Bioactive Sesquiterpenoids from the Peeled Stems ... - ACS Publications

rare 2,2,5,9-tetramethylbicyclo[6.3.0]-undecane (1), a humulane- .... +10 (c 0.1, MeOH), compound 1 was initially assumed to be ..... 16.0 Hz suggeste...
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Bioactive Sesquiterpenoids from the Peeled Stems of Syringa pinnatifolia Ruifei Zhang,† Xiao Feng,† Guozhu Su,† Zejing Mu,‡ Hexinge Zhang,† Yanan Zhao,† Shungang Jiao,† Lan Cao,‡ Suyile Chen,§ Pengfei Tu,† and Xingyun Chai*,†

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†

Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330000, People’s Republic of China § Alashan Mongolian Hospital, East Banner of Alashan, Inner Mongolia 750306, People’s Republic of China S Supporting Information *

ABSTRACT: Fourteen new sesquiterpenoids, alashanoids A−H (1, 2, and 4−9), (+)-2,9-humuladien-6-ol-8-one (3b), and five pairs of enantiomers (1 and 4−7), along with eight known analogues (3a and 10−16) were isolated from the stems of Syringa pinnatifolia. The structures were established using IR, UV, MS, and NMR data. The absolute configurations of the new compounds were resolved by X-ray diffraction, a modification of Mosher’s method, and experimental and calculated ECD data analysis. The new sesquiterpenoids represent three skeletons: a rare 2,2,5,9-tetramethylbicyclo[6.3.0]-undecane (1), a humulanetype (2−8), and a caryophyllene-type (9) skeleton. Compounds 6a, 7, and 11 showed protective effects against hypoxia-induced injury to H9c2 cells at a concentration of 40 μM, and 5−7, 11, and 13 inhibited NO production in LPS-induced RAW264.7 macrophage cells with IC50 values ranging from 13.6 to 70.6 μM. These compounds decreased the TNF-α and IL-6 levels in RAW264.7 cells in a concentration-dependent manner at 20−80 μM.

A

known analogues (3a and 10−16) (Chart 1). This paper reports the isolation, structural determination, and biological evaluation, including the protective effects against hypoxia injury to H9c2 cells and ability to inhibit NO production in LPS-induced RAW264.7 cells, for these sesquiterpenoids.

n endemic Chinese species belonging to the Oleaceae family, Syringa pinnatifolia (SP), is limitedly distributed in the Helan Mountain region between the Ningxia and Inner Mongolia provinces of China. The peeled roots, stems, and twigs, called “Shan-chen-xiang” in Chinese, are a traditional Mongolian folk medicine used to treat cardiovascular symptoms, asthma, and pain, a remedy that has been used for hundreds of years.1,2 SP is traditionally used as a powder, and its purple resin in the wood is believed to contribute to its therapeutic effects.3 Previous pharmacological research revealed that SP essential oils containing mainly zerumbone (11, a humulane-type sesquiterpenoid) showed a cardioprotective effect against acute myocardial ischemia (AMI) in rats.4,5 In addition, the EtOH extract of SP, containing mainly sesquiterpenoids and lignans with moderate to low polarities, showed an antimyocardial ischemic effect in mice.6 Consequently, although terpenoids, particularly sesquiterpenoids, are rarely reported from SP,7,8 they are prominent among the constituents with anti-AMI effects. Therefore, a 1H NMR-guided phytochemical investigation was performed on the extract of the SP stems to clarify the contribution of the terpenoids to its anti-AMI effect. This investigation afforded 14 new sesquiterpenoids, namely, alashanoids A−H (1, 2, and 4−9), (+)-2,9-humuladien-6-ol-8one (3b), five pairs of enantiomers (1 and 4−7), and eight © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION A scalemic mixture of alashanoid A (1), isolated as a paleyellow oil, exhibited an HRESIMS ion at m/z 249.1487 [M − H]− (calcd for C15H21O3, 249.1496) and, combined with the 13C NMR data, suggested a molecular formula of C15H22O3. The IR absorption bands at 3442, 1748, 1635, and 1098 cm−1 indicated the presence of a hydroxy, carbonyl, and conjugated carbonyl group. Its 1H NMR data (Table 1) showed signals for three methyl groups at δH 0.86 (3H, s), 1.00 (3H, s), and 1.13 (3H, s), an olefinic proton at δH 6.70 (1H, dd, J = 6.5, 10.5 Hz), and a formyl proton at δH 9.50 (1H, s). The 13C NMR and HSQC data (Table 1) displayed 15 carbon resonances, which were classified as three methyl, four methylene, three methine (including an olefinic carbon), two carbonyl, two quaternary, and an oxygenated tertiary carbon. Received: December 23, 2017

