Ngo Anh Bangab,
Duong Thi Hai Yena,
Dan Thi Thuy Hanga,
Pham Hai Yena,
Nguyen Huy Hoanga,
Do Thi Tranga,
Duong Thi Dunga,
Nguyen Thi Cuca,
Nguyen The Cuongc,
Nguyen Xuan Nhiemab,
Bui Huu Taiab and
Phan Van Kiem*ab
aInstitute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam. E-mail: phankiem@yahoo.com
bGraduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
cInstitute of Ecology and Biological Resources, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
First published on 1st July 2024
Phytochemical studies on Aphanamixis plants have attracted considerable attention over the past few decades due to the structural diversities and significant biological activities of terpenoids produced by these plants. In the present study, five new acyclic diterpene lactone derivatives, aphanamixionolides A–E (1–5), and three known tirucallane-type triterpenes, namely, piscidinol A (6), hispidone (7), and bourjotinolone A (8), were isolated from the leaves of Aphanamixis polystachya. Their structures were elucidated by comprehensive analyses of HR-ESI-MS and NMR spectroscopic data and by comparison with those reported in the literature. Absolute configurations of the new compounds were determined by experimental and TD-DFT calculated ECD spectra. Compounds 1–8 inhibited NO production with IC50 values of 10.2–37.7 μM, which are comparable to positive control L-NMMA (IC50: 31.5 μM).
(5S,11R)-Aphanamixionolide A | (1) |
White amorphous powder; mp 57.2–57.6; [α]28D: +51.6 (c 0.1, MeOH); ECD (MeOH) θ(λ nm): −5.4(215), −6.3(249) mdeg; HR-ESI-MS: m/z 391.2122 [M + H]+ (calcd for [C22H31O6]+, 391.2115); 1H- and 13C-NMR data are given in Table 1.
(5S,11R/11S)-Aphanamixionolide B | (2) |
No. | 1 | 2 | 3 | 4 | 5 | |||||
---|---|---|---|---|---|---|---|---|---|---|
δC | δH (mult., J in Hz) | δC | δH (mult., J in Hz) | δC | δH (mult., J in Hz) | δC | δH (mult., J in Hz) | δC | δH (mult., J in Hz) | |
a Observed in a pair of signals: 74.12/74.08.b Observed in a pair of signals: 122.54/122.51.c Observed in a pair of signals: 142.01/141.77.d Observed in a pair of signals: 39.35/39.27.e Observed in a pair of signals: 20.97/20.90.f Observed in a pair of signals: 40.34/40.30.g Observed in a pair of signals: 76.11/76.05.h Observed in a pair of signals: 89.64/89.61.i Observed in a pair of signals: 48.16/48.13.k Observed in a pair of signals: δC 24.36/δH 0.99 and δC 24.12/δH 0.89. | ||||||||||
1 | 165.2 | — | 165.2 | — | 165.3 | — | 173.2 | — | 173.0 | — |
2 | 116.7 | 5.82 (s) | 116.7 | 5.82 (s) | 116.7 | 5.83 (s) | 117.5 | 5.81 (s) | 117.4 | 5.81 (s) |
3 | 157.0 | — | 157.0 | — | 157.1 | — | 168.1 | — | 168.1 | — |
4 | 35.1 | 2.39 (dd, 18.0, 10.8) | 35.1 | 2.39 (dd, 18.0, 10.8) | 35.1 | 2.39 (dd, 18.0, 10.8) | 84.1 | 4.89 (t, 4.8) | 84.2 | 4.88 (t, 5.4) |
