Hua Zhang‡
a,
Chuan-Rui Zhang‡a,
Ying-Shan Hanb,
Mark A. Wainbergb and
Jian-Min Yue*a
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, P. R. China. E-mail: jmyue@simm.ac.cn; Tel: +86-21-5080-6718
bMcGill University Aids Centre, The Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada
First published on 3rd December 2015
Chemical fractionation of the ethanolic extract of Flueggea virosa yielded a group of Securinega alkaloids including flueggenines E (1) and F (2) as novel hybrid structures, flueggenines G–I (3–5) as new dimers and fluevirosines E–H (6–9) as new trimers, along with six known biosynthetically related compounds. The diverse structures of these isolates were characterized via comprehensive spectroscopic analyses and comparison with literature data. Compounds 1 and 2 are rare Securinega alkaloid hybrids incorporating tryptamine and piperidine residues, respectively, while 3 represents the first example bearing a securinine-type monomeric unit among Securinega alkaloid dimers. An in vitro anti-HIV screening of all available alkaloids revealed weak to moderate activities for over half of these isolates with EC50 values ranging from 7.8 to 122 μM. Among the tested compounds, the known dimer flueggenine D exhibited the best activity with an EC50 of 7.8 ± 0.8 μM.
Our continuous study of the alkaloids from F. virosa collected at different sites since 2006 has led to the identification of 21 new structures from monomer to pentamer.4–8 It is worthwhile to note that the previous discovery of higher level (n > 2) oligomeric alkaloids was based on a biogenetic proposal6 and relied on a MS-guided separation strategy.7,8 In addition to the above-intended search for routine oligomers, we also noticed some irregular MS peaks indicative of the likely presence of new types of alkaloid oligomers. Further fractionation of the remaining fractions returned two new alkaloid hybrids, flueggenines E (1) and F (2), three new dimers, flueggenines G–I (3–5), and four new trimers, fluevirosines E–H (6–9) (Fig. 1), as well as a known dimer and five known monomers. The new compounds were characterized by spectroscopic means and NMR comparison with known alkaloids. Both new and known compounds were tested in vitro for their anti-HIV effects with more than half showing weak to mild activities. A detailed account of the isolation, structure elucidation and anti-HIV evaluation of these Securinega alkaloids is presented below.
No. | 1a | 2a | 3a | 4b | 5a |
---|---|---|---|---|---|
a Measured in CDCl3.b Measured in CDCl3 with 5% CD3OD.c Interchangeable assignments.d Interchangeable assignments.e Interchangeable assignments.f Interchangeable assignments.g Interchangeable assignments. | |||||
2 | 66.8 | 62.8 | 63.2 | 65.1 | 66.2 |
3 | 29.3 | 25.9 | 27.0 | 29.3 | 29.26f |
4 | 26.8 | 24.8 | 24.3 | 26.82c | 26.8g |
5 | 57.4 | 26.8 | 25.6 | 57.8d | 57.3 |
6 | 52.62 | 48.9 | |||
7 | 63.4 | 57.8 | 58.5 | 64.1 | 63.4 |
8 | 33.8 | 66.7 | 42.2 | 31.1 | 30.4 |
9 | 92.4 | 36.5 | 89.7 | 92.6 | 90.6 |
10 | 84.8 | ||||