A

DOI: 10.1021/acs.jnatprod.7b01071 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

moiety, thus establishing its 2D structure as depicted (Figure S1). The NOESY correlations between H-1 and H-7a/H3-13, between H3-15 and H-7a, and between H-8 and H3-12 suggested that these protons were cofacial. The NOESY correlation of H-4/H-14 suggested that the Δ4(5) double bond was E configured, thus defining the relative configuration of 1 (Figure S2). On the basis of its positive specific rotation, [α]25 D +10 (c 0.1, MeOH), compound 1 was initially assumed to be an optically pure compound. However, the large differences between the experimental and calculated ECD curves were indicative of a scalemic mixture. Resolution via chiral-phase HPLC afforded the enantiomers (+)-1 {[α]25 D +100 (c 0.1, MeOH), 1.65 mg} and (−)-1 {[α]25 −100 (c 0.1, MeOH), D 1.33 mg} in a 1.2:1 ratio (Figure S3). Compounds 1a and 1b displayed ECD curves that were mirror images and showed CEs at ∼245 nm. On the basis of a comparison of their experimental and calculated ECD spectra (Figure 1), the respective (1S,8R,9S) and (1R,8S,9R) absolute configurations of 1a and 1b were assigned. Thus, the structures of (+)- and (−) alashanoid A (1a and 1b) were defined as sesquiterpenoids with a rare 2,2,5,9-tetramethylbicyclo[6.3.0]-undecane carbon skeleton.9−11 Compound 2 shows IR absorption bands at 3441, 1635, and 973 cm−1 that indicated the presence of hydroxy and olefinic groups. The 1H and 13C NMR data (Tables 2 and 3) displayed resonances characteristic of sesquiterpenoids including signals for four methyl carbons, four olefinic carbons, and an oxygenated tertiary carbon. These NMR data were similar to those of zerumbone (11)12 with two exceptions. First, the shielded shift of C-8 (δC 76.6; ΔδC −127.5) in 2 relative to that in 11 indicated that a hydroxy group is located at C-8, which was confirmed by the HMBC crosspeaks from H-8 to C-6 and C-10 and from H3-13 to C-8 (Figure S1). Second, the carbons of the Δ6(7) double bond in 11 were replaced by two sp3 carbons in 2, and this assignment was supported by the shielded shifts of

Table 1. 13C and 1H NMR Spectroscopic Data (125/500 MHz) for 1 in CDCl3 (δ in ppm, J in Hz) position

δC

δH

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

42.3, CH 39.5, C 40.6, CH2

1.85, m

151.8, CH 146.2, C 22.2, CH2 29.6, CH2 46.7, CH 80.3, C 218.0, C 35.5, CH2 22.8, CH3 27.2, CH3 193.9, CH 21.8, CH3

2.11, overlap 2.63, overlap 6.70, dd (6.5, 10.5) 2.91, m 2.13, overlap 1.27, m 2.14, overlap 1.97, m

2.61, overlap 2.12, overlap 0.86 (3H, s) 1.00 (3H, s) 9.50, s 1.13 (3H, s)

In the COSY spectrum, homonuclear vicinal coupling correlations between H-1 and H-8/H-11a and between H-7a and H-6a/H-8 confirmed the structural fragments from C-8 to C-11 and from C-6 to C-8 in 1. The HMBC crosspeaks from H3-15 to C-8, C-9, and C-10 and from H-11a to C-10, combined with the aforementioned COSY correlations, established the 2-hydroxy-2-methylcyclopentanone moiety. The HMBC crosspeaks from H-4 to C-2, C-6, and C-14; from H-8 to C-2 and C-6; from H3-12 to C-1; from H3-13 to C-3; and from H-14 to C-5, in combination with the COSY correlations between H-7a and H-6a/H-8, indicated that 1 contained an unusual 4,4-dimethylcycloocta-1-ene-1-carbaldehyde B

DOI: 10.1021/acs.jnatprod.7b01071 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Experimental and calculated ECD spectra of 1 and 4−10.