2.22 (dd, 18.0, 3.6) | 2.22 (dd, 18.0, 3.6) | 2.20 (dd, 18.0, 3.6) | ||||||||
5 | 74.1 | 5.11 (m) | 74.1a | 5.10 (m) | 74.1 | 5.10 (m) | 29.9 | 2.71 (m) | 30.2 | 2.68 (m) |
2.32 (m) | 2.32 (m) | |||||||||
6 | 122.6 | 5.33 (d, 9.0) | 122.5b | 5.32 (d, 8.4) | 122.8 | 5.33 (d, 9.0) | 116.7 | 4.98 (t, 7.8) | 116.9 | 5.05 (t, 7.2) |
7 | 141.6 | — | 142.0c | — | 141.4 | — | 139.2 | — | 138.9 | — |
8 | 39.1 | 2.04 (m) | 39.3d | 2.04 (m) | 39.1 | 2.03 (m) | 39.4 | 2.00 (m) | 39.4 | 2.00 (m) |
2.08 (m) | ||||||||||
9 | 21.1 | 1.46 (m) | 21.0e | 1.30 (m) | 21.0 | 1.40 (m) | 21.3 | 1.32 (m) | 21.3 | 1.40 (m) |
1.57 (m) | 1.66 (m) | 1.58 (m) | ||||||||
10 | 38.1 | 1.90 (m) | 40.3f | 1.62 (m) | 35.9 | 1.68 (m) | 35.9 | 1.61 (m) | 38.0 | 1.84 (m) |
1.73 (m) | 1.75 (m) | 1.68 (m) | ||||||||
11 | 79.2 | — | 76.0g | — | 75.3 | — | 75.3 | — | 79.1 | — |
12 | 190.9 | — | 194.5 | — | 190.4 | — | 190.7 | — | 191.0 | — |
13 | 100.1 | 5.45 (s) | 101.4 | 5.64 (s) | 103.1 | 5.45 (s) | 102.9 | 5.72 (s) | 100.0 | 5.45 (s) |
14 | 206.7 | — | 206.8 | — | 206.6 | — | 206.7 | — | 206.7 | — |
15 | 89.3 | — | 89.6h | — | 90.2 | — | 90.1 | — | 89.3 | — |
16 | 22.6 | 1.38 (s) | 23.0 | 1.39 (s) | 22.9 | 1.40 (s) | 22.9 | 1.40 (s) | 22.6 | 1.36 (s) |
17 | 22.6 | 1.36 (s) | 22.6 | 1.38 (s) | 22.7 | 1.39 (s) | 22.7 | 1.39 (s) | 22.6 | 1.35 (s) |
18 | 22.3 | 1.68 (s) | 48.1i | 1.60 (m) | 51.0 | 3.92 (d, 11.4) | 50.9 | 3.91 (d, 11.4) | 22.3 | 1.67 (s) |
1.77 (m) | 3.61 (d, 11.4) | 3.62 (d, 11.4) | ||||||||
19 | 16.6 | 1.69 (s) | 16.6 | 1.67 (s) | 16.4 | 1.67 (s) | 16.0 | 1.60 (s) | 16.1 | 1.60 (s) |
20 | 23.0 | 2.00 (s) | 23.0 | 1.99 (s) | 23.0 | 1.98 (s) | 13.9 | 2.05 (s) | 13.9 | 2.05 (s) |
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11-Acetoxy | 18-Isopropyl | 11-Acetoxy | ||||||||
1′ | 169.3 | — | 24.3 | 1.75 (m) | 169.3 | — | ||||
2′ | 21.4 | 2.07 (s) | 24.4k | 0.99 (d, 7.0) | 21.4 | 2.07 (s) | ||||
3′ | 24.4k | 0.99 (d, 7.0) |
White amorphous powder; mp 73.1–73.9; [α]28D: +19.3 (c 0.1, MeOH); ECD (MeOH) θ(λ nm): −6.3(215), −6.6(250), +0.9(299), −0.5(338) mdeg; HR-ESI-MS: m/z 391.2492 [M + H]+ (calcd for [C23H35O5]+, 391.2479); 1H- and 13C-NMR data are given in Table 1.
(5S,11S)-Aphanamixionolide C | (3) |
White amorphous powder; mp 78.1–79.0; [α]28D: −26.5 (c 0.1, MeOH); ECD (MeOH) θ(λ nm): −4.1(215), −6.2(252) mdeg; HR-ESI-MS: m/z 383.1627 [M + H]+ (calcd for [C20H2835ClO5]+, 383.1620) and m/z 385.1605 [M + 2amu + H]+ (calcd for [C20H2837ClO5]+, 385.1605); 1H- and 13C-NMR data are given in Table 1.
(4R,11S)-Aphanamixionolide D | (4) |
White amorphous powder; mp 76.5–77.3; [α]28D: −40.2 (c 0.1, MeOH); ECD (MeOH) θ(λ nm): −11.4(211), −2.7(241) mdeg; HR-ESI-MS: m/z 383.1625 [M + H]+ (calcd for [C20H2835ClO5]+, 383.1620) and m/z 385.1600 [M + 2amu + H]+ (calcd for [C20H2837ClO5]+, 385.1605); 1H- and 13C-NMR data are given in Table 1.
