Guaiane-Type Sesquiterpenes from Curcuma phaeocaulis and Their

Jun 11, 2013 - Shenyang 110016, People,s Republic of China. §. School of ..... 1.29 (3H, s), 1.38 (3H, s), and 1.90 (3H, d, J = 0.7 Hz) and one olefi...
0 downloads 0 Views 379KB Size
Download PDF
Article pubs.acs.org/jnp

Guaiane-Type Sesquiterpenes from Curcuma phaeocaulis and Their Inhibitory Effects on Nitric Oxide Production Yue Liu,†,‡ JiangHao Ma,†,‡ Qian Zhao,†,‡ ChunRu Liao,†,‡ LiQin Ding,†,‡ LiXia Chen,†,‡ Feng Zhao,*,§ and Feng Qiu*,†,‡ †

Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China § School of Pharmacy, Yantai University, Yantai 264005, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Ten new guaiane-type sesquiterpenes (1−10), phaeocaulisins A−J, and 18 known guaiane derivatives were isolated from rhizomes of Curcuma phaeocaulis. Their structures were established on the basis of extensive spectroscopic analyses, X-ray crystallographic analysis, and comparison with literature data. Compound 10 is the first example of a norsesquiterpene with this unusual skeleton isolated from the genus Curcuma. All of the isolated compounds were tested for inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages. Compounds 1, 2, 20, and 22−24 inhibited nitric oxide production with IC50 values less than 2 μM. Preliminary structure−activity relationships for these compounds are discussed.

Curcuma phaeocaulis Valeton (Zingiberaceae), common name rhizoma curcumae, is widely distributed in southern regions of the People’s Republic of China including Sichuan, Yunnan, Guangdong, and Fujian Provinces. The rhizomes are an important crude drug frequently listed in prescriptions of traditional Chinese medicine for the treatment of Oketsu syndromes,1 which are caused by the obstruction of blood circulation, such as arthralgia, psychataxia, and dysmenorrhea. Previous studies indicated that the main bioactive constituents of rhizoma curcumae are diarylheptanoids1,2 and sesquiterpenoids,3−5 which possess anti-inflammatory,6,7 antitumor,8−10 antioxidant,11,12 vasorelaxant,13 hepatoprotective,14,15 and neuroprotective16 activities. According to the Chinese pharmacopoeia, three species of rhizoma curcumae (Curcuma phaeocaulis Valeton, Curcuma kwangsiensis S.G. Lee et C.F. Liang, and Curcuma wenyujin Y.H. Chen et C. Ling) are officially approved as Chinese medicine.17 Recently we reported the chemical constituents of C. kwangsiensis and C. wenyujin as well as their inhibitory activities against LPS-induced nitric oxide production.1−5 Sesquiterpenes were the major compounds isolated from C. wenyujin, while diarylheptanoids were the major compounds isolated from C. kwangsiensis. Both compound types exhibited significant inhibitory activity against nitric oxide production. As part of our ongoing research on the genus Curcuma, and in order to provide a potential explanation for usage of these three species as Chinese herbal medicine in the treatment of inflammatory diseases, an ethanol extract of dried rhizomes of C. phaeocaulis was investigated. In this paper, we describe the isolation, structural elucidation, anti-inflammatory evaluation, and structure− activity relationship of 10 new sesquiterpenes and 18 known guaiane derivatives. © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION The rhizomes of C. phaeocaulis were cut into pieces and extracted with 95% aqueous ethanol, and the extract was successively partitioned with cyclohexane, EtOAc, and n-BuOH. The cyclohexane and EtOAc extracts were subjected to silica gel, Sephadex LH-20, ODS open column chromatography (CC), and preparative Received: March 20, 2013

