Clerodane Diterpenoids from Croton crassifolius - Journal of Natural

Nov 29, 2012 - ... Jinan University, Guangzhou 510632, People's Republic of China ... Cui-Ping Liu , Jin-Biao Xu , Jin-Xin Zhao , Cheng-Hui Xu , Lei D...
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Clerodane Diterpenoids from Croton crassifolius Guo-Cai Wang,†,‡,§ Jia-Gui Li,†,‡,§ Guo-Qiang Li,†,‡ Jiao-Jiao Xu,†,‡ Xia Wu,†,‡ Wen-Cai Ye,†,‡ and Yao-Lan Li*,†,‡ †

Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, People's Republic of China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, and JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Guangzhou 510632, People's Republic of China

‡

S Supporting Information *

ABSTRACT: Seven new clerodane diterpenoids (1−7) were isolated from roots of Croton crassifolius, along with six known compounds. The structures were elucidated by extensive spectroscopic methods (IR, UV, HRESIMS, 1D NMR, and 2D NMR), and the structures of 1, 3, 4, and 7 were confirmed by singlecrystal X-ray diffraction analyses. Compounds 1−13 were evaluated for in vitro antiviral activity against herpes simplex virus type 1 using the cytopathic effect reduction assay.

Croton crassifolius Geisel. (Euphorbiaceae) is known as “jiguxiang” in China and distributed mainly in south and southwest China, Vietnam, Laos, and Thailand.1 The roots of C. crassifolius are used as a traditional medicine for treatment of stomachache, sore throat, rheumatism,2 and cancer.3 Previous investigations have shown that the genus Croton is a rich source of clerodane diterpenoids,4 but there are few reported studies of C. crassifolius.5−7 This article reports seven new clerodane diterpenes (1−7), together with six known compounds, spiro[furan-3(2H),1′(2′H)-naphthalene]-5′-carboxylic acid (8),8 chettaphanin I (9),9 penduliflaworosin (10),10 1,4-methano-3-benzoxepin-2(1H)-one (11),11 isoteucvin (12),10 and teucvin (13),10 isolated from the roots of C. crassifolius. Structural elucidation of the compounds was achieved by spectroscopic methods and by comparison with closely related compounds. In the case of compounds 1, 3, 4, and 7, the relative stereochemistry was confirmed by single-crystal X-ray diffraction analyses. All of the compounds were evaluated, in vitro, for antiviral activity against herpes simplex virus type 1 (HSV-1) with the cytopathic effect (CPE) reduction assay.

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RESULTS AND DISCUSSION Compound 1 was assigned the molecular formula C21H30O4 on the basis of its HRESIMS [m/z 347.2211 [M + H]+ (calcd for C21H31O4, 347.2217)]. The IR spectrum showed absorption bands due to lactone (1776, 1237 cm−1) and ester carbonyl groups (1724 cm−1). Signals of three methyl groups [δH 0.85 (3H, d, J = 6.8 Hz), 1.23 (3H, s), and 0.90 (3H, s)], one OCH3 group [δH 3.62 (3H, s)], and one olefinic proton [δH 5.79 (1H, br s)] were observed in the 1H NMR spectrum. The 13C NMR © 2012 American Chemical Society and American Society of Pharmacognosy

spectrum showed 21 carbon signals including three methyl (δC 16.3, 24.6, and 20.9), one OCH3 (δC 52.2), and two carbonyl (δC 174.2 and 178.1) groups. The 1H and 13C NMR spectra of compound 1 showed a number of similarities to those of 5-[2Received: September 18, 2012 Published: November 29, 2012 2188

