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Isolation, Structure Elucidation, and Absolute Configuration of Syncarpic Acid-Conjugated Terpenoids from Rhodomyrtus tomentosa Ya-Long Zhang, Xu-Wei Zhou, Lin Wu, Xiao-Bing Wang, Ming-Hua Yang, Jun Luo, Jian-Guang Luo,* and Ling-Yi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Three new syncarpic acid-conjugated sesquiterpenoids, tomentodiones E−G (1−3), and six new syncarpic acidconjugated monoterpenoids, tomentodiones H−M (4−9), were isolated from the leaves of Rhodomyrtus tomentosa. Compounds 1−3 represent the first examples of β-calacorene-based meroterpenoids. Their structures and absolute configurations were determined by a combination of NMR and ECD spectroscopy and X-ray diffraction analysis. On the basis of ECD data analysis for isolated and synthesized compounds, an empirical rule was proposed to determine the absolute configuration at C-7′ of syncarpic acid-conjugated terpenoids. Additionally, a study of the reversal effect of multidrug resistance in doxorubicin-resistant human breast cancer cells showed that the noncytotoxic (+)-4 exerted the strongest potentiation effect of doxorubicin susceptibility, with an enhancement of 16.5-fold at a concentration of 30 μM.

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ship and to develop a convenient and reliable rule for their stereochemical assignment. In this paper, the isolation, structural elucidation, and multidrug resistance (MDR) reversal activities of these new SACTs are described. Also reported is the first empirical rule to assign the C-7′ AC of SACTs.

ecently, syncarpic acid-conjugated terpenoids (SACTs) from the family Myrtaceae have initiated considerable interest by chemists. 1 The skeleton of this class of meroterpenoids is generally composed of an alkylated syncarpic acid moiety and a terpenoid unit. The structural diversity of mono- or sesquiterpenes instills fascinating chemical structures to this class of meroterpenoids. As part of a continuing program to search for novel SACTs from Myrtaceae,1a further investigation of the leaf extract of Rhodomyrtus tomentosa (Aiton) Hassk., which is the only Rhodomyrtus species in the People’s Republic of China,2 resulted in isolation of three new syncarpic acid-conjugated sesquiterpenoids, tomentodiones E− G (1−3), along with six new syncarpic acid-conjugated monoterpenoids, tomentodiones H−M (4−9). Compounds 1−3 represent the first examples of β-calacorene-based meroterpenoids. Structure−activity relationship studies of the target compounds call for reliable determination of their absolute configurations (ACs). However, it is still a challenge to determine the AC of an SACT unless a quality crystal could be obtained. Biomimetic synthesis and electronic circular dichroism (ECD) data have been applied to assign the ACs of SACTs,1c,d,3 but these methods are tedious and prone to error.3b ECD spectroscopy has been proven to be highly sensitive to minute changes in the geometry and electronic structure of investigated molecules.4 The availability of several members of this class of meroterpenoids allowed the investigation of their structure−chiroptical properties relation© 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Air-dried and powdered leaves of R. tomentosa were extracted with 95% EtOH at room temperature. After removal of the solvent, the EtOH extract was partitioned with petroleum ether (PE), CH2Cl2, and EtOAc, respectively. Further purification of the PE-soluble fraction using various chromatographic techniques resulted in the isolation of nine new SACTs, tomentodiones E−M (1−9). Tomentodione E (1), obtained as colorless crystals, exhibited a positive HRESIMS ion at m/z 451.3204 [M + H]+ (calcd for C30H43O3, 451.3207), indicating its molecular formula to be C30H42O3. In the 1H and 13C NMR data of 1 (Table 1), four tertiary methyl signals at δH 1.45 (3H, s), 1.36 (3H, s), 1.34 (3H, s), and 1.27 (3H, s) and two carbonyl signals at δC 213.5 and 196.2 revealed the presence of a syncarpic acid moiety, which was further confirmed by the HMBC cross-peaks (Figure 1). An isopentyl group [δH 2.77 (1H, m, overlapped), 1.89 (1H, m), 0.92 (1H, m), 1.64 (1H, m, overlapped), 0.87 (3H, d, J = 6.5 Hz), and 0.96 (3H, d, J = 6.5 Hz); δC 26.6, 42.3, 25.6, 24.3, Received: November 3, 2016 Published: February 28, 2017 989

DOI: 10.1021/acs.jnatprod.6b01005 J. Nat. Prod. 2017, 80, 989−998

Journal of Natural Products

Article

Figure 1. Key HMBC and ROESY correlations of 1.

and 20.9] was attached to C-6′ of the syncarpic acid moiety, as indicated by the HMBC cross-peaks from H-7′ (δH 2.77) to C1′ (δC 169.5) and C-6′ (δC 112.9). The presence of an alkylated syncarpic acid moiety indicated that 1 was an SACT. The remaining 15 carbon atoms were classified as a substituted benzene ring and three methyl, three methylene, two methine, and one oxygenated tertiary carbon based on the HSQC crosspeaks, which indicated the presence of an aromatic sesquiterpenoid moiety. Further 2D NMR spectroscopic analysis enabled the construction of the structure of 1 (Figure 1). In the HMBC Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 1−3 in CDCl3 1 position 1 2 3 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14 15a 15b 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′a 8′b 9′ 10′ 11′ 12′ 13′ 14′ 15′ a

δH (J in Hz) 7.11 brs 7.08 brd (8.0) 7.27 d (8.0)

2.13 1.66 1.84 1.58 2.80 2.35 1.05 0.73 2.35 2.31 1.64

2.77 1.89 0.92 1.64 0.87 0.96 1.45 1.27 1.36 1.34

m m ma ma ma ma d (6.5) d (6.5) s dd (14.5, 6.5) m

ma m m m d (6.5) d (6.5) s s s s

2 δC 139.6 128.5 137.4 127.1 126.0 138.1 79.7 29.1 19.2 43.2 31.0 21.0 17.0 21.4 40.6 169.5 48.2 213.5 55.6 196.2 112.9 26.6 42.3 25.6 24.3 20.9 23.8 26.6 22.6 26.3

δH (J in Hz) 7.11 brs 7.09 brd (8.0) 7.33 d (8.0)

