Article pubs.acs.org/jnp
Hydroxylated Daphniphyllum Alkaloids from Daphniphyllum himalense Hua Zhang,† Sajan L. Shyaula,‡ Jing-Ya Li,† Jia Li,† and Jian-Min Yue*,† †
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China ‡ Nepal Academy of Science and Technology, Khumaltar, Lalitpur, GPO Box 3323, Kathmandu, Nepal S Supporting Information *
ABSTRACT: Thirteen new hydroxylated calyciphylline A-type Daphniphyllum alkaloids (1−13) were isolated from an ethanolic extract of Daphniphyllum himalense. These structures were characterized on the basis of spectroscopic data analysis, especially from their 2D NMR spectra. Oxidation at the C-3, C-9, C-11, and C-12 positions is reported for the first time for this class of compounds. Selective compounds showed low inhibitory rates against three kinase enzymes, PTP1B, aurora A, and IKK-β, at a concentration of 20 μg/mL. Daphniphyllum alkaloids are a family of intriguing natural products isolated from Daphniphyllum species (Daphniphyllaceae), and over 200 members have been discovered from 13 members of this genus so far.1 Owing to their complex structures and diverse carbon skeletons, these alkaloids have been investigated continuously by both natural products and synthetic chemists over the past half century.1 Daphniphyllum himalense (Benth.) Muell.-Arg., a tree mainly distributed in Nepal and the northeast of India, also occurs in Xizang and Yunnan Provinces of mainland China.2 Among the 10 native Chinese species, D. himalense is the only one for which the chemical constituents have been studied preliminarily.3−5 As part of a collaboration between the Chinese Academy of Sciences and the Nepal Academy of Science and Technology to explore Nepalese herbal resources, we have investigated the alkaloidal constituents of the Nepalese D. himalense. Thirteen new hydroxylated Daphniphyllum alkaloids (1−13) were isolated and characterized structurally in the current work. Structural assignments of these new compounds were based on detailed spectroscopic data analysis. Hydroxy group substitution at C-3, C-9, C-11, and C-12 in these structures is being reported for calyciphylline A-type alkaloids for the first time. All the new isolates were tested in an array of kinase inhibitory assays [protein tyrosine phosphatase 1B (PTP1B), aurora kinase A, histone deacetylase 6 (HDAC6), and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKK-β)]. This paper deals with the isolation, structure characterization, and biological evaluation of these new alkaloids.
istic resonances for two methyls (δC 17.8 and 24.0), a hydroxyethyl group (δC 33.1 and 62.3), an oxygenated methine (δC 69.0), a tetrasubstituted double bond (δC 127.8 and 159.3), and two carbonyls [δC 168.6 (conjugated) and 212.7]. Three
■
RESULTS AND DISCUSSION Alkaloid 1 was assigned a molecular formula of C22H31NO5 based on its NMR data (Tables 1 and 2) and the (+)-HRESIMS ion at m/z 390.2286 ([M + H]+, calcd 390.2280). Analysis of the NMR data of 1 revealed character© XXXX American Chemical Society and American Society of Pharmacognosy
Received: August 18, 2015
A
DOI: 10.1021/acs.jnatprod.5b00741 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 1. 1H NMR Spectroscopic Data of Alkaloids 1−5 1a