11 | 173.5 | 173.2 | 172.62e | 173.0 | |
12 | 110.3 | 173.4 | 103.6 | 114.7 | 122.9 |
13 | 173.7 | 117.4 | 171.8 | 172.8 | 165.4 |
14 | 27.4 | 173.6 | 134.7 | 44.2 | 28.1 |
15 | 64.7 | 22.7 | 135.4 | 81.5 | 78.7 |
2′ | 121.6 | 52.60 | 67.0 | 65.8 | 66.9 |
3′ | 114.5 | 28.3 | 29.0 | 29.0 | 29.25f |
4′ | 127.6 | 23.2 | 26.6 | 26.76c | 26.9g |
5′ | 118.9 | 23.5 | 57.5 | 57.9d | 57.7 |
6′ | 119.5 | 46.0 | |||
7′ | 122.3 | 65.0 | 64.8 | 64.6 | |
8′ | 111.4 | 35.8 | 30.5 | 30.0 | |
9′ | 136.4 | 91.9 | 91.6 | 92.5 | |
10′ | 22.5 | ||||
11′ | 55.4 | 172.6 | 172.4 | 172.8 | |
12′ | 110.2 | 111.7 | 110.7 | ||
13′ | 172.8 | 172.60e | 174.7 | ||
14′ | 29.1 | 27.2 | 26.3 | ||
15′ | 42.9 | 41.5 | 39.2 | ||
OMe | 56.6 | 57.1 | |||
NMe | 39.5 |
No. | 1a | 2a | 3a | 4b | 5a |
---|---|---|---|---|---|
a Measured in CDCl3.b Measured in CDCl3 with 5% CD3OD. | |||||
2 | 3.12 (dd, 8.9, 7.0) | 2.18 (br d, 10.5) | 2.04 (dd, 11.3, 2.6) | 2.96 (dd, 8.8, 7.0) | 3.05 (dd, 8.9, 6.9) |
3 | 1.88 (m) | β 1.60 (m) | 1.64 (m) | 1.87 (m) | 1.85–1.94 (m) |
1.73 (m) | α 1.38 (m) | 1.54 (m) | 1.77 (m) | 1.71–1.80 (m) | |
4 | 1.93 (m) | 1.86 (m) | 1.89 (m) | 1.95 (m) | 1.90–1.98 (m) |
1.73 (m) | 1.26 (m) | 1.22 (m) | 1.68 (m) | 1.66–1.76 (m) | |
5 | 3.35 (m) | 1.57 (2H, m) | 1.62 (m) | 3.25–3.32 (m) | 3.31 (m) |
2.58 (m) | 1.59 (m) | b 2.52–2.63 (m) | 2.62 (m) | ||
6 | α 2.80 (m) | 2.98 (m) | |||
β 2.63 (m) | 2.41 (m) | ||||
7 | 3.27 (br d, 6.4) | 2.94 (m) | 3.85 (dd, 5.6, 4.2) | 3.21 (dd, 5.6, 3.9) | 3.13 (m) |
8 | a 2.50 (dd, 11.2, 6.4) | 4.13 (dd, 8.7, 5.0) | 2.50 (dd, 9.1, 4.2) | a 2.32 (dd, 11.1, 5.6) | a 2.27 (dd, 11.1, 5.6) |
b 1.29 (d, 11.2) | 1.72 (d, 9.1) | b 1.75 (d, 11.1) | b 1.70 (d, 11.1) | ||
9 | a 2.82 (dd, 13.4, 8.7) | ||||
b 1.19 (d, 13.4) | |||||
12 | 5.63 (d, 2.3) | 5.56 (s) | 5.72 (br s) | ||
14 | α 3.03 (m) | 2.61 (d, 12.8) | 2.93 (d, 16.4) | ||
β 2.69 (ddd, 14.7, 11.1, 2.3) | 2.66 (ddd, 16.4, 5.3, 1.7) | ||||
15 | 2.62 (ddd, 11.1, 5.3, 1.5) | α 3.22 (dd, 19.4, 2.4) | 6.67 (d, 5.6) | 3.48 (d, 3.9) | 3.57 (dd, 5.3, 4.1) |
β 2.76 (dd, 19.4, 2.0) | |||||
1′ | 8.01 (br s) | ||||
2′ | 7.03 (d, 2.2) | 3.86 (dd, 12.3, 3.1) | 3.10 (dd, 8.8, 7.0) | 3.15 (dd, 9.0, 7.0) | 3.20 (dd, 8.8, 6.8) |
3′ | 2.02 (m) | 1.93 (m) | 1.91 (m) | 1.85–1.94 (m) | |
1.87 (m) | 1.79 (m) | 1.75 (m) | 1.71–1.80 (m) | ||
4′ | 1.98 (m) | 1.96 (m) | 1.93 (m) | 1.90–1.98 (m) | |
1.55 (m) | 1.60 (m) | 1.66 (m) | 1.66–1.76 (m) | ||
5′ | 7.60 (br d, 7.9) | 1.83 (m) | 3.22 (m) | 3.25–3.32 (m) | 3.39 (m) |
1.71 (m) | 2.59 (m) | 2.52–2.63 (m) | 2.64 (m) | ||
6′ | 7.12 (ddd, 7.9, 7.2, 0.9) | 3.39 (br d, 12.9) | |||
2.92 (ddd, 12.9, 12.3, 3.2) | |||||
7′ | 7.20 (ddd, 8.2, 7.2, 1.1) | 3.01 (m) | 2.80 (m) | 3.17 (m) | |
8′ | 7.37 (br d, 8.2) | a 2.61 (m) | a 2.42 (dd, 11.3, 5.6) | 2.29 (dd, 11.6, 5.8) | |
b 1.49 (d, 11.0) | b 1.56 (d, 11.3) | 1.54 (d, 11.6) | |||
10′ | 2.95 (2H, m) | ||||
11′ | 3.01 (m), 2.97 (m) | ||||
12′ | 5.67 (d, 2.1) | 5.72 (br s) | 5.74 (d, 2.4) | ||
14′ | 2.96 (m) | 2.91 (m, 2H) | 3.11 (ddd, 17.3, 9.6, 2.4) | ||
2.67 (m) | 2.91 (d, 17.3) | ||||
