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Seven new carolignans, including two pairs of enantiomers (±)-erythro-7′-methylcarolignan E (1a/1b) and (±)-threo-7′-methylcarolignan E (2a/2b), (+)-threo-carolignan E (3a), (+)-erythro-carolignan E (4a), and (−)-erythro-carolignan Z (5), together with four known lignans (3b, 4b, 6, and 7) and six polyphenols (8–13) were isolated from the aerial parts of Euphorbia sikkimensis. The structures of the new compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined by electronic circular dichroism calculations. Seven of the isolates were examined for anti-HIV effects, and compounds 1a and 1b showed moderate anti-HIV activity with EC50 values of 6.3 and 5.3 μM.
The genus Euphorbia belongs to the family Euphorbiaceae, which is characterized by production of a white, milky latex that is somewhat toxic. The family contains about 300 genera and 7000 species. Euphorbia is one of the largest genera in the Euphorbiaceae family, with about 1600 species. Numerous chemical studies on the Euphorbia genus have been performed.1,2 Euphorbia sikkimensis Boiss. has been used to treat poisoning, malaria, rheumatism, and jaundice.3 In 2013, new and known diterpenes, triterpenoids, tocopherol derivatives, and other compounds were isolated.4,5 Two diterpenoids showed antiproliferative effects against the A549 cancer cell line, while (−)-bornyl ferulate exhibited antiangiogenic activity in a zebrafish model.5
In a continuing search for anti-HIV natural products from herbal medicinal plants, anti-HIV screening showed that an EtOAc-soluble fraction from an ethanol extract of the aerial parts of E. sikkimensis had an EC50 value of 2.5 μg/mL and an SI (selective index) value of more than 8.3. Prior literature reported that diterpenoids from Euphorbia showed potent anti-HIV activity at the nanomolar level,6,7 whereas one of these diterpenoids, a phorbol ester, also showed tumor-promoting activity.8 These results encouraged us to investigate the anti-HIV-active constituents of the EtOAc-soluble fraction, which led to the isolation and characterization of seven new lignans (1–7) and six polyphenols (8–13). Compounds 1a and 1b showed moderate anti-HIV activity with EC50 values of 6.3 and 5.3 μM, respectively. Herein are reported details of the isolation, structural elucidation, and anti-HIV activity of these compounds.
The known compounds were identified as (−)-(7′R,8′S)-erythro-carolignan E (3b),9,10 (−)-(7′S,8′S)-threo-carolignan E (4b),9,10 threo-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol (6),11,12 erythro-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol (7),11,12 (E)-ferulic acid (8),13,14 ethyl ferulate (9),14 protocatechuic acid (10),15,16 vanillic acid (11),17,18 methyl gallate (12),19 and ethyl gallate (13),19 based on spectroscopic data analysis and comparison with reported literature values.
Compound 1 (1a/1b) was obtained as a white, amorphous solid. A molecular formula of C41H44O13 was deduced from a quasimolecular ion peak at m/z 743.27039 (calcd 743.27091) observed in the negative HRESIMS. The IR spectrum showed absorption bands for OH (3407 cm−1), carbonyl (1704 cm−1), and aromatic groups (1599, 1514, and 1460 cm−1). The 1H NMR spectrum showed two pairs of doublets at δH 7.59 (1H, d, J = 15.9 Hz, H-7) and 6.27 (1H, d, J = 15.9 Hz, H-8); 7.48 (1H, d, J = 15.9 Hz, H-7) and 6.23 (1H, d, J = 15.9 Hz, H-8), together with six aromatic protons (ring A: δH 7.00 (1H, d, J = 1.8 Hz, H-2), 6.91 (1H, d, J = 8.1 Hz, H-5), 7.05 (1H, dd, J = 1.8, 8.1 Hz, H-6); ring C: δH 7.02 (1H, d, J = 1.9 Hz, H-2), 6.85 (1H, d, J = 8.2 Hz, H-5), 7.05 (1H, dd, J = 1.9, 8.2 Hz, H-6)). The 13C NMR spectrum exhibited two conjugated carbonyl carbons at δC 167.1 and 167.5 ppm. The above data indicated the presence of two feruloyl moieties. The 13C NMR spectrum (Figure S6, Supporting Information) also displayed two oxymethylene (CH2O, δC 63.9), two methylene (CH2, δC 32.1, 30.6), and two oxymethine (CHO, δC 83.5, 82.3) signals, and the COSY correlations (Figure 1) suggested one CH2O–CH2–CH2 fragment and one CH2O–CHO–CHO group. The 1H and 13C NMR data (Tables 1 and and2)2) also indicated two 1,3,4-trisubstituted aromatic units (rings B and D) and five methoxy groups. HMBC correlations (Figure 1) of H-9″ (δH 4.15, 2H, t, J = 6.5 Hz) with C-9 (δC 167.5) and H-9′ (δH 4.11, 1H, dd, J = 4.7, 11.6 Hz; 4.31, 1H, dd, J = 2.8, 11.6 Hz) with C-9 (δC 167.1) showed that two feruloyl units were connected at C-9 and C-9. All of the above data were closely similar to those of the known compound erythro-carolignan E,9,10 except for an additional methoxy group in the new compound. The position of this methoxy group at C-7′ was confirmed from the HMBC correlation of MeO (δH 3.27, s) with C-7′ (δC 83.5, d). Its relative configuration was established on the basis of NOESY correlations (Figure 2) and interpretation of 1H–1H coupling constants. A coupling constant of 15.9 Hz between H-8 (8) and H-7 (7) suggested an E-configuration for Δ7 and Δ7. The coupling constant between H-7′ and H-8′ (3.0 Hz) implied an erythro-configuration of these two protons.20–22 Thus, compound 1 (erythro-7′-methylcarolignan E) was fully identified.
