.80 (q, C-21). The HMBC and 1 H−1H COSY correlations of compound 7 (Figure 1) supported the planar structure proposed. The absolute configuration of (2′S)-hydroxystemofoline was assigned by Xray crystallographic analysis of hydroxystemofoline and the Mosher ester method.10,15 By comparing the NMR, NOE, and optical rotation data of compound 7, [α]D25 +218 (c 0.085, MeOH), to those of (2′S)-hydroxystemofoline, [α]D21 +197 (c 0.5, MeOH), the configurations of the two compounds were found to be the same (C-19 was indicated as C-2′ in the previous literature report).15 Thus, the structure of compound 7 was assigned as (19S)-hydroxy-21-methoxystemofoline. The molecular formula of compound 8 was determined to be C16H23NO2, with six degrees of unsaturation, by its HREIMS (m/z 284.1623 [M + Na]+, calcd for C16H23NO2Na 284.1626). The 13C NMR data (Table 1) of compound 8 were closely comparable to those of dihydrostemonine,25 except for the resonances at δC 134.3 (s, C-3), 26.3 (t, C-13), 31.6 (t, C-14), 22.6 (t, C-15), and 13.9 (q, C-16) replacing those at δC 132.1 (s, C-3), 71.4 (d, C-13), 34.7 (t, C-14), 36.0 (d, C-15), 177.9 (s, C-16), and 13.7 (q, C-17) in dihydrostemonine. This indicated that C-3 is linked to a n-butyl group with a carbon bond in compound 8 instead of the C-13−C-17 lactone moiety found in dihydrostemonine. This inference was supported by the HMBC (Figure 1) correlations of 8 combined with the
a 3R,9aS,12S configuration. This was confirmed by ECD calculations (Figure 3) using the time-dependent density functional theory method at the B3LYP/6-31G(d) level as implemented in the Gaussian 03 program package.22 The ECD calculations were performed after optimization of the selected conformers at the B3LYP/6-31G(d) levels.23 Thus, the structure of compound 1 was assigned as shown, and this compound has been named 3β-n-butylstemonamine. Compound 2 was isolated as an orange amorphous substance and the ESIMS at m/z 410 [M + Na]+ suggested its molecular weight to be 387 Da. The molecular formula of 2 was determined as C22H29NO5 with nine degrees of unsaturation by HRESIMS (m/z 410.1940 [M + Na]+, calcd for C22H29NO5Na 410.1943). By comparing the molecular formula, NMR data (Table 1), and IR spectra with compound 1, compound 2 was related to 1 in having a carbonyl group located at C-8. The key HMBC correlations from H-6 [δH 2.13 (1H, m)] and H-7 [δH 2.81 (1H, ddd, J = 2.6, 7.0, 12.9 Hz) and 2.56 (1H, ddd, J = 5.7, 6.1, 12.9 Hz)] to C-8 [δC 202.1 (s)] and the 1H−1H COSY correlations of compound 2 (Figure 1) confirmed this conclusion. Compound 2 was assigned the same configuration as compound 1 based on the ROESY analysis (Figure 2) and an ECD exciton chirality study (Figure 3), as well as biosynthetic considerations that indicated a 3R,9aS,12S configuration. Thus, the structure of compound 2 was assigned as 8-oxo-3β-nbutylstemonamine. Compounds 3 and 4 were isolated as separate compounds and were both formulated as C22H29NO4 from their HRESIMS data at m/z 394.1994 [M + Na]+, calcd for C22H29NO4Na 394.1988 of 3, and m/z 371.2095 [M]+, calcd for C22H29NO4 371.2097 of 4. These two compounds showed 13C NMR data (Table 1) similar to those of neostemonine.16 However, differences were apparent for the signals at δC 134.3 and 134.1 (s, C-3) in 3 and 4, respectively, compared with δC 122.6 (d, C3) in neostemonine, as well as the additional resonances at δC 26.4 (t, C-18), 31.5 (t, C-19), 22.6 (t, C-20), and 13.9 and 14.0 (q, C-21) in both 3 and 4, corresponding to a n-butyl group in each case. The n-butyl groups were attached at C-3 for both alkaloids as established by the HMBC correlations and 1H−1H COSY correlations shown in Figure 2. A 1H and 13C NMR data comparison between compounds 3 and 4 (Table 1) showed that these alkaloids are configurational isomers. From the NOE (Figure 3) correlations between MeO-13 [δH 4.20 (3H, s)]/H17 [δH 1.53 (H, d, J = 6.5 Hz)], H-17/H-9 [δH 2.95 (H, dd, J = 9.8, 10.2 Hz)], and H-10 [δH 3.52 (H, dd, J = 6.5, 9.8 Hz)]/H8 [δH 3.78 (H, ddd, J = 3.5, 10.2, 11.0 Hz)], the relative configuration of compound 3 with an α-orientation of H-8 