(4R,11S)-Aphanamixionolide E | (5) |
White amorphous powder; mp 55.4–56.1; [α]28D: −33.1 (c 0.1, MeOH); ECD (MeOH) θ(λ nm): −22.0(210) mdeg; HR-ESI-MS: m/z 391.2118 [M + H]+ (calcd for [C22H31O6]+, 391.2115); 1H- and 13C-NMR data are given in Table 1.
Compound 1 was obtained as a white amorphous powder. Its molecular formula, C22H30O6, was determined by protonated ion [M + H]+ at m/z 391.2122 in the HR-ESI-MS (calcd for [C22H31O6]+, 391.2115), indicating eight degrees of unsaturation. The 1H-NMR spectrum of 1 contained six singlet methyl groups [δH 2.07, 2.00, 1.69, 1.68, 1.38, and 1.36 (each, 3H)], three olefinic protons [δH 5.45 (1H, s), 5.33 (1H, d, J = 9.0 Hz), and 5.82 (1H, s)], and one oxygenated methine group [δH 5.11 (1H, m)]. The 13C-NMR and HSQC spectra of 1 showed 22 carbons including six methyl groups, four methylenes, four methines, and eight non-protonated carbons. The methyl [δC 21.4/δH 2.07 (3H, s)] and carbonyl signals (δC 169.3) were assigned to an acetoxy group. Other 20 carbons suggested for a diterpenoid backbone. The COSY spectrum of 1 revealed two coupled systems, including H2-4 (δH 2.39 and 2.22)/H-5 (δH 5.11)/H-6 (δH 5.33) and H2-8 (δH 2.04)/H2-9 (δH 1.46)/H2-10 (δH 1.90). The HMBC correlations between H3-20 (δH 2.00) and C-2 (δC 116.7)/C-3 (δC 157.0)/C-4 (δC 35.1), and H-2 (δH 5.82) and C-1 (δC 165.2) indicated a terminal carboxylate functional group at C-1 and a double bond at C-2/C-3. The HMBC correlations between H3-19 (δH 1.69) and C-6 (δC 122.6)/C-7 (δC 141.6)/C-8 (δC 39.1) indicated another double bond at C-6/C-7. Furthermore, a carbon chemical shift of C-14 (δC 206.7) and the HMBC correlations between H3-16 (δH 1.38)/H3-17 (δH 1.36) and C-15 (δC 89.3)/C-14 (δC 206.7) confirmed a ketone functional group at C-14. Then, the HMBC correlations between H-13 (δH 5.45) and C-14 (δC 206.7)/C-12 (δC 190.9), and H3-18 (δH 1.68) and C-10 (δC 38.1)/C-11 (δC 79.2)/C-13 (δC 100.1) suggested the last double bond at C-12/C-13 and fully established an acyclic diterpene backbone. Carbon chemical shifts of C-5 (δC 74.1), C-11 (δC 79.2), C-12 (δC 190.9), and C-15 (δC 89.3) indicated oxygen-bearing carbons. Additionally, de-shielded signals of C-12 (δC 190.9) and C-15 (δC 89.3) suggested the existence of an ether bridge between these carbons, forming a furan-3-one moiety. The strong downfield shifted signals of oxygenated olefinic carbon (C-12: δC 190.9) and carbinol carbon (C-15: δC 89.3) in the furan-3-one moiety are consistent with those previously reported in novel nemoralisin-type diterpenoids from Aphanamixis plants.9–12 Although interactions between H-5 (δH 5.11) and C-1 (δC 165.2) are not clearly observed in the HMBC spectrum, the chemical shift values at those positions (δH-5 5.11, δC-5 74.1, δC-1 165.2) are consistent with the presence of lactone bridge between C-1 and C-5 as compared to compound 2 (showing weak HMBC correlation between H-5 and C-1). The acetoxy group, therefore, was assigned at the last oxygen bearing carbon C-11. The geometry of double bond at C-6/C-7 was determined to be E-configuration based on NOESY correlations of H3-19 (δH 1.69)/H-5 (δH 5.11) and H-6 (δH 5.33)/H2-8 (δH 2.04). Later, absolute configurations at C-5 and C-11 were elucidated by experimental and TD-DFT calculated ECD spectra. The theoretical ECD spectra of the four possible stereoisomers of 1, namely, (5S,11S)-1a, (5S,11R)-1b, (5R,11R)-1c, and (5R,11S)-1d were computed. The experimental ECD spectrum of 1 displayed negative Cotton effects at wavelengths of 215 nm (−5.4 mdeg) and 249 nm (−6.3 mdeg), which is consistent with those calculated ECD spectrum for (5S,11R)-1b isomers. Consequently, the structure of compound 1 was completely determined and it was named (5S,11R)-aphanamixionolide A.