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structure was confirmed by 2D NMR spectra, and compound 2 was named crassifolin B. Compound 3 was had molecular formula C21H24O5 by HRESIMS. The IR spectrum showed absorptions indicating an ester carbonyl group (1725, 1275 cm−1), conjugated carbonyl groups (1671 cm−1), and a furan ring (3140, 1455, 867 cm−1). The 1H NMR spectrum showed signals of three methyl groups [δH 0.75 (3H, d, J = 8.0 Hz), 1.41 (3H, s), and 1.15 (3H, s)], an OCH3 group [δH 3.54 (3H, s)], and five olefinic protons [δH 5.83 (1H, s), 6.01 (1H, s), 6.66 (1H, m), 7.35 (1H, br s), and 7.92 (1H, br s)], suggesting that the structure of 3 was similar to that of chettaphanin I (9).9 The most notable differences were two extra olefinic carbon signals (δC 133.5 and 128.9) in compound 3 and the absence of C-5 (δC 71.5) and C-6 (δC 24.8) signals in 9. This indicated that 3 had a double bond between C-5 and C-6. The structure was confirmed by HMBC correlations from H-6 to C-4, C-8, and C-10, from H-1, H-3, H-7, and H-19 to C-5, and from H-7 and H-8 to C-6, and compound 3 was named crassifolin C. The molecular formula of compound 4 was established as C21H24O6 (by HRESIMS). The IR spectrum showed characteristic ester carbonyl (1740, 1254 cm−1) and furan ring (3144, 1446, 814 cm−1) absorptions. 1H and 13C NMR spectra of 4 were very similar to those of penduliflaworosin (10).10 However, in contrast to 10, compound 4 had two additional carbon atoms bearing oxygen (δC 77.9, 77.4), and there was no methyl at δC 23.0 (C-19) and no methylene at δC 26.8 (C-6) in the 13C NMR spectrum. In addition, the quaternary C-4 carbon in 4 was at δC 54.1 as compared with that in 10 (δC 47.5). It was reasoned that a five-membered ether ring was present between C-6 and C-19. The existence of the ether ring was confirmed by HMBC correlations from H-19 to C-6 and from H-6 to C-19. Signal assignments were completed by analysis of HSQC, HMBC, and 1H−1H COSY correlations. The relative stereochemistry of 4 was partially established by the application of NOE experiments and confirmed by single-crystal X-ray diffraction analysis. Compound 4 was named crassifolin D. The 13C NMR signals of compound 5 were also similar to those of penduliflaworosin.10 The main differences between them were the presence of two methine carbons bearing oxygen (δC 75.9 and 63.9) in 5 instead of one OCH3 (δC 51.9) and two methylene carbons (δC 18.9 and 26.6) in 10. A lactone ring was formed between C-2 and C-4, on the basis of the HMBC correlation between H-2 and C-18. HMBC correlations from H-6 to C-5, C-8, and C-10 indicated that the OH group was attached to C-6. The relative configuration of compound 5 was established by the NOE interactions (H-19/H-2; H-19/H-6; H-17/H-6; H-8/H-11). The NOE interaction of H-12/H-17 was accepted as a definitive method to determine the configuration at C-12,12 and in this case no NOE interaction was observed. It was reasonable to deduce that the methine proton of C-12 and the methyl protons of C-17 were on the same side of the plane defined by the C-20−C-12 lactone ring. The CD spectrum of compound 5 showed a negative Cotton effect at 220−240 nm, similar to that of the known compound 8. The Cotton effect at 220−240 nm depended on the γlactone chromophore, which had two chiral carbon atoms (C-9 and C-12).13 The relative configurations of C-9 and C-12 in 5 were the same as those in 8, so the similar Cotton effects of the two compounds suggested that the absolute configurations of C-9 and C-12 in 5 and 8 were the same. Thus, the absolute configurations of C-9 and C-12 were assigned as R and S, and compound 5 was named crassifolin E.

(furan-3-yl)ethyl]-1,5,6-trimethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-1-carboxylic acid.11 The main difference between them was the presence of a γ-lactone ring [δC 171.2 (C-13), 115.2 (C-14), 174.2 (C-15), 73.3 (C-16)] in compound 1 instead of the β-substituted furan ring [δC 125.8 (C-13), 111.0 (C-14), 142.6 (C-15), 138.4 (C-16)] in 5-[2-(furan-3-yl)ethyl]1,5,6-trimethyl-1,2,3,4,5,6,7,8-octahydronaphthalene-1-carboxylic acid11 and an extra OCH3 group [δC 52.2 (C-21)] in compound 1. The presence of a γ-lactone ring attached to C-12 was confirmed by HMBC correlations of H-14/C-12, H-16/C12, and H-12/C-13, C-14, C-16. The HMBC spectrum also showed a correlation between H-21 (δH 3.62) and C-18 (δC 178.1), suggesting that the OCH3 group was located at C-18. The signal assignments were completed by analysis of HSQC, HMBC, and 1H−1H COSY correlations. Furthermore, a singlecrystal X-ray diffraction study of 1 determined the relative stereochemistry of 1 (Figure 1). Thus, the structure of 1 was elucidated as shown, and it was named crassifolin A.

