Abietane Diterpenoids from the Roots of Clerodendrum trichotomum

ABSTRACT: Twelve new abietane diterpenoids (1−12) and. 31 known analogues (13−43) were isolated from a medicinal. Chinese herb, Clerodendrum ...
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Article Cite This: J. Nat. Prod. 2018, 81, 1508−1516

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Abietane Diterpenoids from the Roots of Clerodendrum trichotomum and Their Nitric Oxide Inhibitory Activities Hai-Jun Hu,†,‡ Yue Zhou,† Zhu-Zhen Han,† Yan-Hong Shi,† Shu-Sheng Zhang,‡ Zheng-Tao Wang,*,† and Li Yang*,†,‡ †

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The MOE Key Laboratory of Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines, and SATCM Key Laboratory of New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica (ICMM), and ‡Center for Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine (SHUTCM), Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Twelve new abietane diterpenoids (1−12) and 31 known analogues (13−43) were isolated from a medicinal Chinese herb, Clerodendrum trichotomum Thunberg. The absolute configurations of 1−3 were established on the basis of ECD and X-ray crystallography data, whereas that of 4 was elucidated by comparison of experimental and calculated ECD data. Eight diterpenoids, 15,16-dehydroteuvincenone G (1), trichotomin A (4), 2α-hydrocaryopincaolide F (7), villosin C (20), 15-dehydro-17-hydroxycyrtophyllone A (22), demethylcryptojaponol (38), 6β-hydroxydemethylcryptojaponol (39), and trichotomone (43), exerted inhibitory effects against NO production with IC50 values of 5.6−16.1 μM. The structure− activity relationships of the isolated diterpenoids are also estimated. abeo-abietane diterpenoid13 and is similar to teuvincenone G (13),14 except for the deshielded C-15 (ΔδC +68.3) and C-16 (ΔδC +73.3) resonances, which indicated that 1 is a 15,16dehydro derivative of 13. The structure of 1 was further characterized by the HMBC cross-peaks (Figure 2) of H3-17/ C-15 and C-16. The NOESY correlations (Figure 3) of H-5/ H3-18, H-5/H-6α (δH 2.56), H-6β (δH 2.76)/H3-19, and H-6β/ H3-20 suggested that the A/B rings are trans-fused. The (5R,10S) absolute configuration of 1 was assigned by the electronic circular dichroism (ECD) data exhibiting a negative and a positive Cotton effect (CE) at 294 and 326 nm, respectively,13,15 and was unequivocally confirmed through Cu Kα diffraction analysis (Figure 4) [Flack parameter: −0.02(7)] (Table S2, Supporting Information). Accordingly, the structure of 15,16-dehydroteuvincenone G (1) was established as (5R,10S)-12,16-epoxy-11,14-dihydroxy-17(15→16)-abeo-abieta-8,11,13,15-tetraen-3,7-dione. The (+)-HRESIMS analysis revealed that the molecular formula of compound 2 was C20H26O5. The NMR data (Tables 1 and 2) resembled those of teuvincenone G (13).14 In contrast to 13, compound 2 possesses an oxygenated methine carbon (δH 3.10, δC 75.5) but lacks the carbonyl carbon (δC 216.0) in 13. This suggested that a hydroxy group replaced the carbonyl group at C-3 in 13, which was supported by 2D NMR analyses (Figure 2). The diagnostic NOESY cross-peaks (Figure 3) of H-3/H-5 indicated that HO-3 was β-oriented. X-ray diffraction

Clerodendrum trichotomum Thunberg (Lamiaceae), an ornamental shrub or small tree, is popularly known as “Chou-WuTong” in China. Its dried stems, leaves, and roots are utilized as folk medicine for inflammatory diseases and hypertension.1 Several compounds, such as phenylethanoid glycosides,2,3 flavonoids,4 and neo-clerodane diterpenoids,5,6 derived from its leaves exhibit potential anti-inflammatory activities.2,4 Rearranged abietane diterpenoids7,8 have also been isolated from its roots. However, the biological activities of these compounds have not been assayed. In the continuous search for bioactive natural diterpenoids from traditional Chinese medicines,9−12 the present study focused on the isolation, structure elucidation, and evaluation of nitric oxide (NO) inhibitory activities of the 12 new abietanes (1−12) and the 31 known analogues (13−43) from the roots of C. trichotomum.

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RESULTS AND DISCUSSION The chromatographic separation of C. trichotomum extract afforded 10 new abietane diterpenoids (1−10), two new abietane-type glycosides (11 and 12) (Figure 1), and 31 known analogues (13−43) (Table S1 and Figure S1, Supporting Information). The (−)-HRESIMS ion at m/z 341.1407 (calcd 341.1389) indicated that compound 1, yellow needle-like crystals (CHCl3), possessed the molecular formula C20H22O5. An olefinic proton (δH 6.55, d, J = 1.2 Hz), three methyl singlets (δH 1.16, 1.17, and 1.47), and a methyl doublet (δH 2.43, d, J = 1.1 Hz) were evident from the 1H NMR spectrum. Its 1D NMR data (Tables 1 and 2) suggested that 1 is a 17(15→16)© 2018 American Chemical Society and American Society of Pharmacognosy

Received: September 25, 2017 Published: June 20, 2018 1508

DOI: 10.1021/acs.jnatprod.7b00814 J. Nat. Prod. 2018, 81, 1508−1516

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Figure 1. New compounds 1−12 isolated from the roots of C. trichotomum.

