Abstract
Edgeworthianins A–E (1–5) were isolated from Edgeworthia chrysantha as a class of macrocyclic daphnane orthoesters with an unusual macrocyclic ring formed from an C14 aliphatic chain. Their structures were elucidated by extensive physicochemical and spectroscopic analyses. Compounds 2, 4, and 5 exhibited potent anti-HIV activity against HIV-1 infection of MT4 cells with EC50 values of 29.3, 8.4, and 2.9 nM, respectively. These compounds broaden the findings of the structure-activity relationship of macrocyclic daphnane orthoesters for further anti-HIV drug development.
Graphical Abstract
Macrocyclic daphnane orthoesters (MDOs) are a class of 1-alkyldaphnane featuring the structure of a macrocyclic ring constructed from an aliphatic chain at C-1 connecting to the C-9,13,14-orthoester moiety (Figure 1). Macrocyclic daphnane orthoesters are found exclusively in plants of the Thymelaeaceae family. In general, the 1-alkyl group of the macrocyclic ring originates from a C10 aliphatic chain unit (Type Ⅰ). Since gnidimacrin was isolated from Gnidia subcordata,1 about 50 MDOs belonging to type I have been reported from Daphne, Daphnopsis, Dirca, Gnidia, Pimelea, Stellera, Synaptolepis, and Wikstroemia.2 A variation of MDOs with a 1-alkyl group originated from a C16 aliphatic chain (Type Ⅱ) was isolated from plants of the genus Synaptolepis.3 MDOs have attracted much attention for drug discovery, in particular as anticancer drug candidates because of their significant antineoplastic activity.1,4 Several MDOs were also highlighted for their unique ability to eliminate latent HIV-1 cells.5
Figure 1.
Skeleton of macrocyclic daphnane orthoesters.
Edgeworthia chrysantha Lindl., also known as Oriental paperbush, belongs to the family Thymelaeaceae. It is a deciduous shrub, which has distinctive three-pronged branches and yellow flowers that bloom in spring. It is mainly distributed in central and southern China, Nepal, and Japan. In Japan this plant is cultivated, since the barks are used as raw materials for Japanese paper and banknotes. Its buds and roots have been used in traditional Chinese medicines for the treatment of hoarseness, bruise, arthralgia, neuralgia, and eye diseases such as photophobia, epiphora, and visual impairment.6 Chemical constituents of E. chrysantha have been reported, including coumarins, flavonoids, steroids, and phenols.7 A previous phytochemical investigation also reported the presence of MDOs in the branches of this plant, but with insufficient details.8
In our ongoing research for the discovery of anti-HIV diterpenoids from the plants of Thymelaeaceae, a MeOH extract of the flower buds of E. chrysantha showed significant anti-HIV activity (EC50 = 4.1 μg/mL). Herein we report the isolation, structural elucidation, and anti-HIV activity evaluation of a class of MDOs with an unusual macrocyclic ring from E. chrysantha.
RESULTS AND DISCUSSION
A MeOH extract of the flower buds of E. chrysantha was partitioned between EtOAc and H2O. The EtOAc-soluble fraction was fractionated by octadecylsilyl (ODS) and silica gel column chromatography, as well as by preparative HPLC, to afford five MDOs, named edgeworthianins A–E (1–5).
Edgeworthianin A (1) was isolated as a colorless solid, [α]D26 +18.4° (c 0.10, MeOH). The molecular formula of 1 was determined as C36H51O11 by HRESIMS in observing a protonated molecular ion at m/z 659.3416 [M + H]+ (calcd for C36H50O11, 659.3426). The IR absorptions revealed the presence of hydroxy (3434 cm−1) and carbonyl (1743 cm−1) functionalities. In the 1H and 13C NMR spectra, the characteristic resonances for an isopropenyl moiety at δH 4.92 (Ha-16), 4.97 (Hb-16), 1.78 (H3-17), and δC 143.1 (C-15) and two methyl groups at δH 1.38 (H3-18) and 1.72 (H3-19) indicated the presence of a daphnane skeleton (Table 1). The resonances of a methine at δH 2.51 (H-1) and a typical orthoester carbon at δC 119.9 (C-1’) suggested that 1 was an MDO diterpenoid. Additionally, resonances for an acetal carbon at δC 112.1 (C-2) and a lactone carbonyl carbon at δC 173.5 (C-3) were observed. The positions of these functional groups were deduced by HMBC correlations from H-1, H3-19 to C-2, and H-10, H-5 to C-3, indicating the presence of a bicyclo[2.2.1]hexane ring structure in A-ring. Other resonances due to polyoxygenated functionalities in the daphnane skeleton were observed for an epoxy group at δH 3.53 (H-7), δC 59.2 (C-6) and 59.4 (C-7), three oxygenated methines at δH 4.86 (H-5), 4.98 (H-12), and 4.61 (H-14), an oxygenated methylene at δH 3.70 (Ha-20) and 4.10 (Hb-20), and three oxygenated tertiary carbons at δC 86.5 (C-4), 80.3 (C-9), and 83.0 (C-13). Positions of these functionalities were deduced by comprehensive analyses of the DQF-COSY and HMBC spectra (Figure 2). Furthermore, the presence of an acetyloxy moiety was indicated by the resonances of an ester carbonyl carbon at δC 169.8 (OCOCH3) and an acetyl methyl proton at δH 1.97 (OCOCH3). The position of the acetyloxy moiety at C-12 was determined by the HMBC correlation from H-12 to OCOCH3.