Figure 1. Perspective drawings of the X-ray structures of 1, 3, 4, and 7.

The 1H and 13C NMR data of compound 2 were very similar to those of compound 1, except that the OCH3 group at δC 52.2 was missing, suggesting that 2 had a carboxylic acid group at C-4. The molecular formula was determined to be C20H29O4 by HRESIMS, and IR absorption bands confirmed the presence of a carboxylic acid group (3600−3000, 1708 cm−1). The 2189

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Compound 6 had the molecular formula C23H28O7. The NMR spectra of 6 were again similar to those of penduliflaworosin (10),10 except for an additional acetoxyl group at δC 172.2 and 21.2, an additional methine carbon bearing oxygen at δC 77.2, and one less methylene carbon in 6. The additional acetoxyl group was presumed to be attached to C-3. This was confirmed by a 1H−1H COSY correlation between H-2 and H-3 and HMBC correlations from H-3 to C18, C-19, and C-21. The relative configuration of 1 was elucidated based on NOE interactions (H-19/H-3; H-19/Hβ-6; Hα-6/H-17; H-8/H-11), and there was no NOE correlation of H-12/H-17.12 The absolute configuration of 6 was also determined by CD analysis as above-mentioned, in which the negative Cotton effect at 220−240 nm was similar to that of 5. Thus, the structure of 6 was determined to be as shown, and it was named crassifolin F. Compound 7 had an [M + H]+ ion peak at m/z 345.1315, consistent with the molecular formula C19H20O6. The 1H and 13 C NMR spectra were almost identical to those of Jatrophoidin, a known furan diterpene isolated from Croton jatrophoides.10 The most notable differences were the absence of a methoxycarbonyl moiety (δC 170.6 and 52.3) attached to C-10 in Jatrophoidin, and C-10 at δC 56.2 in Jatrophoidin was found at δC 70.3 in compound 7, suggesting that the methoxycarbonyl at C-10 in Jatrophoidin was replaced by an OH in 7. This was consistent with the molecular formula C19H20O6. The relative stereochemistry of compound 7 was confirmed by a single-crystal X-ray diffraction analysis (Figure 2), and compound 7 was named crassifolin G.