Table 1. 1H NMR Data for Compounds 1−5 (δ in ppm, J Values in Hz) 1a

position 1a 1b 2a 2b 3a 3b 5 6a 6b 15a 15b 16 17a 17b 18 19 20 11-OH 14-OH 3-OH OCH3 a

2b

3c

3.43 2.02 2.64 2.62

ddd (13.3, 7.2, 5.5) dt (14.0 8.5) m m

3.32 1.28 1.62 1.58 3.10

dt (13.8, 4.5) td (13.8, 4.0) ov.f m dt (11.3, 4.9)

2.43 2.76 2.56 6.55

dd (14.6, 2.8) dd (16.6, 14.6) dd (16.7, 2.9) d (1.2)

1.61 2.69 2.38 3.23 2.69 5.08 1.43

dd (14.5, dd (17.0, dd (17.1, dd (15.2, dd (15.2, m d (6.3)

2.43 d (1.1) 1.17 s 1.16 s 1.47 s 5.11 s 13.54 s

2.5) 14.6) 2.4) 9.0) 7.5)

0.94 s 0.79 s 1.26 s 8.41 s 13.31 s 4.49 d (5.1)

3.50 1.39 2.00 1.60 1.76 1.45

dt (13.6, 3.7) ov.f m m dt (12.0, 3.6) td (12.2, 4.4)

6.27 s 3.23 2.99 5.00 3.80 3.70 1.27 1.37 1.67

dd dd m dd dd s s s

4d 3.06 1.92 2.49 2.25

ddd (13.0, 5.5, 1.4) td (12.8, 5.6) m dd (19.0, 5.2)

5e 3.28 1.54 2.70 2.50

dd (13.5, 5.0) td (13.0, 5.8) m dd (19.2, 5.5)

5.94 s

6.41 s

(15.5, 9.5) (15.5, 7.0)

6.46 d (0.7)

(12.1, 3.7) (12.1, 6.0)

2.4 d (1.0)

3.38 2.86 5.12 1.50

1.88 s 1.78 s 1.40 s

dd (15.3, 9.0) dd (15.3, 7.3) m d (6.5)

2.15 s 1.51 s 4.98 s 13.42 s 3.78 s

500 MHz in CDCl3. b600 MHz in DMSO-d6. c400 MHz in methanol-d4. d400 MHz in CDCl3. e600 MHz in CDCl3. fov.: overlapped signal.

parameter: 0.12(16)] (Table S4, Supporting Information). Accordingly, the structure of 17-hydroxymandarone B (3) was established as (10S,16R)-12,16-epoxy-11,14,17-trihydroxy17(15→16)-abeo-abieta-5,8,11,13-tetraen-7-one. Diterpenoid 4 had the molecular formula C20H18O5, as deduced from its 13C NMR (Table 2) and (+)-HRESIMS data. Its 1D NMR spectra (Tables 1 and 2) showed four methyls (δH 1.40, 1.78, 1.88, and 2.40), two olefinic protons (δH 5.94 and 6.46), and three carbonyl carbons (δC 177.6, 182.1, and 189.7). This indicated that 4 is a 17(15→16),18(4→3)-diabeo-abieta diterpenoid and is highly similar to 12,16-epoxy-17(15→ 16),18(4→3)-diabeo-abieta-3,5,8,12,15-pentaen-7,11,14-trione (33).7 However, in contrast to 33, compound 4 contains an 8,9-epoxy group. This inference was corroborated by the

analysis of compound 2 confirmed the absolute configuration as (3S,5R,10S,16S) [Flack parameter = −0.02(7)] (Table S3, Supporting Information). The structure of 3-dihydroteuvincenone G (2) was thus defined as (3S,5R,10S,16S)-12,16-epoxy3,11,14-trihydroxy-17(15→16)-abeo-abieta-8,11,13-trien-7-one. The molecular formula of C20H24O5 was established for diterpenoid 3 according to the (−)-HRESIMS. NMR data (Tables 1 and 2) revealed that 3 was also a 17(15→16)-abeoabietane and was structurally similar to mandarone B.16 Compared with mandarone B, the Me-17 in 3 was oxygenated (17-CH2OH: δH 3.70 and 3.80; δC 65.1), which was supported by the HMBC interactions (Figure 2) from H2-17 to C-15 (δC 29.6) and C-16 (δC 87.3). The (10S,16R) absolute configuration of 3 was defined by X-ray diffraction data [Flack 1509

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biosynthetic considerations.7 The calculated ECD spectrum for the (8S,9S,10S) isomer of 4 showed good agreement with the experimental data in the 200−400 nm region (Figure 5). Therefore, the structure of trichotomin A (4) was established as (8S,9S,10S)-8,9;12,16-diepoxy-17(15→16),18(4→3)-diabeoabieta-3,5,12,15-tetraen-7,11,14-trione. The molecular formula of diterpenoid 5 (red, amorphous powder) was deduced as C21H22O6. The NMR data (Tables 1 and 2) resembled those of formidiol,17 with slight differences. The C-15 and C-16 resonances of 5 were shielded by −67.0 and −71.6 ppm, respectively, which, when combined with the COSY correlations of H3-17/H-16/H2-15 and the HMBC interactions of H-15/C-12 and C-14 of 5 (Figure 2), suggested that 5 was a 15,16-dihydro derivative of formidiol. Thus, the structure of 15,16-dihydroformidiol (5) was identified as (10S,16S)-12,16-epoxy-11,14-dihydroxy-17(15→16),18(4→3)diabeo-abieta-3,5,8,11,13-pentaen-7-one-18-carboxylate. Diterpenoid 6 had the molecular formula C20H20O6, as inferred from its 13C NMR and (+)-HRESIMS data. The NMR data of 6 (Tables 3 and 4) resembled those of teuvincenone E (28).18 A vinylic methyl (δH 1.98) in 28 was replaced by a hydroxymethyl group (δH 4.49, δC 55.3) at C-3 in 6. This was verified by the diagnostic HMBC interactions (Figure 2) of H218 (δH 4.49)/C-2 and C-4. Accordingly, the structure of 18hydroxyteuvincenone E (6) was defined as (10S,16S)-12,16epoxy-11,14,18-trihydroxy-17(15→16),18(4→3)-diabeo-abieta3,5,8,11,13-pentaen-2,7-dione. Compound 7 exhibited the same molecular formula (C20H22O5) and similar NMR data (Tables 3 and 4) compared to caryopincaolide F (24),12 indicating that 7 has the same 2D structure, except for the β-oriented HO-2 in 7 (α-oriented HO2 in 24), which was supported by the diagnostic C-1 and C-3

Table 2. 13C NMR Data for Compounds 1−5 (δ in ppm) position

1a

2b

3c

4d

5e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCH3

36.0, CH2 34.7, CH2 216.0, C 47.3, C 49.4, CH 36.4, CH2 204.7, C 110.5, C 130.8, C 39.5, C 131.8, C 150.1, C 117.6, C 153.3, C 101.9, CH 155.5, C 14.2, CH3 21.2, CH3 27.2, CH3 18.3, CH3

