Diketopiperazines and Sesquilignans from the Branches and Leaves of Claoxylon polot

• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolates
• Anti-Coxsackie virus B3 activity assay
• Supporting information
• Acknowledgements
• References

Abstract

Six new diketopiperazines (1–6), two new sesquilignans (7–8), and ten known compounds (9–18) were isolated from the branches and leaves of Claoxylon polot. Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of 1–3 were assigned by computational methods. Compounds 1 and 2 exhibited antiviral activity against Coxsackie B3 virus with IC₅₀ values of 14.6 and 25.9 µM, respectively.

Introduction

Claoxylon A. Juss. is a genus of small trees or shrubs in the family Euphorbiaceae distributed in the tropical regions of the Eastern Hemisphere [1]. Claoxylon polot (Burm.) Merr. (Euphorbiaceae), a tree, is widely distributed in southwest China and used to treat rheumatism, inflammation, and bleeding as Chinese folk medicine [2], [3]. Our previous studies on the branches and leaves of C. polot have led to the isolation of novel prenylbisabolane diterpenes, which showed antiviral activity against the Coxsackie virus [4]. Further chemical research on C. polot led to the isolation of ten alkaloids possessing diketopiperazine-related structures, of which six (1–6) were new ones, and eight lignans, of which two (7, 8) were new ([Fig. 1]). In this paper, we describe the isolation, structural elucidation, and biological evaluation of these compounds.

Results and Discussion

Compound 1 had UV absorption bands at 202, 220, and 297 nm, suggesting a carbonyl chromophore with extended conjunction, and IR absorption bands at 3337 and1687 cm⁻¹, indicating hydroxy and conjugated carbonyl functionalities. The molecular formula C₁₅H₁₆ N₂O₃ was established by HRESIMS (m/z 295.1062 [M + Na]⁺, calcd. 295.1053) combined with ¹H and ¹³C NMR data. The ¹H NMR spectrum showed resonances for one methyl group at δ _H 2.88 (s, 8-NCH₃), three methylene groups at δ _H 3.88 (m, H-3α), 3.61 (m, H-3β), 2.21 (m, H-4α), 2.04 (m, H-4β), and 2.37 (m, H₂-5), one olefinic proton at δ _H 7.16 (s, H-10), and five aromatic protons near δ _H 7.32 (m). The ¹³C NMR spectrum indicated the presence of two carboxyl carbons (δ _C 166.8, 161.8), six aromatic carbons (δ _C 128.4, 128.4, 128.7, 129.5, 129.5, 133.5), two olefinic carbons (δ _C 122.0, 132.6), an oxygenated methine carbon (δ _C 87.5), three methylene carbons (δ _C 20.3, 36.6, 45.4), and a methyl carbon (δ _C 34.8). The ¹H and ¹³C NMR data of 1 revealed structural features similar to those of cyclo (8-N-methyl-R-Pro-9,10-Z-didehydro-Phe) (11) [5], a known compound also isolated from the crude extract, with the exception that the methine at C-6 (δ _H/δ _C 4.22/58.7) in 11 was replaced by an oxygenated methine (δ _C 87.5) in 1.

The absolute configuration of 1 was established by theoretical calculations of its electronic circular dichroism (ECD) using the time-dependent density functional theory (TD-DFT) method. On the basis of one chiral carbon of 1, there were only two possible structures considered for it with an absolute configuration of 6S or 6R. Their optimized geometries were obtained, and then the ECD spectra were calculated at the B3LYP/6–31 G(d) level with the TD-DFT/PCM model in methanol solution [6]. The results showed that the calculated ECD spectra of the (6S)-1 and (6R)-1 exhibited a CD curve similar and opposite to that of the experimental spectrum of 1, respectively ([Fig. 2]). Accordingly, the calculated ECD spectrum of (6S)-1 exhibited diagnostic positive and negative CEs around 290 and 205 nm, respectively, corresponding to the experimental CEs observed around 290 and 200 nm. Therefore, the absolute configuration of 1 was determined as 6S. In addition, the specific rotation value of 1 ([α]_D ²⁰ +50.4) revealed that the hydroxy group at C-6 was β orientated, which has been previously studied in the literature [7], [8], [9], [10], [11], [12]. Thus, compound 1 was determined to be cyclo (D-6-Hyp-9,10-en-Phe).