and β-orientations of Me-17 and H-9 was determined as shown. The relative configuration of compound 4 with an α-orientation of Me-17 was determined by the key NOE (Figure 2) of H-17/ H-8 [δH 3.78 (H, ddd, J = 3.7, 10.1, 10.8 Hz)]. The absolute configurations of compounds 3 and 4 were determined by ECD calculations. The results (Figure 4) showed that the measured ECD curves for 3 and 4 matched well with the calculated data. Therefore, the absolute configurations of 3 and 4 were determined to be 8R,9R,10S and 8R,9R,10R, respectively. Thus, the structures of compounds 3 and 4 (3-n-butylneostemonine and 10-epi-3-n-butylneostemonine) were assigned as shown. Compound 5 was determined as C23H27NO7, with 11 degrees of unsaturation, from its HRESIMS (m/z 452.1690 [M + Na]+, calcd for C23H27NO7Na 452.1680. By comparing the molecular formula and NMR data (Table 2) with oxy2603
DOI: 10.1021/acs.jnatprod.6b00528 J. Nat. Prod. 2016, 79, 2599−2605
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18 (H2O−MeOH, 1:9 to 0:1, v/v) and Sephadex LH-20 (CHCl3− MeOH, 1:1, v/v) columns to yield compounds 1 (17.9 mg, orange amorphous solid), 3 (38.9 mg, orange amorphous solid), 4 (3.1 mg, orange amorphous solid), 8 (9.5 mg, orange amorphous solid), and stemonamine (4.1 mg, orange amorphous solid). Fraction 2b was separated using silica gel (petroleum ether−Me2CO, 2:1, v/v), RP-18 (H2O−MeOH, 1:9 to 0:1, v/v), and semipreparative HPLC (MeOH− H2O, 35:65, v/v) to yield compounds 2 (21.3 mg, orange amorphous solid), 5 (8.7 mg, orange amorphous solid), isostemonamide (32.3 mg, colorless fine crystals), stemonamide (21.5 mg, orange amorphous solid), and 9 (12.0 mg, orange amorphous solid). Fraction 2c was chromatographed, in turn, on RP-18 (H2O−MeOH, 1:9 to 0:1, v/v) and Sephadex LH-20 (CHCl3−MeOH, 1:1, v/v) columns to yield isooxymaistemonine (5.4 mg, colorless fine crystals) and oxymaistemonine (32.3 mg, colorless fine crystals). Fraction 3 (5.2 g) was chromatographed on silica gel (petroleum ether−Me2CO, 4:1, v/ v) to yield compound 10 (312 mg, colorless fine crystals) and fractions 3a−3c. Fraction 3a was separated using silica gel (petroleum ether− Me2CO, 2:1, v/v), RP-18 (H2O−MeOH, 1:9 to 0:1, v/v), and semipreparative HPLC (MeOH−H 2 O, 35:65, v/v) to yield (−)-stemonine (4.3 mg, orange amorphous solid) and (12R)dihydroprotostemonine (11.8 mg, orange amorphous solid). Fraction 3b was chromatographed on both RP-18 (H2O−MeOH, 1:9 to 0:1, v/ v) and Sephadex LH-20 (CHCl3−MeOH, 1:1, v/v) columns to yield isoprotostemonine (6.9 mg, orange amorphous solid) and didehydroprotostemonine (14.1 mg, orange amorphous solid). Fraction 3c was separated by silica gel (petroleum ether−Me2CO, 2:1, v/v), RP-18 (H2O−MeOH, 1:9 to 0:1, v/v), and semipreparative HPLC (MeOH− H2O, 35:65, v/v) to yield compounds 12 (60.4 mg, orange amorphous solid) and methoxystemofoline (24.5 mg, orange amorphous solid). Fraction 4 (1.7 g) was separated by silica gel (petroleum ether− Me2CO, 2:1, v/v), RP-18 (H2O−MeOH, 1:9 to 0:1, v/v), and semipreparative HPLC (MeOH−H2O, 35:65, v/v) to yield compounds 6 (11.1 mg, orange powder), 7 (23.6 mg, orange amorphous solid), (2′R)-hydroxystemofoline (3.3 mg, orange amorphous solid), (2′S)-hydroxystemofoline (9.4 mg, orange amorphous solid), and 11 (14.6 mg, orange amorphous solid). 3β-n-Butylstemonamine (1): orange amorphous solid, [α]D28 −3.4 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 238 (4.26), 300 (3.17), 360 (2.97) nm; IR (KBr) νmax 3450, 1760, 1709, 1661, 1564, 1413, 1327, 1080 cm−1; ECD (c 0.23 mM, MeOH) λmax (Δε) 206 (+1.19), 232 (−3.23), 302 (+1.77), 349 (−2.23) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 396.2150 (calcd for C22H31NO4Na 396.2152). 8-Oxo-3β-n-butylstemonamine (2): orange amorphous solid, [α]D28 −5.4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 236 (4.21) nm; IR (KBr) νmax 3444, 1707, 1642, 1566, 1555, 1413, 1088 cm−1; ECD (c 0.19 mM, MeOH) λmax (Δε) 210 (+2.11), 232 (−2.73), 305 (+0.56) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 410.1940 (calcd for C22H29NO5Na 410.1943). 