Table 2.

13

C NMR Spectroscopic Data (125 MHz) for 2−9 (δ in ppm)

position

2a

3a

4a

5b

6a

7a

8a

9a

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

40.8, CH2 124.9, CH 134.3, C 40.9, CH2 23.7, CH2 26.5, CH2 42.4, CH 76.6, CH 131.3, CH 137.4, CH 37.6, C 16.2, CH3 17.7, CH3 25.1, CH3 29.2, CH3

41.3, CH2 122.1, CH 137.8, C 37.7, CH2 30.6, CH2 73.0, CH 54.3. CH 201.5, C 128.1, CH 152.0, CH 40.0, C 16.4, CH3 6.1, CH3 23.0, CH3 28.9, CH3

40.5, CH2 60.7, CH 61.9, C 37.4, CH2 25.8, CH2 83.6, CH 48.1, CH 202.3, C 128.0, CH 150.8, CH 36.4, C 16.8, CH3 6.0, CH3 23.5, CH3 29.7, CH3 57.2, CH3

42.6, CH2 123.9, CH 138.0, C 41.6, CH2 23.4, CH2 28.6, CH2 56.5, CH2 206.3, C 127.8, CH 155.0, CH 40.4, C 16.7, CH3 61.8, CH2 26.0, CH3 26.8, CH3

41.3, CH2 122.1, CH 138.1, CH 39.0, CH2 29.0, CH2 82.3, CH 56.5, CH 201.1, C 127.8, CH 152.9, CH 40.3, C 16.4, CH3 58.2, CH2 23.1, CH3 29.0, CH3 56.5, CH3

41.6, CH2 123.6, CH 137.0, C 39.8, CH2 32.5, CH2 82.4, CH 60.8, CH 203.2, C 126.3, CH 155.6, CH 39.2, C 16.5, CH3 60.5, CH2 23.8, CH3 28.9, CH3 57.7, CH3

41.7, CH2 56.7, CH 63.6, C 70.7, CH 31.2, CH2 143.9, CH 140.3, C 202.9, C 128.4, CH 158.6, CH 36.2, C 15.0, CH3 12.1, CH3 24.0, CH3 29.8, CH3

54.4, CH 38.8, CH2 78.1, CH 146.8, C 116.0, C 41.0, CH2 203.5, C 158.2, C 40.4, CH 37.9, CH2 33.2, C 29.8, CH3 22.8, CH3 10.6, CH3 108.6, CH2

a

In CDCl3. bIn methanol-d4. C

DOI: 10.1021/acs.jnatprod.7b01071 J. Nat. Prod. XXXX, XXX, XXX−XXX

D

5.05 (dd, 7.0, 8.5)

2

1.08 (3H, s)

15

b

1.10 (3H, s)

14

OMe

0.99 (3H, d, 7.0)

1.50 (3H, s)

12

13

5.17, d (16.5)

8

5.35, dd (6.0, 16.0)

1.24, overlap

4.13, d (5.0)

7

9

1.42, m

6b

10

1.62, m

1.04, m

1.20, overlap

5a

6a

1.86, dd (3.0, 12.0)

4b

5b

2.10, m

4a

In CDCl3. In CD3OD.

a

1.81, overlap

3

3a

1.06, m

2.71, d (11.0)

1.38, dd (14.0, 11.5)

1.92, d (14.0)

4a

1.16 (3H, s)

1.12 (3H, s)

1.03 (3H, s)

1.37 (3H, s)

6.15, d (16.0)

6.10, d (16.0)

2.91, m

4.22, m

1.30, m

1.11, m

3.38 (3H, s)

1.16 (3H, s)

1.28 (3H, s)

0.97 (3H, d 6.5)

1.10 (3H, s)

6.29, s

6.29, s

3.10, m

3.59, dd (8.5, 3.5)

1.46, m

0.96, overlap

2.07, dd (3.5, 12.5) 2.14, dd (14.0, 8.5)

1.95, brt (12.0)

5.08 d (3.5)

2.21, t (12.0)