position 2 3a 3b 4 6 7α 7β 9 10 11α 11β 12α 12β 13α 13β 14 15 16 17α 17β 18 19α 19β 20 21 OMe a
2a
1.93, 2.06, 1.69, 3.06, 2.17, 2.59, 2.84, 3.98, 4.73, 2.05,
brd (4.5) m brdd (14.5, 5.7) brd (5.7) m dd (11.4, 8.1) dd (8.1, 7.2) brs m m (2H)
1.45, 2.03, 3.00, 3.78,
m m d (16.3) brd (16.3)
1.92, 2.01, 1.71, 3.08, 2.14, 2.62, 2.82, 4.06, 4.49, 1.92, 2.00, 1.44, 2.10, 2.83, 3.55,
brd (4.9) brdd (14.5, 4.9) brdd (14.5, 5.7) brd (5.7) m dd (11.2, 7.7) dd (7.7, 6.7) brs m m m m m brd (16.2) brd (16.2)
α 2.58, m β 2.43, m 4.33, m 4.29, m 3.20, m α 2.69, dd (14.0, 8.9) β 2.37, dd (14.0, 9.1) 0.84, d (7.0) 1.59, s
a 3.91, m b 2.84, m 4.41, m (2H) 3.20, 2.69, 2.34, 0.82, 1.68,
3b
m dd (13.8, 8.8) dd (13.8, 8.9) d (6.9) s
2.19, 2.02, 1.97, 3.37, 2.28, 2.83, 2.91,
4c
m ddd (14.8, 4.6, 1.6) brdd (14.8, 4.0) brs m dd (11.5, 9.5) dd (9.5, 6.6)
2.18, 2.34, 2.06, 3.37, 2.11, 2.81, 2.71,
2.16 m 1.86 ddd (17.2, 4.0, 4.0) 1.82, m (2H)
5c
m brdd (15.1, 4.7) ddd (15.1, 4.9, 1.2) brd (4.9) m dd (13.0, 9.7) dd (9.7, 6.4)
1.59, m 1.81, m 1.69, m 1.59, m 2.15, m 2.95, brdd (13.3, 8.3) α 2.22, brdd (16.3, 9.9) β 2.47, m
2.69 m 3.35 brd (17.5) 5.22, brs
4.87, m
4.87, brdd (6.3, 6.0)
3.26, 2.70, 2.74, 2.85, 2.58, 1.00, 1.20,
2.63, 1.98, 2.77, 2.83, 2.52, 1.01, 1.26,
brdd (17.4, 7.3) m m dd (14.0, 7.5) dd (14.0, 9.7) d (6.7) s
dd (13.0, dd (13.0, m dd (14.2, dd (14.2, d (6.7) s
6.0) 6.3) 7.2) 10.3)
2.56, dd (6.2, 1.5) 3.28, 3.73, 1.81, 2.19, 2.62,
dd (6.2, 3.9) d (3.9) m dd (13.0, 12.8) dd (13.0, 3.5)
1.97, m (2H) 1.54, 2.14, 2.18, 3.13, 2.87,
m m dd (15.6, 9.2) dd (15.6, 5.1) ddd (11.1, 9.2, 5.1)
3.82, m α 1.83, m β 1.23, m 2.61, m 2.32, brdd (15.0, 8.3) 2.90, m 3.06, dd (12.2, 8.1) 2.39, dd (12.2, 7.4) 1.12, d (7.0) 1.30, s 3.62, s
Measured in C5D5N. bMeasured in CDCl3. cMeasured in CD3OD.
Table 2. 13C NMR Spectroscopic Data of Alkaloids 1−13
a
position
1a
2a
3b
4c
5c
6b
7b
8b
9b
10b
11c
12b
13b
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 OMe
212.7 44.1 20.1 68.7 52.6 52.3 55.5 62.7 59.3 69.0 32.6 23.5 41.8 127.8 159.3 33.1 62.3 37.7 49.0 17.8 24.0 168.6
212.4 44.0 20.2 68.3 52.7 52.6 55.3 63.7 58.5 68.9 32.9 22.8 38.7 125.7 163.5 24.7 67.4 37.8 49.0 17.9 23.8 164.4
214.5 43.6 19.9 64.5 49.9 52.0 54.5 61.8 146.2 137.4 25.2d 25.1d 47.7 114.1 159.6 67.2 52.1 32.7 49.8 19.3 23.6
226.0 45.4 20.5 65.7 54.2 51.4 54.8 69.1 149.4 80.5 36.1 21.3 40.4 28.3 160.4 72.1 54.6 33.9 50.5 19.4 21.9
217.5 53.8 65.2 73.9 44.3 46.5 51.3 61.3 147.4 133.9 25.8 26.2 44.1 43.5 58.8 30.4 44.4 35.8 63.1 21.2 20.8 177.3 51.8
215.0 43.8 20.2 65.9 51.6 59.0 49.6 60.2 143.6 130.2 34.3 66.5 40.7 42.2 55.3 28.2 43.1 34.8 49.0 18.4 23.1 175.4 51.5
217.2 45.0 21.3 67.3 51.2 59.5 58.5 63.1 141.3 134.4 35.3 73.3 39.4 41.6 51.0 28.3 40.5 32.9 50.1 18.9 26.2 174.9 51.7
216.7 44.6 20.8 66.8 51.9 49.3 59.1 63.1 141.1 143.0 67.5 37.6 40.3 41.7 53.1 28.3 38.2 33.1 49.9 18.9 25.3 174.9 51.7
215.4 43.7 20.1 65.4 50.7 49.8 53.8 61.3 141.8 138.9 67.5 34.1 40.9 42.6 56.1 27.1 37.9 34.8 49.0 18.5 23.1 175.3 51.5
215.8 44.1 20.2 66.5 52.3 51.6 56.2 61.8 143.5 141.2 22.6 27.1 40.0 42.6 49.3 37.3 83.7 33.3 49.5 18.8 24.4 175.4 51.8