Table 1.
1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data of Compounds 1–3 (CDCl3).
| No. | 1 | 2 | 3 | ||||||
|---|---|---|---|---|---|---|---|---|---|
| δH (J in Hz) | δC type | δH (J in Hz) | δC type | δc (J in Hz) | δC type | ||||
| 1 | 2.51, brt (5.4) | 52.5 | CH | 2.73, brt (6.0) | 51.6 | CH | 2.81, brt (5.5) | 51.7 | CH |
| 2 | 112.7 | C | 112.8 | C | 112.9 | C | |||
| 3 | 173.5 | C | 173.7 | C | 173.7 | C | |||
| 4 | 86.5 | C | 86.5 | C | 86.5 | C | |||
| 5 | 4.86, s | 68.6 | CH | 4.87, s | 68.6 | CH | 4.88, s | 68.7 | CH |
| 6 | 59.2 | C | 59.3 | C | 59.3 | C | |||
| 7 | 3.53, brs | 59.4 | CH | 3.53, brs | 58.9 | CH | 3.55, brs | 58.9 | CH |
| 8 | 3.85, dd (2.6, 1.0) | 34.0 | CH | 3.90, dd (2.9, 1.3) | 33.6 | CH | 3.96, dd (2.5, 1.4) | 33.6 | CH |
| 9 | 80.3 | C | 79.2 | C | 79.3 | C | |||
| 10 | 3.05, d (5.7) | 50.3 | CH | 3.06, d (5.2) | 51.1 | CH | 3.08, d (4.8) | 51.5 | CH |
| 11 | 1.91, q (6.8) | 44.7 | CH | 2.04, d (7.5) | 50.4 | CH | 2.25, d (7.0) | 50.1 | CH |
| 12 | 4.98, s | 78.7 | CH | 5.35, s | 74.4 | CH | 5.49, s | 74.5 | CH |
| 13 | 83.0 | C | 83.1 | C | 83.1 | C | |||
| 14 | 4.61, d (2.6) | 79.4 | CH | 4.63, d (2.9) | 79.4 | CH | 4.66, d (2.5) | 79.3 | CH |
| 15 | 143.1 | C | 142.7 | C | 142.7 | C | |||
| 16 | 4.92, s | 113.1 | CH2 | 4.94, s | 113.3 | CH2 | 4.97, s | 113.3 | CH2 |
| 4.97, brs | 4.98, brs | 5.00, brs | |||||||
| 17 | 1.78, brs | 18.5 | CH3 | 1.79, brs | 18.8 | CH3 | 1.82, brs | 18.5 | CH3 |
| 18 | 1.38, d (6.8) | 20.0 | CH3 | 4.23, d (12.8) | 65.9 | CH2 | 4.59, dd (12.8, 7.0) | 67.0 | CH2 |
| 4.55, dd (12.8, 7.5) | 4.65, d (12.8) | ||||||||
| 19 | 1.72, s | 19.0 | CH3 | 1.78, s | 19.1 | CH3 | 1.72, s | 18.9 | CH3 |
| 20 | 3.70, d (12.3) | 63.3 | CH2 | 3.72, d (12.4) | 63.1 | CH2 | 3.73, d (12.5) | 63.1 | CH2 |
| 4.10, d (12.3) | 4.09, d (12.4) | 4.10, d (12.5) | |||||||
| 1’ | 119.9 | C | 120.2 | C | 120.3 | C | |||
| 2’ | 1.90, 1.96, m | 35.0 | CH2 | 1.89, 1.94, m | 34.7 | CH2 | 1.93, m | 34.7 | CH2 |
| 3’ | 1.67, m | 24.5 | CH2 | 1.67, m | 24.2 | CH2 | 1.67, m | 24.2 | CH2 |
| 4’ | 2.03, 2.16, m | 27.0 | CH2 | 2.00, 2.16, m | 26.8 | CH2 | 2.01, 2.19, m | 26.8 | CH2 |
| 5’ | 5.45, dt (10.8, 7.2) | 129.1 | CH | 5.45, dt (10.9, 6.9) | 128.9 | CH | 5.45, dt (10.6, 6.9) | 128.9 | CH |
| 6’ | 5.38, dt (10.8, 7.2) | 131.0 | CH | 5.36, dt (10.9, 6.9) | 131.3 | CH | 5.37, dt (10.6, 6.9) | 131.3 | CH |
| 7’ | 2.02, m | 25.8 | CH2 | 2.02, m | 25.6 | CH2 | 2.02, m | 25.6 | CH2 |
| 8’ | 1.40, m | 28.0 | CH2 | 1.41, m | 28.1 | CH2 | 1.40, m | 28.0 | CH2 |
| 9’ | 1.30, 1.40, m | 25.3 | CH2 | 1.27, 1.42, m | 25.2 | CH2 | 1.24, 1.39, m | 25.0 | CH2 |
| 10’ | 1.39, 1.56, m | 30.8 | CH2 | 1.36, 1.65, m | 30.1 | CH2 | 1.36, 1.70, m | 29.8 | CH2 |
| 11’ | 1.55, m | 37.1 | CH | 1.48, m | 37.9 | CH | 1.46, m | 38.0 | CH |
| 12’ | 1.37, m | 33.0 | CH2 | 1.37, m | 33.2 | CH2 | 1.33, m | 32.8 | CH2 |
| 13’ | 1.28, 1.34, m | 20.1 | CH2 | 1.27, 1.36, m | 19.2 | CH2 | 1.14, 1.27, m | 18.9 | CH2 |
| 14’ | 0.90, t (7.0) | 14.4 | CH3 | 0.88, t (7.0) | 14.4 | CH3 | 0.76, t (7.1) | 14.3 | CH3 |
| Ac-CO | 169.8 | C | 169.0 | C | 169.0 | C | |||
| Ac-Me | 1.97, s | 21.1 | CH3 | 1.95, s | 21.1 | CH3 | 1.95, s | 21.1 | CH3 |
| 1” | 176.9 | C | 166.6 | C | |||||
| 2” | 2.57, sept (7.0) | 34.1 | CH | 129.6 | C | ||||
| 3” | 1.18, d (7.0) | 18.5 | CH3 | 8.07, dd (7.8, 1.2) | 129.6 | CH | |||
