Absolute Configurations and Bioactivities of Guaiane-Type

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Cite This: J. Nat. Prod. 2018, 81, 1919−1927

Absolute Configurations and Bioactivities of Guaiane-Type Sesquiterpenoids Isolated from Pogostemon cablin Qin-Mei Zhou,†,‡,# Ming-Hua Chen,§,# Xiao-Hong Li,†,‡ Cheng Peng,*,†,‡ Da-Sheng Lin,⊥ Xiao-Nian Li,∥ Yang He,† and Liang Xiong*,†,‡ †

State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources and School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People’s Republic of China § Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ⊥ Chengdu Taihe Health Technology Group Inc., Ltd., Chengdu 610075, People’s Republic of China ∥ State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China

J. Nat. Prod. 2018.81:1919-1927. Downloaded from pubs.acs.org by DUQUESNE UNIV on 09/28/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Seven novel guaiane sesquiterpenoids (1−7) and three known seco-guaianes were isolated from the volatile oil of Pogostemon cablin. Their structures including absolute configurations were determined by spectroscopic analyses, a modified Mosher’s method, and X-ray diffraction and ECD data. The results indicated that the ECD Cotton effects arising from one or two nonconjugated olefinic chromophores could be applied to define the absolute configurations of guaiane sesquiterpenoids. Compounds 3 and 6 exhibited significant vasorelaxant activity against phenylephrine-induced and KCl-induced contractions of rat aorta rings [half-maximal effective concentration (EC50) of 3 against PHE-induced contraction, 5.4 μM; EC50 of 6 against PHE- and KCl-induced contractions, 1.6 and 24.2 μM, respectively]. They also showed antifungal activity against Candida albicans (minimum inhibitory concentrations, 500 and 300 μM, respectively). In addition, 2 and 7−9 displayed a neuroprotective effect against glutamate-induced injury in PC12 cells.

G

family Lamiaceae, has led to the isolation of rare nor-βpatchoulene and seco-patchoulene sesquiterpenoids.21,22 In the present study, minor sesquiterpenoids, comprising seven new guaiane analogues (1−7) and three known seco-guaianes, were isolated. A combination of spectroscopic and chemical methods, including NOESY experiments, X-ray diffraction and electronic circular dichroism (ECD) data, and a modified Mosher’s method, was applied to determine the absolute configurations of the isolated compounds. In addition, the vasorelaxant, neuroprotective, antibacterial, antifungal, and cytotoxic activities of all isolates were assessed.

uaianes are natural sesquiterpenoids with a fused [5.3.0]bicyclic ring core. They have been isolated from various plants such as those in the Araliaceae, 1 Asteraceae, 2 Thymelaeaceae,3,4 and Zingiberaceae families.5 Researchers have been investigating the bioactive properties of guaianes since the 1960s. Guaianes have been reported to have significant cytotoxic,6,7 anti-inflammatory,8 antibacterial,9 antimalarial,10 and antidepressant properties.11 Owing to their intriguing structures with a large substitution diversity and impressive bioactivity, many chemists and pharmacologists have focused on the synthesis, biotransformation, and structure−activity relationships of guaiane analogues.12−16 However, most guaianes contain multiple stereogenic centers. The absolute configurations of many guaianes, such as xylaguaianols A−D,17 4,10epizedoarondiol, 15-hydroxyprocurcumenol, 12-hydroxycurcumenol,18 (1β,5β)-1-hydroxyguaia-4(15),11(13)-diene-12,5-lactone, 1,5-epoxy-4-hydroxyguai-11(13)-en-12-oic acid,19 2α,4αdihydroxy-1β-guai-11(13),10(14)-dien-12,8α-olide, 5α,6αepoxy-2α,4α-dihydroxy-1β-guai-11(13)-en-12,8α-olide, and 6α-hydroxyinuchinenolide B,20 have not yet been determined. Our previous investigation of the volatile oil of the aerial parts of Pogostemon cablin (Blanco) Benth, a fragrant plant in the © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Compound 1, colorless crystals, had a specific rotation of [α]25D −133 (c 0.2, MeOH). The positive HRESIMS data (m/z 259.1675 [M + Na]+) suggested that the molecular formula was C15H24O2 with four indices of hydrogen deficiency (calcd for C15H24O2Na, 259.1674). The 13C NMR and DEPT data of 1 showed 15 carbon signals (Table 1) attributed to 3 × CH3, 4 × Received: August 11, 2017 Published: September 6, 2018 1919