15′ | 2.53 (m) | 2.76 (m) | 3.36 (br d, 9.6) | ||
NMe | 2.56 (s) | ||||
OMe | 3.30 (s) | 3.20 (s) |
Flueggenine F (2) was assigned a molecular formula of C18H26N2O3 based on the (+)-HRESIMS ion at m/z 319.2029 ([M + H]+, calcd 319.2022) indicative of an isomer of secu'amamine F.11 Analysis of the NMR data (Tables 1 and 2) for 2 confirmed this hypothesis with characteristic signals for a neosecurinane substructure and a 2-substituted piperidine moiety. Further examination of 2D NMR data (Fig. 3) established the planar structure of 2 to be identical with that of secu'amamine F, and the critical HMBC correlations from H-2′ (δH 2.56) to C-12 (δC 173.4) and C-13 (δC 117.4) corroborated the linkage between C-2′ and C-13. The relative configuration of the neosecurinane part in 2 was considered to be the same as that of virosine B12 via their highly similar NMR data (particularly the comparable proton couplings) at the corresponding stereocenters (C-2, C-7 and C-8). The ROESY correlations (Fig. 3) of H-2/H-15β, H-3α/H-9a and H-9a/H-8 also supported this assignment. Although the relative configuration at C-2′ remained unassigned due to its distance from the core structure, it was apparent from the coupling patterns (J = 12.3, 3.1 Hz) that H-2′ was axially oriented. The absolute configuration of the neosecurinane part in 2 was determined as shown on the basis of its CD data with Cotton effects at 283 (0.3) and 250 (−0.7) nm.12
The molecular formula of C25H28N2O4 for flueggenine G (3) was established by HREIMS analysis at m/z 420.2049 (Δmmu 0). The NMR data (Tables 1 and 2) for 3 revealed diagnostic resonances for two γ-carbons (δC 91.9 and 89.7) of the lactones in securinine-/norsecurinine-type alkaloids, suggestive of its dimeric nature. Analysis of 2D NMR data (Fig. 4) established one securinine-type fragment and one dihydronorsecurinine unit that were connected from C-15′ to C-14 via the key HMBC correlations of H-15′/C-13 & C-14. The relative configuration of 3 was assigned on the basis of ROESY data (ESI Fig. S24†) and NMR comparison (ESI Table S1†) with virosecurinine13 and flueggenine A.6 Excellent comparisons between the resonances of monomeric unit A and virosecurinine, and those of monomeric unit B and the dihydronorsecurinine moiety of flueggenine A, supported common configurations at all corresponding chiral centers. In addition, the configuration at the C-15′ stereocenter in 3 was also confirmed by the ROESY correlation of H-15′ (δH 2.53) with H-8′b (δH 1.49). As with the other alkaloid oligomers from the same species,6 alkaloid 3 is likely to be biosynthesized from virosecurinine and (−)-norsecurinine, and thus retains the absolute configurations from the two monomeric precursors. Meanwhile, the CD spectrum (Fig. 5) of 3 showed Cotton effects arising from the α,β,γ,δ-conjugated lactone chromophore comparable to that of virosecurinine, confirmative of the above-mentioned biogenetic correlation. Alkaloid 3 was thereby elucidated to be the first dimeric example derived from a securinine-type monomeric unit.