A

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

and 250 nm (Δε −0.43) indicated the absolute configuration at C8 to be S. Therefore, the absolute configuration of 1 was determined to be 1R, 4S, 5S, 8S, 10R. Phaeocaulisin B (2) had the molecular formula C15H22O4 based on HRESIMS (m/z 267.1598 [M + H]+) and NMR data. The UV spectrum (220 nm) and the IR spectrum (3447, 1734, 1650, and 1116 cm−1) suggested the presence of an α,βunsaturated γ-lactone ring and OH groups. The 1H NMR spectrum showed methyl signals at δH 1.10 (3H, s), 1.45 (3H, s), and 1.78 (3H, s). The 13C NMR data indicated 15 carbons, including an ester carbonyl at δC 177.1, olefinic carbons at δC 122.3 and 168.2, and oxygenated carbons at δC 74.6, 81.9, and 85.6. The 13C NMR spectrum of 2 was similar to that of zedoalactone A,25 and HMBC correlations from Me-14 to C-3/ C-4/C-5, from Me-15 to C-1/C-9/C-10, from H-1 to C-4/C-5/ C-15, and from H-5 to C-1/C-2/C-4/C-14 suggested that 2 possessed the same planar structure as zedoalactone A. The relative configuration of 2 was elucidated through NOESY experiments, which revealed correlations between H-8 and H-5 and between H-1 and Me-14/Me-15. The absolute configuration at C-8 of 2 was determined to be R on the basis of the Cotton effect at 218 nm (Δε −0.16) and 250 nm (Δε +0.39) due to the α,β-unsaturated γ-lactone moiety.15,18,26 Thus, the absolute configuration of 2 was determined to be 1S, 4R, 5R, 8R, 10R. Phaeocaulisin C (3) was determined to have the same molecular formula as 2 on the basis of the HRESIMS and NMR spectra. The UV, IR, 1H NMR, and 13C NMR spectroscopic data were similar to those of 2, and we speculated that 3 possessed the same planar structure as 2. The 2D NMR (HSQC and HMBC) confirmed this conclusion. The relative configuration of 3 was determined by the NOESY correlations of H-1 with H-8/Me-14, and H-5 with Me-15. The α,β-unsaturated γ-lactone moiety15,18,26 provided strong contributions to the CD spectrum, and the Cotton effect observed [228 nm (Δε +0.81), 248 nm (Δε −4.76)] indicated that the absolute configuration of 3 is 1S, 4R, 5R, 8S, 10S. The guaianolide zedoalactone E (3a, Figure 2) was recently isolated from C. wenyujin.3 However, the authors proposed that

HPLC to yield 10 new guaiane sesquiterpenes (1−10) and 18 known compounds: isocurcumenol (11),14 curcumenol (12),14 4-epicurcumenol (13),18 cis-guai-6-en-10-ol (14),19 alismoxide (15),20 7α,11α-epoxy-5β-hydroxy-9-guaiaen-8-one (16),21 procurcumadiol (17),22 procurcumenol (18),15 zedoarondiol (19),23 isozedoarondiol (20),23 (1S,4S,5S,10R)-zedoarondiol (21),3 zedoalactone B (22),24 zedoalactone D (23),3 zedoalactone A (24),25 zedoalactone C (25),25 zedoarolide B (26),18 zedoarolide A (27),18 and curcumadionol (28).5 The structures of the known compounds can be seen in the Supporting Information. Phaeocaulisin A (1) was assigned the molecular formula C15H20O5 on the basis of HRESIMS (m/z 281.1387 [M + H]+) and NMR data. The IR spectrum showed absorptions indicating OH (3452 cm−1) and carbonyl (1756 cm−1) groups, and the UV spectrum revealed an absorption maximum at 215 nm indicating the presence of an α,β-unsaturated γ-lactone.18 The 1H NMR spectrum had signals corresponding to three methyl groups δH 1.11 (3H, s), 1.34 (3H, s), and 1.74 (3H, d, J = 1.92 Hz). The 13C NMR data indicated the presence of an ester carbonyl carbon (δC 174.3), two olefinic carbons (δC 118.8, 162.4), two oxygenated carbons (δC 76.9, 80.8), and three methyl carbons (δC 7.9, 25.9, 27.9). The 1H NMR and 13C NMR spectra of 1 were similar to those of zedoarolide B, which had been obtained previously from zedoariae rhizoma.18 However, a distinctive difference in the 13C NMR spectrum was observed between these two molecules. The chemical shift of C-1 in 1 was δC 96.3 rather than δC 53.2 as in zedoarolide B. Consideration of the degrees of unsaturation and the chemical shifts suggested that there was an oxygen bridge between C-1 and C-8. This deduction was supported by the HMBC spectra, which showed correlations from Me-13 to C-7/ C-11/C-12, from Me-14 to C-3/C-4/C-5, from Me-15 to C-1/ C-10/C-9, and from H-5 to C-1/C-4/C-6/C-7/C-14. NOESY correlations of Me-14 with H-3β, H-6β, Me-13, Me-15 with H-9β, H-2β, H-2α, H-5 with H-2α, H-3α, H-6α, H-6β, and H-6α with H-9α were also observed. These correlations suggested α-orientation for H-5 and β-orientation for Me-15 and Me-14. The structure and relative configuration of 1 were confirmed unequivocally by X-ray crystallography (Figure 1). Finally, the