Compounds 2 and 3 gave pseudomolecular ions [M + H]⁺ at m/z = 271.1444 and 271.1439, respectively, (calcd. 271.1441) by HRESIMS, which is consistent with a molecular formula of C₁₆H₁₈N₂O₂. Interpretation of their 1D and 2D NMR spectroscopic data ([Tables 1] and [2]) established the same planar structure, which resembled that of 1, with the exception of methine at C-6 (δ _H/δ _C 4.31/59.4, 4.35/57.5) in 2 and 3 rather than an oxygenated methine carbon (δ _C 87.5) in 1, and one more methyl group (δ _H/δ _C 1.15/17.6, 1.13/18.8) in 2 and 3. The location of the methyl group at C-4 was then confirmed by the HMBC correlations from 4-CH₃ to C-3, C-4, and C-5. The aforementioned data combined with the specific rotation values of 2 ([α]_D ²⁰ +227.4) and 3 ([α]_D ²⁰ −266.7) indicated that they could be enantiomers, which was supported by the computational methods. The (4S, 6R)-2 and (4R, 6S)-3 ECD spectra were calculated at the B3LYP/6–31 G(d) level with the TD-DFT/PCM model in methanol solution, and the results showed that the calculated ECD spectrum of the (4S, 6R)-2 exhibited a CD curve similar to that of the experimental spectrum of 2, and the calculated ECD spectrum of (4R, 6S)-3 exhibited a CD curve similar to that of the experimental spectrum of 3 ([Fig. 3]). Therefore, the absolute configurations of 2 and 3 were determined as (4S, 6R) and (4R, 6S), respectively.

Compound 4 gave the pseudomolecular ion [M + H]⁺ at m/z 287.1398, consistent with the molecular formula C₁₆H₁₈N₂O₃. The ¹H and ¹³CNMR data for 4 revealed structural features nearly identical to those of 3, with the exception that the methine at C-6 (δ _H/δ _C 4.35/57.5) in 3 was replaced by an oxygenated methine carbon (δ _C 88.5) in 4 ([Tables 1] and [2]). The specific rotation value of 4 ([α]_D ²⁰ −256.4) indicated the absolute configuration of 4 was 6R [7], [8], [9], [10], [11], [12]. Thus, compound 4 was determined to be cyclo (4R) 4-methyl-(L)-6-Hyp-8-N-methyl-9,10-en-Phe.

Compound 5 was established as C₁₅H₁₆ N₂O₃ based on HRESIMS (m/z 273.1244 [M + H]⁺, calcd 273.1234) and NMR data ([Tables 1] and [2]). The ¹H and ¹³C NMR data of compound 5 were similar to those of compound 1. The differences were the presence of one amino proton at δ _H (7.87, br s) and a methoxy group (δ _H/δ _C 3.30/51.4) in 5. However, the same molecular C₁₅H₁₆ N₂O₃ of 1 and 5 revealed that the N-methyl in 1 changed to methoxy in 5. The specific rotation value of 5 ([α]_D ²⁰ −122.7) revealed that the methoxy group at C-6 was α orientated, indicating the 6R configuration, and compound 5 was determined to be cyclo (L)-6-methoxy-Pro-9,10-en-Phe.

Compound 6 was determined to be C₁₆H₁₈ N₂O₃ based on HRESIMS and NMR data ([Tables 1] and [2]). The ¹H and ¹³C NMR spectra of 5 revealed structural features nearly identical to those of 5, with the exception of one more methyl group (δ _H/δ _C 2.91/32.5) at N-8 in 6 compared with 5. The structural variation was confirmed by ¹H−¹H COSY, HSQC, and HMBC spectra. Then, the specific rotation value of 6 ([α]_D ²⁰ −83.8) indicated the absolute configuration of 6 was 6R. Thus, compound 6 was determined to be cyclo (L)-6-methoxy-Pro-8-N-methyl-9,10-en-Phe.