Flueggenines H (4) and I (5) gave the same molecular formula of C25H30N2O5 as determined from the (+)-HRESIMS ions at m/z 439.2230 and 439.2218 ([M + H]+, calcd 439.2233), respectively. Analysis of the NMR data (Tables 1 and 2) for 4 indicated a MeOH adduct of flueggenine D7 with diagnostic signals for two sp3 methines (δC 44.2 & 81.5; δH 2.61 & 3.48) and a methoxyl group (δC 56.6; δH 3.30) replacing those for Δ14 in the latter. The acquisition of 2D NMR data (Fig. 6) facilitated the aforementioned structural assignment revealing key 1H–1H COSY correlations of H-14/H-15′ and HMBC correlation from the methoxyl protons to C-15 (δC 44.2). The relative configurations at the chiral centers of C-14 and C-15 as shown were assigned via the diagnostic coupling pattern of H-14/H-15 (J14,15 = 0 Hz)8 and the ROESY crosspeaks of OC3/H-8b and H-14/H-8′b. The relative stereostructure of 4 was hence characterized. As with the case of 4 and flueggenine D, the NMR data (Tables 1 and 2) for 5 also suggested it to be a MeOH adduct at Δ14 of flueggenine C,7 and this was supported by the absence of the olefinic resonances and the presence of those for two sp3 methines and one methoxyl group. Examination of 2D NMR data (ESI Fig. Sa†) further confirmed this structural assignment. The relative configuration at the new C-15 stereocenter was suggested by the ROESY correlation of OC
3/H-8b, while the configurations at the other stereocenters were consistent with their counterparts in flueggenine C7 based on excellent NMR comparisons and ROESY data (ESI Fig. S42†).
The molecular formula of C37H44N3O7 for 6 was determined through 13C NMR data and the (+)-HRESIMS ion at m/z 642.3173 ([M + H]+, calcd 642.3179). Analysis of the NMR data (Tables 3 and 4) for 6 established that it was a trimer derived from 5 by the addition of a dihydronorsecurinine unit at C-12′. Indeed, the absence of the H-12′ signal and the presence of those for a dihydronorsecurinine residue were supportive of this structural variation, which was further confirmed by 2D NMR data (ESI Fig. Sa†) with corroborative HMBC correlations from H-15′′ (δH 3.30) to C-11′ (δC 172.4), C-12′ (δC 121.2) and C-13′ (δC 168.6). The new C-15′′ chiral center was considered to possess identical relative configuration with C-15′ based on the shielded C-8′′ signal (δC 30.2),6,7 while those at all other stereocenters were assigned as drawn via NMR comparison with 5 and analysis of ROESY data (ESI Fig. S51†). The structure of 6 was hence elucidated and was named fluevirosine E after the known trimers fluevirosines A–D.6,7
No. | 6 | 7 | 8 | 9 |
---|---|---|---|---|
a Interchangeable assignments.b Interchangeable assignments.c Interchangeable assignments.d Interchangeable assignments.e Interchangeable assignments.f Interchangeable assignments.g Interchangeable assignments.h Interchangeable assignments.i Interchangeable assignments.j Interchangeable assignments.k Interchangeable assignments. | ||||