| 4” | 1.20, d (7.0) | 18.9 | CH3 | 7.44, dd (7.8, 7.5) | 128.4 | CH | |||
| 5” | 7.55, tt (7.5, 1.2) | 133.0 | CH | ||||||
| 6” | 7.44, dd (7.8, 7.5) | 128.4 | CH | ||||||
| 7” | 8.07, dd (7.8, 1.2) | 129.6 | CH | ||||||
Figure 2.
Key 1H–1H COSY and HMBC correlations of compounds 1–3.
In addition to the carbon resonances assignable to the 1-alkyldaphnane skeleton and the acetyloxy moiety, fourteen carbon resonances were observed in the 13C NMR spectrum. Hydrogen resonances corresponding to the macrocyclic ring moiety were observed for two olefinic protons at δH 5.45 (dt, J = 10.8, 7.2, H-5’) and 5.38 (dt, J = 10.8, 7.2, H-6’), and a terminal methyl group at δH 0.90 (t, J = 7.0, H3-14’). The position of the olefin group was deduced to be Δ5’,6’ by the continuous 1H−1H COSY correlations from H-2’ to H2-8’ and the HMBC correlation from H-14 and H-2’ to C-1’. A cis-configuration was assigned to this double bond from the coupling constant J5’,6’ = 10.8 Hz. The continuous 1H−1H COSY correlations from H3-14’ to H-11’ also indicated the presence of a n-propyl group. The connection of C-11’ and C-1 was deduced by analysis of 1H−1H COSY data, as well as from the HMBC correlations from H-10 to C-11’ and from H-1 to C-10’.
Detailed interpretation of the NOESY correlations and proton couplings led to the determination of the relative configurations of 1. The NOESY correlations between H-1/H-11, H-11/H-8, H2-20/H-7, H-7/H-14, H-14/H2-16, H-14/H3-17, H3-18/H-12 indicated that H-1, CH2-20, H-7, H-8, H-11, H-14, and the isopropenyl moiety at C-13 have β-orientations, while H-12 and CH3-18 have α-orientations (Figure 3). These deductions were supported by the characteristic proton resonance of H-12 as a singlet, indicating that the dihedral angle between H-11/H-12 is near to 90°. Since the NMR data for H-10 and H-5 and associated NOESY correlations were similar to known MDOs with the same A-ring structure in the daphnane skeleton, α-orientations of H-10 and H-5 were deduced.9 Furthermore, H-11’ was determined as α-orientation by observing NOESY correlations between H-1/H3-19, H3-19/H2-12’, and H-10/H-11’. Thus, the structure of edgeworthianin A (1) was determined as shown in Figure 3.
Figure 3.
Key NOESY correlations of compounds 1–3.
Edgeworthianin B (2) was isolated as a colorless solid, [α]D26 +24.2 (c 0.10, MeOH). Its molecular formula of C40H57O13 was determined from the HRESIMS positive ion peak at m/z 745.3784 [M + H]+ (calcd for C40H56O13, 745.3794). The NMR spectroscopic data of 2 suggested it is also an MDO diterpenoid. However, the 1H and 13C resonances for an oxymethylene at δH 4.23 (Ha-18), 4.55 (Hb-18), and δC 65.9 (C-18) were observed instead of the signal for the C-18 methyl group in 1 (Table 1). The presence of isobutyryloxy moiety in 2 was indicated by the resonances of geminal dimethyl protons at δH 1.18 (d, J = 7.0, H3-3”) and 1.20 (d, J = 7.0, H3-4”), a methine proton at δH 2.57 (sept, H-2”), and an ester carbonyl carbon at δC 176.9 (C-1”). The position of the isobutyryloxy moiety was confirmed by the HMBC correlation from H2-18 to C-1” (Figure 2). Analysis of 1H-1H couplings and NOE data indicated that the relative configurations of 2 were the same as those of 1 (Figure 3). Thus, the structure of edgeworthianin B (2) was determined as shown.