The specific rotation of 1 approached zero, and no Cotton effect was found in the electronic circular dichroism (ECD) spectrum of 1, indicating a racemic mixture. Subsequent chiral resolution of 1 afforded the anticipated enantiomers 1a and 1b, which showed mirror image-like ECD curves (Figure 3a) and specific rotations (1a: [α]20D −16.2; 1b: [α]20D +17.1). In order to define the absolute configuration of the enantiomers 1a and 1b, ECD calculations were performed for the two configurations (7′R,8′S)- and (7′S,8′R)-1 using the Gaussian 09 program at the TD-DFT-B3LYP/6-311++G(d,p) level in MeCN. The calculation for the 7′R,8′S enantiomer agreed with the experimental ECD data (Figure 3a) of 1a. Thus, 1a has a 7′R,8′S-configuration. The calculated ECD spectrum for the 7′S,8′R-configuration was in good accordance with the experimental spectrum of 1b (Figure 3a). Consequently, the absolute configuration of 1b was unambiguously assigned with the 7′S,8′R-configuration. Thus, compounds 1a and 1b were given the trivial names (−)-(7′R,8′S)-erythro-7′-methylcarolignan E and (+)-(7′S,8′R)-erythro-7′-methylcarolignan E, respectively.
Compound 2 (2a/2b) was isolated as a white, amorphous solid, having the same molecular formula as that of 1 as deduced from HRESIMS. The NMR data of 2 were almost identical to those of 1, except for a slight discrepancy in the positions of the oxygenated methines (C-7′ and C-8′). Detailed 2D NMR analysis (Figures S18–20, Supporting Information) revealed that 2 shares the same planar structure as that of 1, indicating 2 to be a stereoisomer of 1 with the configuration changed at C-7′ or C-8′. A threo-configuration of 2 was further determined by the coupling constants between H-7′ and H-8′ (6.3 Hz).20,23 Compound 2 also has negligible optical activity and an ECD spectrum devoid of Cotton effects. Chiral isolation of 2 afforded the enantiomers 2a and 2b. By comparison of the calculated ECD spectra of 7′S,8′S-2 and 7′R,8′R-2 with the experimental data of 2a and 2b (Figure 3b), the absolute configurations of 2a and 2b were assigned as 7′S,8′S and 7′R,8′R. Compounds 2a and 2b were given the trivial names (−)-(7′S,8′S)-threo-7′-methylcarolignan E and (+)-(7′R,8′R)-threo-7′-methylcarolignan E, respectively.
