Antiviral Triterpenes from the Twigs and Leaves of Lyonia ovalifolia

Article abstract of DOI:10.1021/acs.jnatprod.6b00585

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.

Full text of DOI:10.1021/acs.jnatprod.6b00585

Article
pubs.acs.org/jnp
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 config-
uration 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 Mo (OAc) -induced electronic
2
4
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 IC values from 2.1 to 11.1 μM. In turn, compounds 1, 1a, 2a, 12a, and 13 exhibited potent activity
5
0
against CVB3, with IC values from 2.1 to 11.1 μM.
5
0
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
1
−5
metabolites, such as grayanane diterpenoids,
triterpene
These com-
pounds exhibit diverse biological activities, including cAMP
6
−8
9,10
11,12
glycosides,
lignans,
and flavonoids.
3
4,5
8
regulating, analgesic,
and tyrosinase inhibitory effects.
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.
RESULTS AND DISCUSSION
■
Compound 1 was obtained as a white powder. Its molecular
13
formula was established as C H O based on the C NMR
3
6
58 10
+
and HRESIMS (m/z 673.3944 [M + Na] , calcd for
4
.86 (1H, d, J = 7.8 Hz, H-1′), suggesting that 1 is a triterpene
C H O Na, 673.3928) data, indicating eight degrees of
36
58 10
monoglycoside. Acid hydrolysis of 1 with HCl afforded a new
unsaturation. The IR spectrum exhibited absorption bands for
−
1
−1
hydroxy (3386 cm ) and carbonyl (1707, 1682 cm ) groups.
1
Its H NMR spectrum displayed signals of five three-proton
Received: June 26, 2016
singlets, a three-proton doublet, and an anomeric proton at δH
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H NMR Data of Compounds 1−6 in Pyridine-d (δ in ppm, J in Hz)
5
b
b
b
b
b
c
b
a
c
position
1
1
1a
2
2a
2b
3
4
5
6
2.08 m
.05 m
2.04 m
.73 m
3.76 dd (11.1,
.1)
2.28 m
.13 m
2.00 m
.25 m
2.15 m
2.11 m
1.87 m
1.80 m
2.08 m
2.06 m
2.04 m
1.74 m
2.16 m
2.09 m
1.88 m
1.81 m
2.16 m
2.12 m
1.91 m
1.86 m
3.73 m
2.08 m
2.08 m
2.04 m
1.75 m
2.08 m
2.00 m
2.04 m
1.74 m
3.77 m
2.07 m
2.07 m
2.05 m
1.74 m
2.06 m
2.06 m
2.03 m
1.73 m
2
2
1
3
6
3.71 dd (11.1,
3.3)
3.77 dd (11.3,
2.2)
3.72 dd (11.0,
2.9)
3.74 dd (11.5,
2.0)
3.74 dd (11.9,
2.3)
3.72 d
(10.8)
2
2.39 m
2.22 m
2.04 m
1.31 m
1.83 m
2.21 m
1.72 m
1.55 m
3.02 m
1.67 m
2.74 m
1.31 m
2.37 m
1.54 m
1.79 m
1.01 s
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
2.41 m
2.24 m
2.05 m
1.31 m
1.85 m
2.23 m
1.74 m
1.56 m
3.04 m
1.72 m
2.74 m
1.31 m
2.37 m
1.53 m
1.84 m
1.03 s
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
2.36 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
2.29 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
2.35 m
2.20 m
2.03 m
1.26 m
1.83 m
2.17 m
1.69 m
1.53 m
3.01 m
1.72 m
2.73 m
1.30 m
2.48 m
1.72 m
1.84 m
1.05 s
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
7
1
8
9
1.79 m
2.14 m
1.68 m
11
12
15
16
1
.50 m
2.96 m
.65 m
2.70 m
.30 m
2.36 m
.54 m
1
1
1
1
1
1
7
8
9
1.76 m
0.99 s
2.24 m
2.31 m
1.72 m
1.60 m
0.96 d (6.5)
2.26 m
1.67 m
1.63 m
1.03 m
2.32 m
1.73 m
1.65 m
1.05 d (6.5)
2.25 m
1.69 m
1.58 m
1.01 d
2.26 m
1.68 m
1.63 m
1.04 m
2.26 m
1.67 m
1.64 m
0.99 d (6.4)
2.26 m
1.66 m
1.64 m
0.99 d (6.4)
2.27 m
1.69 m
1.64 m
1
.66 m
2
2
0
1
1.59 m
0.95 d (6.4)
0.99 d
(6.2)
(
6.4)
2
2
2
3
2.02 m
2.02 m
1.39 m
2.94 m
2.94 m
2.22 m
1.18 m
2.03 m
1.52 m
3.66 d (10.1)
1.46 s
2.22 m
1.20 m
2.04 m
1.53 m
3.67 d (9.6)
1.47 s
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
2.23 m
1.19 m
2.04 m
1.52 m
3.66 d (10.0)
1.47 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
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
1
.38 m
2.93 m
.93 m
2
2
2
2
2
2
3
4
6
7
8
9
1
1.52 s
1.51 s
1.04 s
1.23 s
1.53 s
1.52 s
1.15 s
1.27 s
1.49 s
1.50 s
1.49 s
1.04 s
1.16 s
1.05 s
1.24 s
1.28 s
1.32 s
1′
4.86 d (7.8)
4.87 d (7.7)
4.81 d (7.7)
4.87 d (7.7)
4.82 m
4.72 d
(
6.7)
2
′
′
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
4.36 t (7.6)
3
4.18 d
(
7.8)
4
′
′
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
4.32 m
4.31 m
5
3
.77 m
6′
4.53 dd (11.5,
4.55 dd (11.6,
2.2)
4.95 d (11.5)
4.54 dd (11.5,
2.3)
4.95 dd (11.5,
1.9)
2.1)
4
.38 dd (11.5,
4.39 dd (11.6,
5.2)
4.79 dd (11.6,
6.7)
4.38 dd (11.6,
5.3)
4.79 m
5.2)
8′
1.96 s
1.96 s
1″
4.78 d (7.3)
4.80 m
4.80 d
7.2)
(
2
″
″
4.44 t (7.7)
4.45 t (7.4)
4.45 t (8.1)
3
4.12 dd (9.1,
4.13 dd (9.1,
3.3)
4.13 d
(7.9)
3
.0)
4
″
″
4.27 m
4.28 m
4.28 m
4.29 m
3.80 m
4.29 m
4.30 m
3.80 m
5
3
.79 m
a
b
c
Recorded at 400 MHz. Recorded at 500 MHz. Recorded at 600 MHz.