Article

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 micro-melting point apparatus (Tech, Beijing, P. R. China). Optical rotations were measured by a Jasco P-1020 digital polarimeter. UV spectra were recorded on a Jasco V-550 UV/vis instrument. A Jasco FT/IR-480 Plus spectrometer was used for scanning the IR spectra with KBr pellets. CD spectra were obtained using a Jasco J-810 circular dichroism spectrometer. 1D and 2D NMR spectra were recorded on a Bruker AV-400 spectrometer with TMS as internal standard, and chemical shifts (δ) are expressed in ppm with reference to the solvent signals. HRESIMS were determined on a Micromass Q-TOF mass spectrometer. Column chromatography (CC) was performed with silica gel (200−300 mesh, Qingdao Marine Chemical Plant, Qingdao, P. R. China) or Sephadex LH-20 (25−100 μm, Fluka, Switzerland). TLC was performed on precoated silica gel 60 F254 plates (0.2 mm thick, Merck). Preparative HPLC was performed on an Agilent system equipped with a preparative Cosmosil C18 (20 × 250 mm) column. Plant Material. The roots of C. crassifolius were collected in Conghua City, Guangdong Province, China, in August of 2010, and were authenticated by Prof. Zhou Guang-Xiong of Jinan University. A voucher specimen (No. 20100811) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, P. R. China. Extraction and Isolation. The dried and powdered roots of C. crassifolius (9.0 kg) were percolated with 95% EtOH at room temperature. Removal of EtOH from the extract under reduced pressure yielded a dark residue (1015 g), which was then suspended in H2O and extracted with petroleum ether, EtOAc, and n-butanol. The EtOAc extract was chromatographed on a silica gel column with gradient mixtures of petroleum ether−acetone (100:1 → 100:50). Ten fractions were collected and examined by TLC on silica gel. Fraction 2 was crystallized from hexane−acetone to yield the compounds 1 (3.1 g) and 11 (40 mg). Fraction 3 was subjected to silica gel CC (petroleum ether−acetone, 100:1 → 10:4) to give subfractions 3-1 to 3-4. Subfraction 3-4 was crystallized from hexane−EtOAc to afford compound 10 (20 mg). Fraction 7 was rechromatographed on silica gel (petroleum ether−acetone, 1000:5 → 100:15) to give subfractions 7-1 to 7-8. Subfraction 7-6 was a combination of compounds 3 (400 mg), 4 (200 mg), and 6 (50 mg), which were separated by preparative RP-HPLC (73% MeOH−H2O), on a Cosmosil C18 column (5 μm, 20 × 250 mm). Fraction 8 was rechromatographed on silica gel (petroleum ether−EtOAc, 100:7 → 100:15) to give subfractions 8-1 to 8-5. Fraction 8-5 was separated over silica gel (petroleum ether− EtOAc, 100:15 → 100:20) and further purified over Sephadex LH-20 (MeOH−CHCl3, 1:1) to afford 2 (500 mg). Subfraction 8-4 was a combination of compounds 12 (60 mg) and 13 (100 mg), which were separated by the same preparative RP-HPLC system (55% MeOH− H2O). Fraction 9 was purified by silica gel CC with a gradient of petroleum ether−acetone (1000:5 → 100:4) to give 9 (10 g). Fraction 10 was subjected to silica gel CC (petroleum ether−acetone, 100:1 → 10:8) to give subfractions 10-1 to 10-4. Subfraction 10-4 was crystallized from acetone−MeOH to afford 8 (200 mg). Subfraction 10-3 was separated by RP-HPLC (45% MeOH−H2O) to afford compounds 5 (40 mg) and 7 (50 mg). Compound 1: colorless crystals (MeOH); mp 135−139 °C; [α]25D +35.9 (c 1.1, MeOH); UV (MeOH) λmax (log ε) 206 (0.85) nm; IR (KBr) νmax 2940, 1776, 1724, 1465, 1237, 1111 cm−1; CD (c 1.7 × 10−3 M, MeOH) λmax (Δε) 239 (+2.76) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 347.2211 [M + H]+ (calcd for C21H31O4, 347.2217). Compound 2: colorless crystals (MeOH); mp 205−208 °C; [α]25D +33.6 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 205 (0.83) nm; IR (KBr) νmax 3600−3000, 2937, 1708, 1231, 1157 cm−1; CD (c 1.1 × 10−3 M, MeOH) λmax (Δε) 235 (+1.84) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 333.2067 [M + H]+ (calcd for C20H29O4, 333.2060). Compound 3: colorless crystals (MeOH); mp 198−205 °C; [α]25D +94.1 (c 0.99, MeOH); UV (MeOH) λmax (log ε) 203 (0.6), 281

Figure 2. CD spectra of 5, 6, and 8.

During the course of our antiviral screening of C. crassifolius against herpes simplex virus type 1 (HSV-1), respiratory synsytium virus (RSV), and coxsackievirus B3 (CVB3), the ethyl acetate extract of the roots of C. crassifolius was found to possess anti-HSV-1 activity (no data shown). Therefore, the isolated compounds from this extract were submitted to in vitro antiviral evaluation against HSV-1 using the cytopathic effect reduction assay.14 However, most of the compounds were inactive against HSV-1 at their maximal nontoxic concentrations, and only compounds 3, 4, 6, 8, and 10 showed weak effects, with IC50 values of 50, 50, 50, 25, and 50 μg/mL, respectively. Meanwhile, the IC50 value of acyclovir (positive control in the experiment) was 0.2 μg/mL. 2190