Compound 7 was obtained as yellow gum, and the molecular formula was assigned as C₃₁H₃₄O₁₁ based on HRESIMS (m/z 605.1989 [M + Na]⁺, calcd. 605.1993) and NMR data ([Table 3]). The IR spectrum showed strong bands at 3387 cm⁻¹, indicating the existence of a hydroxy group. The ¹H NMR spectrum showed the presence of one aromatic ring with three coupled protons in an ABX system and two aromatic rings, each one with two protons, which were located at meta sites relative to each other. The presence of a trans double bond was confirmed by the resonances at δ _H 6.73 (d, J = 16.0 Hz, H-7′′) and 6.41 (dd, J = 16.0, 5.6 Hz, H-8′′). Analysis of the ¹H and ¹³C NMR data for compound 7 resembled that of (7R, 8R)-7-(4-hydroxy-3-methoxyphenyl)-8-(4-(3-(hydroxy-methyl)-5-(3-(E)-hydroxyprop-1-enyl)-7-methoxybenzofuran-2-yl)-2-methoxyphenoxy)propane-7,9-diol [13] and buddlenol B [14]. The coupling constant value of 4.8 Hz between H-7′ and H-8′ suggested the erythro relative configuration. In addition, the Δδ _C8-C7 values [Δδ _C8-C7 (threo) >Δδ _C8-C7 (erythro)] were also applicable to threo and erythro aryl glycerols without substituent(s) at C-7 or/and C-8 of the glycerol moiety, the small difference of a chemical shift (Δδ _C8′-C7′, 13.3 ppm) of 7 indicated the erythro aryl glycerol moiety ([Table 3]) [15], [16]. The absolute configuration at the C-8′ position was considered to S, as the CD spectrum showed a positive Cotton effect at 249.5 nm according to the reported literature [17], [18]. (Fig. S47, Supporting Information). Thus, compound 7 was determined to be (7′R, 8′S)-7-(4-hydroxy-3-methoxyphenyl)-8-(4-(2,6-dimethoxyphenyl)-5-(3-(E)-hydroxyprop-1-enyl)-7-methoxybenzofuran-2-yl)-2-methoxyp-henoxy) propane-7,9-diol.

For compound 8, the HRESIMS gave the pseudomolecular ion [M + Na]⁺ at m/z 605.2002, corresponding with the molecular formula C₃₁H₃₄O₁₁. The ¹H and ¹³CNMR spectra of 8 were very similar to that of 7, indicating the same planar structure for 8 as that of 7. The threo relative configuration between C-7′ and C-8′was indicated by the ³ J _{H7′, H8′} (6.4 Hz) combined with the large value of Δδ _C8′-C7′ (14.5 ppm) ([Table 3]). Meanwhile, the positive Cotton effect at 248 nm in the CD spectrum revealed the 8′S configuration, and the absolute configuration of 8 was determined to be 7′S, 8′S.

The known compounds 9–18 isolated from the crude extract were identified as cyclo (L)-Pro-(L)-Phe (9) [9], [10], cyclo (D)-Pro-9,10-en-Phe (10) [19], cyclo (D)-Pro-8-N-methyl-9,10-en-Phe (11) [5], neoechinulin A (12) [20], (−)-(7R, 7′R, 7′′S, 8S, 8′S,8′′S)-4′,4′′-Dihydroxy-3,3′,3′′,5-tetramethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (13) [15], (−)-(7R, 7′R, 7′′R, 8S, 8′S, 8′′S)-4′,4′′-Dihydroxy-3,3′,3′′,5-tetramethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (14) [15], (−)-(7R, 7′R, 7′′S, 8S, 8′S, 8′′S)-4′,4′′-Dihydroxy-3,3′,3′′,5,5′-pentamethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (15) [15], (−)-(7R, 7′R, 7′′R, 8S, 8′S, 8′′S)-4′,4′′- Dihydroxy-3,3′,3′′,5,5′-pentamethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (16) [15], (−)-(7R, 7′R, 7′′S, 8S, 8′S, 8′′S)-4′,4′′-Dihydroxy-3,3′,3′′,5,5′,5′′-hexamethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (17) [15], and (−)-(7R, 7′R, 7′′R, 8S, 8′S, 8′′S)-4′,4′′-Dihydroxy-3,3′,3′′,5,5′,5′′-hexamethoxy-7,9′:7′,9-diepoxy-4,8′′-oxy-8,8′-sesquineolignan-7′′,9′′-diol (18) [15], respectively, by comparison of their NMR and MS data with the reported values.

The anti-Coxsackie virus B3 activity assay was carried out to evaluate the bioactivity of the isolated compounds. Compounds 1 and 2 exhibited antiviral activities against Coxsackie virus B3 with IC₅₀ values of 14.6 and 25.9 µM, respectively, whereas the TC₅₀ values for compounds 1–8 were higher than 100 µM, as shown in [Table 4]. Interestingly, the orientation of the substituent at C-6 may play the key role in anti-Coxsackie virus B3 activity. For example, compounds 1 and 2 with the β orientation of the substituent at C-6 showed anti-Coxsackie virus B3 activity, whereas compounds 3–6 with the α orientation of the substituent at C-6 exhibited no detectable activity.