1
triterpene aglycone (1a: C H O ), named lyonifolic acid A,
the anomeric proton in the H NMR spectrum (Table 1)
3
0
48
5
and glucose. The relatively large coupling constant (7.8 Hz) for
suggested a β-configuration for the glucopyranosyl moiety. The
B
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Data of Compounds 1−6 in Pyridine-d (δ in ppm)
5
b
b
b
b
b
c
b
a
c
position
1
1a
2
2a
2b
3
4
5
6
1
2
3
4
5
6
7
8
9
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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
3
4
5
6
7
8
′
′
′
′
′
′
′
′
103.1
75.4
79.0
72.3
78.6
63.5
103.1
75.5
79.1
72.3
78.6
63.5
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
1″
2″
3″
4″
5″
106.5
73.0
74.9
69.8
67.8
106.5
73.1
75.0
69.9
67.9
a
b
c
Recorded at 100 MHz. Recorded at 125 MHz. Recorded at 150 MHz.
absolute configuration of D-glucose was determined by GC
analysis. The H NMR spectrum of 1a showed five tertiary
(ring B). The structure was fully determined using its HMBC
1
1
1
and H− H COSY spectra. The key HMBC correlations from
methyl groups (δ 1.53, 1.52, 1.27, 1.15 and 1.01), a secondary
H -19 to C-1, C-5, C-8, C-9, C-10, and C-11, and from H -18
H
2
3
methyl group at δ 0.96 (d, J = 6.5 Hz), and an oxymethine
to C-12, C-13, C-14, and C-17, together with the
H
1
1
proton at δ 3.71 (dd, J = 11.1, 3.3 Hz). With the aid of HSQC
corresponding cross-peaks in the H− H COSY spectrum,
H
1
3
analysis, the 30 signals in the C NMR spectrum (Table 2)
could be assigned as eight quaternary carbons (one carbonyl at
δ 216.6, one carboxylic acid at δ 178.7, two olefinics at δ
shown in Figure 1a, provided evidence of the presence of a 6/
14−20
7/6/5 tetracyclic
fused ring system. This spectrum also
showed long-range correlations from H -28/29 to C-3, C-4,
C
C
C
3
1
41.5 and 132.5, and one oxygenated at δ 77.1), five methines
and C-5, and from H -26/27 to C-24 and C-25, confirming the
C
3
(
one oxygenated at δ 75.6), 11 methylenes, and six methyls.
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 H -1, H -6, H -7, H -19, H -28,
C
13
By comparison with the triterpene lanostane, 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 C-
2
2
2
2
3
and H -29 to C-5 (δ 141.5), and from H -1, H -2, H -6, H-9,
3
C
2
2
2
10 is cleaved, and the molecule forms a seven-membered ring
and H -19 to C-10 (δ 132.5) and was located between C-5
2 C
C
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
confirmed by the HMBC correlations of H -26/27 with C-24
3
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
Mo (OAc) -induced circular dichroism (ICD) method devel-
2
4
21−26
oped by Snatzke and Frelek for vicinal diols.
During the
application of the method, the aglycone (2a) obtained from
acid hydrolysis of 2 with HCl was treated with the methylation
reagent CH I and converted into 2b to avoid interference from
3
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
1
1
Figure 1. (a) Selected HMBC (H → C) and H− H COSY ()
correlations of 1a. (b) Selected NOESY correlations of 1a.
and C-10. Besides, HMBC correlations from H-8 and H -15 to
2
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 H -18/H-8, H-20; H-19a/H-8 showed that H-
Figure 3. Circular dichroism spectrum of 2b in DMSO solution of
dimolybdenum tetracetate (the inherent CD spectrum of 2b was
subtracted).
3
8
and H -18 are β-oriented, whereas H -21/H-17, H-9/H-19b
3
3
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
configuration for compound 2b. Thus, compound 2 was
determined as 3α-[(β-D-glucopyranosyl)-oxy]-24S,25-dihy-
droxy-9,10-seco-cycloartan-5(10)-en-30-oic acid.
2). Thus, compound 1a was established as 3α,25-dihydroxy-
6,7,16
9
,10-seco-cycloartan-24-oxo-5(10)-en-30-oic acid.
The HMBC correlation in 1 was observed from H-1′ (δ
Compounds 3−6 (lyonifolosides C−F) were assigned the
molecular formulas of C H O , C H O , C H O , and
H
38 62 11
41 68 14
43 70 15
1
3
4
.86) of the glucose to C-3 (δ 83.1) of the aglycone, which led
C H O , respectively, on the basis of their respective
C
C
40 66 13
NMR and HRESIMS data. Acid hydrolysis of 3−6 with HCl
produced the same aglycone (2a) and D-glucose, D-glucose/L-
arabinose, 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 D-
glucose 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.
D
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H NMR Data of Compounds 7−11 in Pyridine-d (δ in ppm, J in Hz)
5
a
b
c
a
b
b
a
position
7
7a
7b
8
9
10
11
1
2
5.36 s
2.50m
5.42 s
2.50m
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
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
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
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
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
.38 m
3.75 dd (4.2, 4.2)
2.28 m
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
3
5
6
1.86 m
1
.24 m
2.15 m
.41 m
7
1
8
9
1.60 m
2.45 m
1.63 m
11
12
15
16
1
.52 m
2.83 m
.69 m
2.64 m
.27 m
2.40 m
.53 m
1
1
1
1
1
1
7
8
9
1.84 m
0.98 s
2.31 m
2.41m
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
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
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
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
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
2.25 m
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
2
2
2
0
1
2
1.63 m
1.03 d (6.4)
2.23 m
1
.20 m
2.02 m
.53 m
23
1
2
2
2
2
2
3
4
6
7
8
9
1
3.67 dd (10.1, 1.6)
1.47 s
1.50 s
1.50 s
1.54 s
1.40 s
1.40 s
1.47 s
1.40 s
1.21 s
1.19 s
1.25 s
1.22 s
1.19 s
1.17 s
1.18 s
0.91 s
0.87 s
0.86 s
0.91 s
0.86 s
0.84 s
0.89 s
3.53 s
1′
2′
3′
4′
5′
4.77 d (7.8)
3.82 m
4.77 d (7.8)
3.83 m
4.84 d (7.8)
3.85 m
4.82 d (6.3)
3.84 m
4.69 d (6.9)
4.27 m
4.18 t (8.6)
3.99 m
4.18 m
4.22 m
4.21 m
4.14 m
4.00 m
4.23 m
4.22 m
4.29 m
3.96 m
3.98 m
3.91 m
3.89 m
4.28 m
3
.76 m
6
′
′
4.93 dd (11.5, 1.7)
4.94 dd (11.5, 1.7)
4.80 m
4.52 dd (11.5, 2.3)
4.40 dd (11.6, 5.0)
4.51 m
4.39 m
4.79 m
8
2.03 s
2.04 s
1″
2″
3″
4″
5″
4.81 m
4.80 d (7.3)
4.45 dd (8.7, 7.7)
4.13 dd (9.1, 3.0)
4.28 m
5.14 d (5.9)
4.06 m
4.80 d (7.3)
4.46 dd (8.9, 7.4)
4.14 m
4.46 dd (9.0, 7.4)
4.14 m
4.21 m
4.29 m
4.28 m
4.29 m
4.30 m
4.29 m
3.89 m
4.30 m
3
.81 m
3.81 m
3.81 m
6″
4.51 m
4
.45 m
a
b
c
Recorded at 400 MHz. Recorded at 500 MHz. Recorded at 600 MHz.
the glucose group at C-3 in 4 on the basis of the HMBC
correlations between H -6′ (δ 4.95 and 4.79) and C-7′ (δ
[(6-O-acetyl-β-D-glucopyranosyl)-oxy]-24S,25-dihydroxy-9,10-
seco-cycloartan-5(10)-en-30-oic acid, 3α-[(β-D-glucopyranosyl)-
oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy-9,10-seco-
cycloartan-5(10)-en-30-oic acid, 3α-[(6-O-acetyl-β-D-glucopyr-
anosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hydroxy-
9,10-seco-cycloartan-5(10)-en-30-oic acid, and 3α-[(α-L-arabi-
2
H
C
1
71.1). 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 3α-
E
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
nopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-oxy]-25-hy-
droxy-9,10-seco-cycloartan-5(10)-en-30-oic acid, respectively.
