Minor Nortriterpenoids from the Twigs and Leaves of

Article Cite This: J. Nat. Prod. 2018, 81, 1721−1733

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Minor Nortriterpenoids from the Twigs and Leaves of Rhododendron latoucheae Fei Liu,†,§ Ya-Nan Wang,†,§ Yong Li,† Shuang-Gang Ma,† Jing Qu,† Yun-Bao Liu,† Chang-Shan Niu,† Zhong-Hai Tang,† Yu-Huan Li,‡ Li Li,† and Shi-Shan Yu*,† State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, and ‡Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China

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†

S Supporting Information *

ABSTRACT: A hyphenated NMR technique (analytical HPLC with a DAD connected to MS, SPE, and NMR) has proven effective for the full structural analysis and identification of minor natural products in complex mixtures. Application of this hyphenated technique to the CH2Cl2soluble fraction of Rhododendron latoucheae led to the identification of 15 new minor ursane-type 28-nortriterpenoids (1−15). Compounds 1 and 12 inhibited HSV-1 with IC50 values of 6.4 and 0.4 μM, respectively.

N

noids that were rapidly screened through an HPLC−MS− SPE−NMR method and their biological activities are discussed.

ew bioactive compounds from plant sources continue to serve as templates for the development of novel drug scaffolds. In particular, some constituents with significant physiological and biological activities are present in extremely trace levels.1−4 The use of traditional chromatographic techniques often failed to separate closely related structures and the isolation of minor constituents from complex plant extracts. However, the application of HPLC−MS−SPE− NMR,5−14 which enables structural analysis of minor constituents in complex mixtures, may successfully overcome these challenges. HPLC−MS−SPE−NMR was developed from the shortcomings of HPLC−NMR/MS and is used in an increasing number of natural products studies.15−20 This hyphenated technique can yield purified compounds using SPE columns and further concentrate these compounds by collecting fractions from multiple HPLC injections. Thus, it has obvious advantages in the analysis and identification of minor constituents. Furthermore, the UV, MS, and 1H NMR data obtained from HPLC−MS−SPE−NMR offer a screening technique for the efficient elimination of known constituents and the full structural characterization of new constituents. Rhododendron latoucheae Franch (Ericaceae) is used in traditional folk medicine for relieving cough, reducing sputum, and removing blood stasis.21 It has been reported that triterpenoids are also active against these conditions.22,23 Previously, we reported the rapid isolation of four rare triterpenoids from R. latoucheae through this hyphenated NMR method.24 However, the biologically active components of this plant have been poorly studied. Thus, a comprehensive study of the triterpenoids of the CH2Cl2-soluble fraction of R. latoucheae was undertaken. Herein, the new minor nortriterpe© 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Further study of R. latoucheae showed that the CH2Cl2-soluble fraction contained a variety of triterpenoids. These triterpenoids were preliminarily identified by TLC as blue spots after spraying with 10% H2SO4 in EtOH followed by heating. Some of these triterpenoids were found in higher concentrations, whereas others were present in lower concentrations. Initial studies have shown that the major compounds are known triterpenoids. Thus, it is necessary to further study the unknown minor constituents in this fraction. UV spectra, mass spectra (m/z 400−600), and 1H NMR data of the minor constituents obtained from HPLC−MS−SPE−NMR indicated that some of the minor triterpenoids have different spectroscopic characteristics than the common triterpenoids. These triterpenoids, containing conjugated diene moieties (compounds 1, 2, 3, 4, 5, and 16), were screened via UV absorption at approximately 240 nm (Figure 1), and their molecular weights ranged from 442 to 486. The ursane-type 28-nortriterpenoids, which have aromatic E-rings (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 17), were screened via their aromatic proton signals at δH 6.5−7.1, as shown in Figure 2. Thus, 15 new minor ursane-type 28-nortriterpenoids (1−15) and two known triterpenoids (16, 17) were rapidly isolated Received: December 25, 2017 Published: August 14, 2018 1721

DOI: 10.1021/acs.jnatprod.7b01074 J. Nat. Prod. 2018, 81, 1721−1733

Journal of Natural Products

Article

Figure 1. UV spectra of compounds 1, 2, 3, 4, 5, and 16.

Figure 2. 1H NMR spectra of compounds 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 17.

represents the first 19,20-seco-ursane-type 28-nortriterpenoid. Compounds 6−15 are ursane-type 28-nortriterpenoids with an aromatic E-ring. Natural products of this kind are rare and have

from the extract of this plant through the hyphenated NMR method. Compounds 1−3 possess a unique peroxide linkage between C-17 and C-20 to form ring F, whereas compound 5 1722



Article

Chart 1

by a unique peroxide linkage between C-17 and C-20, was established based on the only remaining index of hydrogen deficiency. The presence of a peroxide bridge in this new nortriterpenoid was also inferred from the appearance of a blue-black spot on a silica gel GF254 plate after spraying with KI−HOAc−starch reagent. Thus, the 2D structure (Figure 3) of compound 1, an ursane-type 28-nortriterpenoid, was elucidated. The relative configuration of compound 1 was defined through NOE data (Figure 3). The NOE correlations of H-2/ H3-24/H-3, H3-24/H3-25/H3-26, and H-9/H3-27/H-22 (δH 1.86) suggested that H-2, H-3, H3-24, H3-25, and H3-26 are cofacial, whereas H-9, H3-27, and H3-30 are on the opposite face. Therefore, 1 has only two possible structures, 1a (2R,3S,4R,5R,8R,9R,10R,14S,17R,20S) and 1b (2S,3R,4S,5S,8S,9S,10S,14R,17S,20R). The absolute configuration of 1 was defined using experimental and calculated ECD data via TDDFT at the B3LYP/6-31G(d) level. The data showed that the experimental ECD spectrum of 1 agreed well with those of 1a (Figure 4), suggesting an absolute configuration of (2R,3S,4R,5R,8R,9R,10R,14S,17R,20S) for 1. The 3D structure of 1 was also defined through the antihelicity rule. The positive Cotton effect (CE) at 240 nm based on the antihelicity rule for heteroannular cisoid conjugated dienes31 confirmed the absolute configuration of 1. Thus, the structure of 1 was defined as (2R,3S,17R,20S)-2,3,23-trihydroxy-28-norursane12,18-diene-17,20-peroxide. Compound 2 (2.2 mg), screened via its UV absorption at 239 nm and found to have a molecular weight of 456, was purified from the same fraction as compound 1. The molecular formula of 2, C29H44O4, was obtained via its (+)-HRESIMS data (m/z 479.3138 [M + Na]+, calculated for C29H44O4Na, 479.3132). Based on the 1D NMR data (Tables 1 and 3) of 2 and 1, it is evident that the compounds are similar except for the substituents of their A-rings. Considering both the HSQC and HMBC spectra, the differences involved C-2 (δH 1.94, 1.57, δC 26.2), C-23 (δH 1.05, δC 22.8), and C-24 (δH 3.69, 3.42, δC 66.3) in 2 versus C-2 (δH 3.90, δC 67.2), C-23 (δH 3.55, 3.41, δC 71.3), and C-24 (δH 0.81, δC 17.6) in 1. Finally,