The molecular formula of edgeworthianin C (3) was established as C43H54O13 from the positive ion HRESIMS data, showing a protonated molecule at m/z 779.3626 [M + H]+ (calcd for C43H55O13, 779.3637). Comparison of the NMR data between 3 and 2 indicated that they have a similar MDO structure, but a benzoyloxy moiety was attached to C-18 in 3. The presence of the benzoyloxy moiety was evident from the proton resonances for five aromatic protons at δH 8.07 (dd, J = 7.8, 1.2, H-3”,7”), 7.44 (dd, J = 7.8, 7.5, H-4”,6”), and 7.55 (tt, J = 7.5, 1.2, H-5”). The position of the benzoyloxy moiety was confirmed by the HMBC correlations from H2-18 to δC 166.6 (C-1”). Thus, the structure of edgeworthianin C (3) was determined as shown.
Edgeworthianins D (4) and E (5) were obtained as colorless solids, and revealed characteristic NMR resonances for an isopropenyl moiety, an orthoester moiety, and a methine proton at H-1, suggesting that they were also MDO diterpenoids (Table 2). However, the resonances due to the A-ring of the daphnane skeleton showed large difference from those of 1−3. On the other hand, the 1H and 13C spectroscopic data of 4 and 5 were in accordance with two MDOs which were previously isolated from the same plant but lacked full NMR resonances assignments.8
Table 2.
1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data of Compounds 4 and 5 (CDCl3).
| No. | 4 | 5 | ||||
|---|---|---|---|---|---|---|
| δH (J in Hz) | δC type | δΗ (J in Hz) | δC, type | |||
| 1 | 2.15, ddd (10.9, 10.0, 2.0) | 48.5 | CH | 2.40, ddd (10.7, 9.7, 1.4) | 47.3 | CH |
| 2 | 2.28, dq (10.0, 6.8) | 42.8 | CH | 2.29, dq (9.7, 6.9) | 42.7 | CH |
| 3 | 221.6 | C | 222.1 | C | ||
| 4 | 75.8 | C | 75.9 | C | ||
| 5 | 4.09, s | 71.6 | CH | 4.13, d (3.4) | 71.5 | CH |
| 6 | 60.5 | C | 60.6 | C | ||
| 7 | 3.48, brs | 64.4 | CH | 3.48, brs | 64.0 | CH |
| 8 | 3.50, d (2.9) | 35.6 | CH | 3.56, d (3.0) | 35.1 | CH |
| 9 | 80.0 | C | 79.3 | C | ||
| 10 | 3.28, d (10.9) | 43.9 | CH | 3.31, d (10.7) | 44.0 | CH |
| 11 | 2.49, q (7.0) | 43.5 | CH | 2.62, dd (7.7, 1.5) | 50.0 | CH |
| 12 | 4.96, s | 78.5 | CH | 5.41, s | 74.5 | CH |
| 13 | 82.9 | C | 83.2 | C | ||
| 14 | 4.61, d (2.9) | 80.8 | CH | 4.63, d (3.0) | 80.6 | CH |
| 15 | 143.5 | C | 143.1 | C | ||
| 16 | 4.91, s | 112.8 | CH2 | 4.93, s | 113.0 | CH2 |
| 4.96, brs | 4.97, brs | |||||
| 17 | 1.78, brs | 18.5 | CH3 | 1.80, brs | 18.5 | CH3 |
| 18 | 1.41, d (7.2) | 19.1 | CH3 | 4.34, dd (12.3, 1.5) | 65.7 | CH2 |
| 4.56, dd (12.3, 7.7) | ||||||
| 19 | 1.16, d (6.8) | 16.2 | CH3 | 1.22, d (6.8) | 17.1 | CH3 |
| 20 | 3.84, brs | 65.1 | CH2 | 3.83, dd (12.8, 6.7) | 65.0 | CH2 |
| 3.87, dd (12.8, 6.7) | ||||||
| 1’ | 120.0 | C | 120.1 | C | ||
| 2’ | 1.86, 1.99, m | 35.3 | CH2 | 1.86, 1.97, m | 35.1 | CH2 |
| 3’ | 1.65, m | 25.8 | CH2 | 1.52, 1.67, m | 25.5 | CH2 |
| 4’ | 1.99, 2.06, m | 27.6 | CH2 | 1.98, 2.05, m | 27.6 | CH2 |
| 5’ | 5.51, ddd (10.6, 6.0, 4.2) | 130.4 | CH | 5.50, ddd (10.8, 7.5, 6.0) | 130.1 | CH |
| 6’ | 5.54, ddd (10.6, 7.2, 3.5) | 130.4 | CH | 5.55, dt (10.8, 7.0) | 130.6 | CH |
| 7’ | 1.89, 1.99, m | 27.6 | CH2 | 1.90, 1.97, m | 27.6 | CH2 |
| 8’ | 1.36, m | 28.3 | CH2 | 1.36, m | 28.5 | CH2 |
| 9’ | 1.25, 1.53, m | 25.4 | CH2 | 1.24, 1.52, m | 25.3 | CH2 |
| 10’ | 1.23, 1.47, m | 35.1 | CH2 | 1.23, 1.42, m | 35.0 | CH2 |
| 11’ | 2.26, m | 35.9 | CH | 2.19, m | 36.6 | CH |
| 12’ | 1.12, m | 33.0 | CH2 | 1.11, m | 33.3 | CH2 |
| 13’ | 1.44, 1.54, m | 23.6 | CH2 | 1.45, 1.52, m | 23.7 | CH2 |
| 14’ | 0.95, t (7.3) | 15.2 | CH3 | 0.94, t (7.3) | 15.1 | CH3 |
| Ac-CO | 169.8 | C | 168.9 | C | ||
| Ac-Me | 1.97, s | 21.2 | CH3 | 1.96, s | 21.2 | CH3 |
| 1” | 176.9 | C | ||||