Figure 2. Structure revision of 3a.

structure should be revised as 3. This revision is based on the following evidence: (1) The compound (4S,5S)-germacrone4,5-epoxide is well known as the key biosynthetic precursor of guaiane-type sesquiterpenes in this genus. The cyclization reaction is initiated by protonation at an epoxide oxygen atom, followed by cleavage of the epoxide ring and the formation of a C−C bond between C-1 and C-5 to give guaiane-type derivatives having a cis configuration between the OH at C-4 and H-5.27,28 (2) NOESY correlations of H-1 with H-8/Me-14 and of H-5 with Me-15 were detected, but no cross-peak was observed between Me-14 and H-5 in our current study (Supporting Information Figure S.19-1). (3) The 13C NMR data of 3a are in accordance with those of 3 (Table 1). (4) The absolute configuration of 3 was determined by the CD spectrum. Phaeocaulisin D (4) showed IR absorptions attributed to OH (3417 cm−1) and conjugated carbonyl (1703 cm−1) groups.

Figure 1. X-ray crystal structure of 1.

absolute configuration of 1 was assessed using the empirical rule for α,β-unsaturated-γ-lactones in the circular dichroic (CD) spectrum.15,18,26 The characteristic Cotton effect at 225 nm (Δε +2.2) B

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Data for Compounds 1−4 in Methanol-d4a 1 position

δC

1 2

96.3 28.5

3

39.8

4 5 6

80.8 50.4 21.1

7 8 9

162.4 107.1 47.1

10 11 12 13 14 15

76.9 118.8 174.3 7.9 25.9 27.9

δH (J in Hz) 1.58, m 2.02, m 1.82, m 1.76, m 2.56, d (8.8) 2.87, d (16.8) 2.78, ddd (16.8, 8.8, 2.0)

2.22, d (13.8) 2.12, d (13.8)

1.74, d (1.92) 1.11, s 1.34, s

2 δC 54.8 25.3 41.7 74.6 50.9 22.5 168.2 85.6 42.6 81.9 122.3 177.1 8.2 21.9 28.2

3

δH (J in Hz) 1.30, t (10.5) 2.02, m 1.73, m 2.00, m 1.66, m

54.1 24.3 41.4 81.0 49.5 30.2

2.38, m 2.68, m 2.59, m 5.05, brd (10.7) 1.63, m 1.74, m

1.78, s 1.10, s 1.45, s

δC

166.2 80.9 47.0 73.1 122.4 176.7 8.3 23.0 23.3

δH (J in Hz) 2.00, m 1.64, m 1.83, m 1.62, m 1.69, m 1.56, td (12.4,2.5) 3.03, dd (15.0, 2.5) 2.16, m 5.17, brd (10.5) 1.73, m 2.24, dd (14.6,2.8)

1.79, s 1.22, s 1.18, s

4 δC 148.5 32.7 40.1

δH (J in Hz) 2.77, m 2.91, m 2.17, m 2.04, m

84.9 151.9 131.7

7.87, s

157.5 188.6 140.9

6.97, s

148.5 75.5 29.1 29.3 27.6 24.6

1.54, s 1.55, s 1.40, s 2.28, s

a1 H NMR data of 1 and 3 were measured at 600 MHz, while 1H NMR data of 2 and 4 were measured at 300 MHz. 13C NMR spectra of 1−4 were measured at 75 MHz.