1.97 m 1.82 m 1.77 ma 2.61 2.30 1.06 0.79 2.36 2.20 1.74

2.77 1.90 0.90 1.62 0.86 0.93 1.43 1.30 1.36 1.34

m m d (7.0) d (7.0) s dd (14.5, 6.5) m

m m m m d (6.5) d (6.5) s s s s

3 δC 140.2 128.9 137.4 127.3 126.7 136.9 79.3 29.0 19.7 42.9 32.1 21.8 18.1 21.4 40.9 169.6 48.2 213.6 55.7 198.2 112.9 26.7 42.6 25.5 24.4 20.8 24.1 26.1 22.7 26.2

δH (J in Hz) 7.09 brs 7.02 brd (8.0) 7.11 d (8.0)

2.29 1.86 1.86 1.67 2.86 2.34 1.07 0.73 2.33 2.14 1.94

2.86 1.58 1.35 1.76 0.96 0.98 1.42 1.37 1.36 1.39

ma ma ma m ma ma d (7.5) d (6.5) s dd (15.0, 1.0) dd (15.0, 7.0)

ma m ma m d (6.5) d (6.5) s sa sa s

δC 139.5 128.6 137.3 127.1 125.9 139.0 79.8 31.8 19.2 43.2 31.5 21.0 17.1 21.4 35.3 170.0 48.1 213.4 55.9 198.1 111.6 27.0 41.6 26.4 24.0 21.4 25.3 25.3 21.8 27.2

Overlapped signals. 990



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Figure 2. ORTEP representations of 1 and 6.

spectrum, the cross-peaks between H-8 and C-6/C-7/C-9/C10, between H-9 and C-1/C-7/C-8/C-10, between H3-12/H313 and C-11/C-10, and between H3-14 and C-2/C3/C-4 proved the presence of a sesquiterpenoid moiety with a 6/6 bicyclic ring system, an isopropyl group at C-10, and a methyl group at C-3. A β-calacorene moiety in compound 1 thus emerged from the above spectroscopic data. Additionally, the HMBC cross-peaks between H-5/H-9 and C-7 and between H2-15 and C-6/C-7/C-8 established an oxa-spiro[5.5] ring. The relatively deshielded C-1′ at δC 169.5 and the heteroatombearing C-7 that resonated at δC 79.7, combined with the indices of hydrogen deficiency, suggested the presence of an ether bridge between C-7 and C-1′. The 2D structure of 1 was thus established as depicted. The relative configuration of 1 was established on the basis of a ROESY experiment. The ROESY correlations between H-7′ and H-8a/H-9b, between H-8b and H-10, between H-10/H-11 and H-2, and between H-8′b/H-5 and H-15b indicated that these protons were cofacial, respectively. Thus, the relative configuration of 1 was established as shown in Figure 1. Finally, the structure and AC of 1 were unambiguously confirmed by a Cu Kα X-ray crystallographic analysis. As shown in Figure 2, the AC of 1 was defined as (7S,7′S,10S) via the Flack absolute structure parameter of −0.07(6). Tomentodione F (2) was assigned a molecular formula of C30H42O3 by its HRESIMS ion at m/z 451.3208 [M + H]+ (calcd 451.3207). The NMR data of 2 (Table 1) resembled those of 1, suggesting that compound 2 possesses the same 2D structure as 1. This deduction was corroborated by an HMBC experiment. The relative configuration of 2 was determined by a ROESY experiment. As shown in Figure 3, the ROESY correlations of H-7′ to H2-8, H-8b to H-15a/H-10, H-15b to H-5/H-8′b, and H-2 to H-10/H-11 indicated that these protons were cofacial. Tomentodione G (3) gave the same molecular formula as 1 and 2 by HRESIMS. 2D NMR experiments were used to establish the same 2D structure. The relative configuration of 3 was deduced by a ROESY experiment. The ROESY spectrum showed cross-peaks from H-15b to H-7′/H-5, from H-10 to H2/H-8b, and from H-8a to H-8′b. On the basis of the above

Figure 3. Key ROESY correlations of 2 and 3.

results the relative configuration of 3 was elucidated as shown in Figure 3. Tomentodione H (4) possessed a molecular formula of C25H38O3 corresponding to the [M + H]+ ion at m/z 387.2890 in its HRESIMS data. Comparison of the 1D NMR data of 4 (Table 2) and 1−3 (Table 1) indicated that compound 4 was also an SACT. Besides the isopentylsyncarpic acid moiety, the remaining 10 carbon atoms including one oxygenated tertiary carbon, one quaternary carbon, and three olefinic, three methylene, and two methyl carbons were assigned to a monoterpene unit. The HMBC cross-peaks (Figure 4) from Me-10/Me-9 to C-7, from H-6 to C-7/C-5/C-3, from H-5 to C-4/C-3/C-2, and from H2-1 to C-3/C-2 suggested that the monoterpene unit was a disubstituted myrcene moiety. Furthermore, the HMBC cross-peaks from H-7′ to C-4/C-3 and from H-4 to C-6′/C-7′ indicated that the myrcene unit was linked to the isopentylsyncarpic acid moiety at C-3 and C-4, forming a dihydropyran ring. Thus, the 2D structure of 4 was established as depicted. The relative configuration of 4 was determined by the key ROESY correlations depicted in Figure 4. The correlation between H-2 and H-8′b indicated that the ethenyl and the isobutyl groups were cofacial and arbitrarily assigned to be α-oriented. Tomentodione I (5) gave the same molecular formula as compound 4 by HRESIMS. The 1D NMR data of 5 (Table 2) showed a close resemblance to those of 4, which indicated that compound 5 possesses the same 2D structure as 4. The ROESY correlation between H-7′ and H-2 suggested that compound 5 was an epimer of 4 with a different absolute configuration at C3. 991



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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 4 and 5 in CDCl3 4

5

position

δH (J in Hz)

δC

δH (J in Hz)

δC

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

5.21 brd (16.5) 5.17 brd (11.0) 5.85 dd (17.0, 11.0)

113.7

5.16 brd (11.5) 5.07 brd (17.0) 5.66 dd (17.5, 11.0)