DOI: 10.1021/acs.jnatprod.7b00690 J. Nat. Prod. 2018, 81, 1919−1927

Journal of Natural Products

Article

H-8a, OH-3, and OH-10 were cofacial, whereas H-3, isopropenyl-7, H-8b, H3-14, and H3-15 occupied the opposite face. ECD calculations 24 of (3S,4R,5S,7R,10R)-1 and (3R,4S,5R,7S,10S)-1 were performed to define the absolute configuration (Supporting Information, Experimental Section). Conformational analysis of (3S,4R,5S,7R,10R)-1 was performed via Monte Carlo searching with the MMFF94 molecular mechanics force field using MOE 2008 software. Twenty-five conformers possessing a relative energy within 10 kcal/mol were considered for density functional theory (DFT) optimization at the ωB97XD/def2-TZVP level in MeCN with the Gaussian 09 program25 (Table S2, Supporting Information). The energies, rotational strengths, and oscillator strengths of the first 10 electronic excitations were calculated using the TDDFT methodology at the CAM-B3LYP/def2-TZVP level in MeCN. Finally, the ECD spectrum of (3S,4R,5S,7R,10R)-1 was simulated using the Gausssum 2.25 program26 (σ = 0.3 eV). The calculated ECD spectrum of (3S,4R,5S,7R,10R)-1 matched the experimental ECD spectrum of 1 (Figure 2). The X-ray diffraction data of 1 confirmed its absolute configuration [Cu Kα radiation, Flack parameter = −0.03(6)] (Figure 3). Therefore, the structure of patchouliguaiol A (1) was defined as (−)-(3S,4R,5S,7R,10R)-guaia-1(2),11(12)-dien-3,10-diol. Compound 2 had similar IR and NMR data to 1. The molecular formula of C15H26O2 determined by HRESIMS indicated that 2 possessed two more hydrogen atoms than 1. The 1H, 13C, and DEPT NMR spectra of 2 suggested that it was also a diol-type guaiane; however, a tertiary hydroxy group [δH 2.85 (s); δC 84.3 (C)] in 2 replaced the secondary hydroxy group (OH-3) in 1 (Table 1). Furthermore, the Δ1(2) double bond in 1 was reduced in 2 based on replacement of the olefinic methine (C-2) in 1 by a methylene (δC 27.3) in 2. These observations were verified by 2D NMR data analysis. In particular, HMBC correlations of OH-5/C-1, C-4, C-5, and C-6 and of OH-10/C-1, C-9, C-10, and C-15, together with the 1 H−1H gCOSY signals for two isolated proton spin systems of H-1/H-2/H-3/H-4/H-14 and H-6/H-7/H-8/H-9, indicated that the hydroxy groups were substituted at C-5 and C-10. In the NOESY experiment of 2, H-1, H-4, and OH-5 were enhanced when H3-15 was irradiated; OH-5 and H3-15 were enhanced when H-7 was irradiated. Thus, H-1, H-4, H-7, H3-15, and OH-5 were cofacial, placing H3-14 and OH-10 at the opposite face.

CH2 (one olefinic methylene), 5 × CH (one olefinic methine and one oxygenated methine), and 3 × C (two olefinic carbons and one oxygenated carbon). Thus, two of the four indices of hydrogen deficiency were accounted for, and the remaining two indices were indicative of a bicyclic system. The 1H NMR and 13 C NMR data (Tables 1 and 2) assignable to an isopropenyl group [δH 4.66 (brs), 4.61 (brs), and 1.68 (s); δC 152.5 (C), 108.0 (CH2), 20.8 (CH3)], a methyl group attached to a carbinol carbon [δH 1.38 (s); δC 28.5 (CH3)], and a secondary methyl group [δH 1.03 (d, J = 7.2 Hz); δC 13.4 (CH3)], together with the 1H−1H gCOSY correlations for the vicinally coupled protons (H-2/H-3/H-4/H-5/H2-6/H-7/H2-8/H2-9 and H-4/ H3-14) (Figure 1) indicated that 1 was a guaiane-type sesquiterpenoid with a C-7 isopropenyl unit.23 In addition, the 1 H NMR signals attributed to an olefinic proton [δH 5.56 (brs)], an oxygenated methine [δH 4.35 (m)], and two exchangeable hydroxy protons [δH 3.64 (d, J = 6.0 Hz) and 3.38 (s)] revealed the presence of two hydroxy groups and a trisubstituted double bond. Confirmation of the positions of these substituents was accomplished via HMBC data analysis. Correlations of H-2 with C-1, C-3, C-4, C-5, and C-10; of H-3 with C-1, C-2, and C-14; and of OH-3 with C-2, C-3, and C-4 (Figure 1) confirmed the Δ1(2) trisubstituted double bond and that the secondary hydroxy group was located at C-3. The other hydroxy group resided at C10 based on HMBC correlations of OH-10/C-1, C-9, C-10, and C-15. The relative configuration was established by analysis of NOESY data. Correlations of H-3 with H3-14; of H-5 with H-4, H-6a, H-7, and OH-10; of H-7 with H-6a and H-8a; and of H315 with H-8b (Figure 1) demonstrated that H-4, H-5, H-6a, H-7,

Table 1. 13C NMR (150 MHz, δ in ppm) Data for Compounds 1−7 no.