1
3
According to its C NMR data and HRESIMS m/z
+
7
17.4281 [M + Na] , compound 7 (lyonifoloside G) gave
1
13
the same molecular formula as 3, C H O . The H and
C
3
8
62 11
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 (δ 5.36) to C-3, C-
H
5
, and C-19, and from H-5 and H -6 to C-10 (δC 140.9)
2
indicated that the double bond is located between C-1 and C-
0. The hydroxy group at C-3 in 7a (the aglycone of 7) was
1
Table 4. 13C NMR Data of Compounds 7−11 in Pyridine-d5
δ in ppm)
(
a
b
c
a
b
b
a
position
7
7a
7b
8
9
10
11
1
1
1
2
3
4
5
6
7
8
9
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
Figure 4. (a) Selected HMBC (H → C) and H− H COSY ()
correlations of 7. (b) Selected NOE correlations of 7a.
assigned as α-oriented by comparison with that of cimifoeti-
danol A. Large coupling constants of H-3 (δ 3.78, dd, J =
9.5, 6.6 Hz) and NOESY correlations of H-3/H -29 and H-3/
H-5 were observed in cimifoetidanol A. 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 H -28/H-3, H-5 and H -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 H and
C NMR spectra compared to that between 7 and 3. To
confirm this, a Mo (OAc) -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,
14
H
3
14
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
1
13
2
4
compound 7a was treated with CH I and converted into the
3
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
b. Accordingly, compound 7 was assigned as 3α-[(6-O-acetyl-
β-D-glucopyranosyl)-oxy]-24S,25-dihydroxy-9,10-seco-cycloar-
2
tan-1(10)-en-30-oic acid.
1′
2′
3′
4′
5′
6′
7′
8′
1″
2″
3″
4″
5″
6″
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 5. Circular dichroism spectrum of 7b in DMSO solution of
dimolybdenum tetracetate (the inherent CD spectrum of 7b was
subtracted).
a
b
c
Recorded at 100 MHz. Recorded at 125 MHz. Recorded at 150
MHz.
F
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 5. H NMR Data of Compounds 12−18 in Pyridine-d (δ in ppm, J in Hz)
5
b
b
c
b
b
a
a
c
a
position
1
12
2.23 m
.58 m
2.11 m
.81 m
12a
12b
13
14
15
16
17
18
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
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
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
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
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
2.20 m
1.56 m
2.06 m
1.83 m
3.62 brs
2.00 m
1.62 m
1.55 m
2.32 m
2.24 m
2.39 m
2.17 m
2.75 m
1.79m
2.25 m
1.58 m
2.11 m
1.81 m
3.70 brs
2.03 m
1.57 m
1.57 m
2.32 m
2.22 m
2.44 m
2.17 m
2.76 m
1.81m
2.16 m
1.58 m
2.08 m
1.84 m
3.62 brs
1.98 m
1.63 m
1.58 m
2.38 m
2.24 m
2.40 m
2.19 m
2.80 m
1.81m
1
2
1
3
5
6
3.69 brs
2.01 m
1.63 m
1
.56 m
2.31 m
.21 m
2.44 m
.18 m
2.75 m
.81 m
2.47 m
.69 m
2.41 m
.56 m
7
2
1
1
1
1
1
2
5
6
2
1
2.50 m
1.71 m
2.50 m
1.69 m
2.06 m
0.94 s
2.50 m
1.70 m
2.50 m
1.69 m
2.06 m
0.94 s
2.55 m
1.68 m
2.57 m
1.68 m
2.03 m
0.87 s
2.51 m
1.74 m
2.52 m
1.68 m
2.11 m
0.95 s
1
1
1
1
1
2
2
2
7
8
9
0
1
2
2.02 m
0.92 s
1.13 s
1.18 s
1.13 s
1.14 s
1.17 s
1.12 s
1.13 s
1.22 s
1.14 s
1.61 m
1.64 m
1.58 m
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,
1.53 m
1.05 d (6.5)
2.47 m
1.22 m
1.83 m
1.53 m
3.71 d (9.1)
1.60 m
1.01 d (6.4)
1.86 m
1.86 m
1.71 m
1.71 m
3.80 m
1.61 m
1.52 m
1.63 m
1.06 d (6.5)
2.25 m
1.10 d (6.4) 1.06 d (6.4)
1.01 d (6.3) 1.03 d (6.3) 1.04 d (6.4)
2.28 m
1.23 m
2.24 m
1.20 m
2.07 m
1.55 m
3.71 m
1.84 m
1.84 m
1.75 m
1.73 m
3.80 m
2.48 m
1.21 m
1.82 m
1.53 m
3.71 m
1.88 m
1.88 m
1.74 m
1.74 m
3.83 m
1
.20 m
2.06 m
.54 m
2
2
3
4
2.09 m
1
1.56 m
3.68 m
3.69 d (9.9)
1
.1)
2
2
2
2
3
6
7
8
9
1
1.47 s
1.50 s
1.11 s
0.88 s
1.49 s
1.52 s
1.16 s
0.96 s
1.52 s
1.54 s
1.20 s
0.95 s
3.48 s
1.48 s
1.51 s
1.17 s
0.95 s
1.38 s
1.50 s
1.16 s
0.95 s
1.35 s
1.39 s
1.11 s
0.87 s
1.35 s
1.38 s
1.15 s
0.94 s
1.38 s
1.50 s
1.22 s
0.87 s
1.37 s
1.41 s
1.14 s
0.93 s
1′
2′
3′
4′
5′
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)
4.83 d (7.8)
3.86 m
4.73 m
3.82 m
4.85 d (7.7) 4.69 d (6.7)
3.89 m
4.24 m
4.20 m
3.90 m
4.28 m
4.16 m
4.33 m
4.29 m
4.21 t (9.0)
4.22 d (8.8)
4.19 d (9.1)
3.92 m
4.17 t (8.4)
3.96 d (9.1)
3.95 m
4.30 t (9.2)
3.89 dt (9.2, 3.6)
3
.78 m
6′
4.52 dd (8.1, 2.3)
4.93 dd (11.7,
4.53 dd (11.4,
2.6)
4.53 dd (11.6,
1.9)
4.90 d (11.8)
4.74 m
4.54 m
4.47 m
1
.8)
4
.35 dd (11.7,
4.77 m
4.46 dd (11.4,
4.2)
4.36 dd (11.7,
5.3)
5.3)
8
′
1.99 s
1.98 s
4.78 m
1″
2″
3″
4″
5″
4.79 d (7.3)
4.44 t (7.8)
4.12 dd (9.0, 3.2)
4.27 m
5.17 d (7.7) 4.82 d (7.3)
4.42 t (8.1)
4.11 m
4.08 m
4.22 m
4.30 m
3.94 m
4.47 t (7.4)
4.16 m
4.26 m
4.33 m
4.28 m
4.27 m
4.29 m
3
.78 m
3.78 m
3.80 m
6″
4.54 m
4
.38 m
a
b
c
Recorded at 400 MHz. Recorded at 500 MHz. Recorded at 600 MHz.
