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Antiviral Triterpenes from the Twigs and Leaves of Lyonia ovalifolia Xiao-Jing Lv,† Yong Li,† Shuang-Gang Ma,† Jing Qu,† Yun-Bao Liu,† Yu-Huan Li,‡ Dan Zhang,† Li Li,† and Shi-Shan Yu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Eleven new 9,10-seco-cycloartan triterpene glycosides (1−11), seven new lanostane triterpene glycosides (12−18), and two new ursane triterpenoids (19−20) were isolated from the twigs and leaves of Lyonia ovalifolia. The structures of these compounds were elucidated by extensive MS and NMR spectroscopic analysis. The absolute configuration of compound 1a (the aglycone of 1) was established by X-ray crystallography, and that of C-24 in compounds 2, 7, and 12 was established by Mo2(OAc)4-induced electronic circular dichroism experiments. All compounds were evaluated for their antiviral [herpes simplex virus-1 (HSV-1), influenza A/95−359 (A/95−359), and Coxsackie B3 (CVB3)] activity. Compounds 1, 1a, 2a, 12a, 13, and 16 exhibited potent activity against HSV-1, with IC50 values from 2.1 to 14.3 μM, while compounds 1a, 2a, 12a, 13, and 12−2a exhibited potent activity against A/95−359, with IC50 values from 2.1 to 11.1 μM. In turn, compounds 1, 1a, 2a, 12a, and 13 exhibited potent activity against CVB3, with IC50 values from 2.1 to 11.1 μM. Lyonia ovalifolia (Wall.) Drude (Ericaceae) is a deciduous tree distributed mainly in southern and southwestern mainland China, Taiwan, Pakistan, Nepal, northern India, and Thailand. It has been used as a traditional Chinese medicine for the treatment of swelling, dermatitis, and bowel inflammation. Previous phytochemical studies on plants of the Ericaceae family have led to the isolation of several types of secondary metabolites, such as grayanane diterpenoids,1−5 triterpene glycosides,6−8 lignans,9,10 and flavonoids.11,12 These compounds exhibit diverse biological activities, including cAMP regulating,3 analgesic,4,5 and tyrosinase inhibitory effects.8 However, the bioactive components of L. ovalifolia have not been well-defined. In the present investigation, 20 new triterpenes (1−20) were isolated from the twigs and leaves of L. ovalifolia. Herein, the isolation and structural elucidation as well as the antiviral activities of these compounds are described.

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RESULTS AND DISCUSSION Compound 1 was obtained as a white powder. Its molecular formula was established as C36H58O10 based on the 13C NMR and HRESIMS (m/z 673.3944 [M + Na]+, calcd for C36H58O10Na, 673.3928) data, indicating eight degrees of unsaturation. The IR spectrum exhibited absorption bands for hydroxy (3386 cm−1) and carbonyl (1707, 1682 cm−1) groups. Its 1H NMR spectrum displayed signals of five three-proton singlets, a three-proton doublet, and an anomeric proton at δH © 2016 American Chemical Society and American Society of Pharmacognosy

4.86 (1H, d, J = 7.8 Hz, H-1′), suggesting that 1 is a triterpene monoglycoside. Acid hydrolysis of 1 with HCl afforded a new Received: June 26, 2016 Published: October 28, 2016 2824

DOI: 10.1021/acs.jnatprod.6b00585 J. Nat. Prod. 2016, 79, 2824−2837

Journal of Natural Products

Article

Table 1. 1H NMR Data of Compounds 1−6 in Pyridine-d5 (δ in ppm, J in Hz) position

1b

1ab

2b

2ab

1

2.08 m 2.05 m 2.04 m 1.73 m 3.76 dd (11.1, 2.1) 2.28 m 2.13 m 2.00 m 1.25 m 1.79 m 2.14 m 1.68 m 1.50 m 2.96 m 1.65 m 2.70 m 1.30 m 2.36 m 1.54 m 1.76 m 0.99 s 2.24 m 1.66 m 1.59 m 0.95 d (6.4)


2.08 m 2.06 m 2.04 m 1.74 m 3.77 dd (11.3, 2.2) 2.31 m 2.15 m 2.02 m 1.25 m 1.80 m 2.17 m 1.69 m 1.53 m 2.99 m 1.70 m 2.70 m 1.29 m 2.35 m 1.52 m 1.81 m 1.01 s 2.26 m 1.67 m 1.63 m 1.03 m


2.22 m 1.18 m 2.03 m 1.52 m 3.66 d (10.1) 1.46 s 1.49 s 1.04 s 1.24 s

2.22 m 1.20 m 2.04 m 1.53 m 3.67 d (9.6) 1.47 s 1.50 s 1.16 s 1.28 s

2 3 6 7 8 9 11 12 15 16 17 18 19 20 21 22 23

2.02 1.38 2.93 2.93

m m m m

2.02 1.39 2.94 2.94

m m m m

1.52 1.51 1.04 1.23

s s s s

1.53 1.52 1.15 1.27

s s s s

2bb m m m m m

2.36 m 2.18 m 1.87 m 0.90 m 1.75 m 1.96 m 1.67 m 1.48 m 2.74 m 1.65 m 2.47 m 1.24 m 2.10 m 1.43 m 1.43 m 0.95 s 2.25 m 1.69 m 1.58 m 1.01 d (6.4) 2.20 m 1.19 m 2.02 m 1.55 m 3.69 m 1.54 s 1.51 s 1.22 s 1.28 s 3.58 s

4b 2.08 2.00 2.04 1.74 3.77

m m m m m

5a

6c 2.06 m 2.06 m 2.03 m 1.73 m 3.72 d (10.8) 2.32 m 2.18 m 2.06 m 1.25 m 1.82 m 2.17 m 1.69 m 1.54 m 3.02 m 1.72 m 2.73 m 1.33 m 2.49 m 1.70 m 1.85 m 1.04 s 2.27 m 1.69 m 1.64 m 0.99 d (6.2) 1.85 m 1.85 m 1.82 m 1.73 m 3.81 m 1.36 s 1.40 s 1.04 s 1.27 s

2.08 m 2.08 m 2.04 m 1.75 m 3.74 dd (11.5, 2.0) 2.36 m 2.19 m 2.02 m 1.26 m 1.82 m 2.17 m 1.69 m 1.52 m 3.00 m 1.70 m 2.70 m 1.29 m 2.36 m 1.52 m 1.82 m 1.01 s 2.26 m 1.68 m 1.63 m 1.04 m

2.29 m 2.14 m 2.02 m 1.25 m 1.81 m 2.16 m 1.69 m 1.54 m 3.00 m 1.70 m 2.72 m 1.33 m 2.47 m 1.71 m 1.83 m 1.04 s 2.26 m 1.67 m 1.64 m 0.99 d (6.4)


2.23 m 1.19 m 2.04 m 1.52 m 3.66 d (10.0) 1.47 s 1.49 s 1.05 s 1.32 s

1.85 m 1.85 m 1.77 m 1.71 m 3.81 m 1.35 s 1.39 s 1.04 s 1.23 s

1.85 m 1.85 m 1.74 m 1.72 m 3.82 m 1.36 s 1.40 s 1.05 s 1.32 s

24 26 27 28 29 31 1′

4.86 d (7.8)

4.87 d (7.7)

4.81 d (7.7)

4.87 d (7.7)

4.82 m

2′ 3′

3.96 m 4.25 m

3.96 m 4.25 m

3.94 t (8.1) 4.21 t (8.9)

3.96 t (8.1) 4.25 m

3.95 m 4.22 m

4′ 5′

4.23 m 3.93 m

4.24 m 3.94 m

4.05 t (9.3) 3.98 m

4.23 m 3.93 m

4.06 t (9.1) 3.99 m

6′

4.53 dd (11.5, 2.1) 4.38 dd (11.5, 5.2)

4.55 dd (11.6, 2.2) 4.39 dd (11.6, 5.2)

4.95 d (11.5)

4.54 dd (11.5, 2.3) 4.38 dd (11.6, 5.3)

4.95 dd (11.5, 1.9) 4.79 m

4.79 dd (11.6, 6.7) 1.96 s

8′ 1″ 2″ 3″ 4″ 5″ a

2.16 2.12 1.91 1.86 3.73

3c

4.78 d (7.3)

1.96 s 4.80 m

4.44 t (7.7) 4.12 dd (9.1, 3.0) 4.27 m 4.28 m 3.79 m

4.45 t (7.4) 4.13 dd (9.1, 3.3) 4.28 m 4.29 m 3.80 m

4.72 d (6.7) 4.36 t (7.6) 4.18 d (7.8) 4.32 m 4.31 m 3.77 m

4.80 d (7.2) 4.45 t (8.1) 4.13 d (7.9) 4.29 m 4.30 m 3.80 m

Recorded at 400 MHz. bRecorded at 500 MHz. cRecorded at 600 MHz.

the anomeric proton in the 1H NMR spectrum (Table 1) suggested a β-configuration for the glucopyranosyl moiety. The

triterpene aglycone (1a: C30H48O5), named lyonifolic acid A, and glucose. The relatively large coupling constant (7.8 Hz) for 2825



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Table 2. 13C NMR Data of Compounds 1−6 in Pyridine-d5 (δ in ppm)

a

position

1b

1ab

2b

2ab

2bb

3c

4b

5a

6c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″

32.0 24.9 83.1 40.1 141.3 28.5 32.0 51.2 36.1 132.4 31.9 34.2 48.0 63.7 30.3 29.5 52.6 18.1 43.2 35.9 19.1 30.7 33.7 216.6 77.1 27.6 27.5 21.6 25.5 178.6

32.4 29.2 75.6 41.2 141.5 28.7 32.1 51.4 36.3 132.5 32.1 34.3 48.1 63.8 30.4 29.6 52.7 18.2 43.4 36.0 19.1 30.8 33.7 216.6 77.1 27.6 27.6 21.0 25.7 178.7

32.1 24.9 83.1 40.2 141.3 28.6 32.0 51.3 36.2 132.4 32.0 34.3 48.1 63.7 30.4 29.6 52.8 18.1 43.3 36.8 19.6 34.7 29.5 80.3 73.1 26.5 26.1 21.7 25.5 178.7

32.4 29.2 75.6 41.2 141.5 28.8 32.1 51.4 36.3 132.6 32.1 34.4 48.2 63.8 30.4 29.7 52.8 18.2 43.5 36.9 19.6 34.8 29.6 80.3 73.1 26.5 26.2 21.0 25.7 178.7

32.3 29.2 75.6 41.2 141.4 28.6 31.9 51.3 36.2 132.6 31.8 34.2 48.4 64.2 29.7 29.5 52.9 18.0 43.3 36.7 19.5 34.7 29.4 80.1 73.1 26.5 26.3 21.1 25.7 176.3 51.3

32.1 25.2 83.8 40.2 141.3 28.6 32.0 51.3 36.2 132.4 32.0 34.3 48.1 63.7 30.4 29.7 52.8 18.2 43.3 36.8 19.6 34.7 29.6 80.3 73.1 26.5 26.1 21.6 25.5 178.7

32.0 24.9 83.1 40.1 141.3 28.5 32.0 51.2 36.2 132.4 32.0 34.3 48.1 63.7 30.4 29.6 52.8 18.2 43.3 36.2 19.2 33.6 29.5 90.0 72.3 27.2 25.7 21.7 25.4 178.7

32.1 25.2 83.8 40.2 141.4 28.6 32.0 51.3 36.2 132.4 32.0 34.3 48.2 63.7 30.4 29.6 52.9 18.2 43.3 36.3 19.2 33.6 29.6 90.0 72.3 27.2 25.7 21.6 25.4 178.7

32.0 24.8 83.0 40.2 141.3 28.6 32.0 51.3 36.2 132.5 32.0 34.3 48.1 63.7 30.4 29.6 52.9 18.2 43.3 36.3 19.2 33.6 29.6 90.1 72.3 27.2 25.7 21.6 25.6 178.8

103.4 75.3 78.8 72.0 75.4 65.1 171.1 21.2

103.1 75.2 79.1 72.1 78.6 63.5

103.4 75.3 78.8 72.0 75.3 65.1 171.1 21.1 106.5 73.1 75.0 69.9 67.8

103.5 72.8 75.1 69.9 67.3

103.1 75.4 79.0 72.3 78.6 63.5

103.1 75.5 79.1 72.3 78.6 63.5

106.5 73.0 74.9 69.8 67.8

106.5 73.1 75.0 69.9 67.9


absolute configuration of D-glucose was determined by GC analysis. The 1H NMR spectrum of 1a showed five tertiary methyl groups (δH 1.53, 1.52, 1.27, 1.15 and 1.01), a secondary methyl group at δH 0.96 (d, J = 6.5 Hz), and an oxymethine proton at δH 3.71 (dd, J = 11.1, 3.3 Hz). With the aid of HSQC analysis, the 30 signals in the 13C NMR spectrum (Table 2) could be assigned as eight quaternary carbons (one carbonyl at δC 216.6, one carboxylic acid at δC 178.7, two olefinics at δC 141.5 and 132.5, and one oxygenated at δC 77.1), five methines (one oxygenated at δC 75.6), 11 methylenes, and six methyls. By comparison with the triterpene lanostane,13 the absence of a methyl group at C-19 and the presence of an additional methylene in 1a suggested that the bond between C-9 and C10 is cleaved, and the molecule forms a seven-membered ring

(ring B). The structure was fully determined using its HMBC and 1H−1H COSY spectra. The key HMBC correlations from H2-19 to C-1, C-5, C-8, C-9, C-10, and C-11, and from H3-18 to C-12, C-13, C-14, and C-17, together with the corresponding cross-peaks in the 1H−1H COSY spectrum, shown in Figure 1a, provided evidence of the presence of a 6/ 7/6/5 tetracyclic14−20 fused ring system. This spectrum also showed long-range correlations from H3-28/29 to C-3, C-4, and C-5, and from H3-26/27 to C-24 and C-25, confirming the positions of a carbonyl at C-24 and two hydroxy groups at C-3 and C-25, respectively. One double bond was determined from the HMBC correlations from H2-1, H2-6, H2-7, H2-19, H3-28, and H3-29 to C-5 (δC 141.5), and from H2-1, H2-2, H2-6, H-9, and H2-19 to C-10 (δC 132.5) and was located between C-5 2826



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confirmed by the HMBC correlations of H3-26/27 with C-24 and C-25 and H-24 with C-22, C-23, C-25, and C-27. The absolute configuration of C-24 was determined using the Mo2(OAc)4-induced circular dichroism (ICD) method developed by Snatzke and Frelek for vicinal diols.21−26 During the application of the method, the aglycone (2a) obtained from acid hydrolysis of 2 with HCl was treated with the methylation reagent CH3I and converted into 2b to avoid interference from the intrinsic carboxylic acid group. In the experiment, the Cotton effect observed at 314 nm was positive (shown in Figure 3), which allowed assignment of a 24S absolute

Figure 1. (a) Selected HMBC (H → C) and 1H−1H COSY () correlations of 1a. (b) Selected NOESY correlations of 1a.

and C-10. Besides, HMBC correlations from H-8 and H2-15 to C-30 indicated the location of the carboxylic acid at C-30. Thus, the planar structure of compound 1a could be defined. The relative configuration of compound 1a was deduced from the NOESY spectrum (Figure 1b). The NOESY correlations of H3-18/H-8, H-20; H-19a/H-8 showed that H8 and H3-18 are β-oriented, whereas H3-21/H-17, H-9/H-19b are in α-disposed. The X-ray crystallography data for compound 1a allowed the assignment of its absolute configuration as 3R, 8R, 9R, 13R, 14R, 17R, and 20R [with a Flack parameter of −0.03(6)] (see ORTEP diagram in Figure 2). Thus, compound 1a was established as 3α,25-dihydroxy9,10-seco-cycloartan-24-oxo-5(10)-en-30-oic acid.6,7,16 The HMBC correlation in 1 was observed from H-1′ (δH 4.86) of the glucose to C-3 (δC 83.1) of the aglycone, which led to the assignment of the β-D-glucose moiety at C-3. Thus, the structure of 1 was fully determined as 3α-[(β-D-glucopyranosyl)-oxy]-25-hydroxy-9,10-seco-cycloartan-24-oxo-5(10)-en30-oic acid and was given the trivial name lyonifoloside A. The molecular formula of 2 (lyonifoloside B), C36H60O10, was determined by HRESIMS (m/z 675.4082 [M + Na]+ calcd for C36H60O10Na, 675.4084), in conjunction with the 13C NMR data. The 1H and 13C NMR spectra of 2 (Tables 1 and 2) resembled those of 1, except for evidence of an oxymethine group (δH 3.66, δC 80.3) at C-24 in 2 instead of the keto carbonyl resonance (δC 216.6) in 1. This assignment was

Figure 3. Circular dichroism spectrum of 2b in DMSO solution of dimolybdenum tetracetate (the inherent CD spectrum of 2b was subtracted).

configuration for compound 2b. Thus, compound 2 was determined as 3α-[(β-D-glucopyranosyl)-oxy]-24S,25-dihydroxy-9,10-seco-cycloartan-5(10)-en-30-oic acid. Compounds 3−6 (lyonifolosides C−F) were assigned the molecular formulas of C38H62O11, C41H68O14, C43H70O15, and C40H66O13, respectively, on the basis of their respective 13C NMR and HRESIMS data. Acid hydrolysis of 3−6 with HCl produced the same aglycone (2a) and D-glucose, D-glucose/Larabinose, D-glucose/L-arabinose, and L-arabinose, respectively. Compound 3 was assigned a 6-O-acetyl-glucose group at C-3, as confirmed by the HMBC correlations from H-6′ to C-7′ and from H-1′ to C-3 of the aglycone. For compound 4, the HMBC correlations from H-1′ of the glucose to C-3 and from H-1″ of the arabinose to C-24 of the aglycone indicated that the Dglucose and L-arabinose units are attached at C-3 and C-24, respectively. By NMR data comparison, compound 5 was determined as containing a 6-O-acetyl-glucose group instead of

Figure 2. ORTEP diagram of 1a. 2827



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7a

7ab

7bc

8a

9b

10b

11a

1 2

5.36 s 2.50m 2.38 m 3.75 dd (4.2, 4.2) 2.28 m 1.86 m 1.24 m 2.15 m 1.41 m 1.60 m 2.45 m 1.63 m 1.52 m 2.83 m 1.69 m 2.64 m 1.27 m 2.40 m 1.53 m 1.84 m 0.98 s 2.31 m 2.25 m 1.63 m 1.03 d (6.4) 2.23 m 1.20 m 2.02 m 1.53 m 3.67 dd (10.1, 1.6) 1.47 s 1.50 s 1.21 s 0.91 s

5.42 s 2.50m 2.32 m 3.70 brs 2.47 m 1.86 m 1.35 m 2.24 m 2.21 m 1.64 m 2.53 m 1.70 m 1.57 m 2.81 m 1.69 m 2.69 m 1.30 m 2.42 m 1.52 m 1.83 m 1.00 s 2.41m 2.29 m 1.64 m 1.03 d (6.4) 2.24 m 1.21 m 2.04 m 1.54 m 3.67 d (9.9) 1.48 s 1.50 s 1.19 s 0.87 s

5.41 s 2.47 m 2.32 m 3.71 brs 2.42 m 1.81 m 1.29 m 2.03 m 1.07 m 1.54 m 2.22 m 1.61 m 1.46 m 2.47 m 1.61 m 2.42 m 1.20 m 2.15 m 1.40 m 1.39 m 0.92 s 2.35 m 2.23 m 1.57 m 0.99 d (6.5) 2.20 m 1.19 m 2.03 m 1.53 m 3.69 m 1.51 s 1.54 s 1.25 s 0.86 s 3.53 s

5.36 s 2.52 m 2.41 m 3.76 dd (4.1, 4.1) 2.29 m 1.86 m 1.27 m 2.15 m 1.44 m 1.61 m 2.45 m 1.62 m 1.54 m 2.84 m 1.69 m 2.67 m 1.31 m 2.52 m 1.68 m 1.88 m 1.01 s 2.30 m 2.27 m 1.63 m 0.99 d (6.4) 1.86 m 1.86 m 1.76 m 1.70 m 3.82 m 1.36 s 1.40 s 1.22 s 0.91 s

5.37 s 2.58 m 2.38 m 3.78 m 2.31 m 1.80 m 1.24 m 2.12 m 1.42 m 1.61 m 2.47 m 1.63 m 1.52 m 2.82 m 1.70 m 2.66 m 1.31 m 2.52 m 1.65 m 1.86 m 1.01 s 2.32 m 2.24 m 1.64 m 0.99 d (6.4) 1.87 m 1.87 m 1.78 m 1.72 m 3.82 m 1.36 s 1.40 s 1.19 s 0.86 s

5.35 s 2.57 m 2.36 m 3.76 brs 2.31 m 1.79 m 1.22 m 2.10 m 1.40 m 1.57 m 2.47 m 1.62 m 1.50 m 2.78 m 1.66 m 2.68 m 1.26 m 2.52 m 1.58 m 1.82 m 0.90 s 2.30 m 2.23 m 1.52 m 0.97 d (6.3) 2.43 m 1.21 m 1.78 m 1.51 m 3.68 d (9.1) 1.35 s 1.47 s 1.17 s 0.84 s

5.36 s 2.52 m 2.40 m 3.75 m 2.28 m 1.81 m 1.25 m 2.14 m 1.44 m 1.61 m 2.44 m 1.59 m 1.51 m 2.82 m 1.68 m 2.67 m 1.31 m 2.52 m 1.65 m 1.87 m 1.01 s 2.30 m 2.24 m 1.63 m 0.99 d (6.4) 1.87 m 1.87 m 1.75 m 1.72 m 3.82 m 1.36 s 1.40 s 1.18 s 0.89 s

4.77 d (7.8) 3.82 m 4.18 t (8.6) 3.99 m 3.96 m

4.77 d (7.8) 3.83 m 4.18 m 4.00 m 3.98 m

4.84 d (7.8) 3.85 m 4.22 m 4.23 m 3.91 m

4.82 d (6.3) 3.84 m 4.21 m 4.22 m 3.89 m

4.69 d (6.9) 4.27 m 4.14 m 4.29 m 4.28 m 3.76 m

4.93 dd (11.5, 1.7) 4.79 m 2.03 s

4.94 dd (11.5, 1.7) 4.80 m 2.04 s 4.81 m 4.46 dd (9.0, 7.4) 4.14 m 4.29 m 4.30 m 3.81 m

4.52 dd (11.5, 2.3) 4.40 dd (11.6, 5.0)

4.51 m 4.39 m

4.80 d (7.3) 4.45 dd (8.7, 7.7) 4.13 dd (9.1, 3.0) 4.28 m 4.29 m 3.81 m

5.14 d (5.9) 4.06 m 4.21 m 4.28 m 3.89 m

3 5 6 7 8 9 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 31 1′ 2′ 3′ 4′ 5′ 6′ 8′ 1″ 2″ 3″ 4″ 5″ 6″ a

4.80 d (7.3) 4.46 dd (8.9, 7.4) 4.14 m 4.29 m 4.30 m 3.81 m

4.51 m 4.45 m


[(6-O-acetyl-β-D-glucopyranosyl)-oxy]-24S,25-dihydroxy-9,10seco-cycloartan-5(10)-en-30-oic acid, 3α-[(β-D-glucopyranosyl)oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy-9,10-secocycloartan-5(10)-en-30-oic acid, 3α-[(6-O-acetyl-β-D-glucopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy9,10-seco-cycloartan-5(10)-en-30-oic acid, and 3α-[(α-L-arabi-

the glucose group at C-3 in 4 on the basis of the HMBC correlations between H2-6′ (δH 4.95 and 4.79) and C-7′ (δC 171.1). In the HMBC spectrum of 6, long-range correlations observed from H-1′ to C-3 and H-1″ to C-24 suggested that two arabinose moieties are located at C-3 and C-24, respectively. Thus, compounds 3−6 were established as 3α2828



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nopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy-9,10-seco-cycloartan-5(10)-en-30-oic acid, respectively. According to its 13C NMR data and HRESIMS m/z 717.4281 [M + Na]+, compound 7 (lyonifoloside G) gave the same molecular formula as 3, C38H62O11. The 1H and 13C NMR spectroscopic data (Tables 3 and 4) of 7 were similar to those of 3, except for the location of the double bond. The key HMBC correlations (Figure 4a) from H-1 (δH 5.36) to C-3, C5, and C-19, and from H-5 and H2-6 to C-10 (δC 140.9) indicated that the double bond is located between C-1 and C10. The hydroxy group at C-3 in 7a (the aglycone of 7) was Table 4. 13C NMR Data of Compounds 7−11 in Pyridine-d5 (δ in ppm) position

7a

7ab

7bc

8a

9b

10b

11a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″ 6″

118.2 29.8 81.9 37.4 48.3 28.0 34.5 48.3 36.4 140.9 31.9 34.0 47.8 63.6 30.9 29.9 52.6 17.8 46.8 36.7 19.5 34.7 29.4 80.2 73.1 26.5 26.1 25.0 22.2 178.9 104.1 74.9 78.8 71.9 75.3 65.1 171.1 21.2

118.2 33.4 73.6 38.3 47.1 27.4 35.2 48.8 36.4 141.1 32.1 34.1 47.9 63.8 31.0 29.9 52.8 17.9 47.1 36.8 19.6 34.8 29.5 80.3 73.1 26.5 26.2 25.3 21.5 178.8

118.4 33.4 73.7 38.3 47.0 27.1 35.2 48.8 36.3 140.9 31.8 33.9 48.1 64.2 30.3 29.6 52.9 17.7 46.9 36.6 19.5 34.7 29.4 80.3 73.1 26.5 26.3 25.5 21.4 176.4

118.3 29.9 81.9 37.4 48.3 28.0 34.6 48.4 36.5 140.9 31.9 34.0 47.9 63.7 30.9 29.6 52.8 17.9 46.8 36.3 19.2 33.6 29.7 90.1 72.3 27.2 25.7 25.1 22.3 178.9 104.2 75.0 78.9 72.0 75.3 65.1 171.1 21.2 106.5 73.1 75.0 69.9 67.8

118.2 29.8 81.9 37.4 48.0 27.8 34.8 48.5 36.5 141.0 32.0 34.0 47.9 63.7 31.0 29.6 52.8 17.9 47.0 36.3 19.2 33.7 29.8 90.1 72.3 27.2 25.7 25.2 22.1 178.9 104.2 75.2 79.1 72.1 78.6 63.4

118.2 29.7 81.9 37.4 47.9 27.7 34.7 48.6 36.4 141.0 31.9 34.0 47.7 63.7 30.9 29.7 52.7 17.8 47.0 36.2 19.3 34.0 29.2 92.7 73.9 27.1 24.5 25.2 22.0 179.0 104.1 75.1 79.0 72.0 78.5 63.3

118.2 29.6 81.4 37.4 48.3 27.9 34.6 48.2 36.4 140.9 31.9 34.0 47.8 63.7 30.9 29.7 52.8 17.9 46.7 36.2 19.2 33.6 29.6 90.1 72.3 27.2 25.7 25.1 22.2 178.9 104.3 72.5 75.1 69.7 67.2

106.5 73.1 75.0 69.9 67.8

107.2 76.5 79.0 72.0 78.4 63.3

106.5 73.1 75.0 69.9 67.8

Figure 4. (a) Selected HMBC (H → C) and 1H−1H COSY () correlations of 7. (b) Selected NOE correlations of 7a.

assigned as α-oriented by comparison with that of cimifoetidanol A.14 Large coupling constants of H-3 (δH 3.78, dd, J = 9.5, 6.6 Hz) and NOESY correlations of H-3/H3-29 and H-3/ H-5 were observed in cimifoetidanol A.14 However, differences in compound 7a (i.e., the broad single peak of H-3 indicating that it is in an equatorial position; and the NOE correlations of H-3/H-2a and H-2b) revealed the configuration of H-3 to be opposite to that of cimifoetidanol A. In addition, the NOE correlations of H3-28/H-3, H-5 and H3-29/H-3, H-2a indicated that H-5 is α-oriented. The absolute configuration of C-24 should be S for the same chemical shift of C-24 in the 1H and 13 C NMR spectra compared to that between 7 and 3. To confirm this, a Mo2(OAc)4-induced circular dichroism (ICD) experiment was conducted. However, acid hydrolysis with HCl afforded aglycone 2a instead of 7a. Enzymatic hydrolysis of 7 with snailase generated the expected aglycone (7a), named lyonifolic acid B, and D-glucose. For an ICD experiment, compound 7a was treated with CH3I and converted into the methylated product of 7b. As a result, a positive Cotton effect was observed at 307 nm (shown in Figure 5), and confirmed this compound as having the same 24S configuration as that of 2b. Accordingly, compound 7 was assigned as 3α-[(6-O-acetylβ-D-glucopyranosyl)-oxy]-24S,25-dihydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid.


a

Recorded at 100 MHz. bRecorded at 125 MHz. cRecorded at 150 MHz. 2829



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12b

12ab

12bc

13b

14b

15a

16a

17c

18a

1

2.23 m 1.58 m 2.11 m 1.81 m 3.69 brs 2.01 m 1.63 m 1.56 m 2.31 m 2.21 m 2.44 m 2.18 m 2.75 m 1.81 m 2.47 m 1.69 m 2.41 m 1.56 m 2.02 m 0.92 s 1.13 s 1.61 m 1.06 d (6.5) 2.25 m 1.20 m 2.06 m 1.54 m 3.68 m

2.21 m 1.61 m 2.06 m 1.82 m 3.63 brs 2.16 m 1.73 m 1.64 m 2.44 m 2.29 m 2.55 m 2.27 m 2.87 m 1.86 m 2.52 m 1.75 m 2.43 m 1.60 m 2.09 m 0.96 s 1.18 s 1.64 m 1.10 d (6.4) 2.28 m 1.23 m 2.09 m 1.56 m 3.69 d (9.9)

2.15 m 1.57 m 2.05 m 1.83 m 3.64 brs 2.04 m 1.70 m 1.59 m 2.14 m 2.14 m 2.40 m 2.19 m 2.44 m 1.78 m 2.23 m 1.63 m 2.23 m 1.49 m 1.73 m 0.87 s 1.13 s 1.58 m 1.06 d (6.4) 2.24 m 1.20 m 2.07 m 1.55 m 3.71 m

2.23 m 1.60 m 2.08 m 1.85 m 3.65 brs 2.01 m 1.66 m 1.58 m 2.34 m 2.23 m 2.42 m 2.18 m 2.77 m 1.81 m 2.48 m 1.71 m 2.42 m 1.57 m 2.05 m 0.92 s 1.14 s 1.62 m 1.07 d (6.5) 2.26 m 1.21 m 2.07 m 1.54 m 3.68 dd (10.0, 1.1) 1.48 s 1.51 s 1.17 s 0.95 s

2.21 m 1.59 m 2.05 m 1.82 m 3.62 brs 2.15 m 1.71 m 1.62 m 2.41 m 2.26 m 2.55 m 2.24 m 2.83 m 1.84 m 2.56 m 1.73 m 2.57 m 1.67 m 2.07 m 0.89 s 1.17 s 1.53 m 1.05 d (6.5) 2.47 m 1.22 m 1.83 m 1.53 m 3.71 d (9.1)

2.24 m 1.57 m 2.08 m 1.79 m 3.68 brs 2.01 m 1.58 m 1.58 m 2.34 m 2.24 m 2.43 m 2.18 m 2.76 m 1.81m 2.50 m 1.71 m 2.50 m 1.69 m 2.06 m 0.94 s 1.12 s 1.60 m 1.01 d (6.4) 1.86 m 1.86 m 1.71 m 1.71 m 3.80 m




2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 26 27 28 29 31 1′ 2′ 3′ 4′ 5′

1.47 1.50 1.11 0.88

s s s s

4.82 d (7.8) 3.86 t (8.3) 4.22 t (8.8) 4.17 t (9.1) 3.92 m

4.76 m 3.85 t (8.5) 4.19 t (8.6) 4.00 t (8.2) 3.97 m

5.17 d (7.9) 4.08 t (8.5) 4.21 t (9.0) 4.30 t (9.2) 3.89 dt (9.2, 3.6)

4.83 d (7.8) 3.86 m 4.22 d (8.8) 4.19 d (9.1) 3.92 m

4.73 m 3.82 m 4.17 t (8.4) 3.96 d (9.1) 3.95 m

4.85 d (7.7) 3.89 m 4.24 m 4.20 m 3.90 m

6′

4.52 dd (8.1, 2.3)

4.93 dd (11.7, 1.8) 4.77 m

4.53 dd (11.4, 2.6) 4.46 dd (11.4, 4.2)

4.53 dd (11.6, 1.9) 4.36 dd (11.7, 5.3)

4.90 d (11.8)

4.54 m

4.74 m

4.47 m

1.98 s 4.78 m 4.42 t (8.1) 4.11 m 4.26 m 4.27 m 3.78 m

5.17 d (7.7) 4.08 m 4.22 m 4.30 m 3.94 m

4.35 dd (11.7, 5.3) 8′ 1″ 2″ 3″ 4″ 5″

1.49 1.52 1.16 0.96

s s s s

1.52 1.54 1.20 0.95 3.48

s s s s s

1.38 1.50 1.16 0.95

1.35 1.39 1.11 0.87

s s s s

1.99 s 4.79 d (7.3) 4.44 t (7.8) 4.12 dd (9.0, 3.2) 4.27 m 4.28 m 3.78 m

6″ a

s s s s

1.35 1.38 1.15 0.94

s s s s

1.38 1.50 1.22 0.87

s s s s

1.37 1.41 1.14 0.93

s s s s

4.69 d (6.7) 4.28 m 4.16 m 4.33 m 4.29 m 3.78 m

4.82 d (7.3) 4.47 t (7.4) 4.16 m 4.33 m 4.29 m 3.80 m

4.54 m 4.38 m


HRESIMS and the 13C NMR data indicated the molecular formulas of compounds 8−11 (lyonifolosides H−K) to be C43H70O15, C41H68O14, C42H70O15, and C40H66O13, respectively. Comparison of their NMR data with those of 7 indicated that all these compounds share the same aglycone. Compound 8 was found to possess a 6-O-acetyl-glucose and an arabinose

moiety located at C-3 and C-24, respectively, on the basis of the HMBC correlations from H-1′ of the glucose to C-3 and H-1″ of the arabinose to C-24 of the aglycone. Compound 9 was assigned a glucose and an arabinose moiety located at C-3 and C-24, respectively, as a result of the same HMBC correlations as 8. Compounds 10 and 11 were shown to contain two 2830



Article

glucose and two arabinose units each, and the two sugar moieties were attached at C-3 and C-24 of the aglycone, respectively. Thus, compounds 8−11 were determined as 3α[(6-O-acetyl-β-D-glucopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid, 3α-[(β-D-glucopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid, 3α-[(β- D -glucopyranosyl)-oxy]-24S-[(β- D -glucopyranosyl)oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid, and 3α-[(α-L-arabinopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid, respectively. Compound 12 was obtained as a white powder. The molecular formula was determined to be C36H60O10 based on the sodiated molecular ion peak, [M + Na]+, at m/z 675.4078 (calcd for C36H60O10Na, 675.4084) by HRESIMS, and was supported by its 13C NMR data. The IR spectrum revealed hydroxy and carbonyl groups on the basis of absorption bands at 3394 and 1687 cm−1, respectively. The 1H NMR spectrum (Table 5) exhibited seven methyl groups at δH 1.50, 1.47, 1.13, 1.11, 1.06, 0.92, and 0.88, an anomeric proton at δH 4.82 (1H, d, J = 7.8 Hz), and several oxymethine protons between δH 3.68 and δH 4.82. The 13C NMR spectrum of the aglycone moiety of 12 was very similar to that of inoterpene A,27 except for a glycosylation shift of C-3 and the carboxylic acid in 12 instead of a methyl group at C-30. These observations were supported by the HMBC correlations of H2-15 to C-8/C-13/C-14/C-30/ C-16/C-17 and H2-16 to C-13/C-14/C-17 (Figure 6). The H-3

Figure 6. Selected HMBC (H→C) and correlations of 12.

1


Compounds 13−18 (lyonifolosides M−R) exhibited molecular formulas of C38H62O11, C36H60O10, C41H68O14, C43H70O15, C42H70O15, and C40H66O13, respectively, as revealed by HRESIMS and their 13C NMR data. Analysis of the NMR data for compounds 13−18 clearly indicated that all of these compounds possess the same aglycone as 12. Compound 13 was assigned a 6-O-acetyl-glucose group at C-3, and the glucose unit in 14 was found to be attached to C-24. Compound 15 showed a glucose and an arabinose moiety located at C-3 and C-24, respectively. A 6-O-acetyl-glucose and an arabinose moiety were located also at C-3 and C-24, respectively, in 16. Compound 17 gave evidence of containing two glucose units, wherease two arabinose units were found in 18, and the two sugar moieties were attached at C-3 and C-24 of the aglycone, in each case. Thus, compounds 13−18 were determined as 3α[(6-O-acetyl-β-D-glucopyranosyl)-oxy]-24(S),25-dihydroxylanost-8-en-30-oic acid, 24(S)-[(β-D-glucopyranosyl)-oxy]-3α,25dihydroxylanost-8-en-30-oic acid, 3α-[(β-D-glucopyranosyl)oxy]-24(S)-[(α-L-arabinopyranosyl)-oxy]-25-hydroxylanost-8en-30-oic acid, 3α-[(6-O-acetyl-β-D-glucopyranosyl)-oxy]24(S)-[(α-L-arabinopyranosyl)-oxy]-25-hydroxylanost-8-en-30oic acid, 3α-[(β-D-glucopyranosyl)-oxy]-24(S)-[(β-D-glucopyranosyl)-oxy]-25-hydroxylanost-8-en-30-oic acid, and 3α-[(α-Larabinopyranosyl)-oxy]-24(S)-[(α-L-arabinopyranosyl)-oxy]25-hydroxylanost-8-en-30-oic acid, respectively. The molecular formula of compound 19 was determined as C30H48O7 based on the [M + H]+ ion peak at m/z 521.3479 (calcd for C30H49O7 521.3478) by its 13C NMR data and HRESIMS, indicating seven indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy (3480 cm−1) and carbonyl (1740 cm−1) absorptions. Its 1H NMR spectrum (Table 7) displayed characteristic signals for four tertiary methyl groups (δH 1.67, 1.66, 1.36, and 1.08), two secondary methyl groups (δH 1.30 and 0.87), four oxygenated methines (δH 4.47, 4.55, 4.20, and 4.24), and an oxygenated methylene (δH 4.06 and 3.88). The 13C NMR spectrum (Table 7), when combined with an analysis of the HSQC spectrum, showed the presence of 30 carbon signals, including six methyls, eight methylenes (one oxygenated at δC 65.7), nine methines (four oxygenated at δC 76.3, 74.7, 70.0, and 66.8), and seven quaternary carbons (one carbonyl at δC 180.4 and one oxygenated at δC 96.2). These structural features were shown to be consistent with an ursane triterpenoid skeleton,28,29 with six sites of oxygenation and one carbonyl group. In the HMBC spectrum of 19, correlations from H3-25 to C-1, C-5, C-9, and

H−1H COSY ()

in the 1H NMR spectrum displayed a broad single peak, indicating that the H-3 is in an equatorial position, with a halfchair conformation of the six-membered ring. Owing to the present of a vicinal diol unit, the absolute configuration of C-24 was established using the ICD method. However, the aglycone 12−2a obtained by acid hydrolysis with HCl was the isomer of 12a. The reason could be that the double bond in 12 readily underwent an addition reaction with H2O under acid conditions, and then a five-membered lactone ring was formed from the carboxylic acid group at C-30 with the hydroxy group at C-9. Enzymatic hydrolysis of 12 with snailase afforded the expected aglycone (12a) and β-D-glucose, after GC analysis. To obtain compound 12b, 12a was methylated with CH3I. As shown in Figure 7, the Cotton effect observed at 319 nm was positive, which verified the S configuration of C-24. Thus, compound 12a (lyonifolic acid C) was identified as 3α,24(S),25-trihydroxylanost-8-en-30-oic acid. The glucose group in 12 was placed at C-3 based on the glycosylation shift of C-3 at δC 82.1 in the 13C NMR spectrum and from the HMBC correlation from H-1′ of the glucose unit to C-3 of the aglycone moiety. Thus, 12 (lyonifoloside L) was identified as 3α-[(β-Dglucopyranosyl)-oxy]-24(S),25-dihydroxylanost-8-en-30-oic acid. 2831



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Table 6. 13C NMR Data of Compounds 12−18 in Pyridine-d5 (δ in ppm)

a

position

12b

12ab

12bc

13b

14b

15a

16a

17c

18a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″ 6″

30.9 22.5 82.1 37.9 45.5 18.9 28.1 128.3 140.7 38.2 23.1 32.1 47.5 63.3 28.9 30.1 51.7 18.4 20.2 37.4 19.4 34.7 29.7 80.2 73.1 26.4 26.1 29.2 23.1 178.9

30.9 27.3 75.3 38.6 44.7 19.2 28.3 128.5 140.9 38.4 23.2 32.3 47.6 63.5 29.0 30.2 51.8 18.6 20.2 37.5 19.5 34.8 29.8 80.3 73.1 26.5 26.2 29.3 23.1 178.8

30.9 27.3 75.2 38.6 44.7 19.1 28.2 127.6 141.6 38.4 23.0 32.3 47.9 63.6 28.5 30.0 52.0 18.4 20.0 37.4 19.5 34.8 29.8 80.3 73.1 26.5 26.3 29.4 23.1 176.7 51.9

31.1 22.9 82.6 38.0 45.7 19.0 28.1 128.5 140.7 38.3 23.2 32.1 47.6 63.4 29.0 30.2 51.8 18.5 20.3 37.5 19.5 34.8 29.8 80.3 73.1 26.5 26.2 29.2 23.3 178.9

30.9 27.3 75.3 38.6 44.6 19.0 28.3 128.5 140.7 38.4 23.2 32.3 47.5 63.4 28.9 30.0 51.9 18.6 20.1 37.0 19.3 34.1 29.6 92.9 73.9 27.1 24.5 29.3 23.1 179.0

30.9 22.5 82.1 37.9 45.5 18.9 28.0 128.3 140.7 38.2 23.0 32.1 47.5 63.3 28.9 30.0 51.7 18.5 20.1 36.9 19.1 33.6 29.7 89.9 72.2 27.1 25.6 29.2 23.1 178.9

30.9 22.8 82.6 37.8 45.5 18.9 28.0 128.4 140.6 38.2 23.0 32.0 47.5 63.2 28.9 30.0 51.7 18.4 20.1 36.9 19.1 33.6 29.7 89.9 72.2 27.1 25.6 29.1 23.2 178.9

31.0 22.5 82.1 38.0 45.5 19.0 28.1 128.5 140.6 38.3 23.2 32.3 47.5 63.5 29.0 30.2 52.0 18.5 20.2 37.0 19.3 34.2 29.6 92.9 73.9 27.1 24.5 29.3 23.2 179.3

31.0 22.5 82.3 38.0 45.6 19.0 28.1 128.6 140.7 38.3 23.1 32.2 47.6 63.4 29.0 30.1 51.8 18.5 20.2 37.0 19.2 33.4 29.8 90.0 72.3 27.2 25.8 29.3 23.3 179.3

103.0 75.1 79.0 72.0 75.4 65.1 171.1 21.2

107.3 76.5 79.1 72.0 78.4 63.3

102.7 75.2 79.1 72.2 78.6 63.3

102.9 74.9 78.8 71.9 75.2 65.0 171.1 21.1 106.3 72.9 74.8 69.8 67.7

102.7 75.3 79.2 72.3 78.5 63.4

103.2 72.5 75.1 69.7 67.0

107.3 76.5 79.1 72.1 78.7 63.5

106.5 73.1 75.0 69.9 67.8

102.7 75.2 79.1 72.2 78.6 63.4

106.4 73.0 74.9 69.8 67.7


the configurations of H-2, H-3, and H2-24 as β-oriented, while those between H-7/H-5, H-9, H3-27, and H-12/H-9, H3-27 suggested that H-7 and H-12 are α-oriented (Figure 8b). Therefore, compound 19 was proposed as 2α,3α,7β,12β,24βpentahydroxyurs-28,13β-olide and was given the trivial name lyonifolide A. Compound 20 (lyonifolide B) was assigned the same molecular formula as 19 of C30H48O7 on the basis of its 13C NMR and HRESIMS data. The NMR spectra of 20 and 19 were closely comparable, with the major difference being that the hydroxy group signal at C-7 in 19 (C-7: δH 4.20, δC 76.3; C23: δH 1.67, δC 24.2) was transformed to C-23 in 20 (C-7: δH 1.64 1.22, δC 35.1; C-23: δH 4.61 4.40, δC 69.4). This was

C-10; from H-1 to C-2, C-9, C-10, and C-25; from H-3 to C-1 and C-5; from H2-24 to C-3, C-4, and C-23; from H3-23 to C-3, C-4, C-5 and C-24; from H3-26 to C-7, C-8, C-9; from H-11 to C-8, C-12, and C-13; and from H3-27 to C-8, C-13, C-14, and C-15 indicated that the six sites of oxygenation are located at C2, C-3, C-7, C-12, C-13, and C-24. The ursane triterpenoid skeleton and the carbonyl group accounted for six of the seven degrees of unsaturation, indicating the presence of another ring in the structure. In the 13C NMR spectrum, the relatively downfield chemical shift of C-13 (δC 96.2) suggested C-30/C13 lactonization. Thus, the planar structure of compound 19 could be defined. NOESY correlations observed between H-2/ H3-25, H-3/H-2, H3-23, H2-24, and H2-24/H3-25 supported 2832



Article

EtOH fraction (740 g) was then further separated on a macroporous resin column and eluted in a gradient of EtOH-H2O (30:70, 60:30, 95:5 v/v) in order of increasing concentrations of EtOH. The 60% EtOH fraction (300 g) was subjected to Si gel CC (86 × 14 cm, 200− 300 mesh) and eluted with a CH2Cl2−CH3OH (100:1 → 1:1 v/v) gradient system to yield fractions G1−G8 on the basis of TLC analysis. Fraction G2 was applied to a Sephadex LH-20 column to obtain the terpenoid-containing fraction G2A (8.6 g), which was further resolved on a MCI gel column and eluted in a gradient of MeOH-H2O (30:70, 60:40, 70:30, 80:20, 100:0 v/v) to obtain five subfractions (G2A1−G2A5). Fraction G2A4 (1.5 g) was separated by preparative HPLC with MeCN-H2O (58:42 v/v, 7 mL/min) to afford nine fractions, G2A4a−G2A4i, and compound 13 (160 mg, tR = 44 min). Fraction G2A4c (102 mg) was purified by semipreparative HPLC with MeCN-H2O (45:55 v/v, 4 mL/min) to yield compound 1 (64 mg, tR = 32 min). Purification of fraction G2A4e (86 mg) by semipreparative HPLC with MeCN-H2O (46:54 v/v, 4 mL/min) afforded compound 3 (8.9 mg, tR = 45 min). G2A4i yielded compound 7 (11 mg, tR = 43 min), which was repeatedly purified by semipreparative HPLC with MeCN-H2O (48:52 v/v, 4 mL/min). Fraction G2A3 (1.3 g) was separated by semipreparative HPLC with MeCN-H2O (30:70 v/v, 4 mL/min) to afford 10 fractions, G2A3a− G2A3j. Fraction G2A3g (48 mg) yielded compound 20 (9.0 mg, tR = 39 min) with MeOH-H2O (58:42 v/v, 4 mL/min) by semipreparative HPLC. With the same procedure as for G2, fraction G6 (21 g) was separated with Sephadex LH-20 and MCI gel columns eluted using a gradient of MeOH-H2O (30:70, 60:40, 70:30, 80:20, 100:0 v/v) to produce five subfractions (G6A1−G6A5). Fraction G6A5 (3.7 g) was separated by preparative HPLC with MeCN-H2O (55:45 v/v, 7 mL/ min) to afford eight fractions, G6A5a−G6A5h. Fraction G6A5h (140 mg) yielded compounds 15 (30.8 mg, tR = 12 min) and 16 (52 mg, tR = 17 min) with MeOH-H2O (87:13 v/v, 4 mL/min) by semipreparative HPLC. Fraction G6A5g (291 mg) was purified to obtain compound 5 (7 mg, tR = 11 min) and a mixture of 8 and 9 using the above HPLC system (MeOH-H2O, 87:13 v/v, 4 mL/min). The mixture was further purified by semipreparative HPLC with MeOHH2O (85:15 v/v, 4 mL/min) to afford 9 (50 mg, tR = 18 min) and 8 (73 mg, tR = 23 min). Compounds 11 (15.7 mg, tR = 27 min) and 18 (15 mg, tR = 32 min) were isolated from fraction G6A5f (86 mg) by semipreparative HPLC with MeOH-H2O (84:16 v/v, 4 mL/min). Purification of fraction G6A5d (703 mg) by semipreparative HPLC (MeOH-H2O, 81:19 v/v, 4 mL/min) afforded compounds 2 (34 mg, tR = 16 min) and 12 (20 mg, tR = 27 min) as well as two mixtures, G6A5d1 and G6A5d2. The G6A5d1 mixture was subsequently purified by HPLC with MeOH-H2O (78:22 v/v, 4 mL/min) to yield compound 4 (25 mg, tR = 28 min). Compound 6 (3.4 mg, tR = 25 min) was obtained from the other mixture, G6A5d2, by semipreparative HPLC (MeCN-H2O, 46:54 v/v, 4 mL/min). Fraction G6A5c (409 mg) was purified by HPLC with MeOH-H2O (78:22 v/v, 4 mL/min) to obtain compound 10 (17 mg, tR = 31 min) and six subfractions (G6A5c1−G6A5c6). Compound 17 (6 mg, tR = 38 min) was obtained from subfraction G6A5c6 by semipreparative HPLC with MeOH-H2O (76:24 v/v, 4 mL/min). Fraction G6A2 (2.6 g) was separated by semipreparative HPLC with MeCN-H2O (24:76 v/v, 4 mL/min) to afford eight fractions, G6A2a−G6A2h. Fraction G6A2g (142 mg) was purified by semipreparative HPLC with MeOH-H2O (43:57 v/v, 4 mL/min) to obtain compound 19 (3.8 mg, tR = 39 min). Through the same isolation methods as for fractions G2 and G6, fraction G5 (22 g) was also separated on Sephadex LH-20 and MCI gel columns to produce five subfractions (G5A1−G5A5). The subsequent purification of fraction G5A4 (2.3 g) by semipreparative HPLC system (MeOH-H2O, 80:20 v/v, 4 mL/min) resulted in the isolation of compound 14 (35 mg, tR = 37 min). Lyonifoloside A (1). White powder; [α]25D + 9 (c 0.3, MeOH); IR (KBr) νmax 3386, 2927, 1708, 1683, 1459, 1380, 1080, 1046 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 673.3944 [M + Na]+ (calcd for C36H58O10Na, 673.3928). Lyonifolic Acid A (1a). Colorless crystals (MeOH-H2O); mp 205− 206 °C; [α]25D + 35 (c 0.3, MeOH); 1H and 13C NMR data, see

Figure 8. (a) Selected HMBC (H→C) and 1H−1H COSY () correlations of 19. (b) Selected NOESY correlations of 19.

confirmed by the HMBC correlations from H2-23 to C-3, C-4, C-5 and C-24 and the 1H−1H COSY correlations for C(7)H2− C(6)H 2 . Th us , c o mpound 2 0 was identified as 2α,3α,12β,23α,24β-pentahydroxyurs-28,13β-olide. All compounds were tested for antiviral (HSV-1, influenza A/ 95−359 and Coxsackie B3) activities. Compounds 1, 1a, 2a, 12a, 13, and 16 exhibited potent activity against HSV-1, with IC50 values from 2.1 to 14.3 μM, compounds 8, 12−2a, and 14 showed moderate activity, with IC50 values from 19.3 to 23.1 μM. Compounds 1a, 2a, 12a, 13, and 12−2a displayed potent activity against influenza A/95−359, with IC50 values from 2.1 to 11.1 μM, and compounds 1, 2, 3, 7a, 10, and 16 showed moderate activity against A/95−359, with IC50 values from 11.1 to 33.3 μM. In turn, compounds 1, 1a, 2a, 12a, and 13 exhibited potent activity against CVB3, with IC50 values from 2.1 to 11.1 μM.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an XT5B micromelting point apparatus and are uncorrected. Optical rotations were obtained using a JASCO P-2000 automatic digital polarimeter. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer. 1D and 2D NMR spectra were obtained on an INOVA-500, a Bruker-600, or a Bruker-400 NMR spectrometer. Chemical shifts are given in δ (ppm) with the solvent (pyridine-d5) peaks used as references. HRESIMS data were measured using an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with SPD-20A and RID-10A detectors, using a YMC Pack ODS-A column (250 × 20 mm, 5 μm, Kyoto, Japan). Polyamide resin (30−60 mesh, Jiangsu Linjiang Chemical Reagents Factory, Linjiang, People’s Republic of China), macroporous resin (D101 type, Chemical Plant of Nankai University, Naikai, People’s Republic of China), MCI gel (Mitsubishi Chemical Corporation), Sephadex LH-20 (GE Chemical Corporation), Si gel (160−200, 200−300 mesh, Qingdao Marine Chemical Factory, Qingdao, People’s Republic of China), and ODS (50 μm, Merck, Germany) were used for column chromatography (CC). TLC was carried out with precoated glass Si gel GF254 plates (Qingdao Marine Chemical Factory). Spots were visualized under UV light or by spraying with 10% H2SO4 in EtOH followed by heating. Plant Material. Twigs and leaves of Lyonia ovalifolia were collected from Zhangjiajie, Hunan Province, People’s Republic of China, in August 2014 and identified by Prof. Lin Ma of the Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (ID-s-2623) was deposited in the herbarium at the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences. Extraction and Isolation. Air-dried twigs and leaves of L. ovalifolia (100 kg) were extracted (2 h each time; 1000 L) with EtOH-H2O (95:5 v/v) under reflux conditions. After the removal of the solvent under reduced pressure, the crude extract (4200 g) was suspended in 35 L of H2O and then partitioned with petroleum ether, CH2Cl2, EtOAc and n-butanol (3 × 35 L). The EtOAc extract (1200 g) was subjected to passage over a polyamide resin (30−60 mesh) column and eluted with EtOH-H2O (50:50, 95:5 v/v). The 50% 2833



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Lyonifoloside N (14). White powder; [α]25D −33 (c 0.3, MeOH); IR (KBr) νmax 3367, 2931, 1688, 1457, 1378, 1077, 1048 cm−1; 1H and 13 C NMR data, see Tables 5 and 6; HRESIMS m/z 675.4080 [M + Na]+ (calcd for C36H60O10Na, 675.4084). Lyonifoloside O (15). White powder; [α]25D −34 (c 0.3, MeOH); IR (KBr) νmax 3360, 2971, 2922, 1678, 1456, 1378, 1079, 1049 cm−1; 1 H and 13C NMR data, see Tables 5 and 6; HRESIMS m/z 807.4508 [M + Na]+ (calcd for C41H68O14Na, 807.4507). Lyonifoloside P (16). White powder; [α]25D −36 (c 0.3, MeOH); IR (KBr) νmax 3392, 2922, 1741, 1647, 1465, 1372, 1074 cm−1; 1H and 13 C NMR data, see Tables 5 and 6; HRESIMS m/z 849.4591 [M + Na]+ (calcd for C43H70O15Na, 849.4612). Lyonifoloside Q (17). White powder; [α]25D −50 (c 0.3, MeOH); IR (KBr) νmax 3390, 2934, 1685, 1595, 1458, 1375, 1077 cm−1; 1H and 13 C NMR data, see Tables 5 and 6; HRESIMS m/z 837.4603 [M + Na]+ (calcd for C42H70O15Na, 837.4612). Lyonifoloside R (18). White powder; [α]25D −31 (c 0.3, MeOH); IR (KBr) νmax 3397, 2933, 1679, 1458, 1378, 1073 cm−1; 1H and 13C NMR data, see Tables 5 and 6; HRESIMS m/z 777.4389 [M + Na]+ (calcd for C40H66O13Na, 777.4401). Lyonifolide A (19). White powder; [α]25D −6 (c 0.1, MeOH); IR (KBr) νmax 3480, 1740 cm−1; 1H and 13C NMR data, see Table 7; HRESIMS m/z 521.3479 [M + H]+ (calcd for C30H49O7, 521.3478). Lyonifolide B (20). White powder; [α]25D −13 (c 0.05, MeOH); IR (KBr) νmax 3387, 1752 cm−1; 1H and 13C NMR data, see Table 7; HRESIMS m/z 521.3487 [M + H]+ (calcd for C30H49O7, 521.3478). Crystallographic Data for Compound 1a. C30H48O5, M = 488.68, monoclinic, space group P21; a = 11.3157(4) Å, b = 38.8550(13) Å, c = 13.6376(6) Å, α = 90.00°, β = 113.036(5)°, γ = 90.00°; V = 5517.9(4) Å3, Z = 8, ρcalc = 1.176 mg/mm−3, T = 104.7 K, μ (Cu Kα) = 0.616 mm−1, F(000) = 2144, 40576 reflections measured, 20912 unique (Rint = 0.0293). Final R indexes [I > 2σ (I) i.e., Fo > 4σ (Fo)]: R1 = 0.0506, wR2 = 0.1316. Final R indexes [all date]: R1 = 0.0529, wR2 = 0.1340, Flack Parameters = −0.03 (6). Crystallographic data have been deposited at the Cambridge Crystallographic Data Centre and allocated the deposition number CCDC 1484731. The data can be obtained free of charge via www.ccdc.cam.ac.uk/products/ csd/request. Acid Hydrolysis and Determination of Absolute Configuration of Sugar Moieties of Compounds 1−18. Compound 1 (30 mg) was dissolved in MeOH (3 mL) and added to 3 M HCl (3 mL). The solution was heated at 70 °C for 6 h. After the removal of MeOH and HCl by evaporation, the reaction mixture was diluted with H2O (10.0 mL) and extracted with EtOAc. The organic phase was concentrated and purified by semipreparative HPLC with MeCN-H2O (56:44 v/v, 4 mL/min) to yield aglycone 1a (15.6 mg, tR = 53 min). The H2O-soluble phase was again evaporated and dried in vacuo to furnish the monosaccharide residue. The residue (1 mg) was dissolved in pyridine (1 mL), to which 2 mg L-cysteine methyl ester hydrochloride was added. The mixture was kept at 60 °C for 2.5 h. After the removal of the solvent under reduced pressure and drying in vacuo, N-trimethylsilylimidazole (0.2 mL) was added to trimethylsilylate for 2.5 h at 60 °C. The mixture was partitioned between nhexane and H2O (1 mL each), and the n-hexane extract obtained was analyzed using gas chromatography (GC) under the following conditions: Agilent 19091J-216 (60 m × 0.32 mm × 1 μm) column; FID detector; injection temperature of 210 °C; detector temperature of 250 °C; initial column temperature of 200 °C raised to 280 °C at the rate of 60 °C/min and maintained at 280 °C for 35 min under N2 carrier gas. In the acid hydrolysate of 1, D-glucose was verified by comparison of the retention times of its derivative with that of the corresponding control sample of the derivative prepared in the same way, which exhibited retention times at 30.85 and 30.88 min, respectively. The constituent sugars of compounds 2−18 were also identified by the same method, with the result showing the presence of D-glucose and/or L-arabinose in compounds 2−18. The retention times of the derivatives for L-arabinose in 2−18 and the corresponding control sample were 21.31 and 21.78 min, respectively. Enzymatic Hydrolysis of 7 and 12. Compound 7 (5.0 mg) was dissolved in MeOH-H2O (7:10 v/v, 3.4 mL) and then treated with

Tables 1 and 2; HRESIMS m/z 511.3405 [M + Na]+ (calcd for C30H48O5Na, 511.3399). Lyonifoloside B (2). White powder; [α]25D + 3 (c 0.3, MeOH); IR (KBr) νmax 3391, 2934, 1682, 1460, 1382, 1202, 1076 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 675.4082 [M + Na]+ (calcd for C36H60O10Na, 675.4084). Lyofoligenic Acid (2a). White powder; [α]25D + 34 (c 0.3, MeOH); 1 H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 513.3546 [M + Na]+ (calcd for C30H50O5Na, 513.3556). Methyl Lyofoligenate (2b). White powder; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 527.3716 [M + Na]+ (calcd for C31H52O5Na, 527.3712). Lyonifoloside C (3). White powder; [α]25D + 5 (c 0.3, MeOH); IR (KBr) νmax 3375, 2973, 2933, 1686, 1458, 1379, 1204, 1140, 1081, 1046 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 717.4185 [M + Na]+ (calcd for C38H62O11Na, 717.4190). Lyonifoloside D (4). White powder; [α]25D + 9 (c 0.3, MeOH); IR (KBr) νmax 3400, 2969, 2928, 1691, 1459, 1382, 1077, 1048 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 807.4507 [M + Na]+ (calcd for C41H68O14Na, 807.4507). Lyonifoloside E (5). White powder; [α]25D + 8 (c 0.3, MeOH); IR (KBr) νmax 3397, 2978, 2922, 1681, 1647, 1467, 1380, 1085, 1048 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 849.4605 [M + Na]+ (calcd for C43H70O15Na, 849.4612). Lyonifoloside F (6). White powder; [α]25D + 20 (c 0.3, MeOH); IR (KBr) νmax 3364, 2926, 1684, 1458, 1382, 1073 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 777.4407 [M + Na]+ (calcd for C40H66O13Na, 777.4401). Lyonifoloside G (7). White powder; [α]25D −32 (c 0.3, MeOH); IR (KBr) νmax 3364, 2921, 1688, 1460, 1371, 1078, 1042 cm−1; 1H and 13 C NMR data, see Tables 3 and 4; HRESIMS m/z 717.4281 [M + Na]+ (calcd for C38H62O11Na, 717.4190). Lyonifolic Acid B (7a). White powder; [α]25D −32 (c 0.1, MeOH); 1 H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 513.3556 [M + Na]+ (calcd for C30H50O5Na, 513.3556). Lyonifolic Acid B Methyl Ester (7b). White powder; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 527.3718 [M + Na]+ (calcd for C31H52O5Na, 527.3712). Lyonifoloside H (8). White powder; [α]25D −23 (c 0.3, MeOH); IR (KBr) νmax 3395, 2921, 1692, 1647, 1468, 1421, 1373, 1081 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 849.4610 [M + Na]+ (calcd for C43H70O15Na, 849.4612). Lyonifoloside I (9). White powder; [α]25D −28 (c 0.3, MeOH); IR (KBr) νmax 3348, 2973, 2931, 1692, 1456, 1385, 1202, 1081 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 807.4501 [M + Na]+ (calcd for C41H68O14Na, 807.4507). Lyonifoloside J (10). White powder; [α]25D −22 (c 0.3, MeOH); IR (KBr) νmax 3362, 2973, 2926, 1678, 1453, 1381, 1078, 1047 cm−1; 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z 837.4630 [M + Na]+ (calcd for C42H70O15Na, 837.4612). Lyonifoloside K (11). White powder; [α]25D −32 (c 0.3, MeOH); IR (KBr) νmax 3428, 2967, 2921, 1688, 1460, 1381, 1069 cm−1; 1H and 13 C NMR data, see Tables 3 and 4; HRESIMS m/z 777.4403 [M + Na]+ (calcd for C40H66O13Na, 777.4401). Lyonifoloside L (12). White powder; [α]25D −64 (c 0.3, MeOH); IR (KBr) νmax 3394, 2965, 1687, 1464, 1376, 1078, 1046 cm−1; 1H and 13 C NMR data, see Tables 5 and 6; HRESIMS m/z 675.4078 [M + Na]+ (calcd for C36H60O10Na, 675.4084). Lyonifolic Acid C (12a). White powder; [α]25D −24 (c 0.1, MeOH); 1 H and 13C NMR data, see Tables 5 and 6; HRESIMS m/z 513.3552 [M + Na]+ (calcd for C30H50O5Na, 513.3556). Lyonifolic Acid C Methyl Ester (12b). White powder; 1H and 13C NMR data, see Tables 5 and 6; HRESIMS m/z 527.3729 [M + Na]+ (calcd for C31H52O5Na, 527.3712). Lyonifoloside M (13). White powder; [α]25D −65 (c 0.3, MeOH); IR (KBr) νmax 3441, 2965, 1720, 1701, 1456, 1375, 1250,1081, 1045 cm−1; 1H and 13C NMR data, see Tables 5 and 6; HRESIMS m/z 717.4179 [M + Na]+ (calcd for C38H62O11Na, 717.4190). 2834



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Table 7. 1H NMR and 13C NMR Data of Compounds 19 and 20 in Pyridine-d5 19a

20a

position

δC

δH (J in Hz)

δC

δH (J in Hz)

1

44.3

43.9

2 3 4 5 6

66.8 74.7 45.4 47.4 30.1

2.20 m 1.88 m 4.47 m 4.55 s

2.18 dd (12.0, 4.3) 1.92 m 4.43 m 4.87 s

7

76.3

8 9 10 11

49.4 51.0 39.4 29.4

12 13 14 15

70.0 96.2 46.0 32.2

16

23.9

17 18 19 20 21

46.2 52.8 39.6 40.6 31.6

22

32.7

23

24.2

24

65.7

25 26 27 28 29 30

18.9 13.2 17.6 180.4 16.6 19.9

a

2.05 2.14 2.05 4.20

as that of 2a to obtain products 7b (1.3 mg) and 12b (1.6 mg), respectively. Determination of the Absolute Configuration of the 24,25Diol Moieties in Compounds 2, 7, and 12 by Snatzke’s and Frelek’s Method. According to the published approach,20−24 a 1:1.2 mixture of diol/Mo2(OAc)4 was subjected to ECD measurements at a concentration of 0.5 mg/mL for 2b, 7b, and 12b. The quarta cell was used, and the length of cell was 1 mm. In room temperature, the first ECD spectrum was recorded immediately after mixing, and its evolution was monitored over time until the signal was stationary (approximately 10 min after mixing). The inherent ECD was subtracted. The observed sign of the diagnostic band at 300−340 nm in the induced ECD spectrum was correlated to the absolute configuration of the 24,25-diol moiety. Antiviral Assays. African green monkey kidney cells (Vero) and Madin-Darby canine kidney (MDCK) cells were obtained from the Institute of Virology at the Chinese Academy of Preventive Medicine. Herpes simplex virus-1 (HSV-1 F strain VR 733), influenza A virus (A/95−359), and Coxsackie B3 virus (CVB3) were obtained from the American Type Culture Collection (ATCC) (Tables 8, 9, and 10).

m m m m

1.71 d (12.1) 2.28 dd (12.1, 3.0) 2.20 m 4.24 m

2.59 td (14.4, 6.2) 2.33 dd (15.0, 5.1) 2.18 m 1.36 m 2.88 d (11.4) 1.92 m 0.91 m 1.39 m 1.23 m 1.87 m 1.57 m 1.67 s 4.06 dd (10.8, 4.8) 3.88 dd (10.8, 4.5) 1.08 s 1.66 s 1.36 s 1.30 d (6.4) 0.87 d (5.7)

66.8 74.2 48.0 45.1 18.9 35.1 43.4 50.6 38.7 30.0 69.5 95.5 44.3 28.4 23.4 46.0 52.8 39.2 40.5 31.5 32.7 69.4 64.2 18.4 19.3 17.8 180.2 16.8 19.9

2.14 1.86 1.68 1.64 1.22

m m m m m

1.77 m 2.24 m 2.01 m 4.28 m

2.06 1.13 2.06 1.30

Table 8. Antiviral Activity against HSV-1 and Cytotoxicity of Compoundsa in Vero Cellsb

m m m m

2.73 d (11.4) 1.80 m 0.86 m 1.38 m 1.22 m 1.87 m 1.54 m 4.61 d (10.8) 4.40 m 4.16 d (10.3) 4.04 d (10.8) 1.04 s 1.34 s 1.12 s

compound

TC50c (μmol/L)

IC50 (μmol/L)

SId

1 1a 2a 8 12a 12−2a 13 14 16 acyclovire

23.1 ± 1.96 16.0 ± 0.72 57.7 ± 6.84 >100 16.0 ± 2.30 >100 19.3 ± 0.86 57.7 ± 6.46 >100 >100

11.1 ± 2.31 3.7 ± 1.35 11.1 ± 1.65 19.3 ± 3.31 2.1 ± 1.13 20.6 ± 4.66 6.4 ± 3.32 23.1 ± 7.23 14.3 ± 2.10 0.41 ± 0.10

2.1 4.3 5.2 >5.2 7.6 >4.9 3.0 2.5 >7.0 >244

a Compounds 2−7, 7a, 9−12, 15, and 17−20 all gave IC50 values of ≥33.3 μmol/L. bData represent mean values for three independent determinations. cCytotoxic concentration required to inhibit Vero cell growth by 50%. dSelectivity index value equaled TC50/IC50. ePositive control.

Table 9. Antiviral Activity against Influenza A and Cytotoxicity of Compoundsa in MDCK Cellsb

1.27 d (6.3) 0.85 brs

Recorded at 500 MHz for proton and at 125 MHz for carbon.

snailase (CODE S0100, Beijing Biodee Biotech Co., Ltd., Beijing, People’s Republic of China, 28 mg) at 37 °C for 2 days. The reaction mixture was diluted with H2O (6.0 mL) and extracted with EtOAc (6.0 mL × 3). After the concentration of the EtOAc-soluble phase, it was purified by semipreparative HPLC (MeCN-H2O, 56:44 v/v, 4 mL/min) to yield aglycone 7a (2.7 mg). Compound 12 (8.0 mg) was dissolved in MeOH-H2O (9:10 v/v, 3.8 mL) and then treated with the same procedure as 7, to afford aglycone 12a (3.9 mg). Methylation of Compounds 2a, 7a, and 12a. Compound 2a (4.5 mg) was treated with NaOH (3 mL, 1 mg/mL) for 2 h and then lyophilized. The solid obtained was dissolved in DMF (1 mL) and added to MeI (10 μL), and the mixture was stirred at room temperature for 5 h and detected using TLC. The reaction mixture was neutralized with HCl (0.5 M) and diluted in H2O (10 mL), and then extracted with EtOAc. After concentration, the EtOAc fraction was purified by reversed-phase semipreparative HPLC using MeCN in H2O (70:30) as the mobile phase to yield 2b (3.2 mg). Compounds 7a (2.5 mg) and 12a (3 mg) were methylated with the same method

compound

TC50c (μmol/L)

IC50 (μmol/L)

SId

1 1a 2 2a 3 7a 10 12a 13 12−2a 16 ribavirinf tamifluf

33.3 ± 4.82 6.4 ± 3.20 100.0 ± 1.03 19.3 ± 6.21 48.1 ± 5.23 >100 >100 16.0 ± 4.50 19.3 ± 5.11 >100 57.7 ± 2.08 1164 ± 1.61 1260 ± 2.76

>11.1 2.1 ± 0.56 33.3 ± 2.97 4.8 ± 3.16 >11.1 25.9 ± 4.77 33.3 ± 6.69 3.7 ± 1.08 11.1 ± 3.29 11.1 ± 5.75 33.3 ± 6.31 1.3 ± 0.08 1.7 ± 0.37

-e 3.0 3.0 4.0 -e >3.9 >3.0 4.3 1.7 >9.0 1.7 895 741

a

Compounds 4−9, 11, 12, 14, 15, and 17−20 all gave IC50 values of >33.3 μmol/L. bData represent mean values for three independent determinations. cCytotoxic concentration required to inhibit Vero cell growth by 50%. dSelectivity index value equaled TC50/IC50. eUnder the test conditions, the selectivity index (SI) could not be calculated. f Positive control. 2835



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IR, HRESIMS, 1H, 13C NMR, HSQC, HMBC, and NOESY spectra of compounds 1−20 (PDF) X-ray crystallography data of compound 1a (CIF)

Table 10. Antiviral Activity against CVB3 and Cytotoxicity of Compoundsa in Vero Cellsb compound

TC50c (μmol/L)

IC50 (μmol/L)

SId

1 1a 2a 12a 12−2a 13 pleconarilde ribavirinde

23.1 ± 2.79 16.0 ± 0.73 48.1 ± 5.92 19.3 ± 3.61 >100 19.3 ± 2.38 15.4 ± 0.51 2000 ± 11.29

11.1 ± 1.98 2.1 ± 0.30 4.8 ± 1.20 4.8 ± 1.16 19.2 ± 3.48 11.1 ± 1.17 0.001 ± 0.0001 292 ± 9.04

2.1 7.6 10.0 4.0 >5.2 1.7 15400 6.8

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Corresponding Author

*E-mail: [email protected]. Tel: +8610 63165326. Fax: +8610 63017757. Notes

The authors declare no competing financial interest.

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Compounds 2−12, 7a, and 14−20 all gave IC50 values of ≥33.3 μmol/L. bData represent mean values for three independent determinations. cCytotoxic concentration required to inhibit Vero cell growth by 50%. dSelectivity index value equaled TC50/IC50. e Positive control. a

ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (Nos. 21132009 and 21572274). The authors are grateful to the Department of Instrumental Analysis of Peking Union Medical College for the spectroscopic measurements.

Cytotoxicity Assay. The cytotoxicity of the test compounds toward Vero and MDCK cells was monitored by measuring the cytopathic effect (CPE). Vero and MDCK cells (2.5 × 104/well) were plated into a 96-well plate. Then, 24 h later, the monolayer cells were incubated with various concentrations of the test compounds. After 48 h of culture at 37 °C and 5% CO2 in a carbon dioxide incubator, the cells were monitored by CPE. The median toxic concentration (TC50) was calculated by Reed and Muench analysis. Anti-HSV 1 Activity Assay. The anti-HSV 1 activity of the test compounds was assayed by the CPE inhibition method. Briefly, Vero cells (2.5 × 104 cells/well) were plated into 96-well culture plates for an incubation period of 24 h. The medium was then removed, and the cells were infected with 100 μL of HSV-1 at 100 TCID50 for 2 h. Then, various concentrations of the test compounds were added, and incubation was carried out until the CPE of the control group cells reached a value of 4+. Each experiment was performed in triplicate at least three separate times. The IC50 value is defined as the minimal concentration of inhibitor required to inhibit 50% of the CPE, as determined by the Reed and Muench method. The selectivity index was calculated as the ratio of TC50/IC50. Anti-Influenza A Activity Assay. The anti-influenza A activity of the test compounds was evaluated by the CPE inhibition method. Briefly, MDCK cells (2.5 × 104 cells/well) were plated into 96-well culture plates for a 24 h incubation. The medium was removed, and the cells were washed with PBS and then infected with 100 μL influenza A (A/95−359) at 100 TCID50 for 2 h. Then, various concentrations of the test compounds were added, and incubation was carried out until the CPE of the control group cells reached a value of 4+. Each experiment was performed in triplicate at least three separate times. The IC50 value is defined as the minimal concentration of inhibitor required to inhibit 50% of the CPE, as determined by the Reed and Muench method. The selectivity index was calculated as the ratio of TC50/IC50. Anti-CVB3 Activity Assay. African green monkey kidney (Vero) cells were grown in 96-well microplates, which were infected with 100× the median tissue culture infective dose (100TCID50) of Cox B3 virus. After 2 h of absorption at 37 °C, the monolayers were washed by phosphate buffered saline (PBS) and incubated in the maintenance medium [MEM plus 2% fetal bovine serum (FBS)] at 37 °C with or without various concentrations of test compounds and the positive control. When the viral control group reached value of 4+, the viral cytopathic effect (CPE) was observed and the anti-CVB3 activity of tested compounds were established by the Reed and Muench method.

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AUTHOR INFORMATION

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REFERENCES

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