115.3

a

1.89 1.76 1.63 2.01 5.05

dd (14.0, 7.0) dd (14.0, 6.0) m m m

1.67 s 1.58 s

2.79 1.47 1.20 1.65 0.87 0.95 1.49 1.39 1.33 1.32

m ma m ma d (6.5) d (6.5) s s s s

C-4/C-5/C-6/C-8, and between H2-5 and C-1/C-2/C-3/C-4/ C-6 indicated the presence of a monoterpenoid moiety with a 3/5 bicyclic ring system resembling a disubstituted sabinene moiety. Comparison of the NMR data of 6 with those of myrtucommulone L suggested that their structures were similar except that the isopropyl group at C-7′ in myrtucommulone L was replaced by an isobutyl group.6 The structure of 6 was confirmed on the basis of X-ray crystallography (Figure 2) using Cu Kα radiation. The Flack parameter of 0.01(7) allowed unambiguous assignment of the AC of 6 as (1R,2S,4R,7′R). The 1H and 13C NMR data of tomentodiones K−M (7−9) were similar to those of 6 (Table 3), indicating that 7−9 possessed the same syncarpic acid-conjugated sabinene skeleton. Analyses of the spectroscopic data revealed that the 2D structures of 7−9 were the same as that of 6. The relative configurations of 7−9 were determined by the ROESY experiments shown in Figure 6, respectively. Biosynthetically, compounds 1−9 are hypothesized to arise from hetero-Diels−Alder (HDA) reactions between three different terpenes, β-calacorene, myrcene, or (+)-sabinene, with 2,2,4,4-tetramethyl-6-(3-methylbutylidene)cyclohexane1,3,5-trione (Scheme S1, Supporting Information), respectively, which is consistent with the biosynthesis of rhodomentone A (10) and tomentodiones A−D (11−14).1a Leptospermone and the terpenes are considered to be the biosynthetic precursors of compounds 1−9. Although the putative precursors were not isolated from the title plant, they have been reported from other plants of the family Myrtaceae.7 In these SACTs, a dihydropyran ring with two or three stereogenic centers is thus created by the HDA reaction. Owing to the nonselective HDA reaction, SACTs usually occur as diastereoisomers or racemates in nature. From biosynthetic considerations, the ACs of compounds 2, 3, and 7−9 were inferred as drawn on the basis of the X-ray crystallographic data of compounds 1 and 6. However, it is difficult to determine the ACs of other SACTs conveniently without X-ray crystallographic data.

141.0 81.3 35.0 39.1 22.2 123.7 132.2 25.8 17.8 168.9 48.3 213.3 55.6 198.0 112.7 26.0 41.4 25.5 24.1 20.9 24.8 26.3 22.1 26.7

2.16 dd (14.0, 6.5) 1.46 dd (14.0, 11.0) 1.71 ma 2.11−2.02 m 5.11 ma 1.69 s 1.61 s

2.62 1.84 0.87 1.69 0.89 0.95 1.43 1.39 1.31 1.29

m m ma ma d (6.5) d (6.5) s s s s

139.2 81.7 37.1 41.3 22.1 123.9 132.2 25.8 17.8 169.1 48.3 213.4 55.7 197.9 113.6 26.3 42.5 25.6 24.4 21.0 24.8 26.0 23.1 25.7

Overlapped signals.

Figure 4. Key HMBC and ROESY correlations of 4.

Compounds 4 and 5 were optically inactive, similar to racemic calliviminones C and D.5 Chiral HPLC resolution of 4 and 5 (Figure 5) afforded the anticipated enantiomers (+)- and (−)-4 and (+)- and (−)-5, respectively. The separate pairs of enantiomers displayed mirror image-related ECD curves. Tomentodione J (6) was obtained as colorless crystals. Its molecular formula was established as C25H38O3 from its HRESIMS data (m/z 387.2314 [M + H]+; calcd for C25H39O3, 387.2138). On comparison with data for compounds 1−5, the 1H and 13C NMR data for 6 (Table 3) showed characteristic signals for the isopentylsyncarpic acid moiety, indicating that compound 6 might also be an SACT. On the basis of the molecular formula information, the remaining 10 carbon atoms were assigned to a monoterpenoid moiety. The HMBC cross-peaks between H3-9/H3-10 and C-4/C-8, between H-8 and C-2/C-3/C-5, between H-2 and C-1/C-3/ 992



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Figure 5. Chiral HPLC chromatograms of 4 and 5 and their ECD spectra.

Table 3. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 6−9 in CDCl3 6 position 1 2 3a 3b 4 5a 5b 6a 6b 7a 7b 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′a 8′b 9′ 10′ 11′ 12′ 13′ 14′ 15′ a

δH (J in Hz) 1.22 ma 0.49 dd (8.0, 6.0) 0.33 m 1.86 1.59 1.71 1.21 1.86 1.72 1.40 1.00 0.94

ma ma ma ma ma ma ma d (6.5) d (6.5)

2.71 1.62 1.17 1.72 0.90 0.97 1.40 1.31 1.32 1.32

m m m ma d (6.5) d (6.5) s s sa sa

7 δC 89.6 31.7 13.2 35.5 24.9 33.7 33.9 32.7 19.8 20.3 169.5 48.1 213.7 55.6 198.1 112.9 26.9 42.5 25.8 24.3 21.1 24.7 26.0 22.4 26.6

δH (J in Hz) 1.17 dd (8.5, 3.0) 0.48 dd (8.0, 5.5) 0.30 dd (5.5, 3.5) 1.92 1.68 1.78 1.28 1.94 1.73 1.45 0.99 0.91

ma ma m m ma ma m d (6.5) d (6.5)

2.85 1.56 1.05 1.68 0.88 0.97 1.38 1.35 1.33 1.32

m m m ma d (6.5) d (6.5) s s s s

8 δC 89.5 31.3 12.4 33.9 26.6 34.4 32.9 32.3 20.0 20.2 169.5 48.0 213.6 55.6 198.1 112.6 26.9 42.1 25.6 24.2 21.0 24.9 25.8 22.5 26.5

δH (J in Hz) 1.22 dd (8.0, 3.5) 0.72 m 0.34 dd (8.0, 5.0) 1.70 m 1.70 1.65 2.14 1.51 1.28 0.89 0.89

ma ma dd (14.0, 7.5) dd (14.0, 10.5) ma d (6.5)a d (6.5)a

2.91 1.81 0.92 1.70 0.91 0.97 1.36 1.32 1.33 1.29

m m ma ma d (6.5)a d (6.5) s s s s

9 δC 87.7 27.8 12.2 34.1 24.3 33.9 38.7 32.7 19.6 19.7 171.2 48.3 213.7 55.6 198.4 113.7 26.6 42.9 25.6 24.4 20.7 23.9 26.2 22.7 26.1

δH (J in Hz) 1.20 dd (8.0, 3.5) 0.80 dd (5.0, 3.0) 0.48 dd (8.0, 5.0) 1.71 ma 1.70 ma 1.24 m 2.0 dd (14.0, 8.0) 1.69 ma 1.42 ma 0.95 d (7.0) 0.89 d (7.0)

2.76 1.74 1.05 1.71 0.91 0.98 1.40 1.29 1.30 1.30

m ma m ma d (5.5) d (5.5) s s sa sa

δC 87.6 30.9 11.6 33.3 25.5 31.4 36.9 32.5 19.7 19.8 170.8 48.1 213.7 55.6 198.1 112.9 26.6 43.0 25.7 24.3 21.0 24.5 25.6 22.7 26.2

Overlapped signals.