1.99, dd (10.5, 12.5) 1.86, dd (3.5, 13.0)

2a

1b

1a

position

1.37 (3H, s)

6.23, d (16.0)

6.04, d (16.0)

3.26, m

3.72, dd (4.0, 5.5)

1.21, overlap

1.48, m

1.86, overlap

2.12, dd (7.0, 14.0)

5.07, dd (4.0, 11.5)

1.86, overlap

2.22, t (14.0)

6a

1.56 (3H, s)

6.25, d (16.5)

5.92, d (16.5)

2.82, q (6.0)

3.54, m

1.72−1.75 (2H, m)

2.00, m

2.23, overlap

5.17, dd (4.5, 11.5)

1.92, dd (4.5, 13.5)

2.33, overlap

7a

1.18 (3H, s)

1.17 (3H, s) 3.45 (3H, s)

1.18 (3H, s)

1.14 (3H, s)

3.30 (3H, s)

1.16 (3H, s)

1.17 (3H, s)

3.51, dd (3.5, 12.5), 3.81, dd (3.5, 12.5) 3.77, dd (4.5, 13.5), 3.98, dd (8.0, 12.0) 3.88, dd (5.5, 12.0), 4.03, dd (7.0, 11.5)

1.49 (3H, s)

6.19, d (16.5)

6.09, d (16.5)

2.69, m

1.61 (2H, overlap)

1.34 (2H, overlap)

1.91 (2H, overlap)

5.14, t (7.5)

2.04, dd (7.5, 13.0)

2.15, dd (9.0, 13.0)

5b

Table 3. 1H NMR Spectroscopic Data (500 MHz) of 2−9 (δ in ppm, J in Hz)

1.59, overlap

9a

1.09 (3H, s)

1.30 (3H, s)

1.84 (3H, s)

1.23 (3H, s)

6.00, d (16.5)

6.12, d (16.5)

6.31, d (9.0)

2.62, overlap

2.63, overlap

4.16, brs

5.00 (s), 5.13 (s)

1.59 (3H, s)

0.99 (3H, s)

1.03 (3H, s)

1.80, t (11.0)

1.75, overlap

2.58, q (8.5)

3.20, dd (8.0, 14.5)

3.09, dd (8.5, 14.5)

5.49, t (8.5)

4.21, q (5.0)

3.23, d (12.0) 1.65−1.72 (2H, m)

1.55, dd (12.5, 13.5)

1.94, d (14.0)

8a

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DOI: 10.1021/acs.jnatprod.7b01071 J. Nat. Prod. XXXX, XXX, XXX−XXX

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(ΔδH +0.098) and negative ΔδSR values of H-9 (ΔδH −0.162) and H-10 (ΔδH −0.090) (Figure 2 and Figure S23) revealed the (8R) configuration.13 The (7R,8R) absolute configuration of alashanoid B (2) was confirmed by X-ray diffraction analysis (Figure 3). Its 2D structure was described previously.14 Obtained as colorless needles, compound 3 was assigned a molecular formula of C15H24O2 from its 13C NMR and HRESIMS data. Its IR, UV, and 1H and 13C NMR data were the same as those of 2,9-humuladien-6-ol-8-one;15 however, the absolute configurations of C-6 and C-7 remained undefined. Compound 3 was optically inactive, suggesting that it was a racemate. Resolution via chiral-phase HPLC (Figure S3) afforded the enantiomers (3a and 3b), and their absolute configurations were defined as (6R,7R) and (6S,7S) using single-crystal X-ray diffraction data (Figure 3). Therefore, the structures of (−)-and (+)-2,9-humuladien-6-ol-8-one (3a and 3b) were defined as shown. Compound 4 was obtained as colorless needles. Its 13C NMR and positive-ion HRESIMS data, which exhibited a protonated molecular ion at m/z 267.1957 [M + H]+, corresponded to a molecular formula of C16H26O3 (calcd for C16H27O3, 267.1955). The IR absorption bands at 3447, 1636, and 1060 cm−1 indiated the presence of a conjugated carbonyl group. The 1H and 13C

Figure 2. Δδ (δS − δR) values (in ppm, pyridine-d5) for the MTPA esters of 2 and 9.