2 | 66.5a | 65.1 | 65.1 | 65.5j |
3 | 29.3b | 29.3e | 29.5g | 29.5 |
4 | 26.9c | 26.88f | 27.0h | 27.0k |
5 | 57.55d | 54.8 | 55.2 | 55.3 |
7 | 63.3 | 58.8 | 59.5 | 59.4 |
8 | 30.8 | 36.4 | 36.3 | 35.7 |
9 | 90.4 | 91.4 | 92.3 | 92.2 |
11 | 173.4 | 172.1 | 172.34i | 172.1 |
12 | 122.6 | 120.6 | 107.4 | 107.0 |
13 | 166.8 | 162.9 | 169.3 | 169.4 |
14 | 27.9 | 132.8 | 133.2 | 134.9 |
15 | 78.6 | 139.9 | 139.2 | 141.1 |
2′ | 67.1 | 66.5 | 67.5 | 65.1 |
3′ | 29.21b | 29.22e | 29.4g | 29.2 |
4′ | 26.84c | 26.88f | 26.93h | 26.8k |
5′ | 57.52d | 57.8 | 57.8 | 58.1 |
7′ | 66.0 | 67.5 | 64.3 | 65.4j |
8′ | 30.4 | 30.4 | 35.8 | 31.5 |
9′ | 90.4 | 91.8 | 90.7 | 92.9 |
11′ | 172.4 | 172.8 | 173.9 | 171.8 |
12′ | 121.2 | 109.8 | 122.3 | 113.9 |
13′ | 168.6 | 173.5 | 166.1 | 173.2 |
14′ | 25.1 | 26.76f | 26.9h | 42.9 |
15′ | 37.9 | 38.8 | 43.1 | 47.7 |
2′′ | 66.4a | 67.1 | 67.2 | 66.0 |
3′′ | 29.16b | 29.18e | 29.2g | 29.1 |
4′′ | 26.80c | 26.86f | 26.7h | 26.8k |
5′′ | 57.46d | 58.1 | 57.5 | 57.9 |
7′′ | 65.5 | 65.7 | 65.2 | 64.8 |
8′′ | 30.2 | 36.0 | 35.7 | 30.2 |
9′′ | 92.1 | 91.7 | 91.9 | 91.6 |
11′′ | 172.6 | 173.4 | 173.0 | 172.5 |
12′′ | 110.0 | 110.9 | 110.6 | 112.0 |
13′′ | 174.0 | 172.3 | 172.31i | 171.9 |
14′′ | 25.8 | 28.4 | 27.8 | 27.4 |
15′′ | 38.5 | 42.9 | 39.3 | 44.3 |
OMe | 57.0 |
No. | 6 | 7 | 8 | 9 |
---|---|---|---|---|
2 | 3.20 (dd, 8.9, 6.8) | 3.06 (m) | 3.15 (8.9, 7.1) | 3.07 (m) |
3 | 1.81–1.96 (m) | 1.89–1.99 (m) | 2.01 (m) | 1.99 (m) |
1.66–1.80 (m) | 1.72–1.82 (m) | 1.73–1.83 (m) | 1.70–1.83 (m) | |
4 | 1.90–2.02 (m) | 1.92–2.01 (m) | 1.92–2.03 (m) | 1.92–2.02 (m) |
1.64–1.78 (m) | 1.68–1.79 (m) | 1.63–1.77 (m) | 1.66–1.79 (m) | |
5 | 3.28–3.40 (m) | 3.26 (m) | 3.32 (m) | 3.22 (m) |
2.55–2.69 (m) | 2.52 (m) | 2.54 (m) | 2.49 (m) | |
7 | 3.17 (m) | 3.66 (dd, 6.5, 4.5) | 3.70 (dd, 6.5, 4.5) | 3.61 (dd, 6.5, 4.6) |
8 | 2.28 (m) | 2.54 (dd, 10.6, 4.5) | 2.60 (dd, 10.5, 4.5) | 2.54 (dd, 10.6, 4.6) |
1.72 (d, 11.0) | 1.64 (d, 10.6) | 1.74 (d, 10.5) | 1.57 (d, 10.6) | |
12 | 5.72 (s) | 5.82 (s) | ||
14 | 2.76 (d, 16.6) | |||
2.70 (dd, 16.6, 5.1) | ||||
15 | 3.61 (dd, 5.1, 4.2) | 6.92 (d, 6.5) | 6.73 (d, 6.5) | 6.26 (d, 6.5) |
2′ | 3.15 (m) | 3.17 (dd, 8.9, 6.9) | 3.12 (m) | 3.11 (dd, 8.9, 6.7) |
3′ | 1.81–1.96 (m) | 1.89–1.99 (m) | 1.87–1.95 (m) | 1.88–1.97 (m) |
1.66–1.80 (m) | 1.72–1.82 (m) | 1.73–1.83 (m) | 1.70–1.83 (m) | |
4′ | 1.90–2.02 (m) | 1.92–2.01 (m) | 1.92–2.03 (m) | 1.92–2.02 (m) |
1.64–1.78 (m) | 1.68–1.79 (m) | 1.63–1.77 (m) | 1.66–1.79 (m) | |
5′ | 3.28–3.40 (m) | 3.30 (m) | 3.24 (m) | 3.37 (m) |
2.55–2.69 (m) | 2.53 (m) | 2.52 (m) | 2.54 (m) | |