HRESIMS and the 13C NMR data indicated the molecular
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
formulas of compounds 8−11 (lyonifolosides H−K) to be
C H O , C H O , C H O , and C H O , respec-
43
70 15
41 68 14
42 70 15
40 66 13
tively. Comparison of their NMR data with those of 7 indicated
that all these compounds share the same aglycone. Compound
was found to possess a 6-O-acetyl-glucose and an arabinose
8
G
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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-arabinopyra-
nosyl)-oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic
acid, 3α-[(β-D-glucopyranosyl)-oxy]-24S-[(α-L-arabinopyrano-
syl)-oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid,
3
α-[(β-D-glucopyranosyl)-oxy]-24S-[(β-D-glucopyranosyl)-
oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid, and
α-[(α-L-arabinopyranosyl)-oxy]-24S-[(α-L-arabinopyranosyl)-
3
oxy]-25-hydroxy-9,10-seco-cycloartan-1(10)-en-30-oic acid, re-
spectively.
Compound 12 was obtained as a white powder. The
molecular formula was determined to be C H O based on
3
6
60 10
+
the sodiated molecular ion peak, [M + Na] , at m/z 675.4078
calcd for C H O Na, 675.4084) by HRESIMS, and was
Figure 7. Circular dichroism spectrum of 12b in DMSO solution of
dimolybdenum tetracetate (the inherent CD spectrum of 12b was
subtracted).
(
36
60 10
1
3
supported by its C NMR data. The IR spectrum revealed
hydroxy and carbonyl groups on the basis of absorption bands
−1
1
at 3394 and 1687 cm , respectively. The H NMR spectrum
Table 5) exhibited seven methyl groups at δ 1.50, 1.47, 1.13,
Compounds 13−18 (lyonifolosides M−R) exhibited molec-
(
H
ular formulas of C H O , C H O , C H O , C H O ,
38
62 11
36 60 10
41 68 14
43 70 15
1
.11, 1.06, 0.92, and 0.88, an anomeric proton at δ 4.82 (1H,
H
C H O , and C H O , respectively, as revealed by
42 70 15
40 66 13
13
d, J = 7.8 Hz), and several oxymethine protons between δ 3.68
and δ 4.82. The C NMR spectrum of the aglycone moiety of
1
H
HRESIMS and their C 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-
1
3
H
27
2 was very similar to that of inoterpene A, 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 H -15 to C-8/C-13/C-14/C-30/
2
C-16/C-17 and H -16 to C-13/C-14/C-17 (Figure 6). The H-3
2
[
(6-O-acetyl-β-D-glucopyranosyl)-oxy]-24(S),25-dihydroxyla-
nost-8-en-30-oic acid, 24(S)-[(β-D-glucopyranosyl)-oxy]-3α,25-
dihydroxylanost-8-en-30-oic acid, 3α-[(β-D-glucopyranosyl)-
oxy]-24(S)-[(α-L-arabinopyranosyl)-oxy]-25-hydroxylanost-8-
en-30-oic acid, 3α-[(6-O-acetyl-β-D-glucopyranosyl)-oxy]-
1
1
Figure 6. Selected HMBC (H→C) and H− H COSY ()
correlations of 12.
2
4(S)-[(α-L-arabinopyranosyl)-oxy]-25-hydroxylanost-8-en-30-
oic acid, 3α-[(β-D-glucopyranosyl)-oxy]-24(S)-[(β-D-glucopyr-
anosyl)-oxy]-25-hydroxylanost-8-en-30-oic acid, and 3α-[(α-L-
arabinopyranosyl)-oxy]-24(S)-[(α-L-arabinopyranosyl)-oxy]-
25-hydroxylanost-8-en-30-oic acid, respectively.
1
in the H NMR spectrum displayed a broad single peak,
indicating that the H-3 is in an equatorial position, with a half-
chair 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
2−2a obtained by acid hydrolysis with HCl was the isomer of
2a. The reason could be that the double bond in 12 readily
The molecular formula of compound 19 was determined as
+
C H O based on the [M + H] ion peak at m/z 521.3479
30
48
7
13
1
1
(calcd for C H O 521.3478) by its C NMR data and
30 49 7
HRESIMS, indicating seven indices of hydrogen deficiency.
−1
underwent an addition reaction with H O under acid
The IR spectrum showed the presence of hydroxy (3480 cm )
2
−1
1
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
and carbonyl (1740 cm ) absorptions. Its H NMR spectrum
(Table 7) displayed characteristic signals for four tertiary
methyl groups (δ 1.67, 1.66, 1.36, and 1.08), two secondary
H
methyl groups (δ 1.30 and 0.87), four oxygenated methines
H
obtain compound 12b, 12a was methylated with CH I. As
(δ 4.47, 4.55, 4.20, and 4.24), and an oxygenated methylene
3
H
13
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-
(δ 4.06 and 3.88). The C NMR spectrum (Table 7), when
H
combined with an analysis of the HSQC spectrum, showed the
presence of 30 carbon signals, including six methyls, eight
(
1
S),25-trihydroxylanost-8-en-30-oic acid. The glucose group in
methylenes (one oxygenated at δ 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
C
2 was placed at C-3 based on the glycosylation shift of C-3 at
1
3
δC 82.1 in the C 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-D-
glucopyranosyl)-oxy]-24(S),25-dihydroxylanost-8-en-30-oic
acid.
oxygenated at δ 96.2). These structural features were shown
C
28,29
to be consistent with an ursane triterpenoid skeleton,
with
six sites of oxygenation and one carbonyl group. In the HMBC
spectrum of 19, correlations from H -25 to C-1, C-5, C-9, and
3
H
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 6. 13C NMR Data of Compounds 12−18 in Pyridine-d (δ in ppm)
5
b
b
c
b
b
a
a
c
a
position
12
12a
12b
13
14
15
16
17
18
1
2
3
4
5
6
7
8
9
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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
3
4
5
6
7
8
′
′
′
′
′
′
′
′
102.7
75.2
79.1
72.2
78.6
63.4
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
1″
2″
3″
4″
5″
6″
106.4
73.0
74.9
69.8
67.7
107.3
76.5
79.1
72.1
78.7
63.5
106.5
73.1
75.0
69.9
67.8
a
b
c
Recorded at 100 MHz. Recorded at 125 MHz. Recorded at 150 MHz.