only been isolated from the Melastomataceae, Ebenaceae, Verbenaceae, Rubiaceae, and Rosaceae families.25−29 Some of the compounds showed anticancer activity. This is the first study of the new constituents isolated from the family Ericaceae. The structures of the new compounds were defined via MS, NMR, and electronic circular dichroism (ECD) data. Additionally, some constituents were assessed for their antiviral HSV-1 and influenza A/95−359 activities. Compound 1 (2.1 mg), screened via UV absorption at 239 nm and found to have a molecular weight of 472, was obtained by the hyphenated NMR method from Fr.4-1-3G-6 (sample amount: 50 mg; sample concentration: 100 mg/mL; injection volume: 18 μL). In the HRESIMS data of 1, the [M + Na]+ ion at m/z 495.3069 (calculated as 495.3081) showed its molecular formula to be C29H44O5. This molecular formula showed that compound 1 had eight indices of hydrogen deficiency. The 1H NMR data (Table 1) of 1 indicated that it contained four tertiary methyl groups (δH 0.81, 0.98, 1.01, and 1.07), an allylic methyl group (δH 1.95), a secondary methyl group (δH 1.34), an oxygen-bearing methylene (δH 3.41, d, J = 11.0 Hz; 3.55, d, J = 11.0 Hz), two oxygen-bearing methines (δH 3.62, d, J = 2.9 Hz; 3.90, ddd, J = 11.9, 4.5, and 2.9 Hz), and an olefinic hydrogen (δH 5.66, dd, J = 4.7, 2.9 Hz). A total of 29 carbon signals, including six methyl carbons, nine methylenes, five methines (two oxygen-bearing and one olefinic), two oxygenated tertiary carbons, and seven quaternary carbons (three olefinic), were revealed by its 13C NMR spectrum (Table 3) and DEPT analysis. In comparing the 1D NMR spectra of 1 with those of known compound 16,30 the same A−B−C ring structure could be deduced. This deduction was confirmed via the HMBC cross-peaks from H3-24 to C-3, C-4, C-5, and C23; from H3-25 to C-1, C-5, C-9, and C-10; from H3-26 to C7, C-8, C-9, and C-14; from H3-27 to C-13, C-14, and C-15; and from H-12 to C-14 and C-18 together with the signals from the 1H−1H COSY data shown in Figure 3. The correlations from H-16 to C-18 and C-22; from H-22 to C17, C-18, and C-20; from H-29 to C-18 and C-20; and from H-30 to C-19 and C-21 in the HMBC spectrum revealed the presence of the D/E-ring moiety. Ring F, presumably formed 1723



Article

Table 1. 1H NMR (600 MHz) Data of Compounds 1−8 in Methanol-d4 (δ in ppm, J in Hz) no. 1 2

1 1.68, dd (12.1, 4.5) 1.34 m 3.90 ddd (11.9, 4.5, 2.9)

3

3.62 d (2.9)

5 6

1.59 1.45 1.45 1.62 1.62 1.76

7 9

m m m m m dd (11.5, 5.9)

2 1.45 m 1.43 m 1.94 m

1.57 m 3.77 dd (2.9, 2.9) 1.45 m 1.56 m 1.45 m 1.58 m 1.55 m 1.73 dd (11.6, 5.8) 2.14 m 2.07 m

1.58 m 3.77 dd (2.9, 2.9) 1.45 m 1.58 m 1.45 m 1.58 m 1.55 m 1.77 dd (11.7, 5.8) 2.12 m 2.00 m

11

2.17 m 2.17 m

12

5.66 dd (4.7, 2.9)

5.64 dd (4.7, 2.9)

15

2.12 m 1.08 m

16 19 20 21

22

4

5

1.67 m 1.33 m 3.93 ddd (12.1, 4.4, 2.9)

1.65 dd (12.1, 4.3) 1.32 m 3.92 ddd (12.0, 4.3, 2.9)

3.76 d (2.9)

3.75 d (2.9)

1.39 1.56 1.34 1.55 1.55 1.76

1.39 1.57 1.40 1.57 1.57 1.80

m m m m m dd (11.8, 5.7)

m m m m m dd (11.7, 5.7)

6

7

1.52 m 1.52 m 1.96 m

1.71 dd (12.0, 4.4) 1.39 t (12.0) 3.94 ddd (12.0, 4.4, 2.9)

1.59 m 3.77 dd (2.9, 2.9) 1.50 m 1.59 m 1.41 m 1.52 m 1.52 m 2.05 dd (12.8, 3.2) 2.00 m 1.64 m

3.78 d (2.9) 1.41 1.60 1.48 1.64 1.59 1.86

m m m m m dd (11.7, 5.6)

2.12 m 2.03 m

2.13 m 2.03 m

5.46 dd (4.7, 2.9)

5.62 dd (4.6, 2.7)

5.16 dd (4.9, 3.1)

2.11 m 1.06 m

1.81 m 1.25 m

1.57 m 1.32 m

1.58 m 1.41 m

2.79 td (13.5, 7.6) 2.24 m 1.72 t (12.7) 1.40 m

1.61 m 1.40 m

1.39 m 1.39 m

1.51 m 1.51 m

2.02 m 1.59 m

2.41 t (12.6) 2.34 m

2.01 ddd (13.1, 9.5, 3.8) 1.61 m 1.86 td (12.4, 3.8)

2.00 m

1.94 m

2.01 1.86 2.52 1.69 1.40

2.24 m 0.87 ddd (13.7, 12.1, 5.6) 2.35 m 2.35 m

2.14 m

6.55 d (7.4)

6.87 d (7.4)

1.62 m 1.86 td (12.4, 3.8) 1.79 m 1.05 s

1.58 m 1.86

1.40 m 2.01 m

2.14 m 2.61 m

6.73 d (7.4)

6.72 d (7.4)

1.31 m 1.05 s

2.01 m 1.10 s

2.61 m 1.10 s

1.06 s

1.12 s

3.69 3.42 0.96 0.95 1.01 1.95 1.33

3.68 3.42 0.96 0.94 1.05 1.89 1.33

3.66 3.41 1.02 0.87 0.99 0.80 0.98

3.65 3.41 1.01 0.88 1.08 2.28 2.13

3.64 3.40 0.91 0.82 1.38 2.15 2.20

3.68 3.44 1.02 1.03 0.94 2.26 2.24

24

1.80 3.55 3.41 0.81

m d (11.0) d (11.0) s

25 26 27 29 30

1.07 0.98 1.01 1.95 1.34

s s s s s

23

3

1.44 m 1.44 m 1.94 m

d (11.3) d (11.3) s s s s s

d (11.3) d (11.3) s s s s s

m dd (17.5, 5.6) m m m

d d s s s d d

(11.4) (11.4)

(6.8) (6.9)

d (11.4) d (11.4) s s s s s

d (11.4) d (11.4) s s s s s

2.22 m 2.10 ddd (18.5, 11.7, 2.7) 5.48 dd (4.9, 2.7)

d (11.4) d (11.4) s s s s s

calculated for C29H45O4, 457.3312) and 13C NMR data, compound 3 had the molecular formula C29H44O4, the same as that of compound 2. The 1D NMR data (Tables 1 and 3) of 3 resembled those of 2, except for the signals of ring E. The difference between the two structures involved the orientation of the peroxide bond at C-17 and C-20, defined via the NOE associations of H-22β (δH 1.31)/H-15β (δH 1.81)/H3-26 (Figure 5). The positive CE at 240 nm (Figure S22, Supporting Information) indicated the absolute configuration of 3 as (3R,4S,5R,8R,9R,10R,14S,17S,20R). Consequently, the structure of compound 3 was defined as (3R,17S,20R)-3,24dihydroxy-28-norursane-12,18-diene-17,20-peroxide. Compound 4 (1.7 mg), screened via its UV absorption at 238 nm and possessing a molecular weight of 442, was obtained from Fr.4-1-3J6-8 (sample amount: 50 mg; sample concentration: 100 mg/mL; injection volume: 18 μL). The molecular formula of 4 was determined as C29H46O3 through its (+)-HRESIMS (m/z 465.3337 [M + Na]+, calculated for C29H46O3Na, 465.3339) and 13C NMR data (Table 3). The

the absence of OH-2 in compound 2 and the relative configuration at C-4 were defined via (1) the [−CH2(1)− CH2(2)−CH(3)−] fragment in the 1H−1H COSY data; (2) the correlations of H3-23 with C-3, C-4, C-5, and C-24 and H2 with C-1, C-4, and C-10 in the HMBC data; and (3) the NOE correlations (Figure 5) of H-3/H2-24 (δH 3.69)/H3-25. The positive CE at 240 nm in the experimental ECD spectrum of 2 (Figure S12, Supporting Information) was indicative of the π → π* transition of a heteroannular cisoid conjugated diene moiety, which resembled that of compound 1. This suggested that compound 2 had an absolute configuration of (3R,4S,5R,8R,9R,10R,14S,17R,20S). Thus, the structure of compound 2 was defined as (3R,17R,20S)-3,24-dihydroxy-28norursane-12,18-diene-17,20-peroxide. Compound 3 (2.1 mg), screened via its UV absorption at 238 nm and found to have a molecular weight of 456, was isolated from Fr.4-1-3H2-3 (sample amount: 20 mg; sample concentration: 40 mg/mL; injection volume: 25 μL). According to its (+)-HRESIMS (m/z 457.3323 [M + H]+, 1724