In order to ascertain whether the ECD curves could correctly predict the AC for SACTs, the UV and ECD spectra of

compounds 1−14 were recorded in MeOH and the data are shown in Table 4. Two Cotton effects (CEs) are present in the 993



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Figure 6. Key ROESY correlations of 7−9.

Table 4. UV and ECD Data for Compounds 1−14 Recorded in Methanola compound 1 2 3 (+)-4 (−)-4 (+)-5 (−)-5 6 7 8 9 10 11 12 13 14

UV [ε (λmax)]

ECD [θ (λmax)]

21131 (204) 18882 (204) 21576 (204) 5340 (203)

14831 12582 15890 10172

5481 (204)

9207 (264)

5433 2108 2517 6019 7300 7583 5916 6341

13253 (266) 16870 (266) 9090 (266) 10751 (266) 11571 (267) 10097 (268) 12747 (267) 9832 (267) 8263 (267)

(202) (207) (202) (204) (204) (204) (204) (204)

(265) (267) (266) (265)

−19.2 (200 ) 12.7 (200sh) −15.3 (200sh) 7.3 (212) −8.3 (212) −16.0 (200sh) 16.3 (200sh) sh

7.5 (209) 8.1 (200sh) −1.6 (200sh) 25.4 (210) −8.1 (200sh) 14.9 (209) 27.0 (200sh) 20.7 (200sh)

sh

2.9 (224 )

2.7 (239) −2.7 (239)

−18.0 (260)

1.6 (222)

17.5 (265) −17.9 (264) 4.8 (263) 8.0 (264) −8.3 (265) 38.1 (263) −35.2 (264) −13.6 (265) 13.7 (266) −21.4 (264) 13.8 (265) −8.7 (287) 23.9 (267) −7.8 (271) 10.7 (267) −22.4 (266)

−4.3 (301) 4.7 (302) 2.3 (307) −5.8 (308) 5.6 (308) −10.7 (301) 9.3 (302) 4.9 (304) −6.6 (309) 7.7 (303) −4.5 (302) −13.9 (307) −4.2 (302) 1.5 (301)b −6.8 (305) 3.7 (302)

a UV values are given in M−1 cm−1 as ε (λmax/nm), and ECD values are given in ellipticity (mdeg) as θ (λmax/nm), respectively. bAn additional, lowamplitude (θ = −0.4) CE present at 321 nm.

Figure 7. Comparison of the ECD spectra of 10a−14a with 10−14.

unsaturated carbonyl group, while the β,γ-unsaturated carbonyl group mainly produced a red shift of the absorption bands (weak for 10 and 10a). Thus, the long-wavelength CE near 300 nm can be mainly attributed to the n → π* transition of the α,β-unsaturated carbonyl group, while the one arising at around 265 nm is assigned to the π → π* excitation of the same

ECD spectra in the 220−350 nm region. Additionally, five model compounds (10a−14a) were synthesized using the HDA reaction between isovaleraldehyde, 1,3-cyclohexanedione, and β-caryophyllene via the published method.1a Comparison of the ECD spectra of 11a−14a (Figure 7) with those of 11− 14, respectively, indicated that the CEs were caused by the α,β994



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chromophore.8 Furthermore, the sign of the CEs near 300 nm is opposite the sign of the CEs near 265 nm, except for those of 3 and 10. The exception for compound 3 may be caused by the π → π* transition occurring around 300 nm of the chiral tetralin chromophore, while the exception for compound 10 may be caused by its conformational flexibility in solution.1a,e The ECD band attributed to the n → π* transition near 300 nm is usually subject to the enone helicity rule.8 The data collated in Table 4 showed that the investigated compounds fall into two different groups, A and B. In group A, consisting of compounds 1, (+)-4, (+)-5, 7, 9−11, and 13, the sign of the 300 nm CE is negative, whereas in group B, represented by compounds 2, 3, (−)-4, (−)-5, 6, 8, 12, and 14, the sign is positive. On the basis of the NMR and X-ray data and biomimetic synthesis,1a the ACs at C-7′ of compounds 1, 11, and 13 from group A have been unambiguously determined to be S, while the ACs at C-7′ of compounds 6, 12, and 14 from group B were R. To further confirm that the AC at C-7′ of the SACTs in group A is S, while that of the SACTs in group B is R, ficifolidione (15) and its C-7′ epimer (16), whose ACs have been assigned by a combination of biomimetic synthesis and Xray data,3b were synthesized using the reported method.1a As shown in Figure 8, the CE near 300 nm of 15 is negative,