Compound 2 was obtained as a white amorphous powder. Its molecular formula, C23H35O5, was determined by protonated ion [M + H]+ at m/z 391.2492 in the HR-ESI-MS (calcd for [C23H35O5]+, 391.2479), indicating seven degrees of unsaturation. The 1H and 13C-NMR data of 2 showed close similarity with those of 1, indicating the same acyclic diterpenoid backbone. The NMR data of 2 exhibited signals of an isopropyl group instead of the acetyl group as in compound 1. Additionally, the signals of a methyl group (C-18, δC 22.3/δH 1.68) in 1 were replaced by the signals of a methylene group (C-18, δC 48.1/δH 1.77 and 1.60) in 2, suggesting the position of the isopropyl group at C-18. This deduction was confirmed by COSY correlations of H2-18 (δH 1.77 and 1.60)/H-1′ (δH 1.75)/H3-2′,3′ (δH 0.99) and HMBC correlations between H3-2′,3′ (0.99) and C-1′ (δC 24.3)/C-18 (δC 48.1), H2-18 (δH 1.77 and 1.60) and C-10 (δC 40.3)/C-11 (δC 76.0)/C-12 (δC 194.5). The upfield-shifted signal of C-11 (δC 76.0) in compound 2 compared to that in 1 (C-11: δC 79.2) is consistent with the replacement of an acetoxy group with a hydroxy group at this carbon. The weak HMBC correlation between H-5 (δH 5.10) and C-1 (δC 165.2) further confirmed the lactone bridge between C-1 and C-5. The NOESY correlations of H3-19 (δH 1.67)/H-5 (δH 5.10) and H-6 (δH 5.32)/H2-8 (δH 2.04) confirmed the E-configuration of a double bond at C-6/C-7. Careful examination of the 13C NMR spectra of 2 showed that several carbons significantly divided into a pair of closely chemical shift values (ΔδC < 0.2 ppm, Table 1 and Fig. S16, S17†). This evidence suggested that compound 2 was a pair of dia-stereoisomers at chiral centers C-5 and C-11. The TD-DFT calculation ECD spectra for the four possible stereoisomers (5S,11S)-2a, (5S,11R)-2b, (5R,11R)-2c, and (5R,11S)-2d were performed and compared with the experimental ECD data. The results indicated that compound 2 possessed 5S-configuration (as isomers 2a/2b) and a pair of dia-stereoisomers were formed by difference in absolute configuration at C-11, 11R or 11S. Compound 2, therefore, was determined and it was named (5S,11R/11S)-aphanamixionolide B.
The HR-ESI-MS spectra of 3 showed a pair of isotope protonated molecular ion peaks at m/z 383.1627 [M + H]+ and m/z 385.1605 [M + 2amu + H]+ with an intensity ratio around 3/1, which indicated a molecular formula of C20H27ClO5, containing a chlorine atom (calcd for [C20H2835ClO5]+, 383.1620; and calcd for [C20H2837ClO5]+, 385.1605). The 1H and 13C-NMR data of 3 indicated an acyclic diterpene as compounds 1 and 2. The 1H-NMR spectrum of 3 revealed the presence of four singlet methyl groups [δH 1.98, 1.67, 1.40, and 1.39 (each 3H, s)], suggesting a chlorine atom link to one of the five terminal methyl groups in the diterpene skeleton. Comparison of the NMR data of 3 with those of 1 and 2 showed the difference at signals of methylene group C-18 [δC 51.0/δH 3.92 and 3.61 (each 1H, d, J = 11.4 Hz)], suggesting a chlorine atom link to C-18. This deduction was then confirmed by HMBC correlations between H2-18 (δH 3.92 and 3.61) and C-10 (δC 35.9)/C-11 (δC 75.3)/C-12 (δC 190.4). The NOESY correlations of H3-19 (δH 1.67)/H-5 (δH 5.10) and H-6 (δH 5.33)/H-8 (δH 2.03 and 2.08) confirmed the E-configuration of the double bond at C-6/C-7. Later, absolute configurations 5S and 11S (as isomer 3a) were demonstrated by experimental and TD-DFT calculated ECD spectra, as shown in Fig. 3. Consequently, the structure of 3 was determined and it was named (5S,11S)-aphanamixionolide C.