Article

Table 2. 1H NMR (600 MHz) Data of Compounds 8−15 in Methanol-d4 (δ in ppm, J in Hz) no. 1

8 3.84 dd (11.6, 4.8)

9 1.73 m

10 3.57 d (9.5)

1.40 m 2

3 5 6 7

9 11

12

11

12

1.73 m 1.40 m

1.94 ddd (14.1, 3.95 ddd (12.0, 11.6, 3.3) 4.4, 2.9) 1.75 ddd (14.1, 4.8, 3.3) 3.86 dd (3.3, 3.3) 3.77 d (2.9) 1.43 m 1.43 m

3.63 dd (9.5, 3.0)

3.70 d (3.0) 1.71 m

3.64 d (2.7) 1.63 m

1.59 1.46 1.67 1.61

1.63 1.45 1.66 1.66

1.67 1.46 1.69 1.63

m m m m

2.01 dd (11.6, 5.3) 2.86 dt (19.4, 5.3) 2.21 ddd (19.4, 11.6, 2.8) 5.48 dd (5.3, 2.8)

1.62 1.50 1.65 1.65

m m m m

1.88 dd (11.7, 5.3) 2.25 m 2.14 m

2.04 dd (11.5, 5.3) 2.95 dt (19.4, 5.3) 2.27 ddd (19.4, 11.5, 2.6) 5.49 dd (5.3, 2.6)

m m m m

1.88 dd (11.8, 5.6) 2.26 m 2.15 m

13 3.85 dd (11.2, 4.4)

14 1.49 m

1.92 m

1.93 m

1.67 m

1.58 m

3.71 dd (2.6, 2.6) 1.68 m

4.07 dd (2.9, 2.9) 1.75 m

1.46 m

1.60 1.46 1.66 1.66

1.56 1.56 1.65 1.65

1.70 1.70 1.79 1.68

3.40 d (9.6) 1.37 dd (10.5, 3.2) 1.53 m 1.53 m 1.73 m 1.64 dd (13.2, 3.3) 1.79 dd (11.6, 5.3) 2.21 dt (19.5, 5.3) 2.15 ddd (19.5, 11.6, 2.8) 5.50 dd (5.3, 2.8)

2.04 dd (11.5, 5.3) 2.91 dt (19.0, 5.3) 2.25 ddd (19.0, 11.5, 2.5) 5.49 dd (5.3, 2.5)

1.86 dd (11.8, 5.3) 2.21 dt (18.7, 5.3) 2.09 ddd (18.7, 11.8, 2.7) 5.50 dd (5.3, 2.7)

1.86 dd (11.5, 5.3) 2.23 dt (19.5, 5.3) 2.19 ddd (19.5, 11.5, 2.7) 5.49 dd (5.3, 2.7)

2.26 m 0.87 m

2.29 m 0.88 m

2.25 m 0.86 m

4.26 t (3.4)

2.69 dd (14.2, 3.4) 2.63 dd (14.2, 3.4) 6.92 d (7.4) 6.80 d (7.4) 1.18 s

m m m m

m m m m

2.27 m 0.88 m

2.41 m

2.37 m

2.38 m

2.37 m

2.37 m

2.36 m

2.36 m

2.41 m

2.37 m

2.38 m

2.37 m

2.37 m

2.36 m

21 22 23

6.87 d (7.4) 6.72 d (7.4) 1.04 s

7.10 d (7.4) 6.84 d (7.4) 1.13 s

24

3.69 3.44 1.02 1.06 0.95 2.27 2.24

3.68 3.44 1.04 1.05 0.95 2.37 4.62

6.87 6.72 3.55 3.42 0.85

d d d d s

1.12 1.09 0.95 2.28 2.25

s s s s s

16

25 26 27 29 30

2.26 m 0.86 ddd (13.8, 12.1, 5.9) 2.36 m

d (11.4) d (11.4) s s s s s

d (11.4) d (11.4) s s s s s

(7.4) (7.4) (11.0) (11.0)

6.88 6.73 3.57 3.43 0.83

d d d d s

1.10 1.08 0.95 2.28 2.25

s s s s s

(7.4) (7.4) (11.0) (11.0)

15 2.11 ddd (12.9, 6.5, 3.0) 1.52 td (12.9, 5.7) 2.77 ddd (15.2, 12.9, 6.5) 2.33 ddd (15.2, 5.7, 3.0)

1.49 m

5.51 dd (4.8, 2.6) 2.28 m 0.88 m

15

5.54 dd (4.6, 2.7) 2.27 m 0.88 m

m m m m

3.92 ddd (11.7, 3.8, 2.7)

2.07 dd (12.2, 4.6) 0.99 dd (12.2, 11.4) 3.74 ddd (11.4, 9.6, 4.6)

6.88 6.73 3.53 3.30 0.74

d d d d s

1.11 1.08 0.94 2.27 2.25

s s s s s

(7.4) (7.4) (11.0) (11.0)

6.87 6.72 3.54 3.38 0.78

d d d d s

1.09 1.10 0.96 2.28 2,25

s s s s s

(7.4) (7.4) (11.0) (11.0)

6.88 6.72 3.92 3.75 3.74 3.62 1.01 1.06 0.95 2.27 2.25

d d d d d d s s s s s

(7.4) (7.4) (11.0) (11.0) (11.4) (11.4)

4.04 3.56 1.24 1.08 0.95 2.27 2.25

m m m m

d (11.4) d (11.4) s s s s s

data (Tables 1 and 3) of 5 resembled those of compound 4, with major differences observed for the signals of C-19, C-20, C-21, C-22, C-29, and C-30. In the 1D NMR spectra of 5, two carbonyl carbons at C-19 (δC 211.7) and C-20 (δC 210.6) and two tertiary methyl carbons at C-29 (δH 2.28, δC 30.8) and C30 (δH 2.13, δC 29.8) were seen instead of the two methines at C-19 (δH 2.52, δC 33.3) and C-20 (δH 1.69, δC 34.0) and two secondary methyl groups at C-29 (δH 0.80, δC 13.4) and C-30 (δH 0.98, δC 20.9) for compound 4. This structure was confirmed via the HMBC correlations from H3-29 to C-18 and C-19 and H3-30 to C-20 and C-21. Thus, the structure of compound 5 was defined as a new 19,20-seco-ursane-type 28nortriterpenoid and had the same 2α,3α,24-trihydroxy A-ring as compound 4 based on the NOE correlations of H-2/H2-24 (δH 3.65)/H-3 and H2-24 (δH 3.65)/H3-25. The experimental ECD spectrum of 5 was opposite the calculated ECD spectrum of 5b and was consistent with that of 5a (Figure 6), indicative of an absolute configuration of (2R,3S,4S,5R,8R,9R,10R,14S) for compound 5. Thus, the structure of 5 was assigned as (2R,3S)-2,3,24-trihydroxy-19,20-seco-28-norursane-12,17diene.