Materials and Methods

General experimental procedures

Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter. UV spectra were obtained with a JASCO V650 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission). NMR spectra were recorded on an INOVA-500 spectrometer. HRESIMS data were recorded on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-10A detector using a YMC-Pack ODS-A column (250 × 50 mm, 5 µm). Sephadex LH-20, ODS (45–70 µm), polyamide (30–60 mesh), and silica gel (200–300 mesh) were used for column chromatography. Silica gel GF254 was used for TLC. Ribavirin (purity ≥ 98 %) was purchased from Sigma-Aldrich.

Plant material

The branches and leaves of C. polot (Burm.) Merr. were collected from Guangxi Province, Peopleʼs Republic of China, in July 2010, and identified by Prof. Guang-Zhao Li of the Guangxi Institute of Botany, The Chinese Academy of Sciences. A voucher specimen (ID-S-2504) has been deposited in the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peopleʼs Republic of China.

Extraction and isolation

Air-dried branches and leaves of C. polot (Burm.) Merr. (52.3 kg) were extracted with 95 % EtOH (150 L × 3) and concentrated in vacuo to give the crude extract (2.5 kg), which was suspended in water and then successively partitioned with petroleum ether, CHCl₃, EtOAc, and n-BuOH. The CHCl₃ fraction (220 g) was subjected to silica gel column chromatography (12.0 × 50 cm, 200–300 mesh, 1500 g) and eluted with petro/Me₂CO to obtain seven fractions (Fr. A–Fr. G). Fraction B–D (55.4 g) was applied to a column of polyamide (10.0 × 100 cm, 30–60 mesh, 1500 g) and eluted with 30 %, 50 % and 95 % EtOH to obtain three corresponding subfractions P-a− P-c. Subfraction P-a (3.18 g) was then subjected to column chromatography on ODS (5.0 × 30 cm, MeOH/H₂O, 10 : 90 to 95 : 5) to give eight major fractions (P-a-1–P-a-8). Fraction P-a-1 (210 mg) was separated by RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 30 : 70, 5 mL/min) to afford 1 (6.4 mg). Fraction P-a-2 (640 mg) was separated by a Sephadex LH-20 column (2.0 × 150 cm) using MeOH as the eluent to obtain six subfractions and then was separated by RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 35 : 65, 5 mL/min) to afford 4 (2.3 mg), 5 (5.1 mg), 6 (3.2 mg), and 11 (63.8 mg). Fraction P-a-3 (370 mg) was further separated by Sephadex LH-20 (2.0 × 150 cm, MeOH/H₂O, 90 : 10) and RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 40 : 60, 5 mL/min) to afford 9 (2.5 mg), 10 (6.2 mg), and 12 (24.7 mg). Fraction P-a-4 (110 mg) was further separated by Sephadex LH-20 (2.0 × 150 cm, MeOH) and RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 50 : 50, 5 mL/min) to afford 2 (30.6 mg) and 3 (4.3 mg).

The EtOAc extract (90 g) was subjected to silica gel column chromatography (12.0 × 50 cm, 200–300 mesh, 1000 g) and eluted with a petro/Me₂CO gradient, yielding seven fractions (E1–E7). Fraction E5–E7 (35 g) was subjected to polymide resin CC (8.0 × 120 cm, 30–60 mesh, 500 g) eluting with 30 %, 50 % and 95 % EtOH to yield three fractions (E7-1–E7-3). Fraction E7-1 (18 g, eluted with 30 % EtOH) was purified by ODS CC (5.0 × 30 cm) eluting with MeOH/H₂O (5 : 95 to 90 : 10) to yield six fractions (E7-1-1–E7-1–6). Fraction E7-1–2 (0.85 g) was subjected to separation on a Sephadex LH-20 column (2.0 × 150 cm) using MeOH/H₂O (70 : 30) and was then separated by RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 25 : 75, 5 mL/min) to afford 7 (7.4 mg) and 8 (2.9 mg). Fraction E7-1–4 (3.4 g) was subjected to separation on a Sephadex LH-20 column (2.0 × 150 cm) using MeOH/H₂O (80 : 20) and then separated by RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 30 : 70, 5 mL/min) to afford 13 (29.7 mg) and 14 (5.9 mg). Fraction E7-1–6 (3.4 g) was subjected to separation on a Sephadex LH-20 column (2.0 × 150 cm) using MeOH/H₂O (80 : 20) and then separated by RP-HPLC (2.5 × 25 cm, CH₃CN/H₂O, 30 : 70, 5 mL/min) to afford 15 (40.2 mg), 16 (26.6 mg), 17 (65.3 mg), and 18 (3.8 mg). The purity of all the compounds tested was greater than 95 % as determined by HPLC-ELSD and NMR.