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 H -24 to C-3, C-4, and C-23; from H -23 to C-3,
the configurations of H-2, H-3, and H -24 as β-oriented, while
2
those between H-7/H-5, H-9, H -27, and H-12/H-9, H -27
2
3
3
3
C-4, C-5 and C-24; from H -26 to C-7, C-8, C-9; from H-11 to
suggested that H-7 and H-12 are α-oriented (Figure 8b).
Therefore, compound 19 was proposed as 2α,3α,7β,12β,24β-
pentahydroxyurs-28,13β-olide and was given the trivial name
lyonifolide A.
3
C-8, C-12, and C-13; and from H -27 to C-8, C-13, C-14, and
3
C-15 indicated that the six sites of oxygenation are located at C-
2, 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 C NMR spectrum, the relatively
Compound 20 (lyonifolide B) was assigned the same
1
3
molecular formula as 19 of C H O on the basis of its
C
30
48
7
1
3
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: δ 4.20, δ 76.3; C-
downfield chemical shift of C-13 (δ 96.2) suggested C-30/C-
C
13 lactonization. Thus, the planar structure of compound 19
H
C
could be defined. NOESY correlations observed between H-2/
23: δ 1.67, δ 24.2) was transformed to C-23 in 20 (C-7: δ
H
C
H
H -25, H-3/H-2, H -23, H -24, and H -24/H -25 supported
1.64 1.22, δ 35.1; C-23: δH 4.61 4.40, δ 69.4). This was
3
3
2
2
3
C
C
I
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
EtOH fraction (740 g) was then further separated on a macroporous
resin column and eluted in a gradient of EtOH-H O (30:70, 60:30,
2
9
5:5 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−
00 mesh) and eluted with a CH Cl −CH OH (100:1 → 1:1 v/v)
3
2
2
3
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
Figure 8. (a) Selected HMBC (H→C) and 1H− H COSY ()
1
MeOH-H O (30:70, 60:40, 70:30, 80:20, 100:0 v/v) to obtain five
2
correlations of 19. (b) Selected NOESY correlations of 19.
subfractions (G2A1−G2A5). Fraction G2A4 (1.5 g) was separated by
preparative HPLC with MeCN-H O (58:42 v/v, 7 mL/min) to afford
2
confirmed by the HMBC correlations from H -23 to C-3, C-4,
nine fractions, G2A4a−G2A4i, and compound 13 (160 mg, t = 44
2
R
1
1
C-5 and C-24 and the H− H COSY correlations for C(7)H −
min). Fraction G2A4c (102 mg) was purified by semipreparative
2
HPLC with MeCN-H O (45:55 v/v, 4 mL/min) to yield compound 1
C(6)H . Thus, compound 20 was identified as
2
2
(64 mg, tR = 32 min). Purification of fraction G2A4e (86 mg) by
2
α,3α,12β,23α,24β-pentahydroxyurs-28,13β-olide.
semipreparative HPLC with MeCN-H O (46:54 v/v, 4 mL/min)
2
All compounds were tested for antiviral (HSV-1, influenza A/
5−359 and Coxsackie B3) activities. Compounds 1, 1a, 2a,
2a, 13, and 16 exhibited potent activity against HSV-1, with
afforded compound 3 (8.9 mg, t = 45 min). G2A4i yielded compound
R
9
1
7
(11 mg, tR = 43 min), which was repeatedly purified by
semipreparative HPLC with MeCN-H O (48:52 v/v, 4 mL/min).
2
IC values from 2.1 to 14.3 μM, compounds 8, 12−2a, and 14
50
Fraction G2A3 (1.3 g) was separated by semipreparative HPLC with
showed moderate activity, with IC values from 19.3 to 23.1
5
0
MeCN-H O (30:70 v/v, 4 mL/min) to afford 10 fractions, G2A3a−
2
μM. Compounds 1a, 2a, 12a, 13, and 12−2a displayed potent
activity against influenza A/95−359, with IC values from 2.1
G2A3j. Fraction G2A3g (48 mg) yielded compound 20 (9.0 mg, t =
R
5
0
39 min) with MeOH-H O (58:42 v/v, 4 mL/min) by semipreparative
2
to 11.1 μM, and compounds 1, 2, 3, 7a, 10, and 16 showed
moderate activity against A/95−359, with IC values from 11.1
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
5
0
gradient of MeOH-H O (30:70, 60:40, 70:30, 80:20, 100:0 v/v) to
to 33.3 μM. In turn, compounds 1, 1a, 2a, 12a, and 13
2
produce five subfractions (G6A1−G6A5). Fraction G6A5 (3.7 g) was
exhibited potent activity against CVB3, with IC50 values from
separated by preparative HPLC with MeCN-H O (55:45 v/v, 7 mL/
2
2.1 to 11.1 μM.
min) to afford eight fractions, G6A5a−G6A5h. Fraction G6A5h (140
mg) yielded compounds 15 (30.8 mg, t = 12 min) and 16 (52 mg, t
R
R
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
■
=
17 min) with MeOH-H O (87:13 v/v, 4 mL/min) by semi-
2
preparative HPLC. Fraction G6A5g (291 mg) was purified to obtain
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. Prepara-
tive HPLC was performed on a Shimadzu LC-6AD instrument with
SPD-20A and RID-10A detectors, using a YMC Pack ODS-A column
compound 5 (7 mg, t = 11 min) and a mixture of 8 and 9 using the
R
above HPLC system (MeOH-H O, 87:13 v/v, 4 mL/min). The
2
mixture was further purified by semipreparative HPLC with MeOH-
H O (85:15 v/v, 4 mL/min) to afford 9 (50 mg, t = 18 min) and 8
2
R
(
(
73 mg, t = 23 min). Compounds 11 (15.7 mg, t = 27 min) and 18
R
R
15 mg, t = 32 min) were isolated from fraction G6A5f (86 mg) by
R
semipreparative HPLC with MeOH-H O (84:16 v/v, 4 mL/min).
2
Purification of fraction G6A5d (703 mg) by semipreparative HPLC
(
MeOH-H O, 81:19 v/v, 4 mL/min) afforded compounds 2 (34 mg,
2
t = 16 min) and 12 (20 mg, t = 27 min) as well as two mixtures,
R
R
G6A5d1 and G6A5d2. The G6A5d1 mixture was subsequently purified
(
250 × 20 mm, 5 μm, Kyoto, Japan). Polyamide resin (30−60 mesh,
by HPLC with MeOH-H O (78:22 v/v, 4 mL/min) to yield
2
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
compound 4 (25 mg, t = 28 min). Compound 6 (3.4 mg, t = 25
R
R
min) was obtained from the other mixture, G6A5d2, by semi-
preparative HPLC (MeCN-H O, 46:54 v/v, 4 mL/min). Fraction
2
(
Mitsubishi Chemical Corporation), Sephadex LH-20 (GE Chemical
G6A5c (409 mg) was purified by HPLC with MeOH-H O (78:22 v/v,
2
Corporation), Si gel (160−200, 200−300 mesh, Qingdao Marine
Chemical Factory, Qingdao, People’s Republic of China), and ODS
4
mL/min) to obtain compound 10 (17 mg, t = 31 min) and six
R
subfractions (G6A5c1−G6A5c6). Compound 17 (6 mg, t = 38 min)
(
(
(
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
R
was obtained from subfraction G6A5c6 by semipreparative HPLC with
MeOH-H O (76:24 v/v, 4 mL/min). Fraction G6A2 (2.6 g) was
2
separated by semipreparative HPLC with MeCN-H O (24:76 v/v, 4
light or by spraying with 10% H SO in EtOH followed by heating.