7′ | 2.94 (m) | 2.85 (dd, 5.5, 2.5) | 3.22 (brd, 5.9) | 2.97 (d, 5.4) |
8′ | 2.26 (dd, 11.5, 5.8) | 2.32 (dd, 11.3, 5.5) | 2.62 (dd, 11.1, 5.9) | 2.32 (dd, 11.4, 5.4) |
1.63 (d, 11.5) | 2.17 (d, 11.3) | 1.47 (d, 11.1) | 1.52 (d, 11.4) | |
12′ | 5.66 (br s) | 5.69 (s) | ||
14′ | 2.95 (m, 2H) | 2.99 (m, 2H) | 3.56 (dd, 16.0, 5.0) | 2.78 (d, 10.1) |
2.75 (dd, 16.0, 12.4) | ||||
15′ | 3.34 (m) | 3.42 (m) | 2.51 (m) | 2.98 (s) |
2′′ | 3.01 (m) | 3.14 (dd, 9.0, 7.0) | 3.10 (dd, 9.2, 6.9) | 3.15 (dd, 8.9, 6.9) |
3′′ | 1.81–1.96 (m) | 1.89–1.99 (m) | 1.87–1.95 (m) | 1.88–1.97 (m) |
1.66–1.80 (m) | 1.72–1.82 (m) | 1.73–1.83 (m) | 1.70–1.83 (m) | |
4′′ | 1.90–2.02 (m) | 1.92–2.01 (m) | 1.92–2.03 (m) | 1.92–2.02 (m) |
1.64–1.78 (m) | 1.68–1.79 (m) | 1.63–1.77 (m) | 1.66–1.79 (m) | |
5′′ | 3.28–3.40 (m) | 3.27 (m) | 3.13 (m) | 3.30 (m) |
2.55–2.69 (m) | 2.54 (m) | 2.58 (m) | 2.59 (m) | |
7′′ | 2.96 (m) | 3.00 (m) | 3.01 (m) | 2.82 (m) |
8′′ | 2.30 (dd, 11.3, 6.3) | 2.64 (dd, 10.9, 5.9) | 2.55 (dd, 11.1, 6.1) | 2.40 (dd, 11.2, 5.7) |
1.88 (d, 11.3) | 1.56 (d, 10.9) | 1.53 (d, 11.1) | 1.55 (d, 11.2) | |
12′′ | 5.67 (d, 2.1) | 5.72 (d, 2.0) | 5.66 (d, 2.0) | 5.59 (d, 1.5) |
14′′ | 3.11 (d, 16.0) | 2.86 (m) | 2.99 (dd, 14.7, 5.2) | 2.94 (m) |
3.02 (m) | 2.78 (ddd, 15.9, 12.9, 2.0) | 2.81 (ddd, 14.7, 12.1, 2.0) | 2.59 (m) | |
15′′ | 3.30 (m) | 2.90 (m) | 2.88 (m) | 2.94 (m) |
OMe | 3.26 (s) |
Fluevirosines F (7) and G (8) exhibited quasi-molecular ion peaks in the (+)-HRESIMS spectra at m/z 610.2934 and 610.2910 ([M + H]+), respectively, corresponding to the same molecular formula of C36H39N3O6 and indicative of a pair of isomers. The NMR data (Tables 3 and 4) for 7 were highly similar to those for fluevirosine D7 with the only difference being attributable to signals around C-15′′, which suggested 7 to be a likely 15′′-epimer of the latter. Analysis of 2D NMR data (ESI Fig. Sa†) for 7 confirmed that they possess the same planar structure bearing one norsecurinine and two dihydronorsecurine moieties. Compared to fluevirosine D,7 the coupling pattern of H2-14′′ with H-15′′, the chemical shift (δC 36.0) for C-8′′ and the ROESY correlation of H-8′′b/H-15′′ all supported an inverted configuration at C-15′′. The relative configurations at all other stereocenters were determined to be the same as those in fluevirosine D by their excellent NMR comparisons and analysis of ROESY data (ESI Fig. S60†). Similar to the case of 7 and fluevirosine D, alkaloid 8 was determined to be the 15′′-epimer of fluevirosine A6 by analysis of the NMR data (Tables 3 and 4). The relative configuration at C-15′′ was established as described for 7, and inspection of 2D NMR data (1H–1H COSY, HMBC and ROESY, ESI Fig. Sa & S69†) further corroborated the aforementioned assignment. The structures of 7 and 8 were thus characterized.