C-6 (δC 26.5; ΔδC −100.5) and C-7 (δC 42.4; ΔδC −118.2) and the HMBC crosspeaks from H3-13 to C-6 and C-8 (Figure S1). Thus, the 2D structure of 2 was established. The Δ2(3) and Δ9(10) double bonds were assigned 2E and 9E geometries based on the coupling constant of J9,10 (16.5 Hz) and the NOESY correlations of H-1a/H-2 and H-1b/H3-12. On the basis of the NOESY correlations from H-8 to both H-7 and H3-13, the relative configurations of C-7 and C-8 could not be assigned. A modified Mosher ester analysis was conducted, and the positive ΔδSR (δS − δR) values of H-7 (ΔδH +0.007) and H3-13

Figure 3. ORTEP diagrams of 2, 3a (6R,7R), 3b (6S,7S), and 4. E

DOI: 10.1021/acs.jnatprod.7b01071 J. Nat. Prod. XXXX, XXX, XXX−XXX

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NMR data (Tables 2 and 3) showed high similarity to 13.16 The differences between these two compounds were that the Δ6(7) double bond in 13 was replaced by two sp3 carbons and that a methoxy group was attached to C-6 in 4. These assignments were verified by the shielded shifts of C-6 at δC 83.6 (ΔδC − 64.0), C-7 at δC 48.1 (ΔδC − 91.2), and C-13 at δC 6.0 (ΔδC − 5.8), and the HMBC correlations between H3-13 and C-6/C-8 and between the methoxy protons and C-6 (Figure S1), thus establishing the 2D structure of 4. The J9,10 coupling constant (17.0 Hz) suggested that the Δ9(10) double bond was E configured. The NOESY correlations of H-2/(H-1a and H-6), H-6/H-7, and H3-12/H-1b (Figure S2) indicated the orientation of substituents as (2α,3β,6α,7α). However, 4 was optically inactive, and the X-ray diffraction data revealed it was also a racemate based on the space group of P−1 (crystallographic data, Supporting Information). Resolution by chiral-phase HPLC afforded a pair of enantiomers, 4a and 4b (Figure S3), and they displayed mirror-like ECD curves. The experimental ECD curve of 4a exhibited a negative CE at 235 nm and a positive CE at 308 nm, which agreed well with the calculated ECD spectrum for the (2S,3S,6S,7S) enantiomer. The (2R,3R,6R,7R) configuration for 4b was assigned based on its well-matched experimental and calculated ECD curves (Figure 1). Compounds 4a and 4b were named (+)and (−)-alashanoid C, respectively. Their 2D structure was described as a semisynthetic derivative of 13.17 Compound 5 was obtained as a colorless oil. Its molecular formula was deduced as C15H24O2 from its 13C NMR and positive-ion HRESIMS data at m/z 237.1844 [M + H]+ (calcd for C15H25O2, 237.1849). The IR absorption bands at 3441, 1635, and 1034 cm−1 indicated the presence of a hydroxy group and a conjugated carbonyl. Analysis of the 13C NMR spectroscopic data (Table 2) showed a close resemblance to those of syripinol (12).18 The major difference was that the two carbons of the Δ6(7) double bond in 12 were replaced by two sp3 carbons in 5. This assignment was supported by the shielded shifts of C-6 at δC 28.6 (ΔδC −122.8) and C-7 at δC 56.5 (ΔδC −86.4) and the HMBC crosspeaks from H-13a to C-6 and C-8 (Figure S1). The (E) configuration of the Δ2(3) double bond was defined on the basis of the NOESY correlations of H-1a/(H3-12 and H3-14) and H-1b/ (H-2 and H3-15) (Figure S2), and the Δ9(10) double bond was assigned as the E configuration based on the coupling constant of J9,10 (16.5 Hz). Similar to the aforementioned 4, compound 5 was optically inactive, suggesting it was a racemate. Resolution via chiral-phase HPLC (Figure S3) afforded enantiomers 5a and 5b, and they displayed ECD curves that were mirror images with CEs at 202, 216, 238, and 323 nm. Thus, the (7R) and (7S) absolute configurations of (−)- and (+)-alashanoid D (5a and 5b) were defined via comparison of their experimental and calculated ECD spectra (Figure 1). The molecular formula of compound 6 was defined as C16H26O3 based on its 13C NMR and HRESIMS data. The IR and 1H and 13C NMR spectroscopic data of 6 resembled those of 5 with the major differences being the deshielded resonance of C-6 at δC 82.3 (ΔδC +53.7) and the O-methyl group at δC 56.5, suggesting the presence of a methoxy group at C-6 in 6. This assignment was confirmed by the HMBC crosspeaks from the O-methyl protons to C-6 (Figure S1). The coupling constant of J9,10 (16.0 Hz) suggested that the Δ9(10) double bond was in the E configuration. The NOESY correlations of H-2/H-1b and H3-12/H-1a (Figure S2) suggested that the Δ2(3) double bond also possessed E geometry. The NOESY correlations of

