Bioactive ent-Pimarane and ent-Abietane Diterpenoids from the Whole

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Bioactive ent-Pimarane and ent-Abietane Diterpenoids from the Whole Plants of Chloranthus henryi Chunfeng Xie,*,†,‡ Lingmei Sun,§ Kai Liao,⊥ Sheng Liu,† Meicheng Wang,† Jing Xu,† Mark Bartlam,∥ and Yuanqiang Guo*,† †

State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, and ∥State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, Tianjin 300071, People’s Republic of China ‡ 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 § Department of Pharmacology and ⊥Department of Pathology and Pathophysiology, Medical School of Southeast University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Two new ent-pimarane (1 and 2), eight new ent-abietane (3− 10) diterpenoids, and eight known analogues (11−18) were isolated from the whole plants of Chloranthus henryi. The absolute configuration of 1 was determined on the basis of single-crystal X-ray diffraction data. Compound 8 represents a class of rare naturally occurring C-14 norabietanes, and compounds 9 and 10 feature rare 13,14-seco-abietane skeletons. Compounds 5, 12, 13, and 15 inhibited the yeast-to-hyphae transition of Candida albicans with IC50 values between 97.3 and 738.7 μM.

T

Compound 1 was obtained as colorless needles (MeOH). Its molecular formula was deduced as C20H32O2 from the HRESIMS (m/z 305.2475 [M + H]+, calcd for C20H33O2, 305.2475) and 13C NMR data. The 1H NMR data (Table 1) showed the presence of an olefinic proton (δH 5.57, br s), a vinyl group [δH 5.73 (dd, J = 17.5, 10.4 Hz), 4.97 (d, J = 10.4 Hz), and 4.90 (d, J = 17.5 Hz)], two oxymethine protons [δH 3.27 (dd, J = 11.7, 3.4 Hz) and 4.00 (dd, J = 12.0, 5.6 Hz)], and four tertiary methyl singlets (δH 1.06, 1.04, 0.83, and 0.74). The 13 C NMR data (Table 3) of 1 showed 20 carbon resonances for four methyl groups, five sp3 methylenes, four sp3 methines (two oxygenated at δC 78.9 and 72.6), two double bonds, and three quaternary sp3 carbons, which was supported by DEPT and HMQC spectra. On the basis of these spectroscopic data, compound 1 possessed a pimarane skeleton, which was confirmed by 1H−1H COSY and HMBC experiments (Figure 2). The HMBC cross-peaks of H-15 to C-14 and C-17 and H16 to C-13 showed that a vinyl group was attached to C-13. The Δ8(14) double bond was inferred from the HMBC correlations of H-14 to C-7 and C-9 and H-7 to C-8 and C14. The HMBC correlations of the gem-dimethyl protons and H-1 with C-3 (δC 78.9) indicated that a hydroxy group was attached to C-3, while the correlations of H-7/C-6 and C-14

he genus Chloranthus, belonging to the family Chloranthaceae, comprises 15 species that are distributed widely in eastern and southern Asia.1 The Chloranthus plants produced structurally diverse sesquiterpenoids as well as entkaurane, ent-abietane, and labdane-type diterpenoids, which display a wide array of bioactivities.2,3 Chloranthus henryi, a prerennial herb, has been used in China to promote blood circulation, subdue swelling, and relieve pain.4 Phytochemical investigations of C. henryi indicated the presence of various sesquiterpenoids and labdane-type diterpenoids, which showed antifungal,5 cytotoxic,6−9 hepatoprotective,6 antineuroinflammatory,10 and tyrosinase inhibitory effects.11 As part of the research on bioactive components from medicinal plants or folk medicines,12−14 the chemical constituents of the whole plants of C. henryi have been investigated. Two new ent-pimarane diterpenoids (1 and 2), eight new ent-abietane diterpenoids (3−10), and eight known analogues (11−18) were isolated from C. henryi. Herein, the structural determination and Candida albicans morphogenesis inhibitory activities of these isolates are described.

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RESULTS AND DISCUSSION

The EtOAc-soluble layer from the methanol extract of the whole plants of C. henryi yielded 10 new (1−10) and eight known (11−18) diterpenoids (Figure 1). © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 2, 2015

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DOI: 10.1021/acs.jnatprod.5b00781 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of compounds 1−18.