ketone.31 Thus, the absolute configuration of 5 was determined to be 1R, 4S, 5S, 10S. Phaeocaulisin F (6) was deduced to have the molecular formula C15H22O3 by HRESIMS and NMR spectra. The UV and IR spectra indicated the presence of α,β-unsaturated carbonyl, OH, and carbonyl groups. Comparison of the 1H and 13C NMR data of 6 and the known compound procurcumenol15 indicated a hydroxymethyl moiety in 6 rather than a methyl group in procurcumenol. This was confirmed by the key HMBC correlations of H-15 with C-1/C-9/C-10. NOESY correlation between H-1 and Me-14, but no correlation between H-1 and H-5, suggested that H-1 was α-oriented and H-5 was β-oriented. The CD spectrum indicated that 6 had the same absolute configuration32 as that of procurcumenol. Thus, the absolute configuration of 6 was determined to be 1R, 4S, 5S. Phaeocaulisin G (7) had the molecular formula C15H20O4 (by HRESIMS). The 1H NMR spectrum showed methyl signals at δH 1.29 (3H, s), 1.38 (3H, s), and 1.90 (3H, d, J = 0.7 Hz) and one olefinic signal at δH 5.90 (1H, s). The 13C NMR data indicated 15 carbon resonances, including an ester carbonyl at δC 172.9, four olefinic carbons at δC 121.8, 125.2, 152.8, and 153.9, and two oxygenated carbons at δC 71.7 and 81.1. Comparison of the 1H and 13C NMR data of 7 with those of zedoalactone B24 suggested that the 1-OH group in zedoalactone B was replaced by a proton at δH 2.52 in 7. This deduction was supported by HMBC correlations from Me-14 to C-3/C-4/C-5, from Me-15 to C-1/C-9/ C-10, from H-5 to C-1/C-4/C-6/C-7/C-8/C-10/C-14, and from H-9 to C-1/C-7/C-8/C-10/C-15. NOESY correlations of H-1 with H-5/Me-15 determined the relative configuration of 7. Therefore, the structure of 7 was concluded to be 4β,10αdihydroxy-5βH-guai-7(11),8-dien-12,8-olide. The UV, IR, 1H NMR, and 13C NMR data of phaeocaulisin H (8, C15H20O5) were similar to those of zedoalactone B.24 Key HMBC correlations from Me-14 to C-3/C-4/C-5, from Me-15 to C-1/C-9/C-10, from H-5 to C-1/C-4/C-6/C-7/C-10/C-14, and from H-6 to C-1/C-5/C-7/C-8/C-11 confirmed this conclusion. The relative configuration of 8 was determined by

The molecular formula was determined to be C15H20O3 from its HRESIMS and NMR data. The 1H NMR spectrum showed four methyl signals at δH 1.40 (3H, s), 1.54 (3H, s), 1.55 (3H, s), and 2.28 (3H, s) and two olefinic protons at δH 6.97 (1H, s) and 7.87(1H, s). The 13C NMR spectrum revealed 15 carbon resonances, including a conjugated carbonyl carbon (δC 188.6), six olefinic carbons (δC 131.7, 140.9, 148.5, 148.5, 151.9, 157.5), and two oxygenated carbons (δC 75.5, 84.9). The 1H and 13C NMR spectra of 4 were similar to those of curcumadionol.5 The major difference between 4 and curcumadionol was the absence of one carbonyl carbon and one olefinic proton, while a new oxygenated carbon was detected in 4. The degrees of unsaturation and chemical shifts suggested a ring between C-4 and C-5, consistent with a guaiane skeleton. This was confirmed by the 2D NMR data of 4, especially by the key HMBC correlations of Me-14 with C-4/C-5 and H-6 with C-4/C-5. The S-configuration of C-4 was supported by a positive Cotton effect at 350 nm in the Rh2(OCOCF3)4-induced CD spectrum based on the empirical bulkiness rule proposed by Snatzke.29,30 Phaeocaulisin E (5) had the molecular formula C15H24O3, as indicated by HRESIMS and NMR data. The UV spectrum (252 nm) indicated the presence of an α,β-unsaturated carbonyl moiety, and the IR spectrum showed bands corresponding to OH (3422 cm−1) and carbonyl (1669 cm−1) groups. Comparison of the 1H and 13C NMR data of 5 with zedoarondiol23 indicated that 5 possessed the same planar structure as zedoarondiol. HMBC correlations from Me-14 to C-3/C-4/C-5, from Me-15 to C-1/C-9/C-10, from H-6 to C-1/C-4/C-5/C-7/C-8/C-11, and from H-9 to C-1/C-7/C-8/C-10/C-15 confirmed the above conclusion. NOESY correlations of H-1 with Me-14/Me-15 suggested that 5 should be a 10-epimer of zedoarondiol. The absolute configuration of 5 was determined by the CD spectrum. The CD curve of 5 showed a negative Cotton effect at 318 nm (Δε −0.2) that was attributed to the n−π* transition of an α,βunsaturated ketone and a positive Cotton effect (250 nm, Δε +1.2) that was attributed to the π−π* transition of an α,β-unsaturated C