1a

2a

3a

4b

5b

6a

7a

7c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

157.9 129.9 81.7 49.7 46.8 37.5 50.8 30.5 42.8 71.5 152.5 108.8 20.8 13.4 28.5

61.8 27.3 31.6 49.5 84.3 41.4 42.0 31.4 43.2 72.6 154.0 107.8 20.1 15.2 32.7

84.4 74.0 41.4 32.8 54.6 31.9 49.7 38.0 32.9 154.7 152.5 108.6 20.5 16.8 111.9

146.1 127.4 39.8 37.6 49.0 35.1 42.2 41.5 75.3 149.5 151.8 108.9 20.8 15.9 111.4

136.6 197.1 133.7 171.7 49.3 35.9 51.0 30.8 37.9 151.0 150.8 109.5 20.6 16.8 20.6

73.4 78.5 40.6 40.4 74.1 33.9 43.9 29.7 32.1 32.0 152.6 108.9 20.9 18.8 17.2

75.5 28.9 36.8 80.5 72.1 29.6 44.3 30.1 31.9 33.6 152.6 108.8 20.8 22.7 16.4

74.6 27.7 35.6 78.9 71.1 28.6 42.9 29.0 30.7 32.1 151.2 108.6 20.4 22.4 15.9

a

Data were measured in acetone-d6. bData were measured in CDCl3. cData were measured in DMSO-d6. 1920



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Table 2. 1H NMR (600 MHz, δ in ppm, J in Hz) Data for Compounds 1−7 1a

no. 1 2a

5.56 brs

2b 3a

2a 1.90 dd (9.6, 4.8) 1.92 m

3a 4.46 q (6.6)

5b

5.87 dd (4.8, 2.4)

6a

4.35 m

2.00 m 1.23 m

1.80 ddd (12.6, 7.8, 6.6) 1.67 ddd (12.6, 7.2, 6.6) 2.49 m 2.05 md 1.50 brd (13.8)

2.52 ddd (16.8, 7.2, 2.4) 1.89 ddd (16.8, 4.8, 3.0) 2.40 m 2.91 m 1.69 brd (13.2)

3.09 d (12.0) 2.03 brd (12.0)

1.12 ddd (13.8, 12.0, 10.8) 2.09 m 1.87 m 1.36 m 2.47 m

1.26 m

1.01 q (12.0)

2.58 m 2.15 m 1.55 m 4.53 brd (6.0)

2.25 m 1.83 m 1.30 m 2.44 dd (13.2, 12.0) 2.20 ddd (13.2, 6.6, 1.2)

4 5 6a

2.04 md 2.79 m 1.90 brd (12.6)

2.10 dd (13.2, 10.8)

6b

0.97 q (12.6)

1.51 brd (13.2)

7 8a 8b 9a

2.08 md 1.65 m 1.14 m 1.82 m

2.57 m 1.86 m 1.47 m 1.80 m

9b

1.78 m

1.68 m

2.22 ddd (13.2, 6.0, 4.2)

4.66 brs 4.61 brs 1.68 brs 1.03 d (7.2) 1.38 s

4.65 brs 4.56 brs 1.70 brs 0.89 d (7.2) 1.15 s

3.64 d (6.0) (OH-3), 3.38 s (OH-10)

3.06 s (OH-10), 2.85 s (OH-5)

4.65 brs 4.59 brs 1.68 brs 0.87 d (7.2) 5.17 brs 4.91 brs 4.10 d (6.6) (OH-2), 3.44 s (OH-1)

1.81 m

4.73 brs 4.67 brs 1.73 brs 0.85 d (7.2) 5.19 d (1.2) 4.86 d (1.2)

4.70 brs 4.70 brs 1.70 brs 2.06 s 2.36 s

7a

7c

4.16 m

2.14 m

1.93 m

1.54 ddd (13.2, 1.50 dd 7.2, 1.2) (13.2, 8.4) 1.47 m 1.39 m

1.76 m

3b

10 12a 12b 13 14 15a 15b OH

4b

2.05 m

1.12 brd (13.2) 2.04 md

1.47 m

1.34 m

2.06d

2.07d

1.99 brd (13.8) 2.18 m 1.43 m 1.43 m 1.75 m

2.03d 2.08 m 1.45 m 1.39 m 1.74 m

1.96 dd (13.2, 12.0) 1.89 brd (13.2) 2.01 m 1.37 m 1.29 m 1.62 m

1.45 m

1.48 m

1.42 m

2.48 m 4.70 brs 4.63 brs 1.72 brs 1.10 d (7.2) 1.30 d (6.6)

2.34 m 4.69 brs 4.63 brs 1.72 brs 1.19 s 1.12 d (7.2)

2.30 m 4.68 brs 4.62 brs 1.68 brs 1.10 s 1.06 d (7.2)

4.02 d (4.2) (OH-2)

3.55 s (OH-4)

4.48 s (OH4)

a

Data were measured in acetone-d6. bData were measured in CDCl3. cData were measured in DMSO-d6. dThe signals were overlapped.