Table 1. 1H NMR Data (400 MHz, CDCl3, δ in ppm, J in Hz) for Compounds 1−5 position

1

1α 1β 2α 2β 3 5 6α 6β 7 9 11α 11β 12

1.65 m 1.12 m 1.67 m 1.54 m 3.27 dd (11.7, 3.4) 1.06 m 1.40 m 2.03 m 4.00 dd (12.0, 5.6) 1.62 m 1.57 m 1.35 m α1.58 m β 1.21 m 5.57 br s 5.73 dd (17.5, 10.4) 4.97 d (10.4) 4.90 d (17.5) 1.06 s 1.04 s 0.83 s 0.74 s

14 15 16 17 18 19 20 OMe

2 1.78 1.26 1.65 1.73 3.31 1.51 2.39 2.59

m m m m dd dd dd dd

(11.4, (13.5, (18.8, (18.8,

3

4.0) 5.0) 13.5) 5.2)

2.01 m 1.30 m 1.60 m α1.67 m β 1.33 m 6.65 br s 5.69 dd (17.5, 10.4) 5.01 d (10.4) 4.76 d (17.5) 1.13 s 0.99 s 0.87 s 0.82 s

1.85 1.28 1.63 1.73 3.32 1.56 2.42 2.62

m m m m dd dd dd dd

(12.1, 3.9) (13.6, 5.3) (19.1,13.3) (19.1, 5.2)

1.87 m 1.41 m 1.77 m α 1.89 m β 1.60 m 6.98 br s

4 1.77 1.26 1.64 1.71 3.31 1.54 2.39 2.61

m m m m dd dd dd dd

2.07 1.43 1.77 1.82

m m m m

(11.5, (13.6, (18.8, (18.8,

5

3.5) 4.7) 13.6) 4.8)

1.78 1.28 1.63 1.75 3.31 1.61 2.44 2.66

m m m m m dd (13.4, 5.1) dd (18.6, 13.8) dd (18.6, 4.9)

2.33 1.79 1.86 3.97

m m m t (2.0)

1.19 s

6.82 br s 1.83 m 0.95 d (6.9)

6.80 br s 2.07 sept (6.8) 1.10 d (6.8)

1.10 1.01 0.90 0.87 3.29

0.91 0.99 0.88 0.82 3.12

1.04 0.99 0.89 0.88 3.18

s s s s s

d (6.9) s s s s

d (6.8) s s s s

NOE relationships between H3-20/H3-19 and H-1α revealed that Me-20 and Me-19 were both α-oriented. The large values observed for JH2α, 3 (11.7 Hz) and JH6α, 7 (12.0 Hz) further indicated that H-3 and H-7 were both axially β-oriented.15,16 The absolute configuration of 1 was determined by X-ray crystallographic analysis with Cu Kα radiation (Figure 4). On the basis of the Flack parameter [−0.03(5)], the absolute

implied that the other hydroxy group was attached to C-7. Thus, the 2D structure for 1 was established. The relative configuration of compound 1 was determined by NOESY data and the splitting patterns of related protons. The NOE correlations (Figure 3) of H-3/H-1β, H-2β, H-5, and H318, H-5/H-7 and H-9, H3-17/H-9, H-11β, and H-12β, and H318/H-6β implied that the protons were all β-oriented, while the B

DOI: 10.1021/acs.jnatprod.5b00781 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 1H NMR Data (400 MHz, CDCl3, δ in ppm, J in Hz) for Compounds 6−10 position 1α 1β 2 3 5 6α 6β 7α 7β 8 9 11 12 14 15 16 17 18 19 20 OMe

6 1.86 m 1.28 m α 1.62 m β 1.73 m 3.32 dd (11.4, 1.54 dd (13.3, 2.39 dd (18.9, 2.56 dd (18.9,

7

4.3) 5.4) 13.5) 5.4)

2.11 m α 1.25 m β 1.87 m α 1.98 m β 2.67 m 7.70 d (2.1) 1.95 1.82 0.99 0.89 0.81

8

2.32 m 1.55 m 1.83 m 3.31 1.34 1.77 1.93 2.98 2.88

dd (11.2, 5.1) dd (12.3, 2.2) m m br d (17.1, 5.9) m

9

1.93 m 1.16 m α 1.65 m β 1.55 m 3.25 dd (11.3, 4.5) 1.24 dd (11.3, 5.1) 1.92 m 2.06 m 5.70 m 5.36 dd (9.9, 1.5) 1.60 m a 1.10 m b 1.84 m a 2.61 m b 2.39 m