Fluevirosine H (9) gave a molecular formula (C36H39N3O6) same as 8 based on the (+)-HRESIMS ion at m/z 610.2924 ([M + H]+, calcd 610.2917). Compared to 8, the NMR data (Tables 3 and 4) for 9 also exhibited the presence of one norsecurinine and two dihydronorsecurinine fragments with extra signals for a olefinic proton (δH 5.69, H-12′) and a methine group (δH 2.78 & δC 42.9, CH-14′) but absence of those for a methylene and a quaternary carbon, which suggested a distinct oligomerization pattern. Further analysis of 2D NMR data (Fig. 7) confirmed the presence of the aforementioned monomeric substructures and the connections between them with a confirmatory 1H–1H COSY correlation of H-14′/H-15′′ and HMBC correlations from H-15′ (δH 2.98) to C-13 (δC 169.4), C-14 (δC 134.9), C-15 (δC 141.4) and C-15′′ (δC 44.3). The relative configurations at C-14′, C-15′ and C-15′′ were established to be identical with those at the corresponding chiral centers in fluevirosinine E8 via the same coupling patterns of H-14′, H-15′ and H-15′′, while the other stereocenters were assigned on the basis of excellent NMR comparisons with the above-described alkaloids. The absolute configurations of 7–9 were determined to be as shown by analysis of their CD spectra, which displayed Cotton effects at 270 (−10.5), 270 (−14.6) and 267 (−13.2) nm, respectively.8
In addition to the above-mentioned new alkaloids, six known ones, flueggeainol,14 bubbialine,15 (+)-14,15-dihydronorsecurine,16 14,15-epoxynorsecurinine,17 15α-methoxy-14,15-dihydronorsecurine,10 and flueggine B,18 were also obtained and identified by full spectroscopic analyses and comparison with reported data in the literature.
All available Securinega alkaloids from F. virosa, including those (virosecurinine,6 viroallosecurinine,6 (−)-norsecurinine,6 flueggenines C and D,7 and fluevirosine D7) reported previously, were tested for their in vitro anti-HIV activities on HIV-1 NL 4-3 infected MT4 cells and nevirapine was used as the positive control.19 The assay results (Table 5) revealed that more than half of these compounds showed mild protection on MT-4 cells against the HIV-induced cytopathic effect (EC50 10–100 μM) without displaying cytotoxicity (CC50 > 100 μM). The best anti-HIV activity was observed for a known dimer, flueggenine D, with an EC50 of 7.8 ± 0.8 μM (selective index = 12.6).