33.9, CH2 27.2, CH2 75.5, CH 38.3, C 48.7, CH 34.3, CH2 203.9, C 109.2, C 140.2, C 39.6, C 131.4, C 156.9, C 110.4, C 154.1, C 33.1, CH2 81.6, CH 21.3, CH3 27.6, CH3 15.4, CH3 16.9, CH3

35.5, CH2 19.8, CH2 41.7, CH2 39.3, C 178.2, C 123.6, CH 191.6, C 109.8, C 141.8, C 45.1, C 133.0, C 157.4, C 112.4, C 154.7, C 29.6, CH2 87.3, CH 65.1, CH2 33.7, CH3 30.1, CH3 25.3, CH3

29.3, CH2 29.7, CH2 143.2, C 124.7, C 158.8, C 116.5, CH 189.7, C 61.2, C 70.1, C 39.9, C 177.6, C 149.7, C 130.4, C 182.1, C 105.2, CH 161.9, C 14.4, CH3 21.7, CH3 14.8, CH3 21.4, CH3

28.9, CH2 25.1, CH2 132.7, C 136.3, C 163.0, C 123.0, CH 189.7, C 109.5, C 135.7, C 39.3, C 131.3, C 154.6, C 111.5, C 154.6, C 34.6, CH2 83.5, CH 21.4, CH3 169.3, C 17.2, CH3 22.2, CH3 52.1, CH3

a

125 MHz in CDCl3. b150 MHz in DMSO-d6. c100 MHz in methanol-d4. d100 MHz in CDCl3. e150 MHz in CDCl3.

presence of the diagnostic resonances of two oxygenated tertiary carbons [δC 61.2 (C-8) and 70.1 (C-9)] and HMBC cross-peaks (Figure 2) of H-1b (δH 1.92)/C-9, H3-20/C-9, and H-6/C-8. Analysis of HMBCs confirmed the 2D structure of 4 (Figure 1). The Me-20 in 4 was β-oriented based upon

Figure 2. Key HMBC and 1H−1H COSY correlations for compounds 1−12. 1510

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Figure 3. Key NOESY correlations for compounds 1, 2, 7−9, 11, and 12.

Figure 4. ORTEP drawings for compounds 1−3.

resonances (δC 38.6 and 141.1 for 7, and δC 40.1 and 144.0 for 24) and the NOESY cross-peaks of H-2 (δH 4.19)/H-1α (δH 1.66) and H3-20/H-1β (δH 3.66) in 7. Thus, the structure of

2α-hydrocaryopincaolide F (7) was elucidated as (2S,10S,16S)12,16-epoxy-2,11,14-trihydroxy-17(15→16),18(4→3)-diabeoabieta-3,5,8,11,13-pentaen-7-one. 1511

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Table 4. 13C NMR Data for Compounds 6−10 (δ in ppm) position

6a

7b

8c

9a

10c

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

46.7, CH2 198.7, C 138.7, C 152.1, C 162.4, C 126.2, CH 190.2, C 109.8, C 136.7, C 44.2, C 133.6, C 157.7, C 113.6, C 155.6, C 35.0, CH2 84.2, CH 22.3, CH3 55.3, CH2 17.2, CH3 24.7, CH3

38.6, CH2 70.0, CH 141.1, C 128.8, C 168.1, C 120.5, CH 192.0, C 110.1, C 138.8, C 41.0, C 134.1, C 157.6, C 113.1, C 155.3, C 35.5, CH2 84.2, CH 22.7, CH3 18.7, CH3 16.0, CH3 26.4, CH3

29.2, CH2 30.5, CH2 141.0, C 125.5, C 165.8, C 118.6, CH 190.4, C 109.9, C 138.3, C 40.0, C 131.2, C 154.2, C 114.2, C 155.2, C 70.9, CH 86.7, CH 13.7, CH3 20.9, CH3 15.2, CH3 22.3, CH3

46.6, CH2 199.3, C 137.4, C 148.6, C 162.9, C 124.9, CH 190.6, C 109.9, C 138.3, C 44.4, C 134.0, C 157.7, C 115.1, C 157.2, C 76.8, CH 91.5, CH 19.4, CH3 12.0, CH3 17.7, CH3 24.8, CH3

29.7, CH2 18.8, CH2 132.2, C 153.0, C 153.5, C 123.2, CH 188.3, C 109.9, C 133.7, C 39.8, C 131.7, C 155.1, C 112.1, C 155.4, C 34.6, CH2 83.9, CH 22.4, CH3 172.6, C 68.9, CH2 22.2, CH3

a

100 MHz in methanol-d4. b150 MHz in methanol-d4. c150 MHz in CDCl3.

oriented based on the NOESYs (Figure 3) of H3-17/H-15 and H3-17/H-16. Accordingly, the structure of 15α-hydroxyuncinatone (8) was elucidated as (10S,15R,16S)-12,16-epoxy11,14,15-trihydroxy-17(15→16),18(4→3)-diabeo-abieta3,5,8,11,13-pentaen-7-one. The (+)-HRESIMS data demonstrated that compound 9 had the molecular formula C20H20O6 and one more index of hydrogen deficiency than 8. The NMR data of 9 showed the presence of a ketocarbonyl group (δC 199.3) rather than the C2 methylene in 8 (Tables 3 and 4). This assignment was verified by HMBC cross-peaks (Figure 2). The HO-15 of 9 was α-oriented according to the NOESY correlations (Figure 3) of H3-17/H-15 and H3-17/H-16. Therefore, the structure of 15αhydroxyteuvincenone E (9) was established as (10S,15R,16S)12,16-epoxy-11,14,15-trihydroxy-17(15→16),18(4→3)-diabeoabieta-3,5,8,11,13-pentaen-2,7-dione.

Figure 5. Calculated and experimental ECD spectra in MeOH of compound 4 (A) at the WB97XD/6-311+G(d,p) level and (B) at the CAM-B3LYP/6-311+G(d,p) level.