216.3 48.0 23.4 97.4 55.5 49.4 54.4 63.6 149.9 155.2 25.9 24.4 46.8 115.5 172.9 26.4 43.4 34.3 51.8 18.3 20.1 168.2 51.7
214.2 43.9 20.2 64.4 49.4 51.6 53.9 61.3 151.0 151.9 22.2 25.2 44.8 117.6 164.3 36.3 82.7 33.2 49.7 19.1 23.2 166.1 51.5
212.3 44.8 19.4 66.8 52.1 51.2 52.8 62.1 91.0 49.6 18.3 20.8 32.1 24.6 183.9 122.0 212.1 36.3 48.4 18.0 22.5
Measured in C5D5N. bMeasured in CDCl3. cMeasured in CD3OD.
proton resonances that were not resolved in the 1H NMR spectrum were attributable to the presence of three hydroxy
groups, as also supported by HSQC experiment. Further acquisition of 2D NMR 1H−1H COSY and HMBC data B
DOI: 10.1021/acs.jnatprod.5b00741 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
NMR 1H−1H COSY and HMBC data (Figure SI, Supporting Information) confirmed the architecture of 2 as shown, particularly from the diagnostic HMBC correlations from H217 to C-22. The relative configuration of 2 (himalenine B) was consistent with that of 1 by comparing their proton−proton couplings and analyzing the NOESY spectrum (Figure S15, Supporting Information). Alkaloid 3 was assigned a molecular formula of C21H27NO2 from its NMR data and by (+)-HRESIMS analysis at m/z 326.2116 ([M + H]+, calcd 326.2120). The UV absorption maximum at 246 (log ε 3.92) nm supported the presence of a substituted diene group.6 The NMR data of 3 (Tables 1 and 2) exhibited typical signals similar to those observed previously for rings A−D in calyciphylline A-type alkaloids,1 such as the C-1 (δC 214.5) carbonyl and the C-20 (δC 19.3) and C-21 (δC 23.6) methyl signals. The main differences between 3 and daphnilongeranin B7 were attributed to rings E and F, as supported by 2D NMR data (1H−1H COSY and HMBC, Figure SII, Supporting Information). The COSY correlations of H2-13/H-14, together with the HMBC correlations from H2-13 to C-9 and C-15 and from H2-17 to C-9, C-10, and C-15, confirmed the location of the diene moiety across rings E and F, while the correlations of H-16/H2-17 and the chemical shift for C-16 (δC 67.2) showed the presence of an OH-16 substituent. Assignment of the relative configuration of 3 was accomplished by inspection of its ROESY data (Figure SII, Supporting Information). All the other chiral centers had configurations the same as their counterparts in daphnilongeranin B,7 while the OH-16 group was determined to possess a βorientation based on the ROESY correlations of H-11β/H-17β and H-17α/H-16. Alkaloid 3 (himalenine C) was thus characterized structurally as shown. Alkaloid 4 gave a molecular formula of C21H29NO3 as deduced from the (+)-HRESIMS ion at m/z 344.2232 ([M + H]+, calcd 344.2226), indicative of being a dihydro analogue of daphnipaxianine A.8 Analysis of the NMR data (Tables 1 and 2) of 4 confirmed this hypothesis with a diagnostic resonance for the C-16 carbonyl (δC 221.2) in daphnipaxianine A being replaced by those for an oxymethine group (δH 4.87; δC 72.1) in 4. The above-mentioned assignment was corroborated by examination of the 1H−1H COSY and HMBC data (Figure SIII, Supporting Information), in which H2-17 showed correlations with the downfield H-16 signal. The relative configuration of 4 was established by analyzing the NOESY data (Figure