7.19 d (8.3) 7.14 dd (8.3, 1.5) 7.05 br s

s s s s s

1.49 1.49 1.08 0.90 1.19 3.06

2.58 1.09 1.09 0.98 0.87 0.76

s s s s s s

sept (6.8) d (6.9) d (6.9) s s s

10

1.99 m 1.16 m α 1.66 m β 1.55 m 3.26 dd (11.5, 4.4) 1.20 dd (12.5, 5.5) 2.37 m

1.93 m 1.17 m α 1.64 m β 1.57 m 3.27 dd (10.7, 3.2) 1.20 m 2.20 m

6.83 m

6.92 br s

1.92 m a 1.87 m b 1.57 m a 3.10 ddd (16.6, 10.3, 4.8) b 2.43 ddd (22.1, 10.5, 4.9) 9.38 s 2.63 sept (7.0) 1.10 d (7.0) 1.10 d (7.0) 1.01 s 0.91 s 0.80 s

2.03 br s a 1.93 m b 1.47 m a 2.89 m b 2.42 m 2.58 1.07 1.07 0.99 0.88 0.83

sept (6.8) d (6.8) d (6.8) s s s

Table 3. 13C NMR Data (100 MHz, CDCl3, δ in ppm) for Compounds 1−10 position

1

2

3

4

5

6

7

8

9

10

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

36.8 27.5 78.9 38.3 51.2 32.1 72.6 139.9 49.3 38.8 19.1 35.3 38.2 125.2 146.8 113.1 29.5 28.4 15.8 14.8

36.6 27.3 78.6 38.9 49.7 37.0 200.5 136.3 50.8 36.2 18.8 34.6 39.3 143.5 145.5 114.1 28.1 27.4 14.6 13.8

37.0 27.2 78.5 38.9 49.0 36.7 198.9 142.0 50.5 35.7 20.4 29.0 78.5 137.3 76.0 24.4 24.1 27.4 14.8 14.2 53.9

36.4 27.2 78.5 38.9 49.6 37.0 199.5 139.5 50.4 36.0 21.2 25.8 77.5 141.4 36.4 16.8 17.6 27.4 14.7 13.9 50.4

36.2 27.2 78.5 39.0 50.1 37.3 200.0 140.6 45.2 36.1 25.9 70.0 75.7 134.8 32.1 16.4 17.5 27.4 14.6 13.8 51.9

37.0 27.3 78.7 38.9 49.2 36.5 200.0 132.7 50.8 35.6 22.6 26.3 128.3 134.0 139.5 20.6 21.6 27.5 14.7 14.0

36.9 28.0 78.8 39.0 49.8 18.9 30.9 134.6 147.9 37.4 124.3 123.3 142.7 126.2 76.5 27.9 27.8 28.1 15.4 24.9 50.6

37.1 27.4 79.1 38.7 49.4 23.6 126.7 127.8 50.5 35.6 22.3 38.7 214.8

36.7 27.1 78.7 38.6 48.9 25.0 152.9 144.1 49.8 36.7 20.9 42.4 215.3 194.7 40.7 18.2 18.3 27.9 15.1 13.8

37.3 27.0 78.9 38.5 48.8 23.9 140.3 134.0 50.0 36.8 21.8 42.0 215.3 173.1 40.7 18.3 18.3 27.9 15.2 14.1

40.9 18.2 18.3 27.9 15.1 13.4

Figure 2. 1H−1H COSY and selected HMBC correlations of compounds 1, 3, 7, and 8.

C

DOI: 10.1021/acs.jnatprod.5b00781 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Selected NOESY correlations of compounds 1, 3, 7, and 8.

Figure 5. ORTEP drawing of 18.