| 2” | 2.58, sept (7.0) | 34.1 | CH | |||
| 3” | 1.16, d (7.0) | 19.0 | CH3 | |||
| 4” | 1.18, d (7.0) | 19.0 | CH3 | |||
| 5-OH | 3.22, brs | 3.35, d (3.4) | ||||
| 20-OH | 2.13, t (6.7) | |||||
The molecular formula of edgeworthianin D (4) was determined as C36H50O11 from the HRESIMS positive ion peak at m/z 645.3624 [M + H]+ (calcd for C36H53O10, 645.3633). The NMR resonances for a carbonyl carbon at δC 221.6 (C-3) and a methyl proton doublet at δH 1.16 (H3-19) were observed in 4, suggesting the presence of a cyclopentanone structure in the A-ring. This deduction was also confirmed by the HMBC correlations from H-1 to C-19, and H3-19, H-5 to C-3 (Figure 4). The relative configurations of 4 were determined by interpretation of NOESY correlations and characteristic proton multiplicity. The presence of the NOESY correlations between H-2/H10, H-10/H-5, H-1/H-11, H-11/H-8, H2-20/H-7, H-7/H-14, H-14/H2-16, H-14/H3-17, and H3-18/H-12, and the absence of the correlations between H-10/H-11, H-10/H-8, and H-8/H-12, deduced the α-orientations of H-2, H-5, H-10, H-12, and CH3-18, and the β-orientations of H-1, H-11, H-8, CH2-20, H-7, H-14, and the isopropenyl moiety (Figure 5). These deductions were supported by the resonances of H-7 and H-12 as characteristic singlets, indicating that the dihedral angles between H-7/H-8 and H-11/H-12 were about 90°. In the macrocyclic ring moiety, the orientation of H-11’ differs from that of compounds 1−3, which was deduced as a β-orientation by detecting NOESY correlations between H3-18/H-11’, H3-19/H-11’, H-10/H2-12’, and H-2/H2-12’. Thus, the structure of edgeworthianin D (4) was determined as shown.
Figure 4.
Key 1H–1H COSY and HMBC correlations of compounds 4 and 5.
Figure 5.
Key NOESY correlations of compounds 4 and 5.
The molecular formula of edgeworthianin E (5) was determined as C40H58O12 from HRESIMS positive ion peak at m/z 731.3990 [M + H]+ (calcd for C40H59O12, 731.4001). Comparison of the NMR data between 5 and 4 revealed that they have similar structures, except for the presence of an isobutyryloxy moiety at C-18 in 5. The position of the isobutyryloxy moiety was confirmed by the HMBC correlations from Ha,b-18 to C-1”. Thus, the structure of edgeworthianin E (5) was determined as shown in Figure 2.
When measuring the ECD spectra of compounds 1−5, compounds 1−3 showed a positive Cotton effect at around 230 nm, and 4 and 5 showed a positive Cotton effect at around 300 nm (Supporting Information). Based on the structural similarity among these compounds, the Cotton effects were most like due to the n→π* transition of the A-ring functionalities.
Since MDOs have been reported to show excellent anti-HIV activity,2c,e,h,j compounds 1–5 were evaluated for their activity against HIV-1 infection in human T-lymphocyte cell line MT4 (Table 3). Although the activity of the isolated compounds was weaker than that of the positive control gnidimacrin, compounds 2, 4, and 5 showed significant anti-HIV activity with EC50 values of 29.3, 8.4, and 2.9 nM, respectively. This result suggest that the macrocyclic ring formed from an C14 aliphatic chain is a tolerable change for exhibiting anti-HIV activity. Compounds 4 and 5 exhibited more potent anti-HIV activity than 1–3, indicating that a cyclopentanone structure in the A-ring was favorable to enhance the anti-HIV activity, in agreement with our previous study.2c Compounds with an isobutyryloxy moiety at C-18 showed more potent activity than other substituents (5 vs 4; 2 vs 1 and 3). This suggests that an aliphatic substituent may also enhance the anti-HIV activity, structure-activity relationships (SARs) that deserve further investigation.