SIII, Supporting Information), and the configurations at C-2, C-4, C-5, C-6, C-8, and C-18 in 4 were consistent with those of daphnipaxianine A.8 The NOESY correlation of H-12β/H-17β suggested that CH2-12 and CH217 are close to each other above the molecular plane, supportive of an OH-10α substituent, while the correlation of H-17α/H-16 indicated a β-orientation for the OH-16 group. Alkaloid 4 (himalenine D) was thereby elucidated as shown. (+)-HRESIMS analysis of alkaloid 5 (3β-hydroxydaphniyunnine A) revealed a protonated molecular ion at m/z 386.2327 (calcd 386.2331), in accordance with a molecular formula of C23H31NO4 and indicative of this compound being an oxygenated congener of daphniyunnine A.9 Analysis of the NMR data (Tables 1 and 2) of 5 confirmed this assumption and revealed the presence of an oxymethine group (δH 3.28; δC 65.2) in 5 replacing a methylene in daphniyunnine A.9 When compared to daphniyunnine A, the downfield-shifted signals for both the CH-2 and CH-4 groups of 5, owing to an adjacent electron-withdrawing effect, along with the key 1H−1H COSY
(Figure 1) facilitated the construction of the planar structure of 1 with a 10,17-secocalyciphylline A-type backbone.1 Key
Figure 1. Key 2D NMR correlations for 1.
HMBC correlations included those from H2-13 to C-1, C-8, C-14, and C-22 and from H-9 to C-8, C-13, C-14, C-15, and C16, which confirmed the location of the conjugated carboxylic acid and the attachment of the hydroxyethyl group to C-15. The presence of a hydroxy group at C-10 was supported by the chemical shifts for CH-10 (δH 4.73; δC 69.0) and the COSY correlations of H-10 with H-9 and H2-11. The relative configuration of 1 was assigned by examination of its NOESY data (Figure 1). The configurations at C-2, C-4, C-5, C-6, C-8, and C-18 were consistent with those of other calyciphylline Atype alkaloids1 based on similar key NOESY signals, while both H-9 and H-10 were determined to be α-oriented by the diagnostic correlations of H-9 with H-7α, H-12α, and H-10. The structure and relative configuration of 1 was thus characterized as shown, and this compound has been named himalenine A. Alkaloid 2 displayed a quasimolecular ion peak at m/z 372.2180 ([M + H]+, calcd 372.2175) in the (+)-HRESIMS analysis, corresponding to a molecular formula of C22H29NO4 and suggestive of being a dehydrated analogue of 1. The NMR data (Tables 1 and 2) of 2 were closely comparable to those of 1, and the major differences occurred in the chemical shifts for CH2-13 to CH2-17 and C-22. Considering the molecular composition and the aforementioned discussion, alkaloid 2 could be regarded as a lactonization product of 1 where the C17 hydroxy group is dehydrated with the C-22 carboxylic acid to form a new δ-lactone unit. Further examination of the 2D C
DOI: 10.1021/acs.jnatprod.5b00741 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
correlations (Figure 2) of H-3 with H-2 and H-4, were consistent with a hydroxy group substitution at C-3. Further