hydroxylated. Compounds 3 and 18 possessed the same stereostructure based on the NOESY analysis (Figure 3). The structure of compound 3 was therefore elucidated as 3α,15dihydroxy-13α-methoxy-ent-abieta-8(14)-en-7-one and named 15-hydroxysessilifol F. The molecular formula of compound 4 was deduced to be C21H34O3 according to HRESIMS (m/z 357.2403 [M + Na]+, calcd for C21H34NaO3 357.2400) and 13C NMR data. Analysis of NMR data suggested that the structure of 4 resembled that of compound 17,3 but for the presence of a C-13 methoxy group (δH 3.12, δC 50.4) replacing the hydroxy group in 17. This deduction was confirmed by the HMBC spectrum, in which the key correlation from the methoxy protons to C-13 at δC 77.5 was observed. The relative configuration of 4 was the same as that of 17 based on similar NOE relationships (Figure S28, Supporting Information). The absolute configuration of 4 was determined through the comparison of the experimental ECD spectra of 4 and 17 (Figure S73, Supporting Information), in which compound 4 showed Cotton effects at 210 (negative), 256 (positive), and 334 (positive) nm, similar to those of 17. Thus, the structure of compound 4 was elucidated as (3R,5S,9S,10R,13S)-3-hydroxy-13-methoxy-entabieta-8(14)-en-7-one and named 13-O-methylsessilifol D. Compound 5 possessed a molecular formula of C21H34O4 on the basis of HRESIMS (m/z 373.2352 [M + Na]+, calcd for C21H34NaO4 373.2349) and 13C NMR data. The NMR spectra of 5 showed similarities to those of 4, except for an additional secondary hydroxy group (δH 3.97, δC 70.0). The hydroxy group at C-12 was assigned based on the HMBC correlations of H-12/C-9, C-13, and C-14. The NOE interactions between H12/H-16 and H-17 as well as the small coupling constant (J = 2.0 Hz) between H-11 and H-12 indicated the axially βoriented hydroxy group at C-12. Accordingly, the structure of 5 was defined as 3α,12β-dihydroxy-13β-methoxy-ent-abieta8(14)-en-7-one and named chloranhenryin B. Compound 6 was assigned the molecular formula C20H30O2, as determined from HRESIMS (m/z 303.2320 [M + H]+, calcd for C20H31O2 303.2319) and 13C NMR data. The similarity of NMR data of 6 with those of 4 indicated that compound 6 was an analogue of 4. The key differences were the absence of the C-13 methoxy group and the presence of a Δ13(15) double bond (δC 128.3 and 139.5) in 6. The 13C NMR data (Table 3) of 6 showed the signals for C-13 and C-15 deshielded from δC 77.5 and 36.4 in 4 to δC 128.3 and 139.5 in 6, suggesting the Δ13(15)

Figure 4. ORTEP drawing of 1.

configuration of compound 1 was unequivocally assigned as (3R,5S,7R,9S,10R,13R). Therefore, the structure of compound 1 was assigned as (3R,5S,7R,9S,10R,13R)-3,7-dihydroxy-entpimara-8(14),15-diene and named chloranhenryin A. Compound 1 is a stereoisomer of oryzalexin D17 and possesses the opposite configurations at C-7 and C-13. Compound 2 possessed a molecular formula of C20H30O2 on the basis of HRESIMS (m/z 325.2142 [M + Na]+, calcd for C20H30NaO2 325.2138) and 13C NMR data. The 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 3) were similar to those of 1, except for a C-7 ketocarbonyl (δC 200.5) group in 2 instead of an oxymethine (δH 4.00, δC 72.6) group of 1. This assignment was further confirmed by the HMBC correlations from H-14 and H-6 to C-7. The relative configuration of 2 was established as identical to that of 1 by NOESY data (Figure S14, Supporting Information) and by comparing their NMR data. Accordingly, the structure of 2 was defined as 3α-hydroxyent-pimara-8(14),15-dien-7-one and named 13-epioryzalexin A. Compound 2 is a C-13 epimer of oryzalexin A,18−20 a phytoalexin from rice blast leaves. Compound 3 was obtained as a yellow gum. Its molecular formula was determined as C21H34O4 by HRESIMS (m/z 373.2353 [M + Na]+, calcd for C21H34NaO4 373.2349) and 13C NMR data. The 1H NMR data (Table 1) showed six methyl singlets (δH 0.87, 0.90, 1.01, 1.10, 1.19, and 3.29), an oxygenated methine proton [(δH 3.32 (dd, J = 12.1, 3.9 Hz)], and an olefinic proton (δH 6.98, br s). The 13C NMR data (Table 3) of 1 showed 21 carbon resonances, including a methoxy group (δH 3.29, δC 53.9). The additional 20 resonances comprised five methyls, five methylenes, four methines (including one oxygenated and one olefinic methine), two quaternary sp3 carbons, one quaternary sp2 carbon, a carbonyl, and two oxygenated tertiary carbons supported by DEPT and HMQC spectra. The NMR spectra of 3 were similar to those of compound 18, and the structure of the latter was confirmed by X-ray crystallographic analysis with Cu Kα radiation (Figure 5). Compared with 18,3 an extra tertiary (δC 76.0) hydroxy group was present in 3. The key HMBC correlations from Me-16 (δH 1.19) and Me-17 (δH 1.10) to the oxygenated tertiary carbon at δC 76.0 implied that C-15 was D