H-6/H3-12 and H-9/(H-7 and H-2) (Figure S2) defined the relative configuration of 6. Compound 6 was also optically inactive, suggesting it was isolated as a racemate. Resolution using chiral-phase HPLC (Figure S3) afforded enantiomers (+)-6 and (−)-6, and they displayed mirror-like ECD curves that showed opposite CEs at 217 and 312 nm. By comparing their experimental and calculated ECD spectra (Figure 1), the respective (6R,7S)-6a and (6S,7R)-6b absolute configurations were assigned, and the compounds were named (+)-and (−)-alashanoid E, respectively. A scalemic mixture of alashanoid F (7) gave the same molecular formula as 6 on the basis of the HRESIMS and 1D NMR (Tables 2 and 3) spectroscopic data. The major differences were the chemical shifts at C-7 (δC 56.5, 60.8 for 6 and 7, respectively) and H-7 (δH 3.26, 2.82 for 6 and 7, respectively), suggesting 7 is a diastereomer of 6. The NOESY correlations of H-9/H-6, H-10/H-7, H3-12/H-4a, and H-2/ H-4b (Figure S2) assigned the relative configuration of 7. Similar to compound 1, a chiral-phase HPLC resolution afforded enantiomers 7a and 7b in a 1.1:1 ratio (Figure S3). Compounds 7a and 7b displayed ECD curves that were mirror images and showed CEs at approximately 230 and 330 nm (Figure 1), which are consistent with the calculated ECD spectra for the (6S,7S) and (6R,7R) configurations, respectively. Consequently, the structures of (+)- and (−)-alashanoid F (7a and 7b) were established. Compound 8 was obtained as a pale-yellow powder. Its 13 C NMR and positive-ion HRESIMS data, which exhibited a protonated molecular ion at m/z 251.1639, corresponded to a molecular formula of C15H22O3 (calculated for C15H23O3, 251.1642). The 1H and 13C NMR data of 8 showed a close structural resemblance to 1316 with the major difference being the deshielded C-4 at δC 70.7 (ΔδC +32.6). That shift suggested the presence of a hydroxy group at C-13 in 8 that was verified by the HMBC data (Figure S1). The J9, 10 value of 16.0 Hz suggested that the Δ9(10) double bond was E configured, and the NOESY correlations of H-6/H-5b and H3-13/H-5a defined the Δ6(7) double bond as the E geometric isomer. The (2α,3β,4α) orientations of substituents in 8 were deduced from the NOESY correlations of H-2/H3-14, H3-12/[H-4 and H-1b], and H-1b/H3-15. The (2S,3S,4S) absolute configuration was established via comparison of the experimental and calculated ECD spectra, which displayed CEs at 246, 255, and 337 nm (Figure 1). Thus, the structure of alashanoid G (8) was elucidated as depicted. Obtained as a white powder, compound 9 has a molecular formula of C15H22O2, which was deduced from its 13C NMR and HRESIMS data, exhibiting a protonated molecular ion at m/z 235.1688 [M + H]+ (calcd for C15H23O2, 235.1693). Tables 2 and 3 show that the 1H and 13C NMR data are attributable to three methyl groups, three sp3 methylenes, three sp3 methines, a terminal double bond, three quaternary carbons, and a carbonyl carbon. These diagnostic features indicated 9 was a caryophyllene-type sesquiterpenoid. Comparison of the NMR data of 9 with those of 1519 revealed that the Δ4(5) epoxide ring and the C-7 methylene in 15 were replaced by a Δ4(5) double bond and a C-7 carbonyl group in 9, respectively. These assignments were confirmed by analysis of the HMBC data (Figure S1). The (1β,3β,5E,9α) orientations of substituents were deduced from the NOESY correlations of H-1/H-3, H-1/H312, H-5/H-6b, H3-14/H-6a, and H-9/H3-13. A modified Mosher ester analysis was conducted to elucidate the absolute F