3-n-Butylneostemonine (3): orange amorphous solid, [α]D28 −1.3 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 302 (3.85) nm; IR (KBr) νmax 3437, 2958, 2931, 2871, 1740, 1712, 1673, 1619, 1459, 1398, 1365, 1330, 1261, 1221, 1154, 1064, 1020, 800 cm−1; ECD (c 0.01 mM, MeOH) λmax (Δε) 206 (−12.2), 238 (+0.52), 257 (−1.09), 292 (+4.03), 348(−1.42) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 394.1994 (calcd for C22H29NO4Na 394.1988). 10-epi-3-n-Butylneostemonine (4): orange amorphous solid, [α]D28 −2.0 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 300 (3.38) nm; IR (KBr) νmax 3443, 1704,1642, 1412, 1069 cm−1; ECD (c 0.02 mM, MeOH) λmax (Δε) 217 (+0.39), 236 (−1.48), 265 (+0.41) nm; 1 H and 13C NMR data, see Table 1; HREIMS m/z 371.2095 (calcd for C22H29NO4 371.2097). 8-Oxo-oxymaistemonine (5): orange amorphous solid; [α]D28 −3.1 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 232 (4.21) nm; IR (KBr) νmax 3452, 2928, 1762, 1706, 1658, 1565, 1413, 1235, 1091 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [M + Na]+ 452.1690 (calcd for C23H27NO7Na 452.1680). Protostemonine N4-Oxide (6): orange powder; [α]D25 −3.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 302 (3.54), 236 (3.58) nm; IR
sequential 1H−1H COSY cross-peaks observed. The other correlations in the 1H−1H COSY and HMBC spectra confirmed the atom connectivity of the planar structure of compound 8. According to the NOE data (Figure 2) and its possible biosynthetic origin from compound 3, the configuration of compound 8 should be the same as that of 3, with the 8R,9R,10S configuration. Thus, the structure of compound 8 (parvistemonine A) was assigned as shown. All of the isolated alkaloids and the crude extract (at 25 μg/ mL) were tested in a lethality assay against Panagrellus redivevus for nematicidal activity evaluation (Table S2 and Figure S1, Supporting Information). The crude extract showed nematicidal bioactivity with a relative death rate (RDR) of 56.51% at 250 μg/mL. Compounds 1, protostemonamide (9), and (+)-oxystemofoline (11) showed lethality against P. redivevus (with IC50 values of 42.5, 1.95, and 76.4 μM, respectively) comparable to the positive control [albendazole (SigmaAldrich) IC50 = 67.2 μM]. Protostemonine (10) and stemofoline (12) showed nematicidal bioactivity superior to that as the positive control, with IC50 values of 0.10 and 0.46 μM, respectively. Previous studies showed that protostemonine (10) and stemofoline (12) isolated from other Stemona species have potent insecticidal activities.8,17 The present data indicate that alkaloids in S. parviflora may play a defensive role against worms such as P. redivevus.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were obtained on a Cataceo Co., Ltd. Polar-L polarimeter. UV spectra were obtained on a Shimadzu UV-250 spectrometer. ECD were obtained on a Chirascan instrument. IR spectra were obtained on a Thermo Nicolet 380 spectrometer with KBr pellets. NMR spectra were recorded on a Bruker AV-500 NMR spectrometer with TMS as an internal standard. ESIMS, HRESIMS, and HREIMS were recorded with a Micromass Autospec-Uitima-TOF, Waters Autospec Premier spectrometer, or API QSTAR Pulsar 1 spectrometer. Silica gel (200− 300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), and RP-18 (40−70 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan) were used for column chromatography. Semipreparative HPLC was performed on a Dionex P680 liquid chromatograph with a Cosmosil, πNAP 5ϕ5 μm, 10 mm × 250 mm, column. Fractions were detected by TLC, and plates were visualized with Dragendorff reagent. The planar structures were drawn with Chembiodraw 10.0, and 3D structures were drawn with Chembio3D 10.0, and the MM2 method to minimize energy was applied. The ECDs were plotted with OriginPro 2010. Plant Material. The roots of Stemona parviflora were collected in May 2013 from Haikou, Hainan Province, People’s Republic of China, and the plant was identified by Dr. G. Chen (Kunming Institute of Botany, Chinese Academy of Sciences) and S.