DOI: 10.1021/acs.jnatprod.5b00781 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. Plausible Biosynthetic Pathways for Compounds 1−10

methines), two quaternary carbons, and a carbonyl with the aid of DEPT and HMQC spectra. Apart from a ketocarbonyl group and one double bond, compound 8 possessed a bicyclic ring system after deduction of four indices of hydrogen deficiency. The 1H and 13C NMR data of 8 resembled those of 14,3 a bicyclic 14-norabietane diterpenoid, except for the absence of a ketocarbonyl group and the presence of a double bond. The shielding of the C-7 and deshielding of C-8 suggested a Δ7(8) double bond, which was verified by HMBC correlations between H-8/C-6 and C-11 and between H-11/C8. The relative configuration of 8 was determined by the NOE correlations between H-3/H-5, Me-18/H-3 and H-5, H-5/H-9, and Me-19/Me-20 (Figure 3). The structure of compound 8 was therefore elucidated as 13,14-seco-3α-hydroxy-13-oxo-ent14-norabieta-7-ene and named chloranhenryin D. Compounds 9 and 10 were assigned the molecular formulas of C20H32O3 and C20H32O4 on the basis of their HRESIMS m/z 343.2250 [M + Na]+ (calcd for C20H32NaO3 343.2244) and m/ z 337.2378 [M + H]+ (calcd for C20H33O4 337.2373), respectively, and 13C NMR data. Comparison of their NMR spectra with those of 8 showed that they were similar expect for the formyl group (δH 9.38, s; δC 194.7) in 9 or the hydroxycarbonyl group (δC 173.1) in 10. The key HMBC correlation of the formyl proton with C-8 in 9 or H-7 with the carbon signal of the hydroxycarbonyl group in 10 indicated that the formyl and hydroxycarbonyl group were, respectively, attached at C-8 of each compound. The relative configurations of 9 and 10 were the same as that of 8, as determined by NOESY data (Figures S64 and S71, Supporting Information), comparable chemical shifts, and coupling constants. Accordingly, the structures of 9 and 10 were established as 13,14-seco-

location of the second double bond. It was confirmed by the HMBC cross-peaks of H-16, H-17/C-13, and H-14/C-15. The biosynthetic reasoning and similar ECD spectra (Figure S44, Supporting Information) implied that compounds 6 and 4 shared the same configuration. The structure of compound 6 was therefore elucidated as (3R,5S,9S,10R)-3-hydroxy-entabieta-8(14),13(15)-dien-7-one and named chloranhenryin C. Compound 7 displayed a sodium adduct ion [M + Na]+ at 339.2298 in the HRESIMS (calcd for C21H32NaO2 339.2295), consistent with the molecular formula of C21H32O2. The 13C and 1H NMR spectroscopic data of 7 resembled those of sessilifol J.3 The only difference was a methoxy group in 7, instead of a hydroxy group in sessilifol J. The HMBC correlation of the methoxy protons (δH 3.06) with C-15 (δC 76.5) confirmed that the methoxy group was located at C-15 (Figure 2). The structure of compound 7 was therefore determined as 3α-hydroxy-15-methoxy-ent-abieta-8,11,13-triene and named 15-O-methylsessilifol J. Compound 7 is stereoisomeric at C-3, C-5, and C-10 with isolophanthin B isolated from Isodon lophanthoides var. gerardianus.21 Compound 8 was obtained as a colorless oil with the molecular formula C19H32O2, as suggested by the HRESIMS (m/z 315.2298 [M + Na]+, calcd for C19H32NaO2 315.2295) and 13C NMR data. The 1H NMR data (Table 2) of 8 exhibited three methyl singlets (δH 0.76, 0.87, and 0.98), an isopropyl group (δH 1.09, 6H, d, J = 6.9 Hz; δH 2.58, 1H, sept, J = 6.8 Hz), an oxygenated methine proton (δH 3.25, dd, J = 11.3, 4.5 Hz), and two olefinic protons [δH 5.70, 5.36 dd (J = 9.9, 1.5 Hz)]. The 13C NMR data (Table 3) of 8 showed 19 carbon resonances, containing five methyls, five methylenes, six methines (including one oxygenated and two olefinic E