inspection of the HMBC data (Figure 2) supported the abovementioned assignment, so 5 was identified as 3-hydroxydaphniyunnine A. The C-3 configuration was established as shown via the diagnostic ROESY correlation of H-3/H-19β (Figure 2), while configurations at the other stereocenters were consistent with those of daphniyunnine A9 based on the ROESY data and comparison of the NMR data. Alkaloids 6 and 7 were assigned the same molecular formula of C23H31NO4 by (+)-HRESIMS analyses at m/z 386.2332 and 386.2325 (calcd 386.2331), respectively, isomeric with 5. Analysis of the NMR data (Tables 2 and 3) of 6 and 7 suggested them to be regioisomers of 5. Examination of the 2D NMR data (Figure SIV, Supporting Information) confirmed that both alkaloids possess an identical planar structure with an OH-12 substituent, as diagnosed by the key 1H−1H COSY correlation of H-6/H-12 and the chemical shifts for the CH-12 group (6: δH 4.05 and δC 66.5; 7: δH 3.70 and δC 73.3). The relative configurations of 6 and 7 were determined on the basis of the ROESY data (Figure SIV, Supporting Information) and comparison of their NMR data with those of daphniyunnine A.6 The configurations at all chiral centers except C-12 of 6 and 7 were in accordance with those of daphniyunnine A, while the OH-12 group in the two alkaloids was determined to be α- and β-positioned, respectively, via the ROESY correlations of H321/H-12 (for 6) and H-7α/H-12 (for 7). The ROESY correlations of H3-21/H-12 for 6 and H3-21/H-11β for 7 indicated that the seven-membered ring D adopted different conformations in the two compounds. Alkaloids 6 and 7 were thus identified as 12α- and 12β-hydroxydaphniyunnine A, respectively. The (+)-HRESIMS data of alkaloids 8 and 9 showed quasimolecular ion peaks at m/z 386.2322 and 386.2331, respectively, corresponding to the same molecular formula of
Figure 2. Key 2D NMR correlations for 5.
Table 3. 1H NMR Spectroscopic Data of Alkaloids 6−10 in CDCl3 position 2 3a 3b 4 6 7α 7β 11α 11β 12α 12β 13α 13β 14 15 16α 16β 17α 17β 18 19α 19β 20 21 OMe
6 2.09, 2.01, 1.93, 3.29, 2.41, 2.80, 3.07, 2.29, 2.36,
brd (4.4) brdd (14.7, 4.4) m brd (4.9) m m dd (8.4, 6.7) m m
4.05, 2.16, 2.88, 2.77, 3.47, 1.91, 1.25, 2.82, 2.40, 2.91, 2.84, 2.48, 0.97, 1.38, 3.64,
ddd (10.3, 3.7, 3.7) dd (14.5, 8.7) dd (14.5, 5.0) ddd (10.5, 8.7, 5.0) m m m m m m dd (14.2, 5.9) dd (14.2, 9.7) d (6.8) s s
7 2.04, 2.18, 1.99, 3.37, 2.23, 2.65, 3.30, 2.16, 2.47, 3.70,
brd (4.5) brdd (15.0, 4.5) brdd (15.0, 4.6) brd (4.6) ddd (11.5, 7.6, 7.6) dd (11.5, 9.1) dd (9.1, 7.6) brd (16.5) m ddd (10.9, 8.3, 2.5)
2.30, 2.54, 2.72, 3.45, 1.96, 1.27, 2.66, 2.33, 2.85, 2.73, 2.46, 0.97, 1.35, 3.64,
dd (13.5, 7.6) dd (13.5, 9.5) m m m m m brdd (15.8, 9.0) m dd (14.2, 7.7) dd (14.2, 9.9) d (6.8) s s
8 2.11, 2.11, 2.01, 3.36, 2.33, 2.70, 3.08,
m m m brs m dd (11.1, 9.5) dd (9.5, 7.1)
4.26, 1.85, 2.09, 2.29, 2.75, 2.79, 3.50, 1.97, 1.32, 2.91, 2.50, 2.82, 2.78, 2.49, 1.00, 1.29, 3.65,
brd (8.6) ddd (13.8, 9.4, 8.6) ddd (13.8, 5.9, 2.5) dd (12.9, 7.1) m m m m m m m m m m d (6.6) s s
D
9 2.11, 1.98, 1.94, 3.27, 2.33, 2.71, 2.96, 4.30,
brs m m brs m dd (11.8, 8.6) dd (8.6, 6.2) m
2.05, m 2.00, m 2.16, dd (14.6, 8.8) 2.97, dd (14.6, 4.5) 2.81, m 3.47, m 1.89 m 1.19, m 2.71, m 2.82, m 2.84, m 2.80, m 2.49, dd (13.5, 8.7) 0.98, d (6.2) 1.36, s 3.65, s
10 2.12, 2.05, 1.97, 3.36, 2.32, 2.68, 2.99, 2.29, 2.08, 1.72, 1.94, 2.21, 2.75, 2.81, 3.08, 2.49, 1.21, 4.77,