Compds | EC50 | CC50 |
---|---|---|
Flueggenine E (1) | 42.6 ± 4.3 | >100 |
Flueggenine F (2) | — | — |
Flueggenine G (3) | — | — |
Flueggenine H (4) | 122 ± 12 | — |
Flueggenine I (5) | 63.9 ± 6.5 | — |
Fluevirosine E (6) | — | — |
Fluevirosine F (7) | — | — |
Fluevirosine G (8) | 58.7 ± 5.6 | — |
Fluevirosine H (9) | 108 ± 10 | — |
Flueggeainol | 41.9 ± 4.2 | >100 |
Bubbialine | — | — |
14,15-Dihydronorsecurine | — | — |
14,15-Epoxynorsecurinine | 85.5 ± 8.7 | — |
15α-Methoxy-14,15-dihydronorsecurine | 89.0 ± 9.2 | — |
Flueggine B | 79.6 ± 8.1 | — |
Flueggenine C | — | — |
Flueggenine D | 7.8 ± 0.8 | 97.9 |
Fluevirosine D | — | — |
Virosecurinine | 19.3 ± 2.0 | >100 |
Viroallosecurinine | 56.4 ± 5.7 | — |
(−)-Norsecurinine | 43.0 ± 4.4 | >100 |
Nevirapine | 0.119 ± 0.012 | >100 |
A total of 28.2 g crude alkaloids were prepared from the Gongchen species through the same procedures as described formerly.7 Fractionation of the crude alkaloids with a silica gel column (petroleum ether–EtOAc–HNEt2, 5:
1
:
0.1 to 1
:
2
:
0.2) returned six major fractions F1–F6, and F4 (7.76 g) was further subjected to silica gel CC (CHCl3–MeOH, 100
:
1 to 5
:
1) to afford subfractions F4a–F4g. Subfraction F4b was sequentially separated by silica gel CC (petroleum ether–EtOAc–HNEt2, 5
:
1
:
0.1 to 1.5
:
1
:
0.1), amino silica gel CC (petroleum ether–EtOAc, 1
:
1 to 1
:
2), and preparative TLC (petroleum ether–EtOAc–HNEt2, 1.5
:
1
:
0.2) to give 14,15-epoxynorsecurinine (6.6 mg) and 15α-methoxy-14,15-dihydronorsecurine (8.5 mg). Subfraction F4d was processed with amino silica gel (CHCl3–MeOH, 50
:
1) to furnish three elutions, and 4 (1.9 mg) and (+)-14,15-dihydronorsecurine (4.5 mg) were purified from the first and third elutions by HPLC with MeCN–H2O system (20–80% and 20–40%, respectively, over 20 min). Subfraction F4e was fractionated by HPLC with MeCN–H2O system (30–45% over 20 min) to yield 5 (5.6 mg) and a mixture which was further separated by HPLC with MeOH–H2O as mobile phase (35–60% over 20 min) to give 9 (2.5 mg) and 7 (3.7 mg).
Fraction F5 (3.27 g) was first separated over silica gel (CHCl3–MeOH, 50:
1 to 5
:
1) to afford subfractions F5a–F51, and six more elutions (F5a1–F5a6) were further obtained by subsequent fractionation of F5a (2.06 g) on a silica gel column (petroleum ether–EtOAc–HNEt2, 4
:
1
:
0.1 to 2
:
1
:
0.1). Flueggeainol (1.8 mg) was obtained from F5a1 by HPLC purification (30–60% MeCN–H2O over 15 min), while bubbialine (9.6 mg) and 1 (2.3 mg) were acquired from F5a6 also by HPLC separation (MeCN–H2O, 25–30% over 15 min, to 50% in 0.5 min then to 80% over 6 min). Subfraction F5c was further fractionated by HPLC (25–60% MeCN–H2O over 20 min) to furnish 8 (1.9 mg), while subfractions F5g and F5i yielded flueggine B (1.9 mg) and 2 (2.7 mg) via HPLC purifications eluted with gradient (25–60% over 20 min) and isocratic (37%) MeCN–H2O, respectively. All solvent systems used for HPLC separations were at a flow rate of 3.5 mL min−1 and modified with 0.02% HNEt2 unless specified.
Footnotes |
† Electronic supplementary information (ESI) available: Full spectroscopic data including 1D & 2D NMR, IR and MS spectra for all new compounds. See DOI: 10.1039/c5ra22191a |
‡ These authors contributed equally. |
This journal is © The Royal Society of Chemistry 2015 |