DOI: 10.1021/acs.jnatprod.5b00781 J. Nat. Prod. XXXX, XXX, XXX−XXX

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12.2 mg) and 11 (tR = 43 min, 12.6 mg). Fraction F5 (8 g) afforded seven subfractions, F5‑1−F5‑7, by MPLC (70−88% MeOH in H2O). The further purification of F5‑5 yielded compounds 3 (tR = 16 min, 26.4 mg), 5 (tR = 18 min, 25.5 mg), 13 (tR = 31 min, 53.3 mg), and 2 (tR = 33 min, 6.1 mg) through the above HPLC system (85% MeOH in H2O). Fraction F6 (9 g) was chromatographed by MPLC (68−86% MeOH in H2O) to afford six subfractions, F6‑1−F6‑6. The further purification of F6‑4 gave compounds 9 (tR = 19 min, 12.0 mg) and 7 (tR = 36 min, 5.1 mg) by HPLC (85% MeOH in H2O), while F6‑5 gave compound 1 (tR = 24 min, 25.2 mg) purified by HPLC (85% MeOH in H2O). Through a similar isolation method, fraction F7 (10 g) yielded nine subfractions, F7‑1−F7‑9. Compound 14 (tR = 23 min, 35.7 mg) was isolated from F7‑1 (85% MeOH in H2O), compound 15 (tR = 19 min, 48.5 mg) was obtained from F7‑4 (90% MeOH in H2O), while 12 (tR = 24 min, 26.2 mg) was isolated from F7‑8 (90% MeOH in H2O). Compounds 10 (tR = 24 min, 51.7 mg), 16 (tR = 25 min, 65.2 mg), 17 (tR = 27 min, 42.1 mg), 18 (tR = 54 min, 48.5 mg), 4 (tR = 60 min, 19.1 mg), and 6 (tR = 77 min, 8.3 mg) were all isolated from F8‑2 (75% MeOH in H2O), which was obtained from F8 (14 g) by the above MPLC. Chloranhenryin A (1): colorless needles (MeOH); [α]27 D −133 (c 0.2, CH2Cl2); IR (KBr) νmax 3432, 2955, 2927, 2868, 2853, 1718, 1637, 1617, 1458, 1406, 1384, 1264, 1213, 1274, 1184, 11547, 1090, 1026, 997, 943, 923, 869, 736, 684, 629 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 305 [M + H]+; HRESIMS m/z 305.2475 [M + H]+ (calcd for C20H33O2, 305.2475). X-ray crystal data of chloranhenryin A (1): C21H36O3, M = 336.50, monoclinic, space group P2(1), a = 6.7178(11) Å, b = 17.015(2) Å, c = 34.500(6) Å, α = 90°, β = 90.064(6)°, δ = 90°, V = 3943.5(10) Å3, T = 173(2) K, Z = 8, μ(Cu Kα) = 0.573 mm−1, Dcalc = 1.134 g/cm3, F(000) = 1488, crystal size = 0.300 × 0.200 × 0.100 mm. The total number of reflections collected was 33 019, of which 15 064 were unique (R(int) = 0.0249). Final R1 = 0.0373, wR2 = 0.0972 (I > 2σ(I)), Flack parameter = −0.03(5). The deposition of X-ray crystallographic data of 1 has been performed at the Cambridge Crystallographic Data Centre (CCDC 1430575). 13-Epioryzalexin A (2): colorless, amorphous powder; [α]27 D −133 (c 0.1, CH2Cl2); IR (KBr) νmax 3431, 2959, 2929, 2869, 2854, 1686, 1635, 1611, 1457, 1406, 1385, 1371, 1249, 1210, 1111, 1091, 1061, 1035, 1009, 995, 974, 914, 871, 628, 550 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 325 [M + Na]+; HRESIMS m/z 325.2142 [M + Na]+ (calcd for C20H30NaO2, 325.2138). 15-Hydroxysessilifol F (3): yellow gum; [α]27 D −39 (c 0.1, CH2Cl2); IR (KBr) νmax 3442, 2956, 2921, 2850, 1735, 1718, 1685, 1618, 1458, 1375, 1308, 1256, 1215, 1183, 1088, 1025, 953, 907 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 373 [M + Na]+; HRESIMS m/z 373.2353 [M + Na]+ (calcd for C21H34NaO4, 373.2349). 13-O-Methylsessilifol D (4): colorless, amorphous powder; [α]27 D −42 (c 0.1, CH2Cl2); ECD (CH3CN) λmax (Δε) 210 (−5.14), 256 (+4.10), 334 (+0.49); IR (KBr) νmax 3423, 2958, 2919, 2850, 1734, 1687, 1618, 1466, 1373, 1310, 1252, 1213, 1199, 1077, 1029, 959, 894, 634 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 357 [M + Na]+; HRESIMS m/z 357.2403 [M + Na] + (calcd for C21H34NaO3 357.2400). Chloranhenryin B (5): yellow oil; [α]27 D +23 (c 0.1, CH2Cl2); IR (KBr) νmax 3442, 2958, 2921, 2851, 1636, 1457, 1386, 1260, 1216, 1199, 1086, 1034, 906, 697 cm−1; 1H NMR (400 MHz, CDCl3) and 13 C NMR (100 MHz, CDCl3) data, see Tables 1 and 3; ESIMS m/z 373 [M + Na]+; HRESIMS m/z 373.2352 [M + Na]+ (calcd for C21H34NaO4 373.2349). Chloranhenryin C (6): yellow oil; [α]27 D +16 (c 0.1, CH2Cl2); ECD (CH3CN) λmax (Δε) 206 (−0.90), 290 (+0.21), 364 (+0.07); IR (KBr) νmax 3442, 2956, 2925, 2853, 1718, 1676, 1649, 1618, 1459, 1378, 1266, 1216, 1183, 1093, 1032, 962, 734 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 2