Isolates

cyclo (D)-6-Hyp-9,10-en-Phe (1): white amorphous powder, [α]_D ²⁰ +50.4 (c 0.046, MeOH); UV (MeOH) λ _max (log ε) 202 (4.69), 220 (4.42), 297 (4.45) nm; IR ν _max 3337, 2956, 1687, 1624, 1475, 1422, 1389, 1093,772, 701 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, see [Table 1]; ¹³C NMR (125 MHz, CDCl₃) data, see [Table 2]; HRESIMS (positive) m/z 295.1055 [M + Na]⁺ (calcd. for C₁₅H₁₆ N₂NaO₃, 295.1062).

cyclo (4S) 4-methyl-(D)-Pro-8-N-methyl-9,10-en-Phe (2): yellow oil, [α]_D ²⁰ +227.4 (c 0.023, MeOH); UV (MeOH) λ _max (log ε) 205 (4.35), 221 (4.39), 291 (4.53) nm; IR ν _max 3381, 2959, 2876, 1685, 1627, 1446, 1424, 1374, 701 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, see [Table 1]; ¹³C NMR (125 MHz, CDCl₃) data, see [Table 2]; HRESIMS (positive) m/z 271.1444 [M + H]⁺ (calcd. for C₁₆H₁₉ N₂O₂, 271.1441).

cyclo (4R) 4-methyl-(L)-Pro-8-N-methyl-9,10-en-Phe (3): yellow oil, [α]_D ²⁰ −226.7 (c 0.027, MeOH); UV (MeOH) λ _max (log ε) 201 (4.69), 222 (4.61), 289 (4.69) nm; IR ν _max 3377, 2950, 2931, 2875, 1687, 1628, 1445, 1423, 1370, 1116, 700 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, see [Table 1]; ¹³C NMR (125 MHz, CDCl₃) data, see [Table 2]; HRESIMS (positive) m/z 271.1439 [M + H]⁺ (calcd. for C₁₆H₁₉ N₂O₂, 271.1441).

cyclo (4R) 4-methyl-(L)-6-Hyp-8-N-methyl-9,10-en-Phe) (4): white amorphous powder, [α]_D ²⁰ −256.4 (c 0.011, MeOH); UV (MeOH) λ _max (log ε) 201 (4.64), 219 (4.39), 297 (4.38) nm; IR ν _max 3257, 3057, 2984, 2960, 2889, 1695, 1655, 1619, 1469, 1423, 1339, 1101, 1016, 932, 772, 697 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, see [Table 1]; ¹³C NMR (125 MHz, CDCl₃) data, see [Table 2]; HRESIMS (positive) m/z 287.1398 [M + H]⁺ (calcd. for C₁₆H₁₉ N₂O₃, 287.1424).

cyclo (L)-6-methoxy-Pro-9,10-en-Phe (5): white amorphous powder, [α]_D ²⁰ −122.7 (c 0.015, MeOH); UV (MeOH) λ _max (log ε) 201 (4.61), 224 (4.59), 303 (4.70) nm; IR ν _max 3358, 2936, 1686, 1627, 1425, 1065 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, see [Table 1]; ¹³C NMR (125 MHz, CDCl₃) data, see [Table 2]; HRESIMS (positive) m/z 273.1244 [M + H]⁺ (calcd. for C₁₅H₁₇ N₂O₃, 273.1234).

cyclo (L)-6-methoxy-Pro-8-N-methyl-9,10-en-Phe (6): white amorphous powder, [α]_D ²⁰ −83.8 (c 0.013, MeOH); UV (MeOH) λ _max (log ε) 201 (4.68), 223 (4.56), 297 (4.57) nm; IR ν _max 3024, 2951, 1685, 1623, 1414, 1366, 1091, 1056, 771, 704 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, see [Table 1]; ¹³C NMR (125 MHz, CDCl₃) data, see [Table 2]; HRESIMS (positive) m/z 287.1393 [M + H]⁺ (calcd. for C₁₆H₁₉ N₂O₃, 287.1390).