Compound 3 (3a/3b), obtained as a white, amorphous solid, gave a molecular formula C40H42O13 determined from the HRESIMS ion at m/z 729.2542 [M – H]−(calcd C40H41O13). Its NMR data (Tables 1 and and2)2) were almost identical to those of threo-carolignan E.9,10 Compound 3 showed a negligible specific rotation and CD spectrum, indicating it to be a racemic mixture. Subsequent chiral separation of 3 afforded a pair of enantiomer 3a and 3b, which had opposite ECD curves (Figure 3c) and optical rotations (3a: [α]20D +30.4; 3b: [α]20D −29.8). The absolute configurations of 3a and 3b were determined using the same methods as described in 1a and 1b. Thus, 3a was defined as (+)-(7′R,8′R)-threo-carolignan E, and 3b was identified as the known compound (−)-(7′S,8′S)-threo-carolignan E.10
Compound 4 (4a/4b), a white, amorphous solid, showed the same molecular formula as that of 3. The two compounds had similar NMR data, except for the position of the C-7′ and C-8′ oxygenated methines. Detailed 2D NMR analysis confirmed that 4 and 3 shared the same planar structure, indicating that 4 is a stereoisomer of 3 with a different orientation of the substituents at C-7′ or C-8′. The coupling constant J7′,8′ was 3.3 Hz, so therefore the relative configuration at C-7′ and C-8′ was erythro. Compound 4 also showed a negligible optical activity and an ECD spectrum devoid of Cotton effects. Chiral isolation of 4 afforded the enantiomers 4a and 4b. By comparison of the calculated ECD spectra of the 7′R,8′S and 7′S,8′R configurations of 4 with the experimental data of 4a and 4b (Figure 3d), the absolute configurations of 4a and 4b were assigned as 7′S,8′R and 7′R,8′S, respectively. Thus, 4a was identified as (+)-(7′S,8′R)-erythro-carolignan E, and 4b was elucidated as the known compound (−)-(7′R,8′S)-erythro-carolignan E.10
Compound 5, a white, amorphous solid, gave the molecular formula C41H42O14 as determined by the HRESIMS ion at m/z 757.24921 [M – H]−(calcd 757.25018). The NMR data of 5 were similar to those of 1 with an additional methoxy group in the B-ring and two olefinic carbons (δC 134.0, 123.8) rather than two methylene signals. The B-ring proton signals at δH 6.60 (2H, d, J = 1.8 Hz) implied a symmetrical 1,3,4,5-tetrasubstituted aromatic ring, consistent with the location of the additional methoxy group in 5 at C-5″. This assignment was confirmed by the HMBC correlations (Figure 4) of the methoxy protons (δH 3.90) with C-6″ (δC 103.9). The location of the double bond was determined by HMBC correlation from the olefinic protons (δH 6.30, H-8″) with C-1″ (δC 130.9). The overall structure was further established by analysis of its 2D NMR spectra (Figures S46–49, Supporting Information). An erythro-configuration of 5 was further determined by the coupling constants between H-7′ and H-8′ (3.1 Hz).20–22 The experimental ECD spectrum of 5 showed an ECD curve with Cotton effects around 324 (−), 290 (−), and 242 (+) nm (Figure 3e). The absolute configuration of 5 was assigned as R,S through comparison with the calculated ECD spectra of (7′R,8′S)-5 (Figure 3e). Thus, 5 was elucidated as (−)-7′R,8′S-erythro-carolignan Z.
Seven compounds (1a, 1b, 2a, 2b, 5, 8, and 9) were tested for in vitro inhibitory effects against HIV-1 replication in MT4 cell lines, with AZT used as the positive control. One pair of enantiomers, 1a and 1b, showed more potent anti-HIV activity.
Optical rotations were measured on a PerkinElmer 341 automatic polarimeter. UV spectra were recorded using a Shimadzu UV-2450 spectrophotometer. CD spectra were obtained on an Applied Photophysics Chirascan spectrometer. IR spectra were determined on a Bruker Tensor 37 infrared spectrophotometer. The 1H (400 MHz), 13C (100 MHz), and 2D NMR spectra were obtained on a Bruker AM-400 with tetramethylsilane as an internal reference at 25 °C. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. HRESIMS were acquired on a Shimadzu LCMS-IT-TOF instrument, and the ESIMS data were measured on an Agilent 1200 series LC-MS/MS system. Macroporous resin D101 (Sinopharm Chemical Reagent Co. Ltd., Shanghai, People's Republic of China), RP-C18 silica gel (Fuji, 40–75 μm), MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (200–300 mesh, Marine Chemical Ltd., Qingdao, People's Republic of China), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography. Analytical and semipreparative HPLC separation were carried out on an LC-20AT Shimadzu liquid chromatography system with a Zorbax SB-C18 column (250 × 9.4 mm, 5 μm) or an Agilent SB-C18 column connected with an SPD-M20A diode array detector. Semipreparative chiral HPLC separation was carried out on an LC-20AT Shimadzu liquid chromatography system with a Phenomenex Lux cellulose-2 chiral-phase column (250 × 10 mm, 5 μm). Thin-layer chromatography (TLC) analysis was carried out on silica gel plates (Marine Chemical Ltd.). Fractions were monitored by TLC and visualized by heating plates sprayed with 5% H2SO4 in EtOH. All solvents were of analytical grade (Guangzhou Chemical Reagents Company Ltd., Guangzhou, People's Republic of China).