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. 1H and 13C NMR Data for Compounds 5−8a 5

6

position

δC

δH (J in Hz)

δC

1 2

54.7 21.4

1.67, m 1.61, m

49.1 27.2

3

39.9

1.60, m 1.71, m

40.8

4 5 6

80.3 50.1 28.0

7 8 9

135.8 205.3 57.3

10 11 12 13 14 15

71.4 139.7 22.0 22.8 22.0 30.0

1.54, m 1.94, m 2.80, d (15.4)

2.46, d (12.0) 2.88, d (12.0)

1.78, d (0.9) 1.86, d (1.8) 1.13, s 1.23, s

80.5 55.3 29.7 137.9 202.5 126.4 160.4 138.2 22.7 21.4 24.5 64.5

δH (J in Hz) 2.53, m 1.62, m 2.01, m 1.75, m 1.83, m 1.94, td (13.8, 2.5) 2.28, m 2.65, d (15.8)

7 δC 51.9 26.6 42.1 81.1 50.7 24.8

6.16, s

152.8 153.9 121.8

1.77, s 1.81, s 1.24, s 4.21, td (16.3, 6.2)

71.7 125.2 172.9 8.3 23.1 31.2

8b

8

δH (J in Hz) 2.52, m 1.75, m 1.81, m 1.74, m

2.04, m 2.81, dd (14.1, 3.6) 3.12, td (14.1, 1.3)

δC 83.5 35.8 41.4 80.5 51.8 22.3

5.90, s

151.7 148.2 120.0

1.90, d (0.7) 1.29, s 1.38, s

77.7 127.2 172.3 8.7 22.8 25.2

δH (J in Hz) 1.57, m 2.45, m 1.59, m 1.80, m 2.06, dd (11.2, 4.9) 2.69, m

δC 83.2 36.3 42.0 79.7 51.9 22.4

δH (J in Hz) 2.02, m 2.88, m 2.08, m

2.58, dd (12.8, 3.1) 3.00, dd (17.2, 2.8) 3.16, m

5.51, s

151.3 147.6 120.1

6.02, s

1.86, s 1.26, s 1.40, s

77.3 126.5 170.8 9.1 23.8 25.8

1.77, d (0.9) 1.69, s 1.72, s

a1

H NMR spectra were measured at 600 MHz; 13C NMR spectra were measured at 75 MHz; spectrum of compound 5 was obtained in CDCl3, and spectra of 6−8 were obtained in methanol-d4. bObtained in C5D5N.