Table 3.
Anti-HIV and Cytotoxic Activities of Compounds 1–5.a
| Compound | anti-HIV (NL4–3) EC50 (nM) |
cytotoxicity (MT4) IC50 (nM) |
|---|---|---|
| 1 | >32.0 | >32.0 |
| 2 | 29.3 ± 5.5 | >32.0 |
| 3 | >32.0 | >32.0 |
| 4 | 8.4 ± 3.0 | >32.0 |
| 5 | 2.9 ± 0.7 | 21.3 ± 1.9 |
| gnidimacrinb | 0.06 ± 0.02 | >32.0 |
The values are means ± SD (n = 3).
Positive control.
In summary, five macrocyclic daphnane orthoesters (MDOs) 1–5 were isolated from the flower buds of E. chrysantha and three (2, 4, and 5) exhibited potent anti-HIV activity against HIV-1 infection of MT4 cells. MDOs have been paid much attention in anti-HIV drug discovery because of their potent dichotomous activity against both HIV-1 replication and HIV-1 latency.5 The previous structure-activity relationship (SAR) studies have demonstrated that MDOs with a five-membered A-ring structure as well as free 5- and 20-OH groups in the daphnane portion are preferred for exhibiting anti-HIV activity.2c,h,i,5d However, the SAR of the macrocyclic ring portion has not been well discussed due to limited structural diversity. MDOs 1–5 represented a new type of MDOs (Type III), with an unusual macrocyclic ring structure originated from a C14 unsaturated aliphatic chain. The potent anti-HIV-1 activity of these compounds suggested change of macrocyclic ring is tolerable for exhibiting anti-HIV activity, which provided further insight into the structure-activity relationships of MDOs.
EXPERIMENTAL SECTION
General Experimental Procedures
Optical rotations were measured on a JASCO P-2200 polarimeter in a 0.5-dm cell. UV spectra were obtained with a Shimadzu BioSpec-mini spectrophotometer. The ECD spectra were measured on a JASCO J-720W spectropolarimeter in a 10-mm cell. The IR spectra were measured on a JASCO FT/IR-4100 Fourier transform infrared spectrometer using a KBr disk. The NMR spectra were measured on a JEOL ECA-500 spectrometer with deuterated solvent used as the internal reference. The 1H NMR spectra were measured at 500 MHz, and the 13C NMR spectra were measured at 125 MHz. HRESIMS was conducted using a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer. Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Silica gel (Chromatorex PEI MB 100–40/75, Fuji Silysia Chemical Ltd., Aichi, Japan), and ODS (Cosmosil 75C18-PREP, Nacalai Tesque, Inc., Kyoto, Japan) were used for column chromatography. For preparative HPLC, a Waters 515 HPLC pump, equipped with an ERC RefractoMax520 differential refractometer detector and a Shimadzu SPD-10A UV-vis detector were used. For reversed-phase HPLC separations, a RP-C18 silica gel column (YMC-Actus Triart C18, 5 μm, 150×20 mm) was used, at a flow rate of 8.0 mL/min.
Plant Material
The flower buds of Edgeworthia chrysantha Lindl., were collected at Hunan Province, People’s Republic of China in August 2015, and identified by one of the authors, W.L. A voucher specimen (TH-17) has been deposited at the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University, Japan.
Extraction and Isolation
The air-dried flower buds of E. chrysantha (950 g) were extracted ultrasonically with MeOH (4 L × 20 min, 5 times), at room temperature. The MeOH extract was concentrated (53.0 g), suspended in H2O, and then partitioned with EtOAc. The EtOAc fraction (9.22 g) was subjected to ODS column chromatography and eluted with a stepwise gradient of MeOH−H2O (from 7:3 to 10:0, v/v), finally with isopropanol and CHCl3 to afford seven fractions (1 to 7). Fraction 3 (1.02 g) was subjected to silica gel column chromatography eluted with a gradient of n-hexane−EtOAc−MeOH−HCO2H to afford six fractions (3–1 to 3–6). Fraction 3–3 (17.3 mg) was separated by RP-HPLC (85% CH3CN) to afford seven fractions (3-3-1 to 3-3-7). Fraction 3-3-2 was 4 (1.7 mg) and fraction 3-3-4 was 5 (4.6 mg). Fraction 3-3-3 (4.3 mg) was purified by RP-HPLC (90% MeOH) to give 2 (1.7 mg) and 3 (1.6 mg). Fraction 3-3-6 (1.2 mg) was purified by RP-HPLC (90% MeOH) to give 1 (0.9 mg).
Edgeworthianin A (1): Colorless solid; [α]D26 +18.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 225 (3.31) nm; ECD (MeOH): [θ]25 (nm) 9677 (204), 24086 (224); IR (KBr) νmax 3434, 2927, 2856, 1793, 1743, 1632, 1457, 1382, 1230, 1081, 1032 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS (positive) m/z 659.3416 [M +H]+ (calcd for C36H51O11, 659.3426).