remaining compounds can be done via analysis of their ECD data, except for 3 and 10, whose CE signs have been perturbed. The AC at C-7′ in compounds (+)-4, (+)-5, 7, and 9 from group A is assigned as S and as R in compounds 2, (−)-4, (−)-5, and 8 from group B. This is consistent with the results inferred from biosynthetic considerations for compounds 2 and 7−9. Accordingly, the ACs of compounds 1−9 were determined as shown. Compounds (+)-4, (+)-5, 7, 9, 11, and 13 from group A differ from compounds (−)-5, (−)-4, 6, 8, 12, and 14 from group B only by the configuration of C-7′. Comparison of their respective ECD spectra further confirmed that the C-7′ configuration influences the sign of the n → π* CE. Comparison of the ECD spectra of 6−9, 15, and 16 with those of other compounds indicated that the double bonds in the terpenoid moiety do not influence the sign of the n → π* CE. An empirical rule to define the AC at C-7′ of SACTs involving a dihydropyran moiety may be formulated as follows: if the aliphatic chain at C-7′ is assigned as the third Cahn− Ingold−Prelog (CIP) priority substituent, a positive sign of the CE near 300 nm corresponds to a (7′R) AC; if it is assigned as the second CIP priority substituent, a positive sign of the CE near 300 nm corresponds to a (7′S) AC. This empirical rule should be applied with caution because the sign of the ECD band around 300 nm could be influenced by multiple factors. Successful single-crystal X-ray diffraction analysis of 1, 6, and 10−13 provided unambiguous stereochemical data of these compounds.1a The solid-state ECD spectra of compounds 1, 12, and 13 showed the same sign of the n → π* CE as that of the solution ECD spectra, indicating that the X-ray data may provide support for the postulated empirical rule. The X-ray data revealed that these compounds possess inherently dissymmetric chromophores, since the OC-5′−C-6′C-1′ moieties are not planar. As shown in Table 5, the torsion angles of OC-5′−C-6′C-1′ moieties deviate from ±180° by more than 10°. Thus, the observed CEs of the n → π* transition in these SACTs may be a consequence of the inherent dissymmetry of the enone chromophore. Furthermore, the sign of the n → π* CE is consistent with that of the torsion angle of the OC-5′−C-6′C-1′ enoyl moiety. Interestingly, this is opposite Kirk’s orbital helicity rule, which is presumably caused by the adjacent dihydropyran moiety.8a However, whether and how the helicity of the enone chromophore dominates the sign of the n → π* CE needs to be further investigated. After complete structural elucidation of these new SACTs, compounds 1, (+)/(−)-4, (+)/(−)-5, and 6−9 were evaluated

Figure 8. ECD spectra of compounds 15 and 16.

whereas that of 16 is positive, which is consistent with the above results. The mirror-image-related ECD curves for 15 and 16 indicated that the CEs are dominated by the C-7′ configuration. Therefore, defining the ACs at C-7′ of the

Table 5. Selected Torsion Angles (deg) Determined by X-ray Diffraction

compd

the sign of the n → π* CE

C-7′

OC-5′−C-6′C-1′

OC-5′−C-6′−C-7′

O−C-1′C-6′−C-5′

O−C-1′C-6′−C-7′

1 6 10 11 12 13

(−) (+) (−) (−) (+) (−)

S R S S R S

−157.2 153.7 −165.4 −168.2 156.2 −166.3

17.5 −21.1 10.0 9.2 −21.5 11.4

173.6 −168.4 176.0 168.1 −176.8 178.0

−0.9 6.2 0.9 −9.2 1.0 0.4

995



Article

CH2Cl2, and EtOAc. The PE, CH2Cl2, and EtOAc fractions yielded 140, 60, and 110 g after removal of the solvent, respectively. The PE fraction was subjected to silica gel CC, eluted with a gradient of PE− EtOAc (100:1, 50:1, 20:1, 8:1 = v/v), to afford seven fractions (Fr. 1− 7) based on TLC analysis. Fr. 2 (8.1 g) was rechromatographed on an ODS column with MeOH−H2O (80:20, 85:15, 90:10, 95:5, 100:0), to obtain three subfractions (Fr. 2.1−Fr. 2.3). Fr.2.1 (400 mg) was applied to a Sephadex LH-20 column and purified by preparative HPLC (CH3CN−H2O, 91:9) to yield three subfractions (Fr. 2.1.1−Fr. 2.1.3). Fr. 2.1.1 (42 mg) was purified by preparative HPLC (MeOH− H2O, 92:8) and further purified by silica gel preparative HPLC (cyclohexane−EtOAc, 60:1) to afford 4 (8 mg) and 5 (3 mg). Fr. 2.1.2 (40 mg) was further purified by preparative HPLC (MeOH−H2O, 84:16) to obtain 9 (16 mg). Fr. 2.1.3 (200 mg) was subjected to silica gel CC, eluted with cyclohexane−EtOAc (60:1), to yield three subfractions (Fr. 2.1.3.1−Fr. 2.1.3.3). Fr. 2.1.3.1 (40 mg) was separated by recycling preparative HPLC (MeOH−H2O, 88:12, and then CH3CN−H2O, 95:5) to afford 7 (8 mg) and 8 (15 mg). Fr. 2.1.3.3 (20 mg) was purified by preparative HPLC (MeOH−H2O, 84:16) and further separated by recycling preparative HPLC (CH3CN−H2O, 92:8) to give 6 (7 mg). Fr. 2.2 (1.2 g) was chromatographed on an ODS column with MeOH−H2O (82:18) to obtain three subfractions (Fr. 2.2.1−Fr. 2.2.3). Fr. 2.2.2 (130 mg) was subjected to silica gel CC, eluted with PE−EtOAc (50:1), to give four subfractions (Fr. 2.2.2.1−Fr. 2.2.2.4). Fr. 2.2.2.3 (20 mg) was further purified by recycling preparative HPLC (CH3CN−H2O, 95:5) to afford 1 (6 mg), 2 (3 mg), and 3 (2 mg). Tomentodione E (1): colorless needles (EtOH−H2O,10:1); mp 122−125 °C; [α]25D +10 (c 0.2, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2957, 2871, 2848, 1716, 1662, 1616, 1499, 1467, 1383, 1306, 1275, 1202 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 451.3204 [M + H]+ (calcd for C30H43O3, 451.3207). Tomentodione F (2): colorless gum; [α]25D −67 (c 0.2, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2957, 2872, 1716, 1655, 1611, 1467, 1384, 1364, 1304,1280, 1204 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 451.3208 [M + H]+ (calcd for C30H43O3, 451.3207). Tomentodione G (3): colorless gum; [α]25D −25 (c 0.2, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2957, 2870, 1716, 1655, 1616, 1466, 1384, 1335 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 451.3205 [M + H]+ (calcd for C30H43O3, 451.3207). Tomentodione H (4): colorless gum; [α]25D 0 (c 0.4, MeOH); UV data, see Table 4; IR (KBr) νmax 2956, 2922, 2870, 2850, 1716, 1651, 1619, 1468, 1413, 1384, 1313,1278, 1216 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 387.2890 [M + H]+ (calcd for C25H39O3, 387.2894). (+)-4: [α]25D −36 (c 0.2, MeOH); ECD data, see Table 4. (−)-4: [α]25D +36 (c 0.3, MeOH); ECD data, see Table 4. Tomentodione I (5): colorless gum; [α]25D 0 (c 0.3, MeOH); UV data, see Table 4; IR (KBr) νmax 2956, 2922, 2870, 2850, 1717, 1651, 1615, 1468, 1413, 1383, 1299, 1277, 1215 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 387.2892 [M + H]+ (calcd for C25H39O3, 387.2894). (+)-5: [α]25D +92 (c 0.1, MeOH); ECD data, see Table 4. (−)-5: [α]25D −92 (c 0.1, MeOH); ECD data, see Table 4. Tomentodione J (6): colorless needles (EtOH−H2O,10:1); mp 95−99 °C; [α]25D −43 (c 0.2, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2954, 2907, 2869, 1712, 1655, 1619, 1468, 1384, 1360, 1300, 1280 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 387.2895 [M + H]+ (calcd for C25H39O3, 387.2894). Tomentodione K (7): colorless gum; [α]25D −24 (c 0.3, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2957, 2935, 2902, 2870, 1714, 1655, 1601, 1465, 1382, 1344, 1302, 1276, 1208 cm−1; 1H and 13 C NMR data, see Table 3; HRESIMS m/z 387.2893 [M + H]+ (calcd for C25H39O3, 387.2894). Tomentodione L (8): colorless gum; [α]25D −9 (c 0.1, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2957, 2872, 1716, 1655, 1614, 1469, 1383, 1352, 1320, 1281, 1212 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 387.2890 [M + H]+ (calcd for C25H39O3, 387.2894).