The HRESIMS data of 3 indicated that it possessed the same molecular formula as 1. Comparison of the 1H and 13C NMR data of 3 and 1 suggested that 3 was also a guaiane with two hydroxy groups and two double bonds. However, the trisubstituted double bond in 1 was replaced by an exocyclic double bond [δH 4.65 (brs) and 4.59 (brs); δC 154.7 and 111.9] in 3 (Tables 1 and 2). HMBC correlations of OH-1 with C-1, C2, C-5, and C-10; of OH-2 with C-1, C-2, and C-3; and of H2-15 with C-1, C-9, and C-10 established the 2D structure of guaia10(15),11(12)-diene-1,2-diol for 3. The NOESY correlations of OH-1 with OH-2, H-4, and H-5; of H-4 with H-5 and H-7; of H6a (H-eq, brd, J = 13.8 Hz) with H-5 and H-7; and of H-2 with H3-14 and H-6b (H-ax, ddd, J = 13.8, 12.0, 10.8 Hz) indicated that the vicinal hydroxy groups (OH-1 and OH-2) were placed in cis orientation at the same face as H-4, H-5, H-6a, and H-7. In spite of extensive efforts, suitable crystals of 3 could not be obtained for X-ray diffraction. Alternatively, a modified Mosher’s method27,28 was used to determine the absolute configuration. Esterification of 3 with (R)-(−)-α-methoxyphenylacetic acid (MPA) and (S)-(+)-MPA yielded the corresponding esters, 3a [3-(R)-MPA] and 3b [3-(S)-MPA]. The 1H NMR data of these two diastereomers are shown in Table S1 (Supporting Information). Based on the MPA rule of ΔδR−S values,28 the (S) configuration was assigned for C-2 (Figure 4). Furthermore, the experimental and calculated ECD data also supported the (1S,2S,4S,5S,7R) configuration of 3 (Figure 2 and Supporting Information). Therefore, the structure of patchouliguaiol C (3) was defined as (+)-(1S,2S,4S,5S,7R)-guaia10(15),11(12)-dien-1,2-diol.

Figure 1. Selected 1H−1H COSY, HMBC, and NOESY correlations of 1.

In the X-ray diffraction experiment [Flack parameter = 0.02(17)], the (1S,4S,5R,7R,10S) absolute configuration was assigned for 2 (Figure 3). However, the experimental ECD sp ect r u m o f 2 a n d t h e c al c u l a t e d s p e c t r u m o f (1S,4S,5R,7R,10S)-2 calculated by the TDDFT methodology at the CAM-B3LYP/def2-TZVP level in MeCN (Figure S4a, Supporting Information) are not in agreement. The TDDFT calculation of the ECD spectrum of 2 was next conducted at the B3LYP/6-311++G(2d,2p) level. Surprisingly, the result from TDDFT methodology at the B3LYP/6-311++G(2d,2p) level was opposite that from the TDDFT methodology at the CAMB3LYP/def2-TZVP level (Figure S4, Supporting Information), but consistent with that from the X-ray diffraction. Therefore, the structure of patchouliguaiol B (2) was defined as (+)-(1S,4S,5R,7R,10S)-guaia-11(12)-en-5,10-diol. 1921



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Figure 2. Experimental and calculated ECD spectra of 1−7. The TDDFT calculations of ECD spectra of 1 and 3−7 were conducted at the CAMB3LYP/def2-TZVP level. The TDDFT calculations of ECD spectrum of 2 were conducted at the B3LYP/6-311++G(2d,2p) level.

the 1H and 13C NMR data of 3, compound 4 showed resonances for an additional trisubstituted double bond [δH 5.87 (dd, J =

Compound 4 was also a guaiane with a molecular formula of C15H22O, as indicated by the MS and NMR data. Compared to 1922



Article

to C-1, C-2, C-4, and C-5 and from H3-15 and H2-9 to C-1 and C-10. Additional analysis of the HMBC and 1H−1H gCOSY data verified a 2D structure of guaia-1(10),3(4),11(12)-trien-2one for 5. The spatial proximity of H-5 and H-7 was deduced from NOESY correlation between these two protons, while the absolute configuration was defined as (5S,7R) via the experimental and computed ECD data (Figure 2 and Supporting Information). Thus, the structure of patchouliguaiol E (5) was defined as (−)-(5S,7R)-guaia-1(10),3(4),11(12)-trien-2-one. The IR, HRESIMS, and 1D NMR data of 6 revealed that it was another Δ11(12)-guaiene possessing an epoxy unit (δC 74.1 and 73.4) and a secondary hydroxy group [δH 4.16 (m) and 4.02 (d, J = 4.2 Hz, exchangeable OH); δC 78.5]. Analysis of the 2D NMR data showed that 6 was an epimer of (1S,3S,3aR,5R,8S,8aS)-3,8-dimethyl-5-(prop-1-en-2-yl)hexahydro-1H,4H3a,8a-epoxyazulen-1-ol30 [(1S,2S,4S,5R,7R,10S)-guaia-1,5epoxy-11(12)-en-2-ol]. Comparison of their 13C NMR data showed that the C-2 resonance in 6 was deshielded significantly by ΔδC +6.1 ppm, which implied that they were a pair of C-2 epimers. Although no available NOESY data were obtained for determining the relative configuration of the epoxy unit, an Xray diffraction data analysis confirmed the (1S,2R,4S,5R,7R,10S) absolute configuration of 6 (Figure 3). This was consistent with the results of the experimental and computed ECD data (Figure 2 and Supporting Information). Therefore, the structure of patchouliguaiol F (6) was defined as (−)-(1S,2R,4S,5R,7R,10S)guaia-1,5-epoxy-11(12)-en-2-ol. Compound 7 was an isomer of 6, as indicated by HRESIMS, 1 H NMR, and 13C NMR data. Analysis of the 2D NMR data in acetone-d6 or DMSO-d6 defined the 2D structure of 7 as a guaia1,5-epoxy-11-en-4-ol. The NOESY and X-ray diffraction data of 7 (Figure 3) confirmed its relative configuration; however, its absolute configuration could not be determined reliably [Flack parameter = 0.2(6)]. Using the experimental and computed ECD data, the (1R,4S,5R,7R,10S) configuration was established for 7 (Figure 2 and Supporting Information). Therefore, the structure of patchouliguaiol G (7) was defined as (−)-(1R,4S,5R,7R,10S)-guaia-1,5-epoxy-11(12)-en-4-ol. Three known seco-guaianes were defined as 7-epi-chabrolidione A (8),31 1,7-di-epi-chabrolidione A (9),30 and (2R,3S)-3methyl-2-(5-oxo-2-isopropenylhexyl)cyclopentanone32 by comparison of the experimental and reported NMR data. Because single crystals of volatile sesquiterpenoids are difficult to obtain and alternative chemical methods are limited, data on most natural guaianes have been reported without their absolute configurations. In this study, a combination of X-ray diffraction, the experimental and calculated ECD data, and Mosher’s method was used to establish the absolute configurations of the new guaianes. As shown by the experimental ECD spectra of 1− 7 in MeCN, guaianes with only one double bond (2) had no Cotton effect above 185 nm, guaianes with one double bond and an epoxy moiety (6 and 7) showed a Cotton effect at 190−195 nm, and guaianes with two nonconjugated double bonds (1 and 3) displayed a Cotton effect near 200 nm. Although these compounds showed weak ECD Cotton effects in the farultraviolet based upon one or two nonconjugated alkene chromophores, the experimental and calculated ECD data are useful for determining absolute configurations of the class of sesquiterpenoids. This deduction was also supported by the previous studies.33−35 The isolates were investigated for their vasorelaxant, neuroprotective, antibacterial, antifungal, and cytotoxic activities based on the results of previous bioactivity studies on guaianes