above data showed that 4 was closely related to 2α,3β,23trihydroxy-12,17-diene-28-norursane, which was isolated from Rosa laevigata.32 The differences are related to changes in the orientation of the hydroxy groups on the A-ring. An ursanetype 28-nortriterpenoid with a 2α,3α,24-trihydroxy A-ring was deduced from the [−CH2(1)−CH(2)−CH(3)−] fragment in the 1H−1H COSY data and the NOE signals of H-2/H2-24 (δH 3.66)/H-3 and H2-24 (δH 3.66)/H3-25. The negative CE at 248 nm (Figure S32, Supporting Information) was indicative of a π → π* transition33 and confirmed the absolute configuration as (2R,3S,4S,5R,8R,9R,10R,14S,19S,20R). Accordingly, the structure of compound 4 was assigned as (2R,3S)-2,3,24-trihydroxy-28-norursane-12,17-diene. Compound 5 (2.9 mg), screened via its UV absorption at 238 nm and assigned a molecular weight of 472, was purified from Fr.4-1-3F2-2 (sample amount: 30 mg; sample concentration: 60 mg/mL; injection volume: 20 μL). The molecular formula of 5 was established as C29H44O5 via its 13C NMR spectrum and (+)-HRESIMS data (m/z 495.3085 [M + Na]+, calculated for C29H44O5Na, 495.3081), and this formula suggests eight indices of hydrogen deficiency. The 1D NMR 1725


Journal of Natural Products Table 3.

13

Article

C NMR (150 MHz) Data of Compounds 1−15 in Methanol-d4 (δ in ppm)

no.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

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 29 30

42.4 67.2 78.7 42.5 44.5 18.8 33.9 40.8 48.6 39.1 24.5 128.3 136.9 43.9 28.8 31.2 78.8 139.6 137.0 79.8 30.9 28.3 71.3 17.6 17.8 16.7 28.3 13.6 19.8

34.5 26.2 71.3 44.1 50.8 19.3 34.7 40.9 48.6 37.9 24.5 128.4 136.8 43.8 28.7 31.2 78.9 139.7 136.9 79.9 30.9 28.3 22.8 66.3 16.6 16.6 28.2 13.6 19.8

34.5 26.2 71.3 44.2 50.9 19.3 34.7 40.7 48.7 38.0 24.4 126.6 137.5 44.1 28.4 32.1 79.2 141.1 135.8 78.9 31.3 29.6 22.8 66.3 16.7 16.9 27.4 12.6 19.9

43.0 67.1 74.7 45.5 50.1 19.4 35.4 40.1 48.5 39.2 24.8 118.0 138.5 42.0 28.3 29.3 129.9 134.3 33.3 34.0 25.7 32.9 23.1 65.9 17.9 17.4 20.1 13.4 20.9

42.7 67.0 74.6 42.9 50.1 19.3 31.6 39.9 45.5 35.3 24.9 123.9 137.6 39.2 28.1 26.6 133.1 139.9 211.7 210.6 29.5 41.2 23.1 65.9 17.9 17.2 20.9 30.8 29.8

34.9 26.2 71.3 44.2 50.5 19.4 35.5 45.8 51.4 38.7 24.1 38.9 222.0 57.1 31.6 28.7 126.3 153.9 124.0 136.6 121.8 127.4 22.9 66.2 17.3 17.3 17.9 12.0 20.1

43.0 67.1 74.6 45.5 50.1 19.3 35.4 41.3 48.9 39.2 24.6 126.2 140.0 45.3 33.4 32.0 139.5 140.1 134.4 136.0 128.4 123.9 23.1 65.9 17.9 17.5 27.8 17.1 20.9

77.1 36.9 72.3 44.0 50.0 19.3 35.5 41.7 49.8 44.2 27.9 127.5 139.1 45.2 33.2 32.0 139.4 140.2 134.4 135.9 128.2 123.8 22.7 66.3 12.6 17.7 27.8 17.0 20.9

43.0 67.1 74.7 45.5 50.1 19.3 35.4 41.3 48.9 39.2 24.6 126.7 139.8 45.4 33.3 32.2 141.5 140.5 134.4 138.8 127.1 124.0 23.1 65.9 17.9 17.5 27.8 16.0 64.3

81.2 72.1 79.2 42.0 44.3 18.8 34.7 41.7 50.0 44.3 27.8 127.5 139.1 45.3 33.3 32.1 139.4 140.1 134.4 135.9 128.2 123.9 71.1 17.7 14.0 17.9 27.8 17.0 20.9

42.6 67.3 78.7 42.6 44.6 18.9 34.6 41.2 49.0 39.2 24.4 126.3 140.2 45.4 33.4 32.0 139.5 140.0 134.4 136.0 128.4 123.9 71.3 17.6 17.9 17.6 27.8 17.1 20.9

48.2 69.8 78.1 44.2 48.5 18.9 34.4 45.4 49.1 39.0 24.5 126.2 140.2 41.2 33.4 32.0 139.5 140.0 134.4 136.0 128.4 123.9 66.2 14.0 18.1 17.5 27.9 17.1 20.9

77.2 37.2 77.3 41.2 44.0 18.9 34.7 41.7 49.8 44.2 27.9 127.5 139.1 45.2 33.2 32.1 139.4 140.2 134.4 135.9 128.2 123.8 71.2 18.1 12.6 17.8 27.8 17.0 20.9

34.5 26.5 70.8 46.4 45.6 19.4 35.1 41.1 49.1 37.8 24.4 126.3 140.1 45.3 33.4 32.0 139.5 140.1 134.4 136.0 128.3 123.9 68.9 64.9 16.5 17.4 27.8 17.1 20.9

41.5 36.0 217.9 55.5 58.9 20.6 35.2 40.7 49.0 38.0 24.7 125.5 140.2 50.2 68.9 40.5 134.5 139.5 134.3 136.5 128.7 126.3 20.8 65.8 16.8 17.2 19.6 17.1 20.9

Figure 4. Experimental ECD spectrum of 1 and calculated ECD spectra of 1a and 1b.

Figure 3. Selected HMBC, 1H−1H COSY, and NOE correlations of compound 1.

Compound 6 (1.6 mg), screened via its aromatic hydrogen signals at δH 6.55 (d, J = 7.4 Hz) and 6.73 (d, J = 7.4 Hz), was isolated from Fr.4-1-3J4-4-2 (sample amount: 20 mg; sample concentration: 40 mg/mL; injection volume: 25 μL). The (+)-HRESIMS ion at m/z 457.3303 [M + H]+ (calculated for C29H42O3Na, 461.3026) and its 13C NMR data suggested a molecular formula of C29H44O4. This molecular formula showed that compound 6 had eight indices of hydrogen deficiency. The 1H NMR data (Table 1) showed four tertiary methyl groups (δH 0.82, 0.91, 1.06, and 1.38), two aromatic methyl groups (δH 2.15 and 2.20), an oxymethylene (δH 3.40, d, J = 11.4 Hz; 3.64, d, J = 11.4 Hz), an oxymethine (δH 3.77, d, J = 2.9 Hz), and two aromatic hydrogens (6.55, d, J = 7.4;

Figure 5. Key NOE correlations of compounds 2 and 3.

6.73, d, J = 7.4). Its 13C NMR (Table 3) and DEPT spectra revealed 29 carbon signals, including six methyl carbons, nine methylenes (one oxygen-bearing), five methines (two aromatic and one oxygen-bearing), one oxygenated aromatic carbon, seven quaternary carbons (three aromatic), and a carbonyl carbon. By comparing the 1D NMR data of compound 6 with 1726



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concentration: 120 mg/mL; injection volume: 15 μL). Compound 7 had a molecular formula of C29H42O3, as established from its (+)-HRESIMS (m/z 461.3033 [M + Na]+, calcd for C29H42O3Na, 461.3026) and 13C NMR data. Comparing the 1D NMR (Tables 1 and 3) and (+)-HRESIMS data of 7 to those of known compound 1734 suggested that 7 possessed a hydroxy group at C-2 (δH 3.94, δC 67.1). This was confirmed by the [−CH2(1)−CH(2)−CH(3)−] fragment in the 1H−1H COSY data and the HMBC signals from H-2 to C4 and C-10. The NOE associations of H-2/H2-24 (δH 3.68)/ H-3 and H2-24 (δH 3.68)/H3-25 confirmed that H-2, H-3, H224, and H3-25 were cofacial. The X-ray crystallography data (CCDC 1524486) for compound 7 proved the absolute configuration as (2R,3S,4S,5R,8R,9R,10R,14S) (Figure 8). Thus, the structure of compound 7 was assigned as (2R,3S)2,3,24-trihydroxy-28-norursane-12,17,19,21-tetraene.