3α-hydroxy-13-oxo-ent-abieta-7-en-14-al (chloranhenryin E) and 13,14-seco-3α-hydroxy-13-oxo-ent-abieta-7-en-14-oic acid (chloranhenryin F), respectively. The eight known diterpenoids were identified as ent-pimar15-ene-3α,8α-diol (11),22 ent-pimara-8(14),15-diene-3α,7β-diol (12),23 3β-hydroxyabieta-8,11,13-trien-7-one (13),24 sessilifol O (14),3 3β,7α-dihydroxyabieta-8,11,13-triene (15),25 decandrin B (16),15 sessilifol D (17),3 and sessilifol F (18).3 From a biosynthetic standpoint, compounds 1−10 are closely related and might originate from ent-pimaradiene. Proposed biosynthetic routes to 1−10 are illustrated in Scheme 1.3,26−29 Compounds 1−18 were evaluated for their inhibitory abilities on C. albicans cell viability. Compounds 5 and 13 showed weak antifungal activity, with MIC50 values of 731 and 427 μM. Their inhibitory activities against C. albicans yeast-tohyphae conversion, which is an important virulence factor and is crucial to C. albicans infection, were also assessed.30 Compound 13 was found to be the most potent inhibitor of the morphological switch, with an IC50 value of 97.3 μM (Figure S74, Supporting Information), followed by compounds 5, 12, and 15 (IC50 values 333.7, 399.0, and 738.7 μM, respectively). Magnolol, a hyphal morphogenesis inhibitor,31 was used as a positive control (IC50 7.5 μM). This is the first report on the inhibitory effects on C. albicans dimorphism of ent-pimarane and ent-abietane diterpenoids.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations and ECD spectra were recorded on an InsMark IP120 automatic polarimeter (Shanghai InsMark Instrument Technology Co., Ltd., Shanghai, China) and a Jasco J-715 spectropolarimeter, respectively. The infrared (IR) spectra (KBr) were recorded on a Nicolet MAGNA560 instrument. The 1D and 2D NMR spectra were acquired on a Bruker AV 400 instrument (400 MHz for 1H and 100 MHz for 13C) using TMS as a reference at room temperature. The ESIMS data were obtained on an LCQ-Advantage mass spectrometer (Finnigan Co., Ltd., San Jose, CA, USA). HRESIMS spectra were acquired on an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA, USA) or LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). HPLC separations were performed on a Shimadzu LC-6AD system, equipped with a RID-10A refractive index detector and a YMC-pack ODS-AM (250 × 20 mm) column (YMC Co., Ltd., Japan). The flow rate of HPLC was 5.5 mL/min. Silica gel (200−300 mesh) used for column chromatography was from Qingdao Marine Chemical Group Co., Ltd., China. Magnolol was from Xi’an Yuquan Biological Technology Co., Ltd., China. Chemical reagents and other biological reagents were from Tianjin Chemical Reagent Company, China, and Sigma Company, respectively. Plant Material. The whole plants of Chloranthus henryi Hemsl. were purchased from Materia Medica Market of Anguo County, Hebei Province, People’s Republic of China, in December 2012 and identified by one of the authors (C. Xie). A voucher specimen (No. 20121210CH) was deposited at the Laboratory of the Research Department of Natural Medicines, College of Pharmacy, Nankai University, China. Extraction and Isolation. The air-dried whole plants of C. henryi (4.0 kg) were extracted with MeOH (3 × 40 L) under reflux. The solvent was evaporated to obtain a residue (0.4 kg), which was subsequently suspended in H2O (1.5 L) and partitioned with EtOAc (3 × 1.5 L). The resulting EtOAc-soluble part (306 g) was chromatographed on a silica gel column, using mixtures of petroleum ether/acetone of increasing polarity (100:0 to 100:30, 18 L for each gradient elution), to afford nine fractions (F1−F9) on the basis of TLC analyses. F4 (10 g) was fractionated by MPLC over ODS (MeOH in H2O, 72−90%) to give five subfractions (F4‑1−F4‑5). F4‑4 was purified by HPLC (85% MeOH in H2O) to yield compounds 8 (tR = 36 min, F

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*E-mail (Y. Guo): [email protected]. Phone/Fax: 86-2223502595.