The aerial parts of Euphorbia sikkimensis were collected at Longli County of the Qiannan Buyi National Minority Miao National Minority Autonomous Region, Guizhou Province, People's Republic of China, in July 2014. The sample was identified by Dr. Qingwen Sun from Guiyang College of Traditional Chinese Medicine, and a voucher specimen (GZQSY356) has been deposited at the School of Pharmaceutical Science, Sun Yat-sen University.
The air-dried and powdered aerial parts (20 kg) of E. sikkimensis were extracted with 95% EtOH (3 × 40 L) at room temperature for 48 h. The solvent was concentrated under reduced pressure to give a crude extract (2.562 kg). The 95% EtOH extract was then suspended in H2O (2 L) and successively partitioned with petroleum ether (3 × 8 L), EtOAc (3 × 8 L), and n-BuOH (3 × 8 L) to yield three corresponding portions. The EtOAc-soluble extract (334 g) was chromatographed on D101 macroporous resin eluting with a step gradient of EtOH–H2O (2:8, 5:5, 7:3, 10:0) to afford four fractions (A–D). Fraction A was separated using MCI gel CHP20P eluting with an increasing gradient of MeOH–H2O from 30% to 100% and then further purified by silica gel columns to give compounds 8 (4 mg), 10 (6 mg), 11 (20 mg), 12 (2.0 g), and 13 (5.2 g). Fraction B was subjected to an RP-18 column with MeOH–H2O (30–100%) as eluent to afford four subfractions (B1–B4). Fraction B1 (16.4 g) was chromatographed on a silica gel column using CH2Cl2–MeOH (1:0, 100:1, 15:1) to yield six subfractions (B1a–B1f). Fraction B1c was submitted to separation over a Sephadex LH-20 column eluting with CH2Cl2–MeOH (1:1) to give four subfractions (B1c1–B1c4). Fraction B1c2 was further purified by semipreparative HPLC eluting with MeOH–H2O (60:40) to produce compounds 3 (10 mg) and 4 (6.2 mg). Fractions B1d and B1e were combined and further subjected to MPLC using CH2Cl2–EtOAc (50:1) and further purified with semipreparative HPLC eluting with MeCN–H2O (55:45) to give compounds 1 (8.2 mg) and 2 (15.8 mg). Compounds 5 (10.2 mg), 6 (1.0 mg), and 7 (0.8 mg) were isolated from fraction B1f by semipreparative HPLC with MeCN–H2O (50:50). Fraction C was chromatographed over a silica gel-containing column eluting with CH2Cl2–MeOH (200:1, 20:1) to obtain compound 9 (4 mg). Compounds 1–4 were further separated by semipreparative chiral HPLC (CH3OH–H2O, 9:1, 3 mL/min) to give 1a (3.5 mg, tR 30.4 min), 1b (3.4 mg, tR 33.2 min), 2a (7.3 mg, tR 31.2 min), 2b (5.7 mg, tR 34.6 min), 3a (2.9 mg, tR 28.2 min), 3b (5.5 mg, tR 25.7 min), 4a (2.1 mg, tR, 29.4 min), and 4b (2.5 mg, tR, 26.8 min), respectively.
(−)-(7′R,8′S)-erythro-7′-Methylcarolignan E (1a): white, amorphous solid; [α]20D −16.2 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 231 (4.58), 288 (3.87), 327 (4.68) nm; ECD (MeCN) λmax (Δε) 232 (1.03), 211 (−3.72) nm; IR νmax 3407, 2962, 2930, 2853, 1704, 1633, 1599, 1514, 1460, 1428, 1262, 1096, 1027, 803 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and and2;2; HRESIMS m/z 743.27039 [M – H]− (calcd for C41H43O13, 743.27091).
(+)-(7′S,8′R)-erythro-7′-Methylcarolignan E (1b): white, amorphous solid; [α]20D +17.1 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 232 (−1.03), 211 (3.80) nm; UV, IR, NMR, and HRESIMS were the same as those of 1a.
(−)-(7′S,8′S)-threo-7′-Methylcarolignan E (2a): white, amorphous solid; [α]20D −35.1 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 232 (4.36), 288 (2.92), 326 (4.64) nm; ECD (MeCN) λmax (Δε) 325 (−1.78), 295 (−1.80), 240 (−1.14) nm; IR νmax 3402, 2961, 2919, 2850, 1704, 1633, 1597, 1514, 1464, 1428, 1263, 1097, 1028, 803 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and and2;2; HRESIMS m/z 743.26971 [M – H]− (calcd for C41H43O13, 743.27091).