for cytotoxicity using doxorubicin-resistant human breast carcinoma cells (MCF-7/DOX). All compounds were noncytotoxic at the concentration tested against the drug-sensitive and multidrug-resistant cells (IC50 > 30 μM). Therefore, their potential as MDR modulators was evaluated against MCF-7/ DOX cells, and the results are presented as the ability of these noncytotoxic meroterpenoids to potentiate doxorubicin cytotoxicity. All tested compounds at a concentration of 30 μM displayed modulation of doxorubicin susceptibility (Table 6). Compound (+)-4 exerted a high potentiation effect by 16.5 Table 6. MDR Reversal Effects of the Isolates (30 μM) on MCF-7/DOX Cells sample DOX DOX DOX DOX DOX DOX DOX DOX DOX DOX a

+ + + + + + + + + +

1 (+)-4 (−)-4 (+)-5 (−)-5 6 7 8 9 Vera

IC50 (μM)

RF value

± ± ± ± ± ± ± ± ± ±

0.9 16.5 4.7 10.1 7.4 4.5 1.0 1.2 5.6 5.4

38.70 2.14 7.52 3.51 4.76 7.80 34.44 29.99 6.35 6.59

7.20 0.43 1.51 1.10 0.38 1.24 5.57 3.54 0.79 0.06

Verapamil was used as the positive control at 5 μM.

reversal fold (RF) for MCF-7/DOX, while activity was also observed for (+)-5 (10.1-fold), (−)-5 (7.4-fold), 9 (5.6-fold), (−)-4 (4.7-fold), and 6 (4.5-fold). Furthermore, comparing the activities of (+)-4 and (−)-5, (−)-4 and (+)-5, 6 and 7, and 8 and 9, respectively, suggested that the C-7′ absolute configuration may have an influence on their activities.

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EXPERIMENTAL SECTION

General Experimental Methods. Melting points were measured on an X-4 digital display micromelting apparatus and are uncorrected. Optical rotations were recorded on a JASCO P-1020 polarimeter. UV data were collected on a Shimadzu UV-2450 spectrophotometer. ECD spectra were recorded on a JASCO 810 spectropolarimeter in MeOH; the solid-state ECD spectra were recorded on a JASCO 815 spectropolarimeter in a KBr matrix. IR spectra were recorded in KBr-disc on a Bruker Tensor 27 spectrometer. 1D and 2D NMR spectra were acquired on a Bruker AV-500 NMR instrument at 500 MHz (1H) and 125 MHz (13C) in CDCl3. ESIMS and HRESIMS data were recorded on an Agilent 1100 series LC-MSD-Trap-SL mass analyzer and an Agilent 6520B Q-TOF mass instrument, respectively. Column chromatography (CC) was done with silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), ODS (40−63 μm, Fuji, Japan), and Sephadex LH-20 (Pharmacia, Sweden). Preparative HPLC was carried out using a Shimadzu LC-6AD series instrument with a Shim-pack RP-C18 column (20 × 200 mm), a Lux 5u Cellulose-2 column (21.2 × 250 mm), a Zorbax SIL column (4.6 × 250 mm), and a Shimadzu SPD-20A detector. Analytical HPLC was performed on an Agilent 1200 series instrument with a DAD detector using a Shim-pack VP-ODS column (4.6 × 250 mm) and a Lux 5u Cellulose-2 column (4.6 × 250 mm). Plant Material. The leaves of R. tomentosa were collected from Yulin, Guangxi Province, China, in July 2014. A voucher specimen (No. RT201407) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The air-dried and powdered leaves of R. tomentosa (9 kg) were exhaustively extracted with 95% EtOH at room temperature (60 L, 3 × 7 d). The EtOH extract was concentrated under reduced pressure. The crude extract (1200 g) was suspended in H2O (2 L) and successively partitioned with PE, 996