The 13C NMR (Table 4) and (+)-HRESIMS data revealed that the molecular formula of diterpenoid 8 was C20H22O5. Its NMR data (Tables 3 and 4) showed many similarities to those of uncinatone (25).13 Compared to 25, the C-15 resonance of 8 was deshielded (ΔδC +36.4), suggesting the presence of a 15hydroxy moiety in 8.19 This assignment was verified by the 1 H−1H COSY interactions of H-15/H-16/H3-17 and HMBCs of H3-17/C-15 and C-16 (Figure 2). The HO-15 was α-

Table 3. 1H NMR Data for Compounds 6−10 (δ in ppm, J Values in Hz) 6a

position 1a 1b 2a 2b 6 15a 15b 16 17 18 19a 19b 20 11-OH 14-OH a

7b

4.23 d (16.7) 2.40 d (16.7)

3.66 dd (14.5, 1.2) 1.66 dd (14.5, 5.6) 4.19 d (5.1)

6.65 3.36 2.81 5.14 1.51 4.49 2.38

6.19 3.32 2.79 5.11 1.50 1.98 1.96

s ov.d dd (15.4, 7.3) m d (6.3) s s

1.65 s

s ov.d dd (15.2, 7.3) m d (6.3) s s

1.68 s

8c

9a

3.20 1.53 2.50 2.20 6.21 5.38

dt (13.4, 5.5) ddd (13.3, 11.9, 6.0) m dd (19.2, 5.8) s d (5.9)

6.58 s 5.09 d (2.4)

4.80 1.58 1.86 1.89

m d (6.7) s s

4.79 1.39 1.98 2.26

1.48 s 4.79 s 13.97 s

4.24 d (16.7) 2.46 d (16.6)

qd (6.7, 2.4) d (6.7) s s

1.63 s

10c 3.57 1.66 2.59 2.59 6.18 3.41 2.89 5.16 1.53

dt (13.6, 3.6) m m m s dd (15.4, 9.0) dd (15.4, 7.3) m d (6.2)

5.12 d (16.4) 4.90 d (16.8) 1.51 s 4.91 s 13.25 s

400 MHz in methanol-d4. b600 MHz in methanol-d4. c600 MHz in CDCl3. dov.: overlapped signal. 1512

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Table 5. 1H and 13C NMR Data for Compounds 11 and 12 (δ in ppm)a

The (−)-HRESIMS ion at m/z 353.1028 (calcd 353.1025) indicated that compound 10 possessed a molecular formula of C20H18O6. The 1D NMR data (Tables 3 and 4) showed that 10 was also a diabeo-abieta diterpenoid and similar to (R)-6hydroxy-7-isopropyl-9b-methyl-10,11-dihydrophenanthro[1,2c]furan-1,5(3H,9bH)-dione,20 a synthesized intermediate with a butenolide moiety. The HMBC cross-peaks (Figure 2) of H219 (δH 4.90 and 5.12) with C-3, C-4, and C-5 verified the presence of the butenolide group in 10. Analysis of the 2D NMR data (Figure 2) permitted assignment of the structure of trichotomin B (10) as (10S,16S)-12,16-epoxy-11,14-dihydroxy17(15→16),18(4→3)-diabeo-abieta-3,5,8,11,13-pentaen-7-one18,19-olide. The molecular formula of C26H34O9 of compound 11 was elucidated via the (−)-HRESIMS data. The 1D NMR data (Table 5) of 11 revealed signals assignable to an exocyclic Δ4(19) double bond [δH 4.92 (brs, H-19a) and 5.15 (brs, H19b); δC 133.4 (C-4) and 108.4 (C-19)], an olefinic proton (δH 5.63), and a β-glucopyranose moiety [anomeric proton at δH 4.59 (1H, d, J = 7.9 Hz)]. Acid hydrolysis of 11 afforded Dglucose. These characteristics, combined with spectroscopic data, indicated that 11 is an abietane diterpenoid glucoside and is structurally related to 11,16-dihydroxy-12-O-β-D-glucopyranosyl-17(15→16),18(4→3)-abeo-4-carboxy-3,8,11,13-abietatetraen-7-one.21 The structural difference involved the presence of Δ2(3) and Δ4(19) double bonds in the A ring of 11, which were corroborated by the 1H−1H COSY cross-peaks (Figure 2) of H2-1/H-2/H-18 and the HMBCs (Figure 2) of H2-1/C-2, H3-18/C-2 and C-4, and H2-19/C-4 and C-5. Additionally, the glucosidic linkage was determined by the diagnostic HMBC correlation of H-1′ (δH 4.59)/C-12 (δC 150.7). The NOESY cross-peaks (Figure 3) of H3-20/H-1β (δH 1.20); H3-20/H-6β (δH 2.65); H-5/H-1α (δH 2.26); and H-5/H-6α (δH 2.74) suggested that H-5 was α-oriented. Thus, the structure of trichotomside A (11) was defined as (5S,10S)-12-O-β-Dglucopyranosyl-11,16-dihydroxy-17(15→16),18(4→3)-diabeoabieta-2,4(19),8,11,13-pentaen-7-one. The (−)-HRESIMS analysis indicated that compound 12 had a molecular formula of C32H50O13. Its 1D NMR data (Table 5) were similar to those of 3,12-O-β-D-diglucopyranosyl11,16-dihydroxyabieta-8,11,13-triene,21 which displayed signals attributable to three methyls (δH 1.20, 1.05, and 0.84) and two β-glucopyranosyl moieties [anomeric protons at δH 4.23 (d, J = 7.6 Hz, H-1′) and 4.17 (d, J = 7.7 Hz, H-1″)]. The difference involved the isopropyl group at C-13 in 12. This inference was comfirmed by 1H−1H COSY and HMBC correlations (Figure 2). The glucosyl groups were determined as D-glucose by acid hydrolysis, and linkages were established by the HMBCs of H1′/C-12 and H-1″/C-3. The structure of trichotomside B (12) was hence established as (3S,5R,10S)-3,12-O-β-D-diglucopyranosyl-11-hydroxyabieta-8,11,13-triene. Given the biosynthesis of the rearranged abietane diterpenoids in C. trichotomum,7 similar to compounds 1−4, the methyl groups at C-10 in 5−12 and at C-16 in 5−10 were both β-oriented. The methyls at C-10 in 5−10 were confirmed to be β-oriented in view of the negative CE at 287, 270, 299, 296, 284, and 299 nm and the positive CE at 326, 356, 347, 322, 346, and 358 nm in the ECD spectra, respectively.15 Although previous reports have shown that the extract of C. trichotomum leaves inhibits inflammation,2 the active ingredients from its roots remain unreported. On the other hand, abietane diterpenoids have potential anti-inflammatory effects.22 Therefore, the isolates were investigated for their anti-