37.72, 37.75, 42.8), and two methyl (δC 22.7 and 24.9) groups. The 13C NMR data were similar to those of aerugidiol, which was previously isolated from C. aeruginosa,32 and the major difference was the absence of the −CC(CH3)2 unit in 10. The inference was confirmed by HMBC correlations from Me-11 to C-3/C-4/ C-5, from Me-12 to C-1/C-9/C-10, from H-7 to C-5/C-6/C-8, and from H-9 to C-1/C-7/C-10/C-12. In the NOESY spectrum, no cross-peak was observed between Me-11 and H-5, indicating that the orientations of Me-11 and H-5 were the same as in aerugidiol. The CD spectrum of 10 was similar to that of aerugidiol, indicating the same absolute configuration.32 Thus, the absolute configuration of 10 was determined to be 1S, 4S, 5R. Nitric oxide (NO) plays an important role in the inflammatory process, and an inhibitor of NO production may be considered as a potential anti-inflammatory agent. In the present study, all isolated compounds were tested for their inhibitory effects on NO production induced by LPS in macrophages. The IC50 values (Table 4) suggested that most of the compounds exhibited potent inhibitory activities against NO production (IC50 1.3 to 9.8 μM), as expected for α,β-unsaturated-γ-lactone sesquiterpenes. Some sesquiterpenes without an α,β-unsaturated γ-lactone (such as compounds 4 and 20) also showed strong inhibition of NO production. Comparison of the potency of inhibitory effects of the guaianolides revealed that the H atom at C-8 was of pivotal importance. Compound 1 showed a strong inhibitory effect due to the oxygen bridge between C-1 and C-8. A similar phenomenon was observed for compounds 7, 8, 22, and 23 due to the double bond between C-8 and C-9. Furthermore, compound 26 showed potent inhibition since the H-8 was substituted by the hydroxy group. It is noteworthy that the α-orientation of H-8 is beneficial for inhibition of NO production (in compounds such as 2 and 24) and the β-orientation of H-8 might decrease inhibitory effects (in compounds such as 3 and 25). Meanwhile, the epoxide group appears to decrease activity against NO production, in compounds such as 16 and 27, with IC50 values of 57.4 and 87.1 μM, respectively. Cytotoxic activities of these compounds against RAW 264.7 macrophages were also evaluated by

NOESY correlations of Me-15 with H-5/H-6β and of Me-14 with H-6α, suggesting that 8 had the same configuration as zedoalactone B except for the OH at C-1, which indicated that 8 was the 1-epimer of zedoalactone B. This conclusion was supported by the significant pyridine-induced solvent shifts24,33 (Table 2) for H-2α (δ methanol − δ pyridine = −0.45 ppm), H-3α (−0.28 ppm), and H-6α (−0.47 ppm). Therefore, the structure of 8 was determined to be 1α,4β,10α-trihydroxy-5βHguai-7(11),8-dien-12,8-olide. Phaeocaulisin I (9) had the same molecular formula as 7. The 1 H NMR spectrum showed methyl signals at δH 0.97 (3H, d, J = 6.9 Hz), 1.29 (3H, s), and 1.83 (3H, s) and one olefinic signal at δH 5.71 (1H, s). The 13C NMR spectrum indicated 15 carbons, including an ester carbonyl at δC 172.3, four olefinic carbons at δC 121.5, 129.1, 149.0, and 149.4, and two oxygenated carbons at δC 73.0 and 83.2. Comparison of the 1H and 13C NMR data of 9 with those of 7 indicated the absence of one hydroxy group at C-4 or C-10. The HMBC spectrum of 9 showed correlations from Me-14 to C-3/C-4/C-5, from Me-15 to C-1/C-9/C-10, from H-9 to C-1/C-8/C-15, and from H-6 to C-1/C-4/C-5/ C-7/C-8/C-11, confirming that OH groups were located at C-5 and C-10, respectively. NOESY correlations of H-1 with H-2α/ H-3α/Me-14/Me-15 and of H-4 with H-2β/H-3β/H-6β suggested that H-4 was β-oriented and that H-1/Me-14/Me-15 were α-oriented. Finally, the pyridine-induced solvent shifts24,33 (Table 2) for H-4β (δ methanol − δ pyridine = −0.3 ppm), H-2β (−0.4 ppm), H-3β (−0.25 ppm), and H-6β (−0.16 ppm) supported the β-orientation of the OH group at C-5. Thus, the structure of 9 was determined to be 5β,10β-dihydroxy-1α,4βHguai-7(11),8-dien-12,8-olide. Phaeocaulisin J (10) had the molecular formula C12H18O3. The 1H NMR spectrum showed methyl signals at δH 1.31 (3H, s) and 1.97 (3H, s) and an olefinic proton signal at δH 5.70 (1H, s). The 13C NMR and DEPT-135 spectra indicated 12 carbon resonances, including an α,β-unsaturated conjugated carbonyl (δC 203.8), two olefinic (δC 155.2 and 126.2), two oxygenated (δC 84.0 and 87.4), a methine (δC 61.3), four methylene (δC 21.9, D