and 3; ESIMS m/z 303 [M + H]+; HRESIMS m/z 303.2320 [M + H]+ (calcd for C20H31O2 303.2319). 15-O-Methylsessilifol J (7): colorless, amorphous powder; [α]27 D −27 (c 0.1, CH2Cl2); IR (KBr) νmax 3443, 2924, 2851, 1718, 1636, 1497, 1458, 1376, 1363, 1268, 1215, 1173, 1093, 1067, 1034, 1008, 972, 892, 828 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 2 and 3; ESIMS m/z 339 [M + Na]+; HRESIMS m/z 339.2298 [M + Na]+ (calcd for C21H32NaO2 339.2295). Chloranhenryin D (8): colorless oil; [α]27 D −9 (c 0.2, CH2Cl2); IR (KBr) νmax 3452, 2965, 2930, 2871, 1709, 1655, 1464, 1383, 1366, 1247, 1183, 1065, 1038, 1009, 731, 669 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 2 and 3; ESIMS m/z 315 [M + Na]+; HRESIMS m/z 315.2298 [M + Na]+ (calcd for C19H32NaO2 315.2295). Chloranhenryin E (9): yellow oil; [α]27 D −9 (c 0.1, CH2Cl2); IR (KBr) νmax 3445, 2965, 2931, 2870, 2723, 1694, 1633, 1464, 1385, 1366, 1232, 1187, 1160, 1090, 1040, 999, 923, 842, 822, 736, 720, 646 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 2 and 3; ESIMS m/z 343 [M + Na]+; HRESIMS m/z 343.2250 [M + Na]+ (calcd for C20H32NaO3 343.2244). Chloranhenryin F (10): yellow oil; [α]27 D −14 (c 0.1, CH2Cl2); IR (KBr) νmax 3443, 2956, 2924, 2851, 1705, 1646, 1464, 1384, 1247, 1135, 1087, 1040, 998, 962, 926, 893, 858, 822, 737, 718, 703, 647 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 2 and 3; ESIMS m/z 337 [M + H]+; HRESIMS m/z 337.2378 [M + H]+ (calcd for C20H33O4 337.2373). X-ray crystal data of sessilifol F (18): colorless crystals obtained from CH3CN; C21H34O3, M = 334.48, monoclinic, space group P2(1), a = 7.132(2) Å, b = 27.174(6) Å, c = 9.624(2) Å, α = 90°, β = 90.614(7)°, δ = 90°, V = 1865.1(8) Å3, T = 173(2) K, Z = 4, μ(Cu Kα) = 0.605 mm−1, Dcalc = 1.191 g/cm3, F(000) = 736, crystal size = 0.320 × 0.60 × 0.200 mm. The total number of reflections collected was 29 412, of which 7662 were unique (R(int) = 0.0195). Final R1 = 0.0328, wR2 = 0.0901 (I > 2σ(I)), Flack parameter = −0.01(3). The deposition of X-ray crystallographic data of 18 has been performed at the Cambridge Crystallographic Data Centre (CCDC 1430577). In Vitro Antifungal Bioassays. The C. albicans strain SC5314 was from Martin Schmidt (Des Moines University, Des Moines, IA, USA). The antifungal effects of compounds 1−18 were evaluated by the broth microdilution method.32 The MIC50s were determined as the concentrations of the tested compounds that inhibited growth by 50% compared with that of organisms grown without compounds, with fluconazole as positive control (MIC50 1.6 μM). The filamentation assay was performed in RPMI 1640 liquid medium as described previously.31 C. albicans SC5314 (1 × 106 cells/mL) was incubated in RPMI 1640 medium at 37 °C for 4 h, with or without the isolates. The morphogenesis inhibitory activity was quantified through counting the number of budded cells and the number of hyphal cells in the population by optical microscopy. Magnolol was used as a positive control. IC50 values were expressed as the concentrations that gave 50% inhibition of yeast-to-hyphae transition relative to the control.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 81302814), the Ministry of Science & Technology 973 Project (No. 2014CB560709), the Tianjin Science and Technology Program (No. 13JCQNJC13600), Jiangsu Province Natural Science Foundation (No. BK20130640 and BK20140624), Specialized Research Fund for the Doctoral Program of Higher Education Ministry of Education of China (No. 20120031120048), and the Fundamental Research Funds for the Central Universities of China (No. 65011201).

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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.5b00781. 1D and 2D NMR and HRESIMS spectra of compounds 1−10 and the ECD spectra of 4, 6, and 17 (PDF) X-ray data of compound 1 (CIF) X-ray data of compound 18 (CIF)

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Corresponding Authors

*E-mail (C. Xie): [email protected]. G

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