Figure 6. Experimental ECD spectrum of 5 and calculated ECD spectra of 5a and 5b.

those of known compound 17,34 the same A−B-ring structures were identified. This assignment was confirmed via HMBC and 1 H−1H COSY analyses (Figure 7). The 2D structure of 6, a

Figure 8. ORTEP diagram of 7.

Compound 8 (2.0 mg), screened via its aromatic hydrogen signals at δH 6.72 (d, J = 7.4 Hz) and 6.87 (d, J = 7.4 Hz), was purified from Fr.4-1-3I5-5 (sample amount: 50 mg; sample concentration: 100 mg/mL; injection volume: 18 μL). An (+)-HRESIMS ion of 8 at m/z 461.3038 [M + Na]+ (calculated for C29H42O3Na, 461.3026) indicated a molecular formula of C29H42O3. The 1D NMR data (Tables 2 and 3) of 8 resembled those of 17,34 except for a hydroxy group at C-1 (δH 3.84, δC 77.1) in 8. This assignment was confirmed via the 1 H−1H COSY data and the HMBC correlations of H-1 with C-3, C-9, and C-25. The 1β,3α,24-trihydroxyl A-ring moiety in 8 was deduced from the NOE signals of H-1/H-5/H3-23 and H-3/H2-24 (δH 3.69)/H3-25. The experimental ECD spectra (Figure 9) of 8 and 7 showed intense positive CEs at 244 nm, originating from the π → π* electronic transitions of their conjugated chromophores. This allowed the absolute configuration of 8 to be assigned as (1R,3R,4S,5R,8R,9S,10R,14S). Thus, the structure of 8 was defined as (1R,3R)-1,3,24trihydroxy-28-norursane-12,17,19,21-tetraene. Compound 9 (1.8 mg), screened via its aromatic hydrogen signals at δH 6.84 (d, J = 7.4 Hz) and 7.10 (d, J = 7.4 Hz), was obtained from Fr.5-9-8-5 (sample amount: 20 mg; sample concentration: 40 mg/mL; injection volume: 25 μL). Compound 9 had a molecular formula of C29H42O4 based on its (+)-HRESIMS (m/z 477.2986 [M + Na]+, calculated for C29H42O4Na, 477.2975) and 13C NMR data. The 1D NMR data of 9 (Tables 2 and 3) resembled those of 7, except for the presence of a 30-hydroxymethyl group (δH 4.62, δC 64.3) in 9 rather than a methyl group (δH 2.24, δC 20.9). This was

Figure 7. Selected HMBC, 1H−1H COSY, and NOE correlations of compound 6.

19,20-seco-28-nortriterpenoid, was elucidated by (1) the HMBC correlations from H3-26 to C-7, C-8, C-9, and C-14; from H3-27 to C-8, C-13, C-14, and C-15; from H-12 to C-9, C-11, C-13, and C-14; from H3-29 to C-18, C-19, and C-20; from H3-30 to C-19, C-20, and C-21; and from H-22 to C-16, C-18, and C-20 and (2) the corresponding cross-peaks in the 1 H−1H COSY data (Figure 7). The α-orientation of OH-3 was confirmed by comparison of the chemical shift, splitting pattern, and coupling constant of H-3 (δH 3.77, dd, J = 2.9, 2.9 Hz) of compound 6 to those of H-3 (δH 3.77, dd, J = 2.9, 2.9 Hz) of compound 17. The NOE cross-peaks (Figure 7) of H9/H3-27 and H-3/H2-24 (δH 3.64)/H3-25/H3-26 indicated that H2-24, H3-25, and H3-26 are cofacial, whereas H-9 and H3-27 are on the opposite face. The positive CE at 292 nm (Figure S52, Supporting Information) in the experimental ECD spectrum was consistent with the octant rule35 and demonstrated the absolute configuration of 6 as (3R,4S,5R,8R,9R,10R,14S). Thus, the structure of compound 6 was defined as (3R)-3,24-dihydroxy-13-oxo-19,20-seco-28norursane-17,19,21-triene. Compound 7 (3.9 mg), screened via its aromatic hydrogen signals (δH 6.72, d, J = 7.4 Hz; 6.87, d, J = 7.4 Hz), was obtained from Fr.4-1-3J6-5 (sample amount: 60 mg; sample 1727



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Compounds 11 (1.7 mg) and 12 (2.2 mg), screened via aromatic hydrogen data at δH 6.73 (d, J = 7.4 Hz) and 6.88 (d, J = 7.4 Hz), were obtained from Fr.4-1-3J4-4-7 (sample amount: 60 mg; sample concentration: 120 mg/mL; injection volume: 15 μL) and Fr.4-1-3J4-5-4 (sample amount: 21 mg; sample concentration: 42 mg/mL; injection volume: 25 μL). The molecular formulas of compounds 11 (m/z 461.3045 [M + Na]+, calculated for C29H42O3Na, 461.3026) and 12 (m/z 461.3034 [M + Na]+, calculated for C29H42O3Na, 461.3026) were both established as C29H42O3 from their (+)-HRESIMS and 13C NMR data. The 1D NMR data (Tables 2 and 3) of 11 resembled those of 10, except for the absence of OH-1 (δH 1.73 and 1.40, δC 42.6) in 11. This structural assignment was confirmed by the 1H−1H COSY data and the HMBC correlations from H-3 to C-1 and C-5. The NOE cross-peaks of H3-25/H-2/H3-24/H-3 and the proton signals assigned to H-2 (δH 3.92, ddd, J = 11.7, 3.8, 2.7 Hz) and H-3 (δH 3.64, d, J = 2.7 Hz) in 11 constructed a 2α,3α,23-trihydroxy fragment in 11. The experimental ECD spectrum of 11 showed a similar positive CE at 243 nm (Figure S102, Supporting Information) to that of 7, which proved the absolute configuration as (2R,3S,4R,5R,8R,9R,10R,14S). Thus, the structure of 11 was defined as (2R,3S)-2,3,23-trihydroxy-28-norursane12,17,19,21-tetraene. Comparison of the NMR data of 12 and 11 (Tables 2 and 3) showed that the two compounds were structurally quite similar. Compound 12 was identified as the C-3 epimer of compound 11 based on the NOE associations of H3-25/H-2/H3-24 and H-3/H-5/H-9/H3-27 and the proton signals assigned to H-2 (δH 3.74, ddd, J = 11.4, 9.6, 4.6 Hz) and H-3 (δH 3.40, d, J = 9.6 Hz) in 12. The similarities of the experimental ECD spectrun of 12 (Figure S112, Supporting Information) to those of 7 allowed the abosolute configuration to be assigned as (2R,3R,4R,5R,8R,9R,10R,14S). Thus, the structure of compound 12 was determined as (2R,3R)-2,3,23trihydroxy-28-norursane-12,17,19,21-tetraene. Compound 13 (1.8 mg), screened via its aromatic hydrogen data at δH 6.72 (d, J = 7.4 Hz) and 6.87 (d, J = 7.4 Hz), was isolated from Fr.4-1-3J5-3 (sample amount: 16 mg; sample concentration: 32 mg/mL; injection volume: 30 μL). The (+)-HRESIMS data confirmed that compound 13 had the same molecular formula as 8 (C29H42O3). In addition, the 1D NMR data of 13 (Tables 2 and 3) were similar to those of 8. The differences involved the presence of a change of the relative configuration at C-4, which was confirmed via the NOE associations of H-3/H3-24/H3-25 and H-1/H-9. The similarities in the experimental ECD spectrum of 13 (Figure S122, Supporting Information) to those of 7 allowed the absolute configuration to be assigned as (1R,3R,4R,5R,8R,9S,10R,14S). Thus, the structure of compound 13 was defined as (1R,3R)-1,3,23-trihydroxy-28norursane-12,17,19,21-tetraene. Compound 14 (1.6 mg), screened via its aromatic hydrogen signals at δH 6.72 (d, J = 7.4 Hz) and 6.88 (d, J = 7.4 Hz), was purified from the same fraction as compound 12. Compound 14 showed an (+)-HRESIMS ion at m/z 461.3037 [M + Na]+ (calculated for C29H42O3Na, 461.3026), which corresponded to a molecular formula of C29H42O3. The 1D NMR data (Tables 2 and 3) of 14 resembled those of 17,34 except that the tertiary methyl group at C-4 in 17 was substituted by a hydroxymethyl group (δH 3.92, 3.75, δC 68.9) in 14, as confirmed through the HMBC correlations of H2-23 with C-3, C-5, and C-24. The 3α-hydroxy group was determined via comparison of the chemical shift, splitting pattern, and