Figure 3. ORTEP drawings of compounds 1, 2, 6, and 7.

Figure 4. ΔδH values (δR − δS, in ppm) for 3a and 3b.

2.4, 4.8 Hz); δC 146.1 and 127.4] (Tables 1 and 2). In addition, only one oxygenated carbon signal (δC 75.3) suggested a hydroxy group in 4 to match the molecular composition, even though no signal of an exchangeable hydroxy proton was observed. HMBC cross-peaks from the olefinic methine proton (H-2) to C-3, C-4, C-5, and C-10 and from the olefinic methylene protons (H2-15) to C-1, C-9, and C-10 permitted the establishment of Δ1(2),10(15) conjugated double bonds. The hydroxy group was placed at C-9 on the basis of HMBC crosspeaks from the oxymethine proton (H-9) to C-1, C-7, C-8, C-10, and C-15. NOESY cross-peaks of H-5 with H-4 and H-7 indicated α orientations for these protons, whereas the crosspeaks of H-9 with both H-8a and H-8b and of H-7 only with H8a, together with the coupling pattern of H-9 (brd, J = 6.0 Hz), placed H-9 in a β-equatorial orientation. Using the same method as described for 1 and 3, the (4S,5S,7S,9R) absolute configuration of 4 was elucidated via the experimental and calculated ECD data (Figure 2 and Supporting Information). Thus, the structure of patchouliguaiol D (4) was defined as (−)-(4S,5S,7S,9R)-guaia-1(2),10(15),11(12)-trien-9-ol. Compound 5 was another guaiane sesquiterpenoid. The molecular formula (C15H20O) revealed the presence of four unsaturated groups. 1H NMR and 13C NMR data of 5 showed characteristic signals for a carbonyl group (δC 197.1) conjugated with both a trisubstituted double bond (δH 6.04; δC 133.7 and 171.7) and a tetrasubstituted double bond (δC 136.6 and 151.0) (Tables 1 and 2).29 Additional reliable support for this substructure was obtained from HMBC correlations from H-3 1923



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Figure 5. Vasorelaxant activities of (a) 3, 6, and phentolamine mesylate against PHE-induced contraction and (b) 3, 6, 8, and methoxyverapamil against KCl-induced contraction of rat aorta rings.

Figure 6. Protective effects of 2 and 7−9 against glutamate-induced damage in PC12 cells. Data are presented as mean ± standard deviation (n = 6). ## indicates p < 0.01 when compared to the control cells. * indicates p < 0.05 and ** indicates p < 0.01 when compared to the model cells.