(7′R, 8′S)-7-(4-hydroxy-3-methoxyphenyl)-8-(4-(2,6-dimethoxyphenyl)-5-(3-(E)-hydroxyprop-1-enyl)-7-methoxybenzofuran-2-yl)-2-methoxyphenoxy) propane-7,9-diol (7): yellow gum, [α]_D ²⁰ −10.5 (c 0.025, MeOH); UV (MeOH) λ_max 203, 232, 279, 306 nm; IR ν_max 3387, 2940, 1599, 1504, 1464, 1279, 1128 cm⁻¹; ¹H NMR (800 MHz, CD₃OD) and ¹³C NMR (200 MHz, CD₃OD) data, see [Table 3]; HRESIMS (positive) m/z 605.1989 [M + Na]⁺ (calcd. for C₃₁H₃₄O₁₁Na, 605.1993).

(7′S, 8′S)-7-(4-hydroxy-3-methoxyphenyl)-8-(4-(2,6-dimethoxyphenyl)-5-(3-(E)-hydroxyprop-1-enyl)-7-methoxybenzofuran-2-yl)-2-methoxyphenoxy) propane-7,9-diol (8): yellow gum, [α]_D ²⁰ −16.7 (c 0.020, MeOH); UV (MeOH) λ_max 203, 230, 279, 305 nm; IR ν_max 3409, 2937, 2876, 1598, 1505, 1464, 1278, 1128 cm⁻¹; ¹H NMR (800 MHz, CD₃OD) and ¹³C NMR (200 MHz, CD₃OD) data, see [Table 3]; HRESIMS (positive) m/z 605.2002 [M + Na]⁺ (calcd. for C₃₁H₃₄O₁₁Na, 605.1993).

Anti-Coxsackie virus B3 activity assay

Confluent Vero cells (African green monkey kidney cell line) grown in 96-well microplates were infected with 100 median tissue culture infective doses (100 TCID50) of Cox B₃ virus. After 1 h of adsorption at 37 °C, the monolayers were washed with phosphate-buffered saline (PBS) and incubated at 37 °C in maintenance medium [MEM plus 2 % fetal bovine serum (FBS)] in the absence or presence of various concentrations of test compounds. The viral cytopathic effect (CPE) was observed when the viral control group reached 4+, and the antiviral activity of the tested compounds was determined by Reed-Muench analysis [4].

Supporting information

The UV, IR, HRESIMS, and NMR spectra for compounds 1–8 are available as Supporting Information.

Acknowledgements

This project was supported by the National Science and Technology Project of China (No. 20012ZX09301002–002), the Natural Science Foundation of China (No. 21132009), and PCSIRT (No. IRT1007). We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for the IR, UV, NMR, and MS measurements.

Conflict of Interest

The authors declare no competing financial interest.

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• 9 Poisel H, Schmidt U. Asymmetrische Synthese aromatischer α-Aminosauren und N-Methyl-α-Aminosäuren. – Synthese von L-Dopa. – Über die katalytische Hydrierung ungesättigter Cyclodipeptide. Chem Ber 1973; 106: 3408-3420
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• 15 Xiong L, Zhu CG, Li YR, Tian Y, Lin S, Yuan SP, Hu JF, Hou Q, Chen NH, Yang YC, Shi JG. Lignans and neolignans from Sinocalamus affinis and their absolute configurations. J Nat Prod 2011; 74: 1188-1200
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• 17 Liao SG, Wu Y, Yue JM. Alkaloids from Daphniphyllum longeracemosum . Helv Chim Acta 2006; 89: 73-80
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• 20 Li C, Gu QQ. Study on the Antitumor Metabolites of a Sponge-Derived Fungus, Aspergillus repens . Periodical of Ocean University of China 2010; 40: 69-71
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Correspondence

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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 14 Houghton PJ. Lignans and neolignans from Buddleja davidii . Phytochemistry 1985; 24: 819-826
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 20 Li C, Gu QQ. Study on the Antitumor Metabolites of a Sponge-Derived Fungus, Aspergillus repens . Periodical of Ocean University of China 2010; 40: 69-71
  PubMedSearch in Google Scholar

• Supplementary Material