11b δH (J in Hz)

position 1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12 13 14 15a 15b 16

2.85 dd (14.1, 2.8) 2.74 dd (16.1, 3.0) 2.63 dd (16.1, 14.3)

7.46 3.21 2.71 4.12

s dd (13.3, 6.5) dd (13.6, 6.8) m

δH (J in Hz)

δC 39.8, CH2

3.21 m 1.15 m 126.7, CH 1.96 m 1.64 m 147.3, C 3.08 d (8.1) 133.4, C 45.4, CH 1.14 m 37.9, CH2 1.71 m 1.47 m 200.3, C 2.71 m 129.9, C 139.0, C 39.4, C 150.3, C 150.7, C 133.2, C 121.5, CH 6.37 s 41.1, CH2 3.57 m 68.5, CH

1.04 d (6.9)d 1.08 d (6.9)d 1.05 s 0.84 s

17

1.13 d (6.2)

23.0, CH3

18 19

1.88 brs 5.15 brs, 4.92 brs

20 1′

1.20 s 4.59 d (7.9)

2′ 3′ 4′ 5′ 6′

3.54 m 3.45 m 3.46 m 3.30 m 3.85 dd (12.0, 2.4), 3.76 dd (12.0, 4.8)

19.8, CH3 108.4, CH2 15.3, CH3 1.20 s 107.7, CH 4.23 d (7.6) 75.6, CH 3.30 m 78.1, CH 3.23 m 71.0, CH 3.04 m 78.8, CH 3.05 m 62.3, CH2 3.65 m, 3.43 m 4.17 d (7.7) 2.96 m 3.12 m 3.21 m 3.22 m 3.63 m, 3.51 m

1″ 2″ 3″ 4″ 5″ 6″ a

3.61 dd (18.6, 5.9) 2.26 d (18.8) 5.63 d (5.6)

12c δC 34.3, CH2 26.5, CH2 88.1, CH 39.4, C 52.7, CH 18.4, CH2 32.4, CH2 133.2, C 132.4, C 38.4, C 147.4, C 141.0, C 139.1, C 116.1, CH 25.0, CH 23.5, CH3d 23.9, CH3d 28.2, CH3 16.9, CH3 19.4, CH3 106.5, CH 73.8, 76.9, 70.2, 76.6, 61.3,

CH CH CH CH CH2

105.4, CH 74.0, 77.3, 69.6, 76.3, 60.7,

At 400 and 100 MHz for 1H and 13C NMR, respectively. methanol-d4. cIn DMSO-d6. dInterchangeable.

CH CH CH CH CH2 b

In

inflammatory effects. NO production is a main indicator to assess the inflammatory activities. Here, the isolated compounds were assessed for their abilities to inhibit NO production in LPS-stimulated RAW 264.7 cells (Table 6). Diterpenoids 1, 4, 7, 20, 22, 38, 39, and 43 exerted inhibitory effects at noncytotoxic concentrations with IC50 values of 6.0, 10.6, 6.5, 15.5, 16.1, 15.5, 9.4, and 5.6 μM, respectively, better than the positive control, aminoguanidine hydrochloride (IC50 = 26.2 ± 1.1 μM), which is an irreversible NO synthase inhibitor. Compounds 9, 10, 18, 21, and 28 showed moderate or weak activities with IC50 values of 28.9−38.6 μM. 1513