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Table 1. 1H NMR Data (δ) of Compounds 1−7 (J in Hz) 1a

position 1

3a

4b

1.66 m 2.09 m 1.59 m

1.63 m 2.07 m 1.59 m

5.83 s

2.01 1.39 2.01 1.81 1.42

2.25 m 2.92 d (13.2) 6.01 s

7

1.99 1.40 2.01 1.71 1.44

8 11

1.60 m 1.60 m

1.60 m 1.60 m

12

2.27 m 1.99 m 5.79 br s

2.27 m 1.99 m 5.78 br s

4.69 d (1.6) 0.85 d (6.8) 1.23 s 0.90 s

2 3 6

14 15 16 17 19 20 22 OMe a

2a

m m m m m

m m m d (16.3) m

2.04 2.65 2.26 2.66 2.94

m m m m d (13.8)

4.69 d (1.6) 0.83 d (6.8) 1.23 s

6.66 7.35 7.92 0.75 1.41

m br s br s d (6.8) s

0.88 s

1.15 s

3.62 s

3.54 s

2.44 1.82 1.91 1.61 2.35 1.21 4.48

m m m m m m m

1.88 m 1.87 2.78 2.33 5.60

m dd (14.1, 8.2) m dd (8.8, 8.6)

6.53 7.56 7.65 1.11 4.28 3.53

dd (1.7, 0.6) br s br s d (6.0) d (8.6) d (8.6)

6b 1.88 m

7b

2.57 br s 2.50 br s 4.87 m

1.60 dt (13.4, 3.2) 2.16 m 2.29 m

2.04 m

2.22 m

4.95 m

4.26 br s

2.34 m

1.76 m 1.90 m 5.12 m

1.92 1.88 1.93 2.64 2.31 5.61

m m m m m dd (9.1, 4.4)

1.72 1.65 1.84 2.86 2.32 5.61

m m m dd (14.0, 8.9) m dd (8.6, 6.4)

2.07 2.20 2.27 2.30 3.25 5.55

m m m m dd (14.5, 9.8) t (8.6)

6.45 7.53 7.54 1.15 1.50

br s br s br s d (6.9) s

6.50 7.55 7.59 0.99 1.36

dd (1.6, 0.6) br s br s d (6.8) s

6.49 7.55 7.62 1.02

d (0.9) br s br s d (6.8)

2.01 s 3.66 s

3.72 s

Recorded at 400 MHz in CDCl3. bRecorded at 400 MHz in CD3OD. × 10−3 M, MeOH) λmax (Δε) 229 (+12.8) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 373.1641 [M + H]+ (calcd for C21H25O6, 373.1646). Compound 5: white, amorphous solid (MeOH); mp 123−132 °C; [α]25D −37.6 (c 0.91, MeOH); UV (MeOH) λmax (log ε) 204 (0.70) nm; IR (KBr) νmax 3402, 3125, 1759, 1508, 1445, 1275, 1187, 867 cm−1; CD (c 1.2 × 10−3 M, MeOH) λmax (Δε) 235 (−30.3) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 359.1480 [M + H]+ (calcd for C20H23O6, 359.1489). Compound 6: white, amorphous solid (MeOH); mp 145−148 °C; [α]25D +24.1 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 207 (0.38) nm; IR (KBr) νmax 3140, 1747,1672, 1455, 1242, 1036, 877 cm−1; CD (c 1.6 × 10−3 M, MeOH) λmax (Δε) 241 (+0.57), 225 (−2.84) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 417.1907 [M + H]+ (calcd for C23H29O7, 417.1908). Compound 7: colorless crystals (MeOH); mp 235−250 °C; [α]25D +97.1 (c 0.72, MeOH); UV(MeOH) λmax (log ε) 228 (0.80) nm ; IR (KBr) νmax 3407, 2947, 1751, 1345, 1173, 1024, 956 cm−1; CD (c 1.4 × 10−3 M, MeOH) λmax (Δε) 228 (+8.65), 201 (−2.16) nm; 1H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 345.1315 [M + H]+ (calcd for C19H21O6, 345.1333). Compound 8: CD (c 1.8 × 10−3 M, MeOH) λmax (Δε) 225 (−4.21), 280 (−0.84) nm. X-ray Crystallographic Analysis of Compound 1. Colorless blocks, C21H30O4, Mr = 346.45, monoclinic, space group P21, a = 9.2160(4) Å, b = 11.9757 (3) Å, c = 9.9484(4) Å, V = 978.84 (6) Å3, Z = 2, dx = 1.175 Mg/m3, F(000) = 376, μ(Cu Kα) = 0.638 mm−1. Data collection was performed on a Gemini S Ultra using graphitemonochromated radiation (λ = 1.541 84 Å); 2324 unique reflections were collected to θmax = 62.65°, where 3652 reflections were observed [F2 > 2σ(F2)]. The structure was solved by direct methods (SHELXS 97) and refined by full-matrix least-squares on F2. Final R = 0.0477, Rw = 0.1255, and S = 1.054. X-ray Crystallographic Analysis of Compound 3. Colorless blocks, C21H24O5, Mr = 356.40, orthorhombic, space group P212121, a = 8.8343(2) Å, b = 12.0172(4) Å, c = 17.6834(4) Å, V = 1877.33(8) Å3, Z = 4, dx = 1.261 Mg/m3, F(000) = 760, μ(Cu Kα) = 0.729 mm−1. Data collection was performed on a Gemini S Ultra using graphite-