and the essential oil of P. cablin. Compounds 3 and 6 showed significant dose-dependent vasorelaxant activity against both phenylephrine (PHE)- and KCl-induced contractions of rat aorta rings (Figure 5). The vasorelaxant effect of 6 (EC50, 1.6 and 24.2 μM, respectively) was stronger than that of 3 (EC50 against PHE-induced contraction, 5.4 μM). Because PHEinduced contraction is linked to activation of the α1 receptor,36 and a high concentration of K+ opens Ca2+ voltage-sensitive channels,37 the results indicate that 3 and 6 possibly block extracellular Ca2+ influx via both receptor-operated calcium channels (ROCC) and voltage-dependent calcium channels (VDCC); however, the effect on ROCC is greater. Compound 8 showed weak vasorelaxant activity against KCl-induced contraction, and the other compounds were inactive at 50 μM. Phentolamine mesylate and methoxyverapamil were used as positive controls. In the neuroprotective assay, PC12 cells were damaged by Lglutamate in a concentration-dependent manner. At a concentration of 30 mM, glutamate caused a significant decrease in PC12 cell viability (p < 0.01 vs blank control), whereas cotreatment of PC12 cells with glutamate and nimodipine (NDP, 10 μM) significantly attenuated PC12 cell injury (p < 0.01 when compared to the cells treated with glutamate alone). As shown in Figure 6, compounds 2 and 7−9 significantly inhibited glutamate-induced damage in PC12 cells (p < 0.05 or p

< 0.01 when compared to cells treated with glutamate alone), especially at high concentrations. Compared to cells treated with glutamate alone, the cell viability upon treatment with 2 and 7− 9 at 50 μM increased from 49.14 ± 3.15 to 66.14 ± 9.76, 65.51 ± 6.72, 78.68 ± 7.67, and 75.67 ± 7.11%, respectively (p < 0.01), which was higher than the increase provided by NDP at 10 μM. In addition, compounds 3 and 6 exhibited antifungal activity against Candida albicans with MIC values of 500 and 300 μM, respectively. However, the isolated guaianes showed no cytotoxic activity against A549, MCF-7, SGC-7901, or SMMC-7712 human cancer cell lines (IC50 > 50 μM). Additionally, they did not show antibacterial activity against MRSA (ATCC 43300), Staphylococcus aureus (ATCC 25923), or Escherichia coli (ATCC 25922) (MIC > 500 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on an Anton Paar MCP 200 polarimeter. ECD data were obtained using a JASCO J-815 ECD instrument. X-ray crystallographic analyses were performed on a D8 Venture or an APEX DUO diffractometer (Bruker Corporation, Billerica, MA, USA). A Nicolet 5700 FT-IR spectrophotometer was used to measure IR spectra. Highresolution ESI mass spectra were acquired on a Waters Synapt G2 HDMS instrument. NMR spectra were recorded with solvent peaks as internal standard on a Bruker Avance III 600 NMR spectrometer. Melting points were obtained on an MP300 automatic melting point