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were obtained on a Waters UPLC Premier Q-TOF spectrometer. An Agilent 7890B gas chromatograph coupled to a 7693 mass selective detector triple quadrupole mass spectrometer system and an Rxi-1 ms capillary column (30 m × 0.25 mm, i.d., 0.25 μm) were used for GC analysis. MPLC separation was performed on a Büchi sepacore with ODS C18 (45−60 μm, YMC Co., Ltd., Kyoto, Japan). Column chromatography (CC), preparative HPLC, and TLC were conducted using methods similar to those described previously.9,10 Plant Material. The roots of C. trichotomum were obtained in September 2015 from Bozhou, Anhui Province, People’s Republic of China, and were authenticated by one of the authors (L.Y.). A voucher specimen (No. CTR-150712) has been deposited at our institute. Extraction and Isolation. Cut and air-dried roots (38 kg) of C. trichotomum were extracted under reflux with 95% ethanol (3 × 300 L). After filtration and evaporation of the organic solvents, the crude extract (808 g) was suspended in water (7 L) at 55 °C and fractionated with CH2Cl2 (4 × 7 L) and n-BuOH (4 × 7 L) successively to yield the CH2Cl2 (178 g) and n-BuOH (210 g) fractions, respectively. The CH2Cl2 fraction (170 g) was separated into 10 fractions (Frs. 1−10) on a silica gel column eluted with petroleum ether−acetone (1:0 to 0:1, v/v). Fr. 2 (7.6 g) was chromatographed over MPLC eluted with MeOH−H2O (3:7 to 1:0, v/v) to obtain a subfraction containing the abietane diterpenoids. The subfraction was separated using a Sephadex LH-20 column eluted with petroleum ether− CH2Cl2−MeOH (5:5:1, v/v/v) to yield compound 15 (15 mg). Fr. 3 (8.7 g) was also chromatographed by a similar procedure (MPLC), to yield four subfractions. Compounds 4 (6.7 mg), 25 (180.0 mg), 26 (7.2 mg), and 33 (8.7 mg) were obtained from the four subfractions by Sephadex LH-20 and preparative TLC (PTLC, CH2Cl2), respectively. Fr. 4 (6.6 g) was also separated into six subfractions (Fr. 4-1−Fr. 4-6) using MPLC. On further CC on Sephadex LH-20 and PTLC, compounds 5 (21.0 mg), 34 (4.1 mg), and 38 (25 mg) were obtained from Fr. 4-2 (202.1 mg), Fr. 4-3 (131.2 mg), and Fr. 4-5 (162.1 mg), respectively. Fr. 4-4 (586.7 mg) was applied to a Sephadex LH-20 column and purified through PTLC (CH2Cl2) and semipreparative HPLC (MeOH−H2O, 75:25, v/v) to obtain compounds 16 (49 mg) and 17 (42.1 mg). Fr. 5 (5.8 g) was separated through similar procedures (MPLC and Sephadex LH-20) and PTLC (CH2Cl2−EtOAc, 50:1, v/v) to give compounds 13 (8.5 mg) and 18 (8.2 mg). Compounds 1 (10.2 mg), 19 (6.8 mg), 23 (10.2 mg), 27 (11.6 mg), 28 (20.5 mg), 29 (12.6 mg), and 39 (7.8 mg) were obtained from Fr. 6 (6.5 g) through procedures similar to those described for compound 17. Fr. 7 (5.6 g) was fractionated into five subfractions (Fr. 7-1−Fr. 75) through MPLC. Fr. 7-2 (338.4 mg) was separated on a Sephadex LH-20 column and further purified by preparative HPLC (MeCN− H2O−formic acid, 68:32:0.1, v/v/v) and PTLC (CH2Cl2−EtOAc, 20:1, v/v) to yield compounds 2 (9.2 mg), 10 (8.6 mg), and 35 (1.8 mg). Through similar procedures, Fr. 7-3 (251.0 mg) and Fr. 7-4 (311.7 mg) yielded compounds 8 (10.2 mg) and 40 (20.0 mg), respectively. By using processes of separation [MPLC, Sephadex LH20, preparative HPLC (MeCN−H2O, 76:24, v/v)] similar to those described for compound 10, compounds 7 (9.0 mg), 14 (34.5 mg), 20 (10.8 mg), and 36 (6.7 mg) were separated from Fr. 8 (6.2 g). Compounds 3 (14.6 mg), 21 (12.7 mg), 22 (5.4 mg), 24 (16.5 mg), 30 (6.0 mg), 31 (4.5 mg), 32 (1.1 mg), and 43 (3.6 mg) were obtained from Fr. 9 (10.9 g) through similar procedures. Fr. 10 (37.8 g) was subjected to an MCI gel column eluted with MeOH−H2O (8:2, v/v) and further separated through MPLC, Sephadex LH-20 (MeOH), and PTLC (petroleum ether−acetone, 4:1, v/v) to afford compound 37 (2.5 mg) and through preparative HPLC (MeCN− H2O, 55:45, v/v) to give compounds 6 (10.8 mg) and 9 (5.6 mg). The n-BuOH fraction (200 g) was separated into nine fractions (Frs. A−I) by CC with MCI gel (MeOH−H2O, 0:1 to 1:0, v/v). Fr. G (7.3 g) was separated into six subfractions (Frs. G1−G6) by MPLC (MeOH−H2O, 1:9 to 8:2, v/v). Fr. G3 (182.1 mg) was separated over a column of Sephadex LH-20 (MeOH−H2O, 8:2, v/v) and then purified through semipreparative HPLC (MeOH−H2O, 6:4, v/v) to yield compound 41 (7.8 mg). Similarly, Fr. G5 (68.7 mg) yielded

Table 6. Inhibitory Effects of Compounds 1−43 against NO Production in RAW 264.7 cells compound

IC50 ± SD (μM)a

compound

IC50 ± SD (μM)

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

6.0 ± 0.5 −b − 10.6 ± 0.5 >50 − 6.5 ± 0.4 >50 38.6 ± 3.1 28.9 ± 1.9 >50 >50 >50 >50 >50 >50 >50 31.1 ± 1.1 >50 15.5 ± 0.6 35.6 ± 2.7 16.1 ± 1.5

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 AGc

>50 − − − >50 31.4 ± 1.0 − − − >50 − >50 >50 >50 >50 15.5 ± 0.6 9.4 ± 0.3 >50 >50 >50 5.6 ± 0.3 26.2 ± 1.1

IC50 values were expressed as mean ± SD (n = 3). bThe samples showed cytotoxic effects to cells at 25 μM (less than 15% cell survival). c AG = aminoguanidine hydrochloride was used as the positive control. a

The 42 abietane diterpenoids could be classified into three subgroups: (I) 17(15→16)-abeo-abietanes (1−3 and 13−23), (II) 17(15→16),18(4→3)-diabeo-abietanes (4−11 and 24− 37), and (III) abietanes (12 and 38−42) (Table S1 and Figure S1, Supporting Information). As shown in Table 6, more than a third of the compounds in subgroup II (e.g., 24−26, 29−31, and 33) showed cytotoxic activities against cultured RAW 264.7 cells. This result corresponds with a previous report that an A ring rearrangement is necessary for cytotoxicity. 7 On comparison with 2, 13, 14, 17−19, and 23 in subgroup I, the 3-oxo, 5,6-dihydro, and Δ15,16 double bond in compound 1 were required for the inhibitory effects. The presence of the epoxy group of 4 (vs 33) in subgroup II enhances inhibitory activity and reduces the cytotoxicity. Compound 7 shows a significant effect (IC50 = 6.5 μM), while its three close analogues 24, 31, and 32 are inactive or cytotoxic, which indicated that the β-oriented HO-2 is crucial for the inhibitory activities. The substitution of an isopropyl moiety at C-13 in 38 and 39 (vs 40) was also important for the inhibitory activities. In conclusion, abietane and abeo-abietane diterpenoids can partly account for the therapeutic effects of C. trichotomum against inflammatory diseases. The molecular mechanism of these diterpenoids are worthy of further studies.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on a Büchi B-540 apparatus. Optical rotations were recorded using an Autopol VI polarimeter. UV and ECD spectra were acquired on a TU-1901 spectrometer (Beijing Purkinje General Instrument Co., Ltd., China) and a Brighttime Chirascan spectrometer (Applied Photophysics Ltd., UK), respectively. The IR spectra were recorded on a PerkinElmer FT-IR spectrometer. Bruker AVANCE-III instruments (400, 500, and 600 MHz) were utilized to measure NMR spectra, with residual solvent signals as a reference. HRESIMS data 1514