brd (4.3) brdd (15.2, 4.3) brdd (15.2, 4.7) brd (4.7) m dd (11.6, 9.4) dd (9.4, 6.5) m m m m dd (14.3, 8.6) dd (14.3, 4.4) m m m m dd (6.3, 6.3)
2.83, 2.81, 2.52, 0.99, 1.31, 3.65,
m m dd (12.0, 8.9) d (6.4) s s
DOI: 10.1021/acs.jnatprod.5b00741 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 4. 1H NMR Spectroscopic Data of Alkaloids 11−13
C23H31NO4 (calcd 386.2331), suggestive of being isomeric analogues of 5. By careful examination of the NMR data (Tables 2 and 3) of 8 and 9, they were assigned as additional regioisomers of 5−7 with different types of hydroxy group substitution. Further analysis of the 2D NMR data (Figure SV, Supporting Information) confirmed the epimeric nature at C11 of 8 and 9 with both showing key 1H−1H COSY correlations of H-11/H2-12 and diagnostic signals for the CH-11 groups (8: δH 4.26 and δC 67.5; 9: δH 4.30 and δC 67.5). While retaining identical configurations with the corresponding stereocenters of 5, the OH-11 groups of 8 and 9 were established as being α- and β-oriented, respectively, via the key ROESY correlations (Figure SV, Supporting Information) of H3-21/H-11 (for 8) and H-7α/H-11 (for 9). Based on the ROESY data, it was interesting to note that the sevenmembered ring D in 6−9 adopted suitable conformations to keep the hydroxy group in a pseudoequatorial position. Alkaloids 8 and 9 were thus elucidated as 11α- and 11βhydroxydaphniyunnine A, respectively. Alkaloid 10 was assigned a molecular formula of C23H31NO4 based on the (+)-HRESIMS ion at m/z 386.2320 ([M + H]+, calcd 386.2331), indicative of being an isomeric analogue of 5. Analysis of the NMR data (Tables 2 and 3) of 10 revealed that it is also a regioisomer of 5 and the hydroxy group was located at C-17 (δH 4.77 and δC 83.7). Subsequent inspection of the 2D NMR data (Figure SVI, Supporting Information), particularly the 1H−1H COSY correlations of H2-16/H-17, further corroborated this assignment. The OH-17 group was determined to be β-directed via observation of the ROESY correlations of H-15/H-16α and H-16α/H-17 (Figure SVI, Supporting Information), while the configurations at the other stereocenters were consistent with those of 5. Alkaloid 10 was hence characterized as 17β-hydroxydaphniyunnine A. Alkaloids 11 and 12 gave the same molecular formula of C23H29NO4 as deduced from the (+)-HRESIMS ions at m/z 384.2174 and 384.2169 ([M + H]+, calcd 384.2175), respectively, suggestive of being isomeric congeners of daphlongamine F.10 By careful examination of the NMR data (Tables 2 and 4), alkaloids 11 and 12 were identified to be a regioisomer and an epimer of daphlongamine F, respectively. Analysis of the 2D NMR 1H−1H COSY and HMBC data (Figures SVII and SVIII, Supporting Information) confirmed that the hydroxy group of 11 is located at C-4 and alkaloid 12 is the 17-epimer of daphlongamine F.10 The OH-4 group of 11 was determined to be β-oriented, as deduced from the significantly upfield shifted C-21 signal (γ-gauche effect), when compared to other analogues without C-4 substituents, while the OH-17α assignment of 12 was supported by the ROESY correlation of H-11β/H-17. The configurations at