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Fig. 3 Experimental ECD spectra of 1–5 and TD-DFT calculated ECD spectra for their possible stereoisomers. |
Compound 4 was obtained as a white amorphous powder. The HR-ESI-MS spectra of 4 closely resembled those of 3 by a pair of isotope protonated molecular ion peaks at m/z 383.1625 [M + H]+ and m/z 385.1600 [M + 2amu + H]+ with an intensity ratio around 3/1. This evidence indicated that compound 4 had the same molecular formula as that of 3, C20H27ClO5 (calcd for [C20H2835ClO5]+, 383.1620; and calcd for [C20H2837ClO5]+, 385.1605). The 1H and 13C-NMR data of 4 were revealed to be different from those of 3 by signals related to the lactone ring (C-1 to C-7 and C-20, Table 1). The sequencing COSY correlations of H-6 (δH 4.98)/H2-5 (δH 2.71 and 2.32)/H-4 (δH 4.89) and chemical shift values of the methine group C-4 (δC 84.1) indicated that an oxygen-bearing carbon was at C-4 in compound 4 instead of C-5, as shown in compound 3. The HMBC correlations between H3-20 (δH 2.05) and C-2 (δC 117.5)/C-3 (δC 168.1)/C-4 (δC 84.1) confirmed the presence of a double bond at C-2/C-3. The HMBC correlations between H-2 (δH 5.81)/H-4 (δH 4.89) and C-1 (δC 173.2) were demonstrated for a lactone bridge between C-1 and C-4. Compound 4, therefore, contained a γ-lactone instead of a δ-lactone moiety in compounds 1–3. The NOESY correlations of H3-19 (δH 1.60)/H-5 (δH 2.71 and 2.32) and H-6 (δH 4.98)/H-8 (δH 2.00) confirmed the E-configuration of the double bond at C-6/C-7. The absolute configurations 4R and 11S (as isomer 4a) were demonstrated by experimental and TD-DFT calculated ECD spectra, as shown in Fig. 3. Consequently, the structure of 4 was determined and it was named (4R,11S)-aphanamixionolide D.
Compound 5 was obtained as a white amorphous powder. The HR-ESI-MS spectra of 4 closely resembled those of 1 by protonated molecular ion peaks at m/z 391.2118 [M + H]+, indicating the same molecular formula as that of 1, C22H30O6 (calcd for [C22H31O6]+, 391.2115). The NMR spectra data of 5 indicated signals of acetoxy group (δC 169.3 and δC 21.4/δH 2.07) and an acyclic diterpenoid backbone as compounds 1–4 (Table 1). Additionally, the 13C-NMR spectral data of 5 exhibited partially similar carbon chemical shifts with both compounds 1 (from C-8 to C-19 and acetoxy group) and 4 (from C-1 to C-7 and C-20). This evidence indicated that compound 5 possessed two structural fragments, namely, an acetoxylated furan-3-one moiety (as compound 1) and a γ-lactone moiety (as compound 4). The planar structure of 5, including the E-geometric configuration of double bond at C-6/C-7, was then confirmed by analysis of HMBC, COSY, and NOESY correlations, as shown in Fig. 2. Finally, the absolute configurations 4R and 11S (as isomer 5b) were demonstrated by experimental and TD-DFT calculated ECD spectra. Consequently, the structure of 4 was determined and it was named (4R,11S)-aphanamixionolide E.
Other known compounds were determined to be tirucallane-type triterpenes including piscidinol A (6),13,14 hispidone (7),15 and bourjotinolone A15,16 by consistence of their NMR spectral data with those reported in the literature.
All isolates (1–8) were evaluated for their anti-inflammatory activities by inhibiting nitric oxide production in LPS-activated RAW 264.7 cells. First, the effects of compounds on the cell viability were examined. At a concentration of 50 μM, compounds 1–8 did not show significant cytotoxicity towards RAW 264.7 cells by MTT tests. The NO inhibitory activity was then evaluated at serial diluted concentrations of 0.4–50 μM. As shown in Table 2, compounds 1–8 inhibited NO production with IC50 values of 10.2–37.7 μM, comparable to positive control L-NMMA (IC50: 31.5 μM). Our results are consistent with previous reports that terpenoid compositions of Aphanamixis plants potentially inhibit LPS-induced NO production activities.2,5,17,18
Footnote |
† Electronic supplementary information (ESI) available: HR-ESI-MS, NMR, and ECD spectra of new compounds of all isolated compounds. See DOI: https://doi.org/10.1039/d4ra02968b |
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