Edgeworthianin B (2): Colorless solid; [α]D26 +24.2(c 0.10, MeOH); UV (MeOH) λmax (log ε) 221 (3.29) nm; ECD (MeOH): [θ]25 (nm) 5003 (204), 14621 (225); IR (KBr) νmax 3433, 2926, 2855, 1793, 1737, 1632, 1458, 1385, 1227, 1190, 1157, 1078, 1034 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESITMS (positive) m/z 745.3784 [M + H]+ (calcd for C40H57O13, 745.3794).
Edgeworthianin C (3): Colorless solid; [α]D26 +13.8 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 228 (3.85) nm; ECD (MeOH): [θ]25 (nm) 3459 (215), 5989 (232); IR (KBr) νmax 3434, 2926, 2855, 1793, 1718, 1634, 1457, 1385, 1270, 1103 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS (positive) m/z 779.3626 [M + H]+ (calcd for C43H55O13, 779.3637).
Edgeworthianin D (4): Colorless solid; [α]D26 +35.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 285 (2.01) nm; ECD (MeOH): [θ]25 (nm) −5314 (212), 11132 (303); IR (KBr) νmax 3442, 2927, 2857, 1737, 1636, 1458, 1385, 1262, 1231, 1092, 1038 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS (positive) m/z 645.3624 [M + H]+ (calcd for C36H53O10, 645.3633).
Edgeworthianin E (5): Colorless solid; [α]D26 +28.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 285 (1.77) nm; ECD (MeOH): [θ]25 (nm) −2723 (212), 11863 (303); IR (KBr) νmax 3445, 2928, 2857, 1742, 1646, 1636, 1458, 1379, 1261, 1232, 1036 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS (positive) m/z 731.3990 [M + H]+ (calcd for C40H59O12, 731.4001).
Anti-HIV Assay and Cytotoxicity Study
HIV-1 NL4–3 (multiplicity of infection = 0.001) were used to infect MT4 cells in the presence of compounds at various concentrations in 96-well plates. Fresh medium containing appropriate concentrations of the compounds were added to the culture 48 h after infection to maintain normal cell growth. Virus replication was analyzed 4-day postinfection using p24 ELISA kit purchased from Perkin-Elmer.2h The viability of the cells in culture is based on quantitation of ATP in metabolically active cells using the CellTiter-Glo Luminescent Cell Viability Assay kit purchased from Promega. The CellTiter-Glo reagent was added to the MT4 cells that were cultured parallel to the antiviral assays. Cytotoxicity of the compounds to MT4 lymphocytes was performed using the CellTiter-Glo Luminescent Cell Viability Assay kit. A dose-response curve was established from three independent experiments. The anti-HIV-1 activity of each compound was assessed with 4-fold serial dilutions encompassing the inhibitory phase of the dose-response curve. The EC50 was then derived using the Quest Graph™ IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator). Gnidimacrin was isolated from Stellera chamaejasme (Thymelaeaceae) in our previous study and used as a positive control in the assays.2c
Supplementary Material
ACKNOWLEDGMENTS
The investigation was supported by the Japan Society for the Promotion of Science KAKENHI 21K06619 (W.L.) and the Sasakawa Scientific Research Grant from The Japan Science Society (No. 2021–3002) (K.O.). This work was also supported by the United States National Institute of Allergy and Infectious Diseases (NIAID) R01 AI165473 (C.-H.C.). We appreciate the editing of the manuscript by Ms. Jessica Bronchick at the Duke University School of Medicine.
Footnotes
Supporting Information.
The following files are available free of charge.
1D and 2D NMR, HRESIMS, IR, UV, and ECD spectra of compounds 1−5. (PDF)
The authors have no competing financial interests to declare.