2
2
4
mL/min) to afford eight fractions, G6A2a−G6A2h. Fraction G6A2g
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.
(
142 mg) was purified by semipreparative HPLC with MeOH-H O
2
(
43:57 v/v, 4 mL/min) to obtain compound 19 (3.8 mg, t = 39 min).
R
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
Extraction and Isolation. Air-dried twigs and leaves of L.
ovalifolia (100 kg) were extracted (2 h each time; 1000 L) with
HPLC system (MeOH-H
isolation of compound 14 (35 mg, t
Lyonifoloside A (1). White powder; [α]
2
O, 80:20 v/v, 4 mL/min) resulted in the
= 37 min).
R
25
EtOH-H O (95:5 v/v) under reflux conditions. After the removal of
+ 9 (c 0.3, MeOH); IR
D
2
−1 1
the solvent under reduced pressure, the crude extract (4200 g) was
(KBr) νmax 3386, 2927, 1708, 1683, 1459, 1380, 1080, 1046 cm ; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 673.3944 [M
suspended in 35 L of H O and then partitioned with petroleum ether,
2
+
CH Cl , EtOAc and n-butanol (3 × 35 L). The EtOAc extract (1200
+ Na] (calcd for C H O Na, 673.3928).
2
2
36 58 10
g) was subjected to passage over a polyamide resin (30−60 mesh)
Lyonifolic Acid A (1a). Colorless crystals (MeOH-H O); mp 205−
2
25
1
13
column and eluted with EtOH-H O (50:50, 95:5 v/v). The 50%
206 °C; [α]
+ 35 (c 0.3, MeOH); H and C NMR data, see
D
2
J
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Tables 1 and 2; HRESIMS m/z 511.3405 [M + Na]+ (calcd for
Lyonifoloside N (14). White powder; [α]25 −33 (c 0.3, MeOH);
D
−1 1
C H O Na, 511.3399).
Lyonifoloside B (2). White powder; [α] + 3 (c 0.3, MeOH); IR
IR (KBr) ν 3367, 2931, 1688, 1457, 1378, 1077, 1048 cm ; H and
30
48
5
max
25
13
C NMR data, see Tables 5 and 6; HRESIMS m/z 675.4080 [M +
D
−
1
1
+
(
KBr) νmax 3391, 2934, 1682, 1460, 1382, 1202, 1076 cm ; H and
Na] (calcd for C H O Na, 675.4084).
36
60 10
1
3
25
C NMR data, see Tables 1 and 2; HRESIMS m/z 675.4082 [M +
Lyonifoloside O (15). White powder; [α] −34 (c 0.3, MeOH);
D
+
−1
Na] (calcd for C H O Na, 675.4084).
IR (KBr) νmax 3360, 2971, 2922, 1678, 1456, 1378, 1079, 1049 cm ;
36
60 10
2
5
1
13
Lyofoligenic Acid (2a). White powder; [α] + 34 (c 0.3, MeOH);
H and C NMR data, see Tables 5 and 6; HRESIMS m/z 807.4508
D
1
13
+
H and C NMR data, see Tables 1 and 2; HRESIMS m/z 513.3546
[M + Na] (calcd for C41
H
68
O
14Na, 807.4507).
25
+
Lyonifoloside P (16). White powder; [α] −36 (c 0.3, MeOH); IR
[
M + Na] (calcd for C H O Na, 513.3556).
D
3
0
50
5
−1 1
1
13
(KBr) ν 3392, 2922, 1741, 1647, 1465, 1372, 1074 cm ; H and
Methyl Lyofoligenate (2b). White powder; H and C NMR data,
see Tables 1 and 2; HRESIMS m/z 527.3716 [M + Na] (calcd for
max
13
+
C NMR data, see Tables 5 and 6; HRESIMS m/z 849.4591 [M +
+
Na] (calcd for C H O Na, 849.4612).
C H O Na, 527.3712).
Lyonifoloside C (3). White powder; [α] + 5 (c 0.3, MeOH); IR
43 70 15
31
52
5
25
25
Lyonifoloside Q (17). White powder; [α] −50 (c 0.3, MeOH);
D
D
−1 1
IR (KBr) ν 3390, 2934, 1685, 1595, 1458, 1375, 1077 cm ; H and
(
1
7
KBr) ν 3375, 2973, 2933, 1686, 1458, 1379, 1204, 1140, 1081,
max
max
13
−
1
1
13
C NMR data, see Tables 5 and 6; HRESIMS m/z 837.4603 [M +
046 cm ; H and C NMR data, see Tables 1 and 2; HRESIMS m/z
+
+
Na] (calcd for C H O Na, 837.4612).
17.4185 [M + Na] (calcd for C H O Na, 717.4190).
42 70 15
38
62 11
25
25
Lyonifoloside R (18). White powder; [α] −31 (c 0.3, MeOH);
Lyonifoloside D (4). White powder; [α] + 9 (c 0.3, MeOH); IR
D
D
−
1
1
13
−1
1
IR (KBr) νmax 3397, 2933, 1679, 1458, 1378, 1073 cm ; H and C
(
KBr) ν 3400, 2969, 2928, 1691, 1459, 1382, 1077, 1048 cm ; H
max
NMR data, see Tables 5 and 6; HRESIMS m/z 777.4389 [M + Na]+
13
and C NMR data, see Tables 1 and 2; HRESIMS m/z 807.4507 [M
+
+
(calcd for C H O Na, 777.4401).
Na] (calcd for C H O Na, 807.4507).
40 66 13
41
68 14
2
5
25
Lyonifolide A (19). White powder; [α] −6 (c 0.1, MeOH); IR
Lyonifoloside E (5). White powder; [α] + 8 (c 0.3, MeOH); IR
D
D
−1 1 13
(
KBr) νmax 3480, 1740 cm ; H and C NMR data, see Table 7;
(
KBr) ν 3397, 2978, 2922, 1681, 1647, 1467, 1380, 1085, 1048
max
+
−1
1
13
HRESIMS m/z 521.3479 [M + H] (calcd for C H O , 521.3478).
30 49 7
cm ; H and C NMR data, see Tables 1 and 2; HRESIMS m/z
8
2
5
+
Lyonifolide B (20). White powder; [α] −13 (c 0.05, MeOH); IR
−1 1 13
D
49.4605 [M + Na] (calcd for C H O Na, 849.4612).