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

to TMS and expressed in δ values (ppm), with coupling constants reported in Hz. HRESIMS were obtained on an Agilent 6210 TOF mass spectrometer. Silica gel GF254 prepared for TLC and silica gel (200− 300 mesh) for column chromatography (CC) were obtained from Qingdao Marine Chemical Factory (Qingdao, People’s Republic of China). Sephadex LH-20 was a product of Pharmacia. Octadecyl silica gel was purchased from Merck Chemical Company Ltd. RP-HPLC separations were conducted using a LC-6AD liquid chromatograph with a YMC Pack ODS-A column (250 × 20 mm, 5 μm, 120 Å) and SPD-10A VP UV/vis detector. Data collection and X-ray analysis of the crystal were performed on a Rigaku RAXIS-RAPID instrument equipped with a narrow-focus, 5.4 kW sealed tube X-ray source (graphite-monochromated Mo Kα radiation, λ = 0.71073 Å). All reagents were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company. Spots were detected on TLC plates under UV light or by heating after spraying with anisaldehyde-H2SO4 reagent. Plant Material. Rhizomes of C. phaeocaulis were collected from Sichuan Province, China, and identified by Professor Qishi Sun, Department of Pharmaceutical Botany, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (CP-20100715) has been deposited in the herbarium of the Department of Natural Products Chemistry, Shenyang Pharmaceutical University. Extraction and Isolation. The rhizomes of C. phaeocaulis (10 kg) were cut into approximately 2 cm pieces and extracted with 95% EtOH (100 L × 2 h × 2). The resulting extract (0.6 kg) was concentrated in vacuo, suspended in H2O (3 L), and partitioned successively with cyclohexane, EtOAc, and n-BuOH (3 L × 3). The EtOAc extract (105 g) was subjected to silica gel CC (10 × 80 cm) eluted with cyclohexane/ acetone (100:1, 40:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain six fractions (EA−EF). Fraction EA (23 g) was subjected to a silica gel column (6 × 80 cm) and eluted with CH2Cl2/EtOAc (from 40:1 to 0:1) to produce seven fractions (EA1−EA7). Fraction EB (10.6 g) was subjected to a silica gel column (6 × 80 cm) and eluted with CH2Cl2/ acetone (from 40:1 to 0:1) to yield EB1−EB6. Separation of EB4 (1.4 g) on a reversed-phase C18 silica gel column (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) yielded fractions EB4-1 to EB4-5. EB4-4 (90 mg) was purified by preparative TLC (CH2Cl2/acetone, 3:1) to obtain 10 (37.4 mg). EB5 (2.3 g) was subjected to RP-C18 silica gel CC (2.5 × 30 cm) eluted with MeOH/ H2O (1:9 to 8:2) to yield EB5-1 and EB5-2. EB5-1 (400 mg) was separated by HPLC (50% MeOH/H2O) to afford compounds 28 (70 mg), 9 (8 mg), and 5 (160 mg). Fraction EC (15 g) was subjected to silica gel CC (6 × 80 cm) eluted with a gradient of increasing acetone (0−100%) in n-hexane to afford fractions EC1−EC7. EC2 (7.8 g) was chromatographed over RP-C18 silica gel (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) to give five fractions, EC2-1 to EC2-5. EC2-2 (5.6 g) was separated by HPLC (50% MeOH/ H2O) to afford compounds 19 (2.1 g), 20 (1.4 g), and 21 (35 mg). Separation of EC7 (2 g) using an RP-C18 silica gel column (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0 v/v) provided fractions EC7-1 to EC7-6, and subfraction EC7-3 (80 mg) was separated by preparative HPLC (40% MeOH/H2O) to give compound 7 (10 mg). EC7-4 (120 mg) was separated by