Figure 9. Experimental ECD spectra of 7 and 8.

confirmed via the HMBC signals of H-30 with C-19, C-20, and C-21. The similarity of the experimental ECD spectra of 9 (Figure S82, Supporting Information) and 7 indicated a (2R,3S,4S,5R,8R,9R,10R,14S) absolute configuration. Thus, the structure of 9 was defined as (2R,3S)-2,3,24,30tetrahydroxy-28-norursane-12,17,19,21-tetraene. Compound 10 (2.3 mg), screened via its aromatic hydrogen signals (δH 6.72, d, J = 7.4 Hz; 6.87, d, J = 7.4 Hz), was isolated from the same fraction as compound 7. The (+)-HRESIMS data of compound 10 (m/z 477.2984 [M + Na]+, calculated for C29H42O4Na, 477.2975), indicated a molecular formula of C29H42O4. The 1D NMR data of 10 (Tables 2 and 3) resembled those of 8. The differences involved the presence of a hydroxy group at C-2 in 10 and a change of the relative configuration at C-4 and were supported by the HMBC correlations from H-3 to C-1, C-2, C-4, C-5, and C-24 and the NOE associations of H-1/H-5/H-23 and H3-25/H-2/H3-24/ H-3. In addition, the resonance assigned to H-2 in 10 at δH 3.63 (dd, J = 9.5, 3.0 Hz) indicated that OH-2 was αoriented.36 The similarities between the experimental ECD spectrum of 10 (Figure S92, Supporting Information) and those of 7 allowed the absolute configuration to be assigned as (1S,2R,3S,4R,5R,8R,9S,10R,14S). Thus, the structure of compound 10 was defined as (1S,2R,3S)-1,2,3,23-tetrahydroxy-28-norursane-12,17,19,21-tetraene. 1728



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Figure 10. (A) HPLC chromatogram (DAD 230 nm) of Fr.4-1-3G-6 containing four peaks (P1−P4). (B) Total ion chromatogram of Fr.4-1-3G-6; (a) UV spectrum of P1; (b) MS spectrum of P1; (c) UV spectrum of P2; (d) MS spectrum of P2; (e) UV spectrum of P3; (f) MS spectrum of P3; (g) UV spectrum of P4; (h) MS spectrum of P4.

(+)-HRESIMS (m/z 437.3071 [M + H] + , calcd for C29H41O3, 437.305) and 13C NMR spectra. The 1D NMR data (Tables 2 and 3) of 15 resembled those of 17,34 except that a carbonyl carbon (δC 217.9) at C-3 in 15 was substituted by an oxymethine at C-3 in 17. Compound 15 also contains an additional hydroxy group at C-15 (δH 4.26, δC 68.9). This deduction was confirmed by the 1H−1H COSY data and the HMBC correlations from H-1, H-2, H3-23, and H-24 to C-3 and from H3-27 to C-15. The NOE associations of H-15/H326/H3-25/H-24 suggested that OH-15 was α-oriented. The negative CE at 311 nm (Figure S142, Supporting Information) in the experimental ECD spectrum was consistent with the octant rule.35 Thus, the absolute configuration of 15 was

coupling constant of H-3 (δH 4.07, dd, J = 2.9, 2.9 Hz) of 14 with those of H-3 (δH 3.77, dd, J = 2.9, 2.9 Hz) of 17. The similarities between the experimental ECD spectrum of 14 (Figure S132, Supporting Information) and those of 7 allowed the absolute configuration to be assigned as (3R,5R,8R,9R,10R,14S). Thus, the structure of compound 14 was defined as (3R)-3,23,24-trihydroxy-28-norursane12,17,19,21-tetraene. Compound 15 (1.7 mg), screened via its aromatic hydrogen signals at δH 6.80 (d, J = 7.4 Hz) and 6.92 (d, J = 7.4 Hz), was obtained from Fr.4-1-3I1 (sample amount: 20 mg; sample concentration: 40 mg/mL; injection volume: 25 μL). The compound had a formula of C 29 H 40 O 3 through its 1729



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Figure 11. 1H NMR spectra of P1−P4 (methanol-d4), separated from Fr.4-1-3G-6 (HPLC chromatogram, see Figure 10), obtained from HPLCMS-SPE-NMR (600 MHz). TOF instrument (micrOTOF-Q II, Bruker Daltonics, Billerica, MA, USA). A Spark-Holland SPE system was combined with the HPLC system, and the UV absorption of the eluate was monitored at 230 nm. Prior to SPE trapping, the H2O dilution stream (3.0 mL/min) in the eluent was added via a replenishing pump to reduce the concentration of the eluate. The SPE cartridges (Spark, Holland) were conditioned with 500 μL of MeOH and then equilibrated with 500 μL of H2O before each use. After repeated LC runs, the target analytes were trapped on different SPE cartridges. Then, a flow of nitrogen gas was used to dry the SPE cartridges for 40−50 min. Finally, the adsorbed constituents were separately eluted using 150 μL of methanol-d4 into 3 mm NMR capillary tubes. Most of the NMR experiments were performed on a Bruker instrument (AVANCE III HD 600, 5 mm CryoProbe based on a closed cycle helium cryocooler). NOE data were obtained on an Agilent-NMR-vnmrs 600 spectrometer (5 mm CPDCH probe). Plant Material. Rhododendron latoucheae was acquired in Zhangjiajie, Hunan Province, China, in October 2014. The sample was authenticated by Prof. Lin Ma of Materia Medica, Chinese Academy of Medical Sciences (voucher specimen: ID-22815). Extraction and Isolation. The branches and leaves of Rhododendron latoucheae (107 kg) were extracted by refluxing in 95% EtOH/H2O (10 L/kg; 2 h × 3). The resulting extract (6000 g) was dispersed in H2O (30 L) and extracted sequentially with petroleum ether, CH2Cl2, EtOAc, and n-BuOH (3 × 30 L). An MCI gel column (MeOH/H2O, 90:10, 100:0 v/v) was used to purify the CH2Cl2-soluble fraction (500 g), and the 90% MeOH subfraction (350 g) was further purified on a silica gel column (petroleum ether/ acetone, 50:1−1:1, v/v) to afford Fr.1−Fr.7. Four subfractions (Fr.41-1−Fr.4-1-4) were obtained from the purification of Fr.4 (40 g) on a Sephadex LH-20 column. An MCI gel column was used for Fr.4-1-3 (20 g) to obtain 11 subfractions (Fr.4-1-3A−Fr.4-1-3K). Fr.4-1-3F (2 g) was separated via Sephadex LH-20, silica gel, and ODS gel columns and further purified via the hyphenated NMR method to afford compounds 16 (1.8 mg) and 5 (2.9 mg). Compounds 1 (2.1 mg) and 2 (2.2 mg) were isolated from Fr.4-1-3G (1.5 g) following a Sephadex LH-20 column and the hyphenated NMR method. Fr.4-1-3H (1 g) was similarly applied to Sephadex LH-20 and ODS gel columns and