43 70 15
25
(KBr) νmax 3387, 1752 cm ; H and C NMR data, see Table 7;
Lyonifoloside F (6). White powder; [α] + 20 (c 0.3, MeOH); IR
KBr) νmax 3364, 2926, 1684, 1458, 1382, 1073 cm ; H and
D
+
−1
1
13
HRESIMS m/z 521.3487 [M + H] (calcd for C30
Crystallographic Data for Compound 1a. C30
88.68, monoclinic, space group P2 ; a = 11.3157(4) Å, b =
H
49
O
7
, 521.3478).
H O , M =
48 5
(
C
+
NMR data, see Tables 1 and 2; HRESIMS m/z 777.4407 [M + Na]
4
1
(
calcd for C H O Na, 777.4401).
40 66 13
Lyonifoloside G (7). White powder; [α] −32 (c 0.3, MeOH); IR
KBr) νmax 3364, 2921, 1688, 1460, 1371, 1078, 1042 cm ; H and
25
38.8550(13) Å, c = 13.6376(6) Å, α = 90.00°, β = 113.036(5)°, γ =
D
3
−3
9
0.00°; V = 5517.9(4) Å , Z = 8, ρ = 1.176 mg/mm , T = 104.7 K,
−1
1
calc
(
−1
μ (Cu Kα) = 0.616 mm , F(000) = 2144, 40576 reflections measured,
1
3
C NMR data, see Tables 3 and 4; HRESIMS m/z 717.4281 [M +
2
0912 unique (Rint = 0.0293). Final R indexes [I > 2σ (I) i.e., Fo > 4σ
+
Na] (calcd for C H O Na, 717.4190).
38
62 11
(Fo)]: R = 0.0506, wR = 0.1316. Final R indexes [all date]: R =
2
5
1
2
1
Lyonifolic Acid B (7a). White powder; [α] −32 (c 0.1, MeOH);
D
0.0529, wR = 0.1340, Flack Parameters = −0.03 (6). Crystallographic
2
1
13
H and C NMR data, see Tables 3 and 4; HRESIMS m/z 513.3556
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.
+
[
M + Na] (calcd for C H O Na, 513.3556).
30
50
5
1
13
Lyonifolic Acid B Methyl Ester (7b). White powder; H and
C
+
NMR data, see Tables 3 and 4; HRESIMS m/z 527.3718 [M + Na]
(
(
calcd for C H O Na, 527.3712).
Acid Hydrolysis and Determination of Absolute Config-
uration 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
31
52
5
2
5
Lyonifoloside H (8). White powder; [α] −23 (c 0.3, MeOH); IR
D
−
1 1
KBr) ν 3395, 2921, 1692, 1647, 1468, 1421, 1373, 1081 cm ; H
max
13
and C NMR data, see Tables 3 and 4; HRESIMS m/z 849.4610 [M
+
+
Na] (calcd for C H O Na, 849.4612).
43 70 15
25
Lyonifoloside I (9). White powder; [α] −28 (c 0.3, MeOH); IR
H
2
O (10.0 mL) and extracted with EtOAc. The organic phase was
concentrated and purified by semipreparative HPLC with MeCN-H
56:44 v/v, 4 mL/min) to yield aglycone 1a (15.6 mg, t = 53 min).
D
−1 1
(
KBr) ν 3348, 2973, 2931, 1692, 1456, 1385, 1202, 1081 cm ; H
O
2
max
13
(
and C NMR data, see Tables 3 and 4; HRESIMS m/z 807.4501 [M
+
R
+
The H O-soluble phase was again evaporated and dried in vacuo to
Na] (calcd for C H O Na, 807.4507).
2
41
68 14
2
5
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 trimethylsi-
lylate for 2.5 h at 60 °C. The mixture was partitioned between n-
Lyonifoloside J (10). White powder; [α] −22 (c 0.3, MeOH); IR
D
−1 1
(
KBr) ν 3362, 2973, 2926, 1678, 1453, 1381, 1078, 1047 cm ; H
max
13
and C NMR data, see Tables 3 and 4; HRESIMS m/z 837.4630 [M
+
+
Na] (calcd for C H O Na, 837.4612).
42 70 15
2
5
Lyonifoloside K (11). White powder; [α] −32 (c 0.3, MeOH);
D
−
1 1
IR (KBr) ν 3428, 2967, 2921, 1688, 1460, 1381, 1069 cm ; H and
max
1
3
hexane and H O (1 mL each), and the n-hexane extract obtained was
2
C NMR data, see Tables 3 and 4; HRESIMS m/z 777.4403 [M +
+
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.
Na] (calcd for C H O Na, 777.4401).
40
66 13
2
5
Lyonifoloside L (12). White powder; [α] −64 (c 0.3, MeOH); IR
D
−1 1
(
KBr) νmax 3394, 2965, 1687, 1464, 1376, 1078, 1046 cm ; H and
1
3
C NMR data, see Tables 5 and 6; HRESIMS m/z 675.4078 [M +
+
Na] (calcd for C H O Na, 675.4084).
36
60 10
2
5
Lyonifolic Acid C (12a). White powder; [α] −24 (c 0.1, MeOH);
D
1
13
H and C NMR data, see Tables 5 and 6; HRESIMS m/z 513.3552
+
[
M + Na] (calcd for C H O Na, 513.3556).
30
50
5
1
13
Lyonifolic Acid C Methyl Ester (12b). White powder; H and
NMR data, see Tables 5 and 6; HRESIMS m/z 527.3729 [M + Na]
calcd for C H O Na, 527.3712).
C
+
(
31
52
5
2
5
Lyonifoloside M (13). White powder; [α] −65 (c 0.3, MeOH);
D
IR (KBr) ν 3441, 2965, 1720, 1701, 1456, 1375, 1250,1081, 1045
cm ; H and C NMR data, see Tables 5 and 6; HRESIMS m/z
7
max
−1
1
13
Enzymatic Hydrolysis of 7 and 12. Compound 7 (5.0 mg) was
+
17.4179 [M + Na] (calcd for C H O Na, 717.4190).
dissolved in MeOH-H O (7:10 v/v, 3.4 mL) and then treated with
38
62 11
2
K
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
13
as that of 2a to obtain products 7b (1.3 mg) and 12b (1.6 mg),
respectively.
Table 7. H NMR and C NMR Data of Compounds 19 and
0 in Pyridine-d5
2
Determination of the Absolute Configuration of the 24,25-
a
a
1
9
20
Diol Moieties in Compounds 2, 7, and 12 by Snatzke’s and
20−24
Frelek’s Method. According to the published approach,
a 1:1.2
position
δC
δH (J in Hz)
δC
δH (J in Hz)
mixture of diol/Mo (OAc) was subjected to ECD measurements at a
2
4
1
44.3
2.20 m
43.9
2.18 dd (12.0, 4.3)
1.92 m
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
1
.88 m
2
3
4
5
6
66.8
74.7
45.4
47.4
30.1
4.47 m
4.55 s
66.8
74.2
48.0
45.1
18.9
4.43 m
4.87 s
(
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).