-Z. H. A voucher specimen (HUANG0009) was deposited at the Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou, People’s Republic of China. Extraction and Isolation. Air-dried S. parviflora roots (5.4 kg) were powdered and extracted with EtOH (3 × 10 L) under reflux. After evaporation of the solvent, the EtOH extract was partitioned between H2O (0.01% HCl) and petroleum ether. The pH value was adjusted to 11.0 with 2 M NaOH, and then the H2O extract was partitioned by CHCl3. The CHCl3 extract (54 g) was chromatographed on silica gel with gradient mixtures of CHCl3−MeOH (1:0 to 0:1, v/v) to give five pooled fractions. Fraction 2 (3.1 g) was chromatographed on silica gel (petroleum ether−Me2CO, 4:1, v/v) to yield protostemotinine (289 mg, colorless fine crystals) and fractions 2a, 2b, and 2c. Fraction 2a was chromatographed sequentially on RP2604
DOI: 10.1021/acs.jnatprod.6b00528 J. Nat. Prod. 2016, 79, 2599−2605
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(KBr) νmax 3452, 2931, 1756, 1689, 1701, 1632, 1454, 1401, 1189, 1158, 1073, 1013 cm−1; 1H and 13C NMR data, see Table 2; HREIMS m/z [M + H]+ 434.2178 (calcd for C23H32NO7 434.2179). (19S)-Hydroxy-21-methoxystemofoline (7): orange amorphous solid; [α]D25 +218.0 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 298 (3.38), 292 (3.91) nm; IR (KBr) νmax 3444, 2936, 1746, 1708, 1621, 1456, 1398, 1366, 1222, 1079, 1057, 1007, 966 cm−1; 1H and 13 C NMR data, see Table 2; HREIMS m/z [M + H]+ 434.2184 (calcd for C23H32NO7 434.2179). Parvistemonine A (8): colorless amorphous solid; [α]D28 −75.1 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 334 (3.62) nm; IR (KBr) νmax 3444, 1640, 1561, 1412, 1067 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z [M + Na] + 284.1623 (calcd for C16H23NO2Na 284.1626). Antinematode Bioassay. The test compounds were dissolved in dimethylsulfoxide (10 μL) and then diluted with sterilized water (containing 0.3% v/v Tween-20) to obtain a stock solution at 25 mg/ mL. Nematodes [Panagrellus redivevus] were bred at 28 °C for 5−6 days in oat culture medium [oat (Avena sativa) flakes, 20 g, and water, 20 mL, in an Erlenmeyer flask, with sterilization at 121 °C for 30 min]. Then using a 6 cm diameter Petri dish containing 300 μL of nematode suspension (about 200 juveniles), a suitable volume of each test sample solution was added and gently mixed to obtain a final concentration of 25 μg/mL (0.2, 1, 5, 25, and 125 μg/mL for IC50). Dimethylsulfoxide (10 μL) dissolved in water (containing 0.3% v/v Tween-20) was established as a control. All treatments were conducted in triplicate. Nematicidal activity (NA) was assessed by counting the dead nematodes (n > 100) under a microscope after incubation at 28 °C for 24 h. The NA value was calculated using the formula NA = DN/SN × 100% (DN is the number of dead nematodes; SN is the sum of all counted nematodes, SN > 100). The RDR was calculated using the formula RDR = NAn − NA0 (NAn is the nematicidal activity of compounds, and NA0 is the nematicidal activity of the blank control).26 If the RDR of any test compound was greater than 40%, the IC50 of the compound was assayed and calculated by the Reed and Muench method.27
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Menglun, People’s Republic of China) for proofreading this manuscript.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00528. Calculation details, HPLC data, RDR values of the isolated compounds, and the NMR and mass spectra of compounds 1−8 (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax (H.F. Dai): +86-898-66961869. E-mail: daihaofu@ itbb.org.cn. *Tel/Fax (Y.X. Zhao): +86-898-66989095. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the grant from Natural Science Foundation of China (31300294), Special Fund for AgroScientific Research in the Public Interest (201303117), and Fundamental Scientific Research Funds for CATAS (ITBB2016). We thank Dr. Y.L. Huang (Department of Epigenetics and Molecular Carcinogenesis, UT MD Anderson Cancer Center, Houston, TX, USA), Liwen Tian (Southern Medical University, Guangzhou, People’s Republic of China), and Dr. F. Jacques (Xishuangbanna Tropical Garden CAS, 2605
DOI: 10.1021/acs.jnatprod.6b00528 J. Nat. Prod. 2016, 79, 2599−2605