Table 2. 13C NMR Data (δ) of Compounds 1−7 position

1a

2a

3a

4b

5b

6b

7b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 OMe

25.4 20.2 36.5 47.9 133.2 27.7 26.9 33.9 41.2 134.8 33.7 23.8 171.2 115.2 174.2 73.3 16.3 178.1 24.6 20.9

25.3 20.1 36.6 47.6 132.6 27.8 26.9 33.8 41.2 135.2 33.6 23.7 171.4 115.1 174.3 73.3 16.2 183.7 24.4 20.8

123.7 197.2 47.7 49.3 133.5 128.9 31.7 34.4 41.6 159.9 47.0 192.9 129.1 109.2 145.1 147.3 16.0 175.1 23.1 21.4

25.5 21.2 30.1 54.1 138.3 77.4 35.5 37.2 54.4 132.7 40.9 74.5 126.4 109.5 145.7 141.8 16.9 176.5 77.9 179.7

33.4 75.9 42.3 45.7 137.9 63.9 36.5 35.4 53.9 131.9 41.0 73.8 127.9 109.3 145.7 140.6 17.9 181.3 17.8 179.4

35.0 21.1 20.6 128.6 164.4 79.1 36.8 33.7 61.1 70.3 35.9 74.1 126.8 109.4 145.7 141.4 17.9 175.3 178.5

52.7

53.1

25.1 24.8 77.2 52.8 134.5 24.6 27.6 38.2 54.6 130.2 42.2 74.2 127.6 109.5 145.7 141.0 16.3 172.2 22.7 180.5 175.4 21.2 52.4

a

5b

52.2

Recorded at 100 MHz in CDCl3. bRecorded at 100 MHz in CD3OD.

(0.38) nm; IR (KBr) νmax 3140, 2960, 1725, 1671, 1445, 1275, 1157, 867 cm−1; CD (c 1.5 × 10−3 M, MeOH) λmax (Δε) 296 (+2.02), 315 (−5.05) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 357.1696 [M + H]+ (calcd for C21H25O5, 357.1697). Compound 4: colorless crystals (MeOH); mp 201−212 °C; [α]25D +38.3 (c 1.09, MeOH); UV (MeOH) λmax (log ε) 207 (0.55) nm; IR (KBr) νmax 3144, 2925, 2864, 1740, 1446, 1254, 814 cm−1; CD (c 1.7 2191