DOI: 10.1021/acs.jnatprod.7b01071 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. Putative Biosynthetic Pathway Towards the Formation of 1

to H9c2 cells.25 Compounds 6a, 7, and 11 showed protective effects toward H9c2 cells (Figure 4) at a concentration of

configuration of the C-3 stereogenic carbinol carbon. The result showed that the distribution of the ΔδRS values between 9a and 9b indicated a (3S) configuration (Figure 2). The (1R,3S,9S) absolute configuration of 9 was assigned by the well-matched calculated and experimental ECD curves, which showed CEs at 202, 229, 258, and 313 nm (Figure 1). Therefore, the structure of alashanoid H (9) was elucidated as shown. By comparing its experimental and reported MS and NMR data,20 the structure of compound 10 was assigned as 5-hydroxy4,5-dihydrocaryophyllen-3-one, which was previously isolated from Premna integrifolia, but its absolute configuration has not been defined. The NOESY correlations of H-1/H3-11, H-4/H-5, and H-9/[H-5 and H3-13] indicted the orientations of substituents as (1β,4α,5α,9α). The negative CE at 288 nm in the experimental ECD spectrum agreed well with the calculated ECD curves for the (1R,4S,5S,9S) absolute configuration (Figure 1). Zerumbone (11),12 syripinol (12),18 zerumbone epoxide (13),16 mitissimol B (14),21 suberosol A (15),19 and (4E,8E)4,7,7-trimethyl-10-oxododeca-4,8-dienal (16) were also isolated,22 and these compounds were identified by comparison of their physiochemical data with those previously reported. A putative biosynthetic pathway toward compounds 1a/1b is shown in Scheme 1. The precursor and intermediates for the biosynthesis of 1a/1b could be derived from farnesyl diphosphate (FPP).23,24 Given the large amount of zerumbone (11, 30 g) and zerumbone epoxide (13, 600 mg) and the reported synthesis of 2,2,5,9-tetramethylbicyclo[6.3.0]undecane-type products,9,10 11 and 13 might be the precursors of 1a/1b. The first step in their synthesis is catalyzed by α-humulene synthase (ZSS1),24 and the oxidation generates the intermediates zerumbone and zerumbone epoxide, which upon cyclization and oxidization afford compounds 1a/1b. The most important rearrangement is a conrotatory electrocyclic reaction of a pentadienyl cation, which leads to a trans ring junction according to the Woodward−Hoffmann rules. This cyclization of compound 13 was reported previously.10 Among these isolates, 4a/4b, 6a/6b, and 7a/7b possess unique methoxy substituents at C-6. However, these methoxy groups probably come from MeOH during the isolation process as no ESI-MS ion representing the formula of C16H26O3 of compounds 4, 6, and 7 were observed in LC-MS spectra of the acetone extract of SP stems (Figures S91−94). Considering the antimyocardial ischemic effects previously reported for the source material,5,6 the sesquiterpenoids were evaluated for protective effects against hypoxia-induced injury

Figure 4. Protective effects of compounds 6a, 7, and 11 against hypoxia-induced injury to H9c2 cells compared with the model group; **P < 0.01, ***P < 0.001.

40 μM, and their effects were compared to the control. Similarly, the in vitro effect of SP, which contains these compounds, against AMI, which is associated with inflammation, inspired us to explore the anti-inflammatory effects of these constituents against NO production in LPS-induced RAW264.7 macrophage cells.6 The result showed that compounds 5−7, 11, and 13 exhibited inhibitory effects against the production of NO with IC50 values in the range of 13.6−70.6 μM (Table 4); Table 4. Inhibitory Effects of the Isolated Compounds on NO Production in LPS-Induced RAW264.7 Cells compounda

IC50 (μM)

5a 5b 6a 6b 7 11 13 indomethacinb

70.6 33.1 27.2 41.4 18.1 13.6 22.3 33.6

a

Compounds 1−4, 8−10, and 14−16 were inactive (