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(240), −2.07 (269), −3.07 (284), +2.80 (346); IR νmax 2922, 1627, 1455, 1385, 1323, 1212, 1011, 976, 837, 627 cm−1; 1H and 13C NMR data (methanol-d4), see Tables 3 and 4; (+)-HRESIMS m/z 379.1156 [M + Na]+ (calcd for C20H20O6Na, 379.1158). Trichotomin B (10): light yellow, amorphous solid; [α]20 D +5 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 206 (4.02), 286 (3.90), 358 (3.30) nm; ECD (MeOH, c 0.51 mM, 1 mm) Δε (λmax, nm) +4.45 (210), −5.62 (225), +0.51 (238), −2.57 (260), −0.40 (299), +1.92 (358); IR νmax 2923, 1750, 1632, 1454, 1378, 1323, 1205, 1115, 1021, 977, 604 cm−1; 1H and 13C NMR data (CDCl3), see Tables 3 and 4; (−)-HRESIMS m/z 353.1028 [M − H]− (calcd for C20H17O6, 353.1025). Trichotomside A (11): white, amorphous powder; [α]20 D +177 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 226 (4.34), 270 (3.76), 316 (3.37) nm; IR νmax 3332, 1601, 1331, 1233, 1057, 1033, 889, 611 cm−1; 1H and 13C NMR data (methanol-d4), see Table 5; (−)-HRESIMS m/z 489.2111 [M − H]− (calcd for C26H33O9, 489.2125); (+)-HRESIMS m/z m/z 513.2099 [M + Na]+ (calcd for C26H34O9Na, 513.2101). Trichotomside B (12): white gum; [α]20 D +13 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 208 (4.31), 274 (2.81) nm; IR νmax 3354, 2937, 1331, 1017, 613 cm−1; 1H and 13C NMR data (DMSO-d6), see Table 5; (−)-HRESIMS m/z 641.3182 [M − H]− (calcd for C32H49O13, 641.3173); (+)-HRESIMS m/z 665.3157 [M + Na]+ (calcd for C32H50O13Na, 665.3149). X-ray Crystal Structure Analysis of Compounds 1−3. Crystals of compounds 1, 2, and 3 were obtained in CHCl3, CHCl3−MeOH (10:1, v/v), and MeOH, respectively. The crystallographic data of 1−3 were acquired with Cu Kα radiation on a Bruker APEX2 CCD diffractometer. The programs SHELXS-97 and SHELXL-97 were used to resolve structures through direct methods. The crystallographic data of 1−3 have been deposited at Cambridge Crystallographic Data Centre (CCDC) with deposition numbers 1571267 (1), 1571271 (2), and 1571272 (3), respectively (Tables S2−S4, Supporting Information). ECD Calculation of Compound 4. Gaussian 09 was utilized for the calculation of 4.23 SYBYL 2.2.1 was used for conformational analysis. Conformers were optimized using the TDDFT method at the B3LYP/6-31G(d,p) level of theory with a PCM solvent model in MeOH, followed by TDDFT calculations using two levels [WB97XD/ 6-311+G(d,p) and CAM-B3LYP/6-311+G(d,p)]. The ECD spectra of four conformers were obtained using SpecDis 1.70.124 and compared with the experimental data. Acid Hydrolysis of Compounds 11 and 12. A solution of 11 or 12 (1.0 mg each) in 3 M trifluoroacetic acid (3 mL) was hydrolyzed at 120 °C for 2 h. After cooling, each solution was concentrated under vacuum, dissolved with water, and extracted twice with CHCl3. The aqueous parts were treated via a similar method described previously.25 The D configurations of the β-glucopyranosyl moiety in 11 and 12 were defined through comparison with the peaks and retention times (tR) of authentic sugar samples (tR-D-glucose = 55.45 min, and tR-Lglucose = 55.60 min). The CHCl3 extracts afforded 11a and 12a as aglycones, which were identified as C20H24O4 and C20H30O3, respectively, in accordance with HRESIMS ions (11a: [M − H]−, found m/z 327.1578 (calcd for C20H23O4, 327.1596); 12a: [M + Na]+, found m/z 341.2096 (calcd for C20H30O3Na, 341.2093)). Cell Culture and Inhibition of NO Production. RAW 246.7 cells were cultured as previously described.26 Cells were seeded in 96well plates at the density of 50 000 cells/well for 24 h, pretreated with the tested compounds for 30 min at 37 °C, and co-incubated with LPS (100 ng/mL) for 24 h. NO production was analyzed through Griess reaction.26 Briefly, cell culture supernatant (50 μL) and Griess reagent (50 μL) were mixed for 10 min, and absorptions were then monitored at 540 nm using a plate reader. All experiments were performed in triplicate. The IC50 values were calculated using SPSS 20 software. All the tested compounds were prepared as stock solutions with a concentration of 10 mM in DMSO (final DMSO concentration should not exceed 0.1% in each well). Aminoguanidine hydrochloride (IC50 = 26.2 μM) was used as the positive control.