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monochromated radiation (λ = 1.541 84 Å); 2649 unique reflections were collected to θmax = 60.71°, where 4702 reflections were observed [F2 > 2σ(F2)]. The structure was solved by direct methods (SHELXS 97) and refined by full-matrix least-squares on F2. Final R = 0.0350, Rw = 0.0865, and S = 1.050. X-ray Crystallographic Analysis of Compound 4. Colorless blocks, C21H24O6, Mr = 372.40, monoclinic, space group P21, a = 6.1689(3) Å, b = 21.3668(9) Å, c = 7.5017(4) Å, V = 909.59(8) Å3, Z = 2, dx = 1.360 Mg/m3, F(000) = 396, μ(Cu Kα) = 0.820 mm−1. Data collection was performed on a Gemini S Ultra using graphitemonochromated radiation (λ = 1.541 84 Å); 2070 unique reflections were collected to θmax = 60.75°, where 3146 reflections were observed [F2 > 2σ(F2)]. The structure was solved by direct methods (SHELXS 97) and refined by full-matrix least-squares on F2. Final R = 0.0340, Rw = 0.0852, and S = 1.067. X-ray Crystallographic Analysis of Compound 7. Colorless blocks, C19H20O6, Mr = 344.35, orthorhombic, space group P212121, a = 6.7585(2) Å, b = 7.0786(3) Å, c = 35.5061(12) Å, V = 1698.63 (10) Å3, Z = 4, dx = 1.347 Mg/m3, F(000) = 728, μ(Cu Kα) = 0.835 mm−1. Data collection was performed on a Gemini S Ultra using graphitemonochromated radiation (λ = 1.54184 Å); 2200 unique reflections were collected to θmax = 62.61°, where 3467 reflections were observed [F2 > 2σ(F2)]. The structure was solved by direct methods (SHELXS 97) and refined by full-matrix least-squares on F2. Final R = 0.0307, Rw = 0.0842, and S = 1.089. Crystallographic data for these structures have been deposited with the Cambridge Crystallographic Data Center as CCDC 907638 for 1, CCDC 907639 for 3, CCDC 907640 for 4, and CCDC 907641 for 7. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033; e-mail: [email protected]]. Cell and Virus. African green monkey kidney cells (Vero; ATCC CCL81) and HSV-1 F strain (ATCC VR733) were kindly provided by Prof. Yifei Wang of Guangzhou Biomedicine Research & Development Center, Jinan University, Guangzhou, China. Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). HSV-1 was propagated in Vero cells and maintained in DMEM with 1% FBS. Virus titers were determined by the 50% tissue culture-infective dose (TCID50) method. All cells were cultured at 37 °C in a 95% humidified atmosphere supplied with 5% CO2. The virus stock was stored at −80 °C until use. Acycloguanosine (acyclovir, ACV; Sigma) was used as a positive control. Cytopathic Effect Reduction Assay. The CPE reduction assay was adopted to evaluate the antiviral activity of the compounds as described in a previous report.14 First of all, the cytotoxic activity of the compounds on Vero cells was evaluated. Vero cells were cultured in 96-well plates. When the cells were confluent, the tested samples in serial 2-fold dilutions were added to the wells. The culture medium without samples was added to the wells as cell control. All cultures were incubated at 37 °C for 3 days. The morphology of cells was observed daily using a light microscope (Olympus Microscope DP70). The CPE was scored in comparison with the cell control (scores: 0 = 0% CPE, 1 = 0−25% CPE, 2 = 25−50% CPE, 3 = 50−75% CPE, 4 = 75−100% CPE). The maximal noncytotoxic concentration (MNCC) was defined as the maximum concentration of the samples that did not exert a toxic effect (0% CPE) under microscopic monitoring. Then, the antiviral activity of the samples was tested at the beginning concentration of MNCC. Briefly, 0.1 mL of 100 TCID50 virus suspension and serial 2-fold dilutions of the test samples were added simultaneously to the confluent Vero cell monolayers in a 96-well plate. Virus suspension and culture medium without samples were added as the virus control and cell control, respectively. The plates were incubated at 37 °C in a humidified CO2 atmosphere for 3 days. The virus-induced CPE in each well was observed against the virus control under a light microscope. The concentration that reduced 50% of CPE with respect to the virus control was estimated from the plots of the data and was defined as the 50% inhibitory concentration (IC50).

Article

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−7, X-ray data of compounds 1, 3, 4, and 7, and CD spectra of 4−8 and 13 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (86-20) 8522-1728. Fax: (86-20) 8522-1559. E-mail: [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (21612417), the National Natural Science Foundation (Nos. 81001374, 81273390), and the Research Team Program of Natural Science Foundation of Guangdong Province (No. 8351063201000003).

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