assigned as (4S,5R,8R,9R,10R,14S,15S), and the structure of compound 15 was defined as (15S)-15,24-dihydroxy-3-oxo-28norursane-12,17,19,21-tetraene. In addition to the new compounds, two known constituents were also obtained. The structures of the known compounds (16 and 17) were defined as 2α,3α,23-trihydroxyurs-12,18dien-28-oic acid30 and kakidiol.34 Some studies have shown that pentacyclic triterpenoids possess antiviral HSV-1 and influenza A/95−359 activities. Thus, the isolated compounds were tested for these activities.37,38 Owing to the limited quantities of compounds 4, 6, 9, 11, and 13−16, only compounds 1−3, 5, 7, 8, 10, 12, and 17 were tested. Compounds 1 and 12 inhibited HSV-1 with IC50 values of 6.4 and 0.4 μM, respectively. Additionally, 12 showed potential as an inhibitor of influenza A/95−359, with an IC50 value of 1.2 μM.

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EXPERIMENTAL SECTION

General Experimental Procedures. A JASCO P-2000 automatic digital polarimeter was used for the determination of optical rotations, while a JASCO V650 spectrophotometer was used for the measurement of UV spectra. ECD data were collected using a JASCO J-815 spectropolarimeter. A Nicolet 5700 FT-IR instrument was used for acquisition of the IR spectra. HRESIMS data were acquired using an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer. Column chromatography separations were carried out on silica gel (160−200, 200−300 mesh, Qingdao Haiyang Chemical Co., Ltd. China), MCI (Mitsubishi Chemical Corporation), Sephadex LH-20 (GE Chemical Corporation), and ODS (50 μm, Merck, Germany). Spots were visualized by spraying with 10% H2SO4 in EtOH and then heating. The presence of peroxides was detected by spraying with a KI-HOAc-starch reagent. HPLC-MS-SPE-NMR Analysis. The chromatographic separation was performed on an Agilent HPLC instument (1260 series, 1.0 mL/ min, 40 °C column oven temperature, YMC C18 column, 5 μm, 4.6 mm × 250 mm). The ESIMS analysis was performed on an ESI-Qq1730



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(MeOH) λmax (log ε) 203 (4.09), 220 (3.71), 275 (3.04) nm; ECD (MeOH) λmax (Δε) 211 (+0.45), 236 (+0.95), 292 (+1.62) nm; IR (KBr) νmax 3497, 3390, 2945, 1690, 1581, 1453, 1388, 1066, 1039, 981, 950, 828, 590 cm−1; 1D NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 461.3033 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (2R,3S)-2,3,24-Trihydroxy-28-norursane-12,17,19,21-tetraene (7): colorless needles (MeOH); mp 245−246 °C; [α]20 D +35 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 213 (4.30), 244 (3.91) nm; ECD (MeOH) λmax (Δε) 211 (−4.33), 243 (+13.56) nm; IR (KBr) νmax 3373, 2948, 1456, 1374, 1030, 966, 812, 679 cm−1; 1D NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 461.3033 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (1R,3R)-1,3,24-Trihydroxy-28-norursane-12,17,19,21-tetraene (8): white, amorphous powder; [α]20 D +27 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 213 (4.33), 244 (3.95) nm; ECD (MeOH) λmax (Δε) 213 (−4.77), 244 (+15.71) nm; IR (KBr) νmax 3358, 2946, 1722, 1456, 1374, 1284, 1021, 993, 809 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 461.3038 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (2R,3S)-2,3,24,30-Tetrahydroxy-28-norursane-12,17,19,21-tetraene (9): white, amorphous powder; [α]20 D +20 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 213 (4.43), 244 (3.80) nm; ECD (MeOH) λmax (Δε) 215 (−2.61), 243 (+11.58) nm; IR (KBr) νmax 3374, 2931, 1686, 1457, 1376, 1032, 822, 687 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 477.2986 [M + Na]+ (calcd for C29H42O4Na, 477.2975). (1S,2R,3S)-1,2,3,23-Tetrahydroxy-28-norursane-12,17,19,21-tetraene (10): white, amorphous powder; [α]20 D +39 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 213 (4.35), 244 (3.96) nm; ECD (MeOH) λmax (Δε) 212 (−6.57), 244 (+19.22) nm; IR (KBr) νmax 3398, 2946, 1696, 1454, 1378, 1040, 954, 810 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 477.2984 [M + Na]+ (calcd for C29H42O4Na, 477.2975). (2R,3S)-2,3,23-Trihydroxy-28-norursane-12,17,19,21-tetraene (11): white, amorphous powder; [α]20 D +11 (c 0.01, MeOH/H2O, 1:3); UV (MeOH) λmax (log ε) 211 (4.20), 243 (3.73) nm; ECD (MeOH) λmax (Δε) 213 (−3.59), 243 (+13.99) nm; IR (KBr) νmax 3348, 2926, 1598, 1454, 1387, 1124, 1091, 1045, 810, 619 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 461.3045 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (2R,3R)-2,3,23-Trihydroxy-28-norursane-12,17,19,21-tetraene (12): white, amorphous powder; [α]20 D +13 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 213 (4.20), 244 (3.82) nm; ECD (MeOH) λmax (Δε) 212 (−5.66), 243 (+17.01) nm; IR (KBr) νmax 3364, 2935, 1595, 1454, 1387, 1049, 810, 678 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 461.3034 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (1R,3R)-1,2,23-Trihydroxy-28-norursane-12,17,19,21-tetraene (13): white, amorphous powder; [α]20 D +32 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 213 (4.7), 246 (4.3) nm; ECD (MeOH) λmax (Δε) 213 (−1.77), 244 (+5.76) nm; IR (KBr) νmax 3362, 2921, 2852, 1465, 1384, 1202, 1030, 804, 721 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 461.3029 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (3R)-3,23,24-Trihydroxy-28-norursane-12,17,19,21-tetraene (14): white, amorphous powder; [α]20 D +9 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 213 (4.19), 244 (3.79) nm; ECD (MeOH) λmax (Δε) 215 (−3.57), 243 (+12.81) nm; IR (KBr) νmax 3337, 2931, 1453, 1374, 1072, 1034, 1002, 984, 810, 677 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 461.3037 [M + Na]+ (calcd for C29H42O3Na, 461.3026). (15S)-15,24-Dihydroxy-3-oxo-28-norursane-12,17,19,21-tetraene (15): white, amorphous powder; [α]20 D +86 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 206 (4.37), 243 (3.90) nm; ECD (MeOH) λmax (Δε) 214 (−0.93), 243 (+17.26) nm; IR (KBr) νmax 3433, 2935, 1700, 1454, 1382, 1201, 1039, 809 cm−1; 1D NMR data, see Tables 2 and 3; (+)-HRESIMS m/z 437.3071 [M + H]+ (calcd for C29H41O3, 437.305). Crystallographic Data. 7 was recrystallized from MeOH to acquire colorless needles. We obtained the X-ray crystallographic