compound 12 (10.2 mg). Fr. H (5.9 g) was also separated through MPLC under similar conditions to give Fr. H1−Fr. H6. Compounds 11 (6.2 mg) and 42 (12.5 mg) were separately purified from Fr. H5 (373.2 mg) and Fr. H3 (599 mg) on a silica gel column eluted with EtOAc−MeOH−H2O (30:1:0 to 10:2:1, v/v/v) and CH2Cl2−MeOH (30:1 to 9:1, v/v), respectively. Refer to Figures S2 and S3, Supporting Information, for the detailed isolation scheme. 15,16-Dehydroteuvincenone G (1): yellow, needle-like crystals (CHCl3); mp 246.3−248.0 °C; [α]20 D +63 (c 0.02, CHCl3); UV (CHCl3) λmax (log ε) 242 (4.00), 262 (3.96), 364 (3.28) nm; ECD (CHCl3, c 0.29 mM, 1 mm) Δε (λmax, nm) +1.27 (244), −0.69 (294), +1.05 (360); IR νmax 2974, 1708, 1613, 1455, 1373, 1281, 1176, 1073, 966, 931, 888, 819, 672, 602 cm−1; 1H and 13C NMR data (CDCl3), see Tables 1 and 2; (−)-HRESIMS m/z 341.1407 [M − H]− (calcd for C20H21O5, 341.1389). 3-Dihydroteuvincenone G (2): colorless, needle-like crystals (CHCl3−MeOH, 10:1, v/v); mp 250.2−251.8 °C; [α]20 D +51 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 207 (4.27), 298 (4.05), 353 (3.79) nm; ECD (MeOH, c 0.29 mM, 1 mm) Δε (λmax, nm) +3.24 (223), −1.22 (248), −4.38 (294), +3.11 (317); IR νmax 2925, 1608, 1454, 1349, 1238, 1015, 957, 881, 792, 651 cm−1; 1H and 13C NMR data (DMSO-d6), see Tables 1 and 2; (+)-HRESIMS m/z 347.1861 [M + H]+ (calcd for C20H27O5, 347.1858). 17-Hydroxymandarone B (3): colorless, needle-like crystals (MeOH); mp 237.6−239.0 °C; [α]20 D −34 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 208 (4.33), 255 (3.99), 318 (3.71), 377 (3.64) nm; IR νmax 2926, 1634, 1470, 1398, 1326, 1225, 1109, 975, 894, 814, 636 cm−1; 1H and 13C NMR data (methanol-d4), see Tables 1 and 2; (−)-HRESIMS m/z 343.1538 [M − H]− (calcd for C20H23O5, 343.1545). Trichotomin A (4): yellowish, amorphous powder; [α]20 D −34 (c 0.03, CHCl3); UV (MeOH) λmax (log ε) 204 (3.92), 306 (3.93) nm; ECD (MeOH, c 2.96 mM, 0.5 mm) Δε (λmax, nm) −10.6 (207), +6.7 (252), +10.7 (300), −16.1 (348); IR νmax 2916, 2851, 1683, 1543, 1378, 1261, 1033, 801, 754, 626 cm−1; 1H and 13C NMR data (CDCl3), see Tables 1 and 2; (+)-HRESIMS m/z 361.1065 [M + Na]+ (calcd for C20H18O5Na, 361.1052). 15,16-Dihydroformidiol (5): red, amorphous powder; [α]20 D −49 (c 0.1, CHCl3); UV (CHCl3) λmax (log ε) 241 (4.07), 284 (4.21) nm; ECD (CHCl3, c 0.27 mM, 1 mm) Δε (λmax, nm) −1.43 (253 nm), −1.59 (287 nm), +1.53 (326 nm); IR νmax 1722, 1645, 1456, 1392, 1212, 1125, 1054, 976, 886, 807, 630, 614 cm−1; 1H and 13C NMR data (CDCl3), see Tables 1 and 2; (−)-HRESIMS m/z 369.1335 [M − H]− (calcd for C21H21O6, 369.1338). 18-Hydroxyteuvincenone E (6): red, amorphous solid; [α]20 D +100 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 206 (4.25), 294 (4.15) nm; ECD (MeOH, c 0.34 mM, 1 mm) Δε (λmax, nm) +10.18 (222), −6.15 (237), +1.38 (251), −12.54 (270), +5.69 (356); IR νmax 2921, 1652, 1384, 1217, 1033, 627, 611 cm−1; 1H and 13C NMR data (methanol-d4), see Tables 3 and 4; (+)-HRESIMS m/z 379.1163 [M + Na]+ (calcd for C20H20O6Na, 379.1158). 2α-Hydrocaryopincaolide F (7): red, amorphous powder; [α]20 D −130 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.45), 286 (4.23), 390 (3.80) nm; ECD (MeOH, c 0.29 mM, 1 mm) Δε (λmax, nm) −8.28 (218), +4.21 (244), −4.97 (299), +2.34 (347); IR νmax 3297, 1622, 1557, 1468, 1391, 1319, 1279, 1235, 1215, 1014, 879, 818, 629 cm−1; 1H and 13C NMR data (methanol-d4), see Tables 3 and 4; (−)-HRESIMS m/z 341.1371 [M − H]− (calcd for C20H21O5, 341.1389). 15α-Hydroxyuncinatone (8): yellow, amorphous solid; [α]20 D −119 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 210 (4.25), 282 (3.98), 386 (3.62) nm; ECD (MeOH, c 0.38 mM, 1 mm) Δε (λmax, nm) −27.94 (209), +4.59 (231), +8.08 (249), −0.92 (296), +2.75 (322); IR νmax 2923, 1621, 1393, 1279, 1225, 1123, 1033, 967, 875, 811, 602 cm−1; 1H and 13C NMR data (CDCl3), see Tables 3 and 4; (+)-HRESIMS m/z 343.1545 [M + H]+ (calcd for C20H23O5, 343.1545). 15α-Hydroxyteuvincenone E (9): red, amorphous solid; [α]20 D +90 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.23), 293 (4.12) nm; ECD (MeOH, c 0.28 mM, 1 mm) Δε (λmax, nm) +6.24 (220), −7.57 1515

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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.7b00814. UV, ECD, IR, HRESIMS, and 1D and 2D NMR spectra of compounds 1−12 and the names and structures of compounds 13−43 (PDF) Crystallographic data for 1 (CIF) Crystallographic data for 2 (CIF) Crystallographic data for 3 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-21-51322507. Fax: +86-21-51322505. E-mail: [email protected] (Z.-T. Wang). *Tel: +86-21-51322506. Fax: +86-21-51322505. E-mail: [email protected] and [email protected] (L. Yang). ORCID

Li Yang: 0000-0002-5484-0895 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 81530096, 81573581, and 81603156), as well as Young Eastern Scholar Program (No. QD2016038) and Chenguang Program (No. 16CG49) supported by Shanghai Municipal Education Commission. We thank for Prof. G.-Q. Lin of Shanghai Institute of Organic Chemistry for his guidance and assistance in ECD calculation.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00814 J. Nat. Prod. 2018, 81, 1508−1516