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acetone-d6) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 259.1685 [M + Na]+ (calcd for C15H24O2Na, 259.1674). Patchouliguaiol D [(−)-(4S,5S,7S,9R)-Guaia-1(2),10(15),11(12)trien-9-ol] (4): colorless oil; [α]25D −97 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 224 (2.28) nm; ECD (MeOH) 222 (Δε −0.22) nm; IR νmax 3362, 3189, 2957, 2922, 2851, 1723, 1647, 1468, 1422, 1378, 1287, 1134, 1074, 893, 721, 703 cm−1; 13C NMR (150 MHz, CDCl3) and 1H NMR (600 MHz, CDCl3) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 219.1746 [M + H]+ (calcd for C15H23O, 219.1749). Patchouliguaiol E [(−)-(5S,7R)-Guaia-1(10),3(4),11(12)-trien-2one] (5): colorless oil; [α]25D −140 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (2.33), 274 (1.62) nm; ECD (MeOH) 231 (Δε +1.02), 271.5 (Δε −1.46), 353 (Δε +0.08) nm; IR νmax 3390, 3075, 2958, 2934, 2873, 1719, 1695, 1629, 1436, 1379, 1287, 1125, 1075, 891, 746 cm−1; 13C NMR (150 MHz, CDCl3) and 1H NMR (600 MHz, CDCl3) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 239.1412 [M + Na]+ (calcd for C15H20ONa, 239.1412). Patchouliguaiol F [(−)-(1S,2R,4S,5R,7R,10S)-Guaia-1,5-epoxy11(12)-en-2-ol] (6): colorless crystals; mp 78−80 °C (n-hexane); [α]25D −14 (c 0.03, MeOH); ECD (MeCN) 193 (Δε −3.28), 216 (Δε +0.55) nm; IR νmax 3452, 3081, 2964, 2930, 2879, 1645, 1450, 1377, 1318, 1259, 1110, 1036, 1011, 991, 922, 888, 816, 757 cm−1; 13C NMR (150 MHz, acetone-d6) and 1H NMR (600 MHz, acetone-d6) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 259.1674 [M + Na]+ (calcd for C15H24O2Na, 259.1674). Patchouliguaiol G [(−)-(1R,4S,5R,7R,10S)-Guaia-1,5-epoxy11(12)-en-4-ol] (7): colorless needles; mp 72−73 °C (MeOH); ECD (MeCN) 193 (Δε −5.25), 211 (Δε +0.76) nm; [α]25D −23 (c 0.1, MeOH); IR νmax 3516, 3074, 2959, 2927, 2870, 1643, 1451, 1377, 1243, 1202, 1124, 1097, 988, 924, 889, 826, 801 cm−1; 13C NMR (150 MHz, acetone-d6) and 1H NMR (600 MHz, acetone-d6) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 259.1679 [M + Na]+ (calcd for C15H24O2Na, 259.1674). X-ray Crystallographic Analysis. X-ray crystallographic data of 1 and 2 were obtained using a Bruker D8 Venture diffractometer with Cu Kα radiation. X-ray crystallographic data of 6 and 7 were obtained using a Bruker APEX DUO diffractometer with Cu Kα radiation. The CCDC numbers for 1, 2, 6, and 7 contain the supplementary crystallographic data, which can be obtained free of charge via http://www.ccdc.cam.ac. uk/conts/retrieving.html. Crystal data for 1: C15H24O2, M = 236.34, colorless crystals, monoclinic, a = 5.8459(2) Å, b = 7.8647(2) Å, c = 14.9863(5) Å, α = 90.00°, β = 96.3690(10)°, γ = 90.00°, V = 684.76(4) Å3, space group P21, T = 120(2) K, Z = 2, μ(Cu Kα) = 0.576 mm−1, 10 606 reflections measured, 2488 independent reflections (Rint = 0.0328). Final R indices (I > 2σ(I)): R1 = 0.0316, wR2 = 0.0824. Final R indices (all data): R1 = 0.0321, wR2 = 0.0832. Flack parameter: −0.03(6). CCDC number: 1560324. Crystal data for 2: C15H26O2, M = 238.36, colorless crystal, monoclinic, a = 10.280(2) Å, b = 15.493(3) Å, c = 10.270(2) Å, α = 90.00°, β = 119.71(3)°, γ = 90.00°, V = 1420.6(6) Å3, space group P21, T = 104(2) K, Z = 4, μ(Cu Kα) = 0.556 mm−1, 14 544 reflections measured, 4783 independent reflections (Rint = 0.0622). Final R indices (I > 2σ(I)): R1 = 0.0681, wR2 = 0.1732. Final R indices (all data): R1 = 0.0780, wR2 = 0.1853. Flack parameter: 0.02(17). CCDC number: 1560421. Crystal data for 6: C15H24O2, M = 236.34, colorless bulk crystal, monoclinic, a = 11.5078(3) Å, b = 10.0306(3) Å, c = 12.1928(3) Å, α = 90.00°, β = 101.0400(10)°, γ = 90.00°, V = 1381.37(6) Å3, space group P21, T = 100(2) K, Z = 4, μ(Cu Kα) = 0.571 mm−1, 14 185 reflections measured, 4303 independent reflections (Rint = 0.0402). Final R indices (I > 2σ(I)): R1 = 0.0353, wR2 = 0.1017. Final R indices (all data): R1 = 0.0361, wR2 = 0.1026. Flack parameter: 0.13(8). CCDC number: 1542423. Crystal data for 7: C15H24O2, M = 236.34, colorless needles, monoclinic, a = 7.5723(8) Å, b = 9.7359(12) Å, c = 9.3881(11) Å, α = 90.00°, β = 102.926(7)°, γ = 90.00°, V = 674.58(14) Å3, space group P21, T = 100(2) K, Z = 2, μ(Cu Kα) = 0.585 mm−1, 5443 reflections measured, 2069 independent reflections (Rint = 0.0909). Final R indices