(+)-(7′R,8′R)-threo-7′-Methylcarolignan E (2b): white, amorphous solid; [α]20D +32.6 (c 0.2, CHCl3); ECD (MeCN) λmax (Δε) 325 (1.73), 294 (1.76), 240 (1.17) nm; UV, IR, NMR, and HRESIMS were the same as those of 2a.
(+)-(7′R,8′R)-threo-Carolignan E (3a): white, amorphous solid; [α]20D +30.4 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 333 (+1.08), 298 (+1.34), 240 (+1.58) nm; UV (MeOH) λmax (log ε) 230 (4.51), 284 (2.75), 320 (4.83) nm; IR νmax 3413, 2940, 2913, 2852, 1704, 1631, 1597, 1513, 1461, 1430, 1172, 1097, 1031, 801 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and and2;2; HRESIMS m/z 729.2542 [M – H] − (calcd for C40H41O13, 729.2553).
(−)-(7′S,8′S)-threo-Carolignan E (3b): white, amorphous solid; [α]20D −29.8 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 334 (−1.08), 298 (−1.32), 240 (−1.54) nm; UV, IR, NMR, and HRESIMS were the same as those of 3a.
(+)-(7′S,8′R)-erythro-Carolignan E (4a): white, amorphous solid; [α]20D +16.6 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 234 (+3.18), 222 (−1.22) nm; UV (MeOH) λmax (log ε) 232 (4.36), 288 (2.92), 326 (4.64) nm; IR λmax 3512, 2941, 2917, 2850, 1700, 1631, 1592, 1513, 1464, 1430, 1260, 1090, 1030, 802 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and and2;2; HRESIMS m/z 729.2542 [M – H]− (calcd for C40H41O13, 729.2553).
(−)-(7′R,8′S)-erythro-Carolignan E (4b): white, amorphous solid; [α]20D −16.4 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 234 (−3.20), 222 (+1.21) nm; UV, IR, NMR, and HRESIMS were the same as those of 4a.
(−)-(7′R,8′S)-erythro-Carolignan Z (5): white, amorphous solid; [α]23D −34.1 (c 0.24, CHCl3); ECD (MeCN) λmax (Δε) 324 (−2.54), 290 (−2.78), 242 (+1.28) nm; UV (MeOH) λmax (log ε) 219 (6.76), 286 (3.40), 326 (4.31) nm; IR λmax 3427, 2963, 2930, 2853, 1704, 1632, 1588, 1514, 1463, 1427, 1262, 1152, 1097, 1022, 801 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and and2;2; HRESIMS m/z 757.24921 [M – H]− (calcd for C41H41O14, 757.25018).
The anti-HIV-1 assay was performed as previously described.24 HIV-1 NL4-3 Nanolucsec at an infecting dose of 50 TCID50/well was used to infect MT4 cells (1 × 105 cells/mL) in the presence of compounds at various concentrations in 96-well plates. On day 3 postinfection, supernatant samples were harvested and assayed for luciferase activity using the Promega Nano-Glo luciferase assay system. The antiviral potency is defined as the drug concentration that reduces the luciferase activity by 50% (EC50).
A CytoTox-Glo cytotoxicity assay (Promega) was used to determine the cytotoxicity of the isolates. MT4 cells were cultured in the presence of various concentrations of the compounds for 3 days. Cytotoxicity of the compounds was determined by following the protocol provided by the manufacturer. The 50% cytotoxic concentration (CC50) was defined as the concentration that caused a 50% reduction of cell viability.
This study was supported in part by the National High-Tech R&D Program of China (863 Program) (2012AA020307), the Guangdong Innovative Research Team Program (No. 2009010058), and the National Natural Science Foundation of China (No. 81173470). Partial support was also received from the National Institute of Allergy and Infectious Diseases (NIAID) Grant AI33066 (K.H.L.).
The authors declare no competing financial interest.
Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01012.
UV, IR, MS, and 1D and 2D NMR for compounds 1a, 1b, 2a, 2b, 3a, 4a, and 5 (PDF)
Dedicated to Professors John Blunt and Murray Munro, of the University of Canterbury, for their pioneering work on bioactive marine natural products.
Special Issue: Special Issue in Honor of John Blunt and Murray Munro