subsequently purified via the hyphenated NMR method to afford compound 3 (2.1 mg). Compounds 8 (2.0 mg) and 15 (1.7 mg) were obtained from Fr.4-1-3I (5 g) from chromatographic columns and the hyphenated NMR method. Fr.4-1-3J (6 g) was applied to Sephadex LH-20 and ODS columns and further purified via the hyphenated NMR method to afford compounds 4 (1.7 mg), 7 (3.9 mg), 10 (2.3 mg), 11 (1.7 mg), 12 (2.2 mg), 13 (1.8 mg), 14 (1.6 mg), and 17 (2.3 mg). Fr.5-9 (1.8 g), obtained from Fr.5 (16 g) via an ODS column, was further purified via column chromatography and the hyphenated NMR method to afford compound 9 (1.8 mg). All the constituents were obtained via HPLC-MS-SPE-NMR techniques and screened according to their characteristic UV, MS, or 1H NMR signals. The detailed separation information for compounds 1−17 is shown in the Supporting Information. Screening of New Nortriterpenoids by HPLC-MS-SPE-NMR. The triterpenoids initially detected by TLC analysis were concentrated in the CH2Cl2-soluble fraction, and this fraction was further purified via column chromatography to remove the more abundant and known triterpenoids. Finally, the trace-level triterpenoids were screened using UV, MS (m/z 400−600), and 1H NMR data acquired through HPLC-MS-SPE-NMR analyses. The target peaks in each fraction were baseline-resolved using an RP-18 HPLC system. Taking Fr.4-1-3G-6 as an example, the HPLC chromatogram and the total ion chromatogram of Fr.4-1-3G-6 are shown in Figure 10. The online mass spectra (Figure 10) of the four peaks (P1−P4) in this fraction showed that P1, P3, and P4 could be triterpenoids because their molecular weights were greater than 400. Their UV spectra suggested that P3 and P4 were unique triterpenoids with conjugated diene moieties based on their characteristic maximum absorption band at 240 nm. A small amount of sample was isolated and trapped on SPE cartridges for further 1H NMR spectroscopy analysis. The 1H NMR spectra (Figure 11) of the four peaks further confirmed that P1, P3, and P4 were triterpenoids based on their multiple upfield tertiary methyl groups. Thus, the two unique triterpenoids [P3(compound 1) and P4 (compound 2)] were concentrated on two different cartridges, and their structures were elucidated by NMR analysis. (2R,3S,17R,20S)-2,3,23-Trihydroxy-28-norursane-12,18-diene17,20-peroxide (1): white, amorphous powder; [α]20 D +34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 239 (3.67) nm; ECD (MeOH) λmax (Δε) 240 (+14.26) nm; IR (KBr) νmax 3412, 2941, 1708, 1454, 1378, 1045 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 495.3069 [M + Na]+ (calcd for C29H44O5Na, 495.3081). (3R,17R,20S)-3,24-Dihydroxy-28-norursane-12,18-diene-17,20peroxide (2): white, amorphous powder; [α]20 D +26 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 239 (3.65) nm; ECD (MeOH) λmax (Δε) 240 (+11.93) nm; IR (KBr) νmax 3420, 2936, 1708, 1455, 1378, 1034 cm−1; 1D NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 479.3138 [M + Na]+ (calcd for C29H44O4Na, 479.3132). (3R,17S,20R)-3,24-Dihydroxy-28-norursane-12,18-diene-17,20peroxide (3): white, amorphous powder; [α]20 D +79 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (3.85) nm; ECD (MeOH) λmax (Δε) 238 (+6.24) nm; IR (KBr) νmax 3430, 2936, 1708, 1455, 1377, 1032 cm−1; 1D NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 457.3323 [M + H]+ (calcd for C29H45O4, 457.3312). (2R,3S)-2,3,24-Trihydroxy-28-norursane-12,17-diene (4): white, amorphous powder; [α]20 D +43 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 238 (4.14) nm; ECD (MeOH) λmax (Δε) 216 (+0.98), 248 (−0.33) nm; IR (KBr) νmax 3401, 2926, 1710, 1458, 1373, 1036 cm−1; 1D NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 465.3337 [M + Na]+ (calcd for C29H46O3Na, 465.3339). (2R,3S)-2,3,24-Trihydroxy-19,20-seco-28-norursane-12,17-diene (5): white, amorphous powder; [α]20 D +29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (3.93) nm; ECD (MeOH) λmax (Δε) 231 (+0.89), 272 (+0.65), 319 (−0.04) nm; IR (KBr) νmax 3391, 2925, 1696, 1456, 1371, 1262, 1038 cm−1; 1D NMR data, see Tables 1 and 3; (+)-HRESIMS m/z 495.3085 [M + Na]+ (calcd for C29H44O5Na, 495.3081). (3R)-3,24-Dihydroxy-13-oxo-19,20-seco-28-norursane-17,19,21triene (6): white, amorphous powder; [α]20 D −27 (c 0.03, MeOH); UV 1731



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structure of 7 by anomalous scattering of Cu Kα radiation. C29.25H43O3.25, M = 446.63, orthorhombic, a = 45.884(3) Å, b = 7.0425(7) Å, c = 15.7032(17) Å, β = 103.902(9)°, U = 4925.7(8) Å3, T = 106.3, space group C2 (no. 5), Z = 8, μ(Cu Kα) = 0.592, 17 142 reflections measured and 9288 unique reflections (Rint = 0.0444) were used in all calculations. The final wR(F2) was 0.2528, and the Flack parameter = 0.2(2). The complete data were deposited at the Cambridge Crystallographic Data Centre (CCDC 1524486). Antiviral and Cytotoxic Assays. The antiviral (HSV-1 and influenza A/95−359) and cytotoxicity assays of some of the isolates were performed (Tables 4 and 5). The experimental details are shown in the Supporting Information.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165324. ORCID

Yong Li: 0000-0003-1672-6589 Yun-Bao Liu: 0000-0002-1338-0271 Shi-Shan Yu: 0000-0003-4608-1486 Author Contributions §

F. Liu and Y.-N. Wang contributed equally to this paper.

Notes

The authors declare no competing financial interest.

Table 4. Antiviral Activity against HSV-1 and Cytotoxicity for Compounds 1−3, 5, 7, 8, 10, 12, and 17 in Vero Cellsa compound

TC50b (μM)

IC50 (μM)

SIc

1 2 3 5 7 8 10 12 17 acyclovire

23.11 57.74 33.33 69.34 3.70 11.11 1.78 1.78 5.34 >100

6.41 >11.11 >11.11 >33.33 >1.23 >3.70 >0.41 0.41 >1.23 0.41

3.6 −d − − − − − 4.3 − >243.9

Data represent mean values for three independent determinations. Cytotoxic concentration required to inhibit Vero cell growth by 50%. c Selectivity index value equaled TC50/IC50. dThe selectivity index (SI) could not be determined under the test conditions. ePositive control.

Table 5. Antiviral Activity against Influenza A and Cytotoxicity for Compounds 1−3, 5, 7, 8, 10, 12, and 17 in MDCK Cellsa IC50 (μM)

SIc

1 2 3 5 7 8 10 12 17 oseltamivire

57.74 19.25 19.25 >100 7.70 16.02 2.57 3.70 19.25 1260

>33.33 >11.11 >11.11 >33.33 >3.70 >3.70 >1.23 1.23 >11.11 1.63

−d − − − − − − 3.0 − 773.0

a

Data represent mean values for three independent determinations. Cytotoxic concentration required to inhibit MDCK cell growth by 50%. cSelectivity index value equaled TC50/IC50. dThe selectivity index (SI) could not be determined under the test conditions. e Positive control. b

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REFERENCES

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b

TC50b (μM)

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science Foundation of China (No. 21732008) and the CAMS Innovation Fund for Medical Sciences (CIFMS) (No. 2016I2M-1-010 and No. 2016-I2M-3-010). The authors are grateful to the Department of Instrumental Analysis at our institute for the spectroscopic measurements.

a

compound

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01074. X-ray crystallography data for compound 7 (CIF) Experimental details for the isolation of compounds 1− 17 and activity assays; UV, IR, ECD, 1D NMR, HSQC, HMBC, and NOE spectra for compounds 1−15 (PDF) 1732



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Minor Nortriterpenoids from the Twigs and Leaves of

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