apparatus (Jinan Hanon Instruments Co., Ltd., People’s Republic of China). Silica gel (200−300 mesh, Yantai Institute of Chemical Technology, People’s Republic of China) and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden) were used for column chromatography. Silica gel plates (GF254; Qingdao Marine Chemical Inc., People’s Republic of China) were used for TLC. An Agilent 1220 instrument and a Kromasil semipreparative C18 column (250 × 10 mm2, 5 μm) were employed for reversed-phase semipreparative HPLC. A Cometro 6000 instrument and a Kromasil semipreparative silica column (250 × 10 mm2, 5 μm) were employed for normal-phase semipreparative HPLC. Plant Material. P. cablin was collected in Tanshui town, Yangchun city, Guangdong Province, China, in December 2012. The sample was identified by Dr. Fei Long and deposited in the School of Pharmacy in Chengdu University of TCM, Chengdu, China (voucher specimen: SGHX-20121224). Extraction and Isolation of Compounds. The aerial parts of P. cablin (40 kg) were extracted by steam distillation (10 h) in a big selfmade Clevenger-type instrument. A total of 215 g of essential oil was obtained by a water-cooled oil receiver. The oil was dried with anhydrous Na2SO4 and was subjected to column chromatography on silica gel (petroleum ether/EtOAc, 1:0−0:1) to yield 31 major fractions, F1−F31. Separation of fraction F26 (8.5 g) by RP-MPLC with MeOH/H2O (50:50−0:100) afforded 11 fractions (F26‑1−F26‑11). Fraction F26‑4 was further separated into seven fractions (F26‑4a−F26‑4g) by Sephadex LH-20 with petroleum ether/CHCl3/MeOH (5:5:1) as the eluent. Compound 1 (43 mg) was crystallized from F26‑4b in MeOH, and the mother liquor was further purified by column chromatography on silica gel (petroleum ether/EtOAc, 10:0−6:4), followed by preparative TLC (petroleum ether/EtOAc, 12:1), to yield 5 (3.3 mg). Fraction F26‑4 was successively separated by a Sephadex LH-20 column eluted by petroleum ether/CHCl3/MeOH (5:5:1) and preparative TLC developed by petroleum ether/Me2CO (8:1) to give 4 (1.1 mg). Fraction F28 (10.8 g) was further divided into F28‑1−F28‑6 by RPMPLC chromatography using a gradient solvent system (MeOH/H2O, 50:50−0:100). Compound 2 (3.6 mg) was obtained from F28‑2 by successive separation on Sephadex LH-20 (petroleum ether/CHCl3/ MeOH, 5:5:1), preparative TLC (n-hexane/Me2CO, 5:1), and normalphase semipreparative HPLC (n-hexane/isopropyl alcohol, 100:1). Fraction F28‑4 was separated by column chromatography over Sephadex LH-20 eluted with petroleum ether/CHCl3/MeOH (5:5:1) to yield six fractions (F28‑4a−F28‑4f). Fraction F28‑4b was fractionated by repeated CC over silica gel (5−30% EtOAc in petroleum ether), followed by normal-phase semipreparative HPLC (n-hexane/isopropyl alcohol, 100:1), to yield 6 (6.7 mg) and 7 (7.1 mg). Purification of F28‑4e by preparative TLC (n-hexane−acetone, 5:1) and reversed-phase semipreparative HPLC (68% CH3OH in H2O) afforded 3 (9.3 mg). Patchouliguaiol A [(−)-(3S,4R,5S,7R,10R)-Guaia-1(2),11(12)-dien3,10-diol] (1): colorless crystals; mp 186−187 °C (MeOH); [α]25D −133 (c 0.2, MeOH); ECD (MeCN) 203 (Δε −23.5) nm; IR νmax 3287, 3076, 2967, 2936, 2912, 2868, 1644, 1448, 1401, 1373, 1354, 1144, 1104, 1082, 1040, 934, 908, 881, 824, 786, 726, 676 cm−1; 13C NMR (150 MHz, acetone-d6) and 1H NMR (600 MHz, acetone-d6) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 259.1675 [M + Na]+ (calcd for C15H24O2Na, 259.1674). Patchouliguaiol B [(+)-(1S,4S,5R,7R,10S)-Guaia-11(12)-en-5,10diol] (2): colorless needles; mp 100−101 °C (MeOH); [α]25D +17 (c 0.03, MeOH); ECD (MeCN) a negative curve in the range of 215−185 nm; IR νmax 3414, 3071, 2964, 1645, 1447, 1406, 1373, 1338, 1211, 1130, 1063, 1046, 944, 922, 881, 787, 745, 673, 609 cm−1; 13C NMR (150 MHz, acetone-d6) and 1H NMR (600 MHz, acetone-d6) data, see Tables 1 and 2, respectively; (+)-HRESIMS m/z 261.1828 [M + Na]+ (calcd for C15H26O2Na, 261.1830). Patchouliguaiol C [(+)-(1S,2S,4S,5S,7R)-Guaia-10(15),11(12)dien-1,2-diol] (3): colorless oil; [α]25D +62 (c 0.2, MeOH); ECD (MeCN) 199 (Δε +16.4) nm; IR νmax 3425, 3081, 2928, 2855, 1642, 1448, 1378, 1285, 1154, 1119, 1076, 1033, 915, 890, 847, 737, 712 cm−1; 13C NMR (150 MHz, acetone-d6) and 1H NMR (600 MHz, 1925



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(I > 2σ(I)): R1 = 0.0804, wR2 = 0.2031. Final R indices (all data): R1 = 0.1327, wR2 = 0.2664. Flack parameter: 0.2(6). Absolute structure could not be determined reliably. CCDC number: 1542427. Vasorelaxant Activity Assay. The vasorelaxant activity of the isolates against PHE- and KCl-induced contractions of rat aorta rings was measured as described previously.22,27 The experimental details are shown in the Supporting Information. Neuroprotective Activity Assay. The protection of the isolates on glutamate-induced cytotoxicity in PC12 cells was studied using the MTT method.38 The experimental details are shown in the Supporting Information. Antibacterial and Antifungal Activity Assays. The antibacterial and antifungal activities of all isolates were investigated.39 The experimental details are shown in the Supporting Information. Cytotoxicity Assay. The cytotoxicity of the isolates was evaluated by an MTT method using A549, MCF-7, Hey, SGC-7901, and HepG-2 human cancer cell lines.21,40 The experimental details are shown in the Supporting Information.

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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.7b00690. Experimental details of assays; modified Mosher’s experiment for 3; ECD calculation details for 1−7; and 1D NMR, 2D NMR, and HRESIMS spectra for 1−7 (PDF) X-ray crystallographic data for 1, 2, 6, and 7 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C. Peng). *E-mail: [email protected]; [email protected] (L. Xiong). ORCID

Liang Xiong: 0000-0001-6222-8340 Author Contributions #

Q. M. Zhou and M. H. Chen contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support was received from the Outstanding Youth Science Foundation of Sichuan Province (Nos. 2015JQO030 and 2015JQO027), National Major Special Project for the Development of New Drugs in China (2017ZX09201001-008-001), and the Scientific Research Fund of Sichuan Provincial Education Department (No. 15ZA0084).

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Absolute Configurations and Bioactivities of Guaiane-Type

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