the other chiral centers of the two alkaloids were considered to be the same as those of daphlongamine F10 based on favorable NMR spectroscopic comparisons and analysis of the ROESY data (Figures SVII and SVIII, Supporting Information). Alkaloids 11 and 12 were thereby determined, in turn, as 4βhydroxylongistylumphylline A and 17-epidaphlongamine F. Alkaloid 13 showed a protonated molecular ion peak at m/z 342.2068 ([M + H]+, calcd 342.2069) by (+)-HRESIMS analysis, indicative of a molecular formula of C21H27NO3, isomeric with daphniyunnine D.9 Analysis of the NMR data (Tables 2 and 4) of 13 revealed functionalities similar to those in daphniyunnine D, such as the diagnostic resonances for two methyls (δC 18.0 and 22.5), an oxygenated sp3 quaternary carbon (δC 91.0), a double bond (δC 122.0 and 183.9), and two
position 2 3a
11a 2.34, dd (2.9, 2.9) 2.10, d (2.9, 2H)
3b 4 6 7α 7β 10 11α 11β 12α 12β 13α 13β 14 16 17 18 19α 19β 20 21 OMe a
12b
13b
2.18, m 2.04, m 2.00, m
2.49, m 2.91, m (2H)
2.32, m 2.01, ddd (16.3, 3.7, 3.7) 1.80, m (2H) 2.92, brd (17.2) 3.63, brd (17.2)
a 2.69, m b 2.62, m a 2.96, m b 2.92, m 2.74, m 3.04, dd (14.6, 7.9) 2.60, dd (14.6, 9.7) 0.99, d (6.8) 1.14, s 3.68, s
3.35, m 2.33, m 2.88, dd (12.2, 9.0) 2.84, m 2.21, m 2.29, m 1.89, 1.77, 2.84, 3.45,
m m m brd (17.0)
α 2.53, m β 3.14, m 5.14, brd (6.5) 2.78, 2.81, 2.53, 0.98, 1.20, 3.69,
m m m d (6.4) s s
1.96, m 2.01, brdd (14.2, 4.5) 1.89, brdd (14.2, 5.8) 3.06, brd (5.8) 2.27, m 2.74, dd (11.6, 8.7) 2.94, dd (8.7, 7.2) 3.43, dd (9.8, 9.8) 1.78, m (2H)
1.74, m 2.65, m 1.95, m 2.80, m α 2.38, m β 2.81, m 5.71, brs
2.90, 2.80, 2.41, 0.97, 1.36,
m m dd (13.4, 9.5) d (6.8) s
Measured in CD3OD. bMeasured in CDCl3.
carbonyls (δC 212.1 and 212.3). The main differences between 13 and daphniyunnine D9 were attributable to locations of the double bond and the hydroxy group as supported by the 2D NMR data (1H−1H COSY and HMBC, Figure 3). Diagnostic HMBC correlations from H-10 to C-9 and C-17, from H2-13 to C-8 and C-9, and from H-16 to C-9, C-14, C-15, and C-17 were used to establish rings E and F as depicted. The relative configuration of 13 was basically established via the NOESY data (Figure 3). The configurations at C-2, C-4, C-5, C-6, C-13, and C-18 were assigned as being the same as those of daphniyunnine D,9 based on their similar NOESY correlations of H3-21/H-3a, H-4, H-6, and H-13β, H-3b/H-19β, and H19β/H3-20. Subsequent observation of the strong correlation between H-7α and H-10 suggested an α-orientation for H-10. The OH-9 functionality was also determined to be α-positioned via the significantly downfield shifted H-10 signal (ΔδH −0.34) in C5D5N versus that in CDCl3, which indicated a small dihedral angle between H-10 and OH-9.11 The structure of alkaloid 13 (himalenine E) was thus assigned as shown. The inhibitory effects of these alkaloids against four kinases, PTP1B, aurora A, HDAC6, and IKK-β, were evaluated in vitro at an initial concentration of 20 μg/mL (Tables S2−S5, Supporting Information). While compounds 12 and 13 showed around 40% inhibition against PTP1B, only 11 exhibited a >30% inhibitory rate against aurora A. All the tested alkaloids were inactive (