Article

CDCl3) δ 4.91 (s, 1H), 4.90 (s, 1H), 2.78 (m, 1H), 2.43−2.24 (m, 6H), 2.11 (m, 1H), 2.00−1.91 (m, 2H), 1.86 (m, 1H), 1.73 (t, J = 10.0 Hz, 1H), 1.68−1.42 (m, 7H), 1.39−1.29 (m, 3H), 1.33 (s, 3H), 1.10 (m, 1H), 0.97 (d, J = 6.5 Hz, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.83 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 196.9, 170.7, 151.1, 120.2, 111.1, 85.9, 58.5, 46.8, 42.1, 40.6, 40.4, 37.0, 36.3, 35.4, 34.6, 29.9, 29.8, 27.4, 26.5, 25.3, 24.8, 24.0, 23.6, 22.0, 21.8, 20.8; HRESIMS m/z 385.3102 [M + H]+ (calcd for C26H41O2, 385.3101). 13a: [α]25D −51 (c 0.2, MeOH); ECD (MeOH) λmax (θ) 200 sh (+24.4), 222 (−0.6), 265 (+13.9), 297 (−5.9) nm; IR (KBr) νmax 2952, 2866, 1653, 1617, 1461, 1381, 1335, 1279, 1255, 1232, 1207 cm−1; 1H NMR (500 MHz, CDCl3) δ 4.81 (s, 1H), 4.73 (brs, 1H), 2.82 (m, 1H), 2.56 (dd, J = 18.0, 9.0 Hz, 1H), 2.43 (dt, J = 16.5, 5.5 Hz, 1H), 2.36−2.30 (m, 4H), 2.10−2.01 (m, 2H), 1.96−1.92 (m, 3H), 1.79−1.64 (m, 5H) 1.53 (m, 1H), 1.45−1.40 (m, 3H), 1.20 (s, 3H), 1.16 (m, 1H), 1.10 (m, 1H), 0.97 (s, 3H), 0.94 (s, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 197.6, 170.0, 155.2, 118.2, 109.9, 84.7, 57.5, 42.8, 41.9, 41.7, 39.3, 38.3, 37.4, 36.6, 33.5, 29.8, 29.4, 28.8, 28.4, 27.6, 24.2, 23.8, 23.3, 22.5, 22.2, 21.0; HRESIMS m/z 385.3103 [M + H]+ (calcd for C26H41O2, 385.3101). 14a: [α]25D −118 (c 0.2, MeOH); ECD (MeOH) λmax (θ) 200 sh (+41.9), 263 (−54.4), 296 (+9.9) nm; IR (KBr) νmax 2952, 2933, 2896, 2867, 1652, 1630, 1451, 1390, 1380, 1364, 1328, 1283 cm−1; 1H NMR (500 MHz, CDCl3) δ 4.82 (s, 1H), 4.69 (s, 1H), 2.70 (dd, J = 18.5, 9.5 Hz, 1H), 2.47−2.37 (m, 3H), 2.27−2.13 (m, 4H), 2.07 (m, 2H), 1.98−1.85 (m, 4H) 1.71 (m, 1H), 1.63−1.53 (m, 4H), 1.50−1.38 (m, 4H), 1.00 (s, 6H), 0.98 (s, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 197.4, 170.6, 155.4, 116.4, 110.1, 83.1, 55.3, 42.9, 40.3, 39.6, 39.2, 38.5, 37.3, 36.9, 35.1, 34.6, 33.5, 29.8, 29.7, 25.6, 24.5, 24.1, 22.6, 22.4, 20.0, 19.8; HRESIMS m/z 385.3100 [M + H]+ (calcd for C26H41O2, 385.3101). Preparation of Ficifolidione 15 and Its C-7′ Epimer 16. Syncarpic acid1a (54 mg, 0.30 mmol) and ZnI2 (57 mg, 0.18 mmol) in toluene (8 mL) were treated with isovaleraldehyde (66 μL, 0.61 mmol) and β-pinene (141 μL, 0.90 mmol) under reflux at 110 °C. After 1 h, the yellow mixture was cooled to rt and brine (10 mL) and EtOAc (10 mL) were added. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The resulting yellow oil was subjected to silica gel CC, eluted with a gradient of PE− EtOAc (20:1), with the purer fractions further purified by recycling preparative HPLC (MeOH−H2O, 88:12) to yield 15 (38 mg, 33%) and 16 (41 mg, 35%). The 1H and 13C NMR spectra are consistent with reported data.3 15: [α]25D +46 (c 0.1, MeOH); ECD (MeOH) λmax (θ) 200 sh (−16.6), 266 (+24.1), 303 (−8.2) nm. 16: [α]25D −86 (c 0.2, MeOH); ECD (MeOH) λmax (θ) 200 sh (+2.0), 266 (−24.7), 305 (+6.6) nm. Cytotoxicity Assay. MCF-7/DOX cells were cultured in RPMI 1640 containing 10% fetal bovine serum, harvested with trypsin, and resuspended in a final concentration of 5.0 × 104 cells/mL. Aliquots (0.1 mL) of cell suspension were seeded evenly into 96-well culture multiplates and incubated in a 37 °C incubator containing 5% CO2 for 24 h. A series of concentrations for the isolates in DMSO were added to designated wells. After 48 h, an MTT assay was performed as described previously.10 MDR Reversal Assay. The MDR reversal assay was performed as previously reported with slight modifications.11 MCF-7/DOX cells were distributed into 96-well culture plates at 5.0 × 103 cells per well. A full range of concentrations of DOX with or without 30 μM samples or 5 μM verapamil (positive control) were added to the cells. After 48 h, the MTT assay was performed as described above. IC50 values of DOX were calculated from plotted results using untreated cells as 100%. The reversal fold, in terms of potency of reversal, was calculated using the following formula: reversal fold (RF) = IC50(MCF-7/DOX cells)/IC50(MCF-7/DOX cells combined with sample treatment). All assays were performed in triplicate.