REFERENCES
- (1).Kupchan SM; Shizuri Y; Murae T; Swenny JG; Haynes HR; Shen MS; Barrick JC; Bryan AF; vander Helm D; Wu KK J. Am. Chem. Soc 1976, 98, 5719–5720. [DOI] [PubMed] [Google Scholar]
- (2).(a) Liao SG; Chen HD; Yue JM Chem. Rev 2009, 109, 1092–1140. [DOI] [PubMed] [Google Scholar]; (b) Hayes PY; Chow S; Somerville MJ; Fletcher MT; De Voss JJ J. Nat. Prod 2010, 73, 1907–1913. [DOI] [PubMed] [Google Scholar]; (c) Asada Y; Sukemori A; Watanabe T; Malla KJ; Yoshikawa T; Li W; Koike K; Chen CH; Akiyama T; Qian K; Nakagawa-Goto K; Morris-Natschke SL; Lee KH Org. Lett 2011, 13, 2904–2907. [DOI] [PMC free article] [PubMed] [Google Scholar]; (d) Li F; Sun Q; Hong L; Li L; Wu Y; Xia M; Ikejima T; Peng Y; Song S Bioorg. Med. Chem. Lett 2013, 23, 2500–2504. [DOI] [PubMed] [Google Scholar]; (e) Yan M; Lu Y; Chen CH; Zhao Y; Lee KH; Chen DF J. Nat. Prod 2015, 78, 2712–2718. [DOI] [PMC free article] [PubMed] [Google Scholar]; (f) Guo J; Tian J; Yao G; Zhu H; Xue Y; Luo Z; Zhang J; Zhang Y; Zhang Y Fitoterapia 2015, 106, 242–246. [DOI] [PubMed] [Google Scholar]; (g) Li SF; Jiao YY; Zhang ZQ; Chao JB; Jia J; Shi XL; Zhang LW Phytochemistry 2018, 151, 17–25. [DOI] [PubMed] [Google Scholar]; (h) Otsuki K; Li W; Asada Y; Chen CH; Lee KH; Koike K Org. Lett 2020, 22, 11–15. [DOI] [PMC free article] [PubMed] [Google Scholar]; (i) Otsuki K; Zhang M; Kikuchi T; Tsuji M; Tejima M; Bai ZS; Zhou D; Huang L; Chen CH; Lee KH; Li N; Koike K; Li WJ Nat. Med 2021, 75, 1058–1066. [DOI] [PubMed] [Google Scholar]
- (3).(a) Zayed S; Adolf W; Hafez A; Hecker E Tetrahedron Lett. 1977, 39, 3481–3482. [Google Scholar]; (b) Adolf W; Seip EH; Hecker EJ Nat. Prod 1988, 51, 662–674. [DOI] [PubMed] [Google Scholar]; (c) He W; Cik M; Van Puyvelde L; Van Dun J; Appendino G; Lesage A; Van der Lindin I; Leysen JE; Wouters W; Mathenge SG; Mudida FP; De Kimpe N 2002, 10, 3245–3255. [DOI] [PubMed] [Google Scholar]
- (4).(a) Tyler MI; Howden ME J. Nat. Prod 1985, 48, 440–445. [DOI] [PubMed] [Google Scholar]; (b) Yoshida M; Feng W; Saijo N; Ikekawa T Int. J. Cancer 1996, 66, 268–273. [DOI] [PubMed] [Google Scholar]
- (5).(a) Huang L; Ho P; Yu J; Zhu L; Lee KH; Chen CH PLoS One 2011, 6, e26677. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Lai W; Huang L; Zhu L; Ferrari G; Chan C; Li W; Lee KH; Chen CH J. Med. Chem 2015, 58, 8638–8646. [DOI] [PMC free article] [PubMed] [Google Scholar]; (c) Huang L; Lai WH; Zhu L; Li W; Wei L; Lee KH; Xie L; Chen CH ACS Med. Chem. Lett 2018, 9, 268–273. [DOI] [PMC free article] [PubMed] [Google Scholar]; (d) Liu Q; Cheng YY; Li W; Huang L; Asada Y; Hsieh MT; Morris-Natschke SL; Chen CH; Koike K; K. H. Lee J. Med. Chem 2019, 62, 6958–6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- (6).Shirai K Tyuyakudaiziten; Shanghai Scientific & Technical Publishers, SHOGAKUKAN Inc, 1985; Vol. 4, 2474–2475. [Google Scholar]
- (7).(a) Baba K; Tabata Y; Taniguti M; Kozawa M Phytochemistry 1988, 28, 221–225. [Google Scholar]; (b) Baba K; Taniguti M; Yoneda Y; Kozawa M Phytochemistry 1990, 29, 247–249. [Google Scholar]; (c) Hashimoto T; Tori M; Asakawa Y Phytochemistry 1991, 30, 2927–2931. [Google Scholar]; (d) Yan J; Tong S; Chu J; Sheng L; Chen GJ Chromatogr. A 2004, 1043, 329–332. [DOI] [PubMed] [Google Scholar]; (e) Tong S; Yan J; Chen G; Lou JJ Chromatogr. Sci 2009, 47, 341–344. [DOI] [PubMed] [Google Scholar]; (f) Hu XJ; Jin HZ; Zhang WD; Zhang W; Yan SK; Liu RH; Shen YH; Xu WZ Nat. Prod. Res 2009, 23, 1259–1264. [DOI] [PubMed] [Google Scholar]; (g) Zhou T; Zhang SW; Liu SS; Cong HJ; Xuan LJ Phytochemistry Lett 2010, 3, 242–247. [Google Scholar]; (h) Li XN, Tong SQ, Cheng DP, Li QY, Yan JZ Molecules, 2014, 19, 2042–2048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- (8).Ohigashi H; Hirota M; Ohtsuka T; Koshimizu K; Tokuda H; Ito Y Symposium on the Chemistry of Natural Products 1983, 26th, 24–31. [Google Scholar]
- (9).(a) Hayes PY; Chow S; Somerville MJ; De Voss JJ; Fletcher MT J. Nat. Prod 2009, 72, 2081–2083. [DOI] [PubMed] [Google Scholar]; (b) Hayes PY; Chow S; Somerville MJ; Fletcher MT; De Voss JJ J. Nat. Prod 2019, 73, 1907–1913. [DOI] [PubMed] [Google Scholar]
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