2.05 m
2.14 m
2.14 m
1.86 m
1.68 m
1.64 m
2
.05 m
7
76.3
4.20 m
35.1
1
.22 m
8
9
49.4
51.0
39.4
29.4
43.4
50.6
38.7
30.0
1.71 d (12.1)
1.77 m
10
11
2.28 dd (12.1, 3.0)
2.24 m
2.01 m
4.28 m
2
.20 m
Table 8. Antiviral Activity against HSV-1 and Cytotoxicity of
a
b
1
1
1
1
2
3
4
5
70.0
96.2
46.0
32.2
4.24 m
69.5
95.5
44.3
28.4
Compounds in Vero Cells
c
d
compound
TC50 (μmol/L)
IC50 (μmol/L)
SI
1
23.1 ± 1.96
16.0 ± 0.72
57.7 ± 6.84
>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
2.59 td (14.4, 6.2)
2.06 m
1.13 m
2.06 m
1.30 m
1
2
a
a
2
.33 dd (15.0, 5.1)
2.18 m
5.2
16
23.9
23.4
8
>5.2
7.6
1
.36 m
1
2a
16.0 ± 2.30
>100
1
1
1
2
2
7
8
9
0
1
46.2
52.8
39.6
40.6
31.6
46.0
52.8
39.2
40.5
31.5
1
2−2a
>4.9
3.0
2.88 d (11.4)
1.92 m
2.73 d (11.4)
1.80 m
13
14
16
19.3 ± 0.86
57.7 ± 6.46
>100
2.5
0.91 m
0.86 m
>7.0
>244
1.39 m
1.38 m
e
acyclovir
>100
1
.23 m
1.87 m
.57 m
1.67 s
1.22 m
a
2
2
2
2
3
4
32.7
24.2
65.7
32.7
69.4
64.2
1.87 m
Compounds 2−7, 7a, 9−12, 15, and 17−20 all gave IC values of
5
0
b
1
1.54 m
≥33.3 μmol/L. Data represent mean values for three independent
determinations. Cytotoxic concentration required to inhibit Vero cell
growth by 50%. Selectivity index value equaled TC /IC . Positive
c
4.61 d (10.8)
d
e
4
.40 m
50
50
control.
4.06 dd (10.8, 4.8)
4.16 d (10.3)
4.04 d (10.8)
1.04 s
3
.88 dd (10.8, 4.5)
1.08 s
2
2
2
2
2
3
5
6
7
8
9
0
18.9
13.2
17.6
180.4
16.6
19.9
18.4
19.3
17.8
180.2
16.8
19.9
1.66 s
1.34 s
Table 9. Antiviral Activity against Influenza A and
1.36 s
1.12 s
a
b
Cytotoxicity of Compounds in MDCK Cells
c
d
1.30 d (6.4)
0.87 d (5.7)
1.27 d (6.3)
0.85 brs
compound
TC50 (μmol/L)
IC50 (μmol/L)
SI
e
1
1a
2
33.3 ± 4.82
6.4 ± 3.20
100.0 ± 1.03
19.3 ± 6.21
48.1 ± 5.23
>100
>11.1
-
a
Recorded at 500 MHz for proton and at 125 MHz for carbon.
2.1 ± 0.56
33.3 ± 2.97
4.8 ± 3.16
>11.1
3.0
3.0
4.0
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 H O (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-H O, 56:44 v/v, 4
mL/min) to yield aglycone 7a (2.7 mg). Compound 12 (8.0 mg) was
dissolved in MeOH-H O (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 H O (10 mL), and then
extracted with EtOAc. After concentration, the EtOAc fraction was
2a
3
e
-
2
7a
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
>3.9
>3.0
4.3
(
1
0
>100
2
1
2a
16.0 ± 4.50
19.3 ± 5.11
>100
1
3
1.7
2
1
2−2a
>9.0
1.7
1
6
57.7 ± 2.08
1164 ± 1.61
1260 ± 2.76
f
ribavirin
895
741
(
f
tamiflu
a
Compounds 4−9, 11, 12, 14, 15, and 17−20 all gave IC50 values of
b
>33.3 μmol/L. Data represent mean values for three independent
determinations. Cytotoxic concentration required to inhibit Vero cell
growth by 50%. Selectivity index value equaled TC /IC . Under
2
c
d
e
purified by reversed-phase semipreparative HPLC using MeCN in
50 50
H O (70:30) as the mobile phase to yield 2b (3.2 mg). Compounds
the test conditions, the selectivity index (SI) could not be calculated.
Positive control.
2
f
7
a (2.5 mg) and 12a (3 mg) were methylated with the same method
L
DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
IR, HRESIMS, H, 13C NMR, HSQC, HMBC, and
1
Table 10. Antiviral Activity against CVB3 and Cytotoxicity
of Compounds in Vero Cells
a
b
NOESY spectra of compounds 1−20 (PDF)
X-ray crystallography data of compound 1a (CIF)
c
d
compound
TC50 (μmol/L)
IC50 (μmol/L)
SI
1
23.1 ± 2.79
16.0 ± 0.73
48.1 ± 5.92
19.3 ± 3.61
>100
11.1 ± 1.98
2.1 ± 0.30
4.8 ± 1.20
2.1
7.6
1
2
a
a
AUTHOR INFORMATION
■
10.0
4.0
Corresponding Author
12a
4.8 ± 1.16
1
2−2a
19.2 ± 3.48
11.1 ± 1.17
0.001 ± 0.0001
292 ± 9.04
>5.2
1.7
*E-mail: yushishan@imm.ac.cn. Tel: +8610 63165326. Fax:
+8610 63017757.
13
19.3 ± 2.38
15.4 ± 0.51
2000 ± 11.29
e
pleconarild
15400
6.8
Notes
e
ribavirind
The authors declare no competing financial interest.
a
Compounds 2−12, 7a, and 14−20 all gave IC values of ≥33.3
50
b
μmol/L. Data represent mean values for three independent
determinations. Cytotoxic concentration required to inhibit Vero
c
ACKNOWLEDGMENTS
■
d
cell growth by 50%. Selectivity index value equaled TC /IC .
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.
50
50
e
Positive control.
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 × 10 /well) were
4
plated into a 96-well plate. Then, 24 h later, the monolayer cells were
incubated with various concentrations of the test compounds. After 48
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4
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(
(
(
9
(
50
50
(
Anti-Influenza A Activity Assay. The anti-influenza A activity of
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1
(
4
Briefly, MDCK cells (2.5 × 10 cells/well) were plated into 96-well
culture plates for a 24 h incubation. The medium was removed, and
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carried out until the CPE of the control group cells reached a value of
(
7
(
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 TC /IC .
(
(
50
50
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
5
(
°
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.
(
(
(
2
(
2
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00585.
■
*
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010, 46, 36−38.
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DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.6b00585
J. Nat. Prod. XXXX, XXX, XXX−XXX