Tomentodione M (9): colorless gum; [α]25D +94 (c 0.2, MeOH); UV and ECD data, see Table 4; IR (KBr) νmax 2958, 2872, 1716, 1654, 1614, 1469, 1383, 1364, 1317, 1284 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 387.2897 [M + H]+ (calcd for C25H39O3, 387.2894). X-ray Crystallographic Analysis of 1 and 6. Crystal data were obtained on a Bruker Smart-1000 CCD with a graphite monochromator with Cu Kα radiation (λ = 1.541 84 Å) at 291(2) K. The structures were solved by direct methods using SHELXS-97 and expanded using difference Fourier techniques, refined with SHELXL97. Crystallographic data for 1 and 6 have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44 (0)1223-336033 or e-mail: [email protected]). Crystallographic data of 1: C30H42O3 (M = 450.63); orthorhombic crystal (0.37 × 0.25 × 0.22 mm); space group P212121; unit cell dimensions a = 6.12360(10) Å, b = 20.4801(2) Å, c = 21.9533(2) Å, V = 2753.21(6) Å3; Z = 4; Dcalcd = 1.087 mg/mm3; μ = 0.528 mm−1; 24 865 reflections measured (8.054 ≤ 2θ ≤ 139.386); 5152 unique (Rint = 0.0229), which were used in all calculations; the final refinement gave R1 = 0.0415 (>2σ(I)) and wR2 = 0.1234 (all data); Flack parameter = −0.07(6). CCDC number: 1501260. Crystallographic data of 6: C25H38O3 (M = 386.55); orthorhombic crystal (0.36 × 0.21 × 0.20 mm); space group P21; unit cell dimensions a = 6.0388(2) Å, b = 20.9417(8) Å, c = 9.9049(4) Å, V = 1204.88(8) Å3; Z = 2; Dcalcd = 1.065 mg/mm3; μ = 0.529 mm−1; 13 465 reflections measured (9.282 ≤ 2θ ≤ 138.994); 4413 unique (Rint = 0.0215), which were used in all calculations; the final refinement gave R1 = 0.0390 (>2σ(I)) and wR2 = 0.1049 (all data). CCDC number: 1501261. Chiral HPLC Analysis of 4 and 5. Compounds 4 and 5 were resolved by a Lux 5u Cellulose-2 column [detection at 254 nm, eluted with CH3CN−H2O (80:20 for 4; 75:25 for 5) at 1 mL/min]. Preparation of 10a−14a. 1,3-Cyclohexanedione (50 mg, 0.45 mmol) and ZnI2 (86 mg, 0.27 mmol) in toluene (8 mL) were treated with isovaleraldehyde (97 μL, 0.90 mmol) and caryophyllene (306 μL, 1.35 mmol) under reflux at 110 °C. After 1 h, the yellow mixture was cooled to rt, and brine (10 mL) and EtOAc (10 mL) were added. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The resulting yellow oil was subjected to silica gel CC, eluted with a gradient of PE−EtOAc (18:1, 8:1 = v/v), with the purer fractions further purified by recycling preparative HPLC (MeOH−H2O, 95:5) to afford 10a (11 mg, 6%), 11a (23 mg, 13%), 12a (48 mg, 28%), 13a (15 mg, 9%), and 14a (5 mg, 3%). 10a: [α]25D −34 (c 0.1, MeOH); ECD (MeOH) λmax (θ) 215 (+5.2), 259 (−9.0), 288 sh (−2.1), 297 (−1.8) nm; IR (KBr) νmax 2951, 2866, 1651, 1620, 1461, 1387, 1290, 1258, 1228 cm−1; 1H NMR (500 MHz, CDCl3)9 δ 5.30 (brd, 1H), 2.83 (m, 1H), 2.50−2.23 (m, 6H), 2.14−1.77 (m, 10H), 1.69 (s, 3H), 1.65 (m, 1H), 1.56 (m, 1H), 1.47 (m, 1H), 1.19 (m, 1H), 1.05 (d, J = 6.0 Hz, 3H), 0.96−0.91 (m, 10H); 13C NMR (125 MHz, CDCl3)9 δ 197.5, 169.8, 43.1, 37.5, 29.3, 26.0, 24.0, 21.4, 21.1, 20.9; HRESIMS m/z 385.3099 [M + H]+ (calcd for C26H41O2, 385.3101). 11a: [α]25D +106 (c 0.2, MeOH); ECD (MeOH) λmax (θ) 200 sh (−17.3), 264 (+59.8), 297 (−11.2), 307 (−13.9) nm; IR (KBr) νmax 2951, 2867, 1654, 1628, 1446, 1384, 1330, 1280, 1257, 1207 cm−1; 1H NMR (500 MHz, CDCl3) δ 4.88 (s, 1H), 4.86 (s, 1H), 2.44−2.37 (m, 4H), 2.26−1.98 (m, 7H), 1.94−1.72 (m, 6H), 1.70−1.60 (m, 2H), 1.49−1.30 (m, 4H), 1.01 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.79 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 197.5, 170.6, 152.7, 116.6, 110.6, 82.8, 52.2, 42.3, 40.4, 38.2, 37.5, 37.3, 36.7, 35.5, 34.4, 33.8, 33.4, 30.6, 29.7, 25.4, 24.5, 24.2, 22.34, 22.31, 20.8, 20.0; HRESIMS m/z 385.3098 [M + H]+ (calcd for C26H41O2, 385.3101). 12a: [α]25D +47 (c 0.3, MeOH); ECD (MeOH) λmax (θ) 200 sh (+23.4), 266 (−13.4), 297 (+4.0), nm; IR (KBr) νmax 2952, 2930, 2865, 1651, 1621, 1456, 1383, 1306, 1280 cm−1; 1H NMR (500 MHz, 997



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Phytochemistry 2000, 53, 975−979. (c) Brophy, J. J.; Goldsack, R. J. J. Essent. Oil Res. 2007, 19, 26−27. (8) (a) Kirk, D. N. Tetrahedron 1986, 42, 777−818. (b) Frelek, J.; Butkiewicz, A.; Gorecki, M.; Wojcieszczyk, R. K.; Luboradzki, R.; Kwit, M.; Rode, M. F.; Szczepek, W. J. RSC Adv. 2014, 4, 43977−43993. (c) Frelek, J.; Szczepek, W. J.; Weiss, H. P. Tetrahedron: Asymmetry 1995, 6, 1419−1430. (d) Frelek, J.; Szczepek, W. J.; Weiss, H. P. Tetrahedron: Asymmetry 1993, 4, 411−424. (e) Masnyk, M.; Butkiewicz, A.; Gorecki, M.; Roman, L.; Bannwarth, C.; Grimme, S.; Frelek, J. J. Org. Chem. 2016, 81, 4588−4600. (9) Owing to the atropisomerism of 10a in solution, several proton signals were broadened and several carbon resonances were absent, which was consistent with rhodomentone A (10). (10) Xia, Y. Z.; Yang, L.; Wang, Z. D.; Guo, C.; Zhang, C.; Geng, Y. D.; Kong, L. Y. RSC Adv. 2015, 5, 13972−13984. (11) (a) Li, Q. M.; Luo, J. G.; Wang, R. Z.; Wang, X. B.; Yang, M. H.; Luo, J.; Kong, L. Y. Sci. Rep. 2016, 6, 29744. (b) Yuan, W. Q.; Zhang, R. R.; Wang, J.; Ma, Y.; Li, W. X.; Jiang, R. W.; Cai, S. H. Oncotarget 2016, 7, 31466−31483.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01005. NMR spectra, ECD spectra in methanol, IR spectra, and HRESIMS spectra of compounds 1−9, 10a−14a, 15, and 16; solid-state ECD spectra of compounds 1, 12, and 13; UV spectra of compounds 1−9; specific rotations of compounds 1, 6−8, 10−13, 15, and 16 at 405 nm (PDF) Crystallographic data for 1 (CIF) Crystallographic data for 6 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.-G. Luo). *E-mail: [email protected] (L.-Y. Kong). ORCID

Ling-Yi Kong: 0000-0001-9712-2618 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Sciences Foundation of China (Program No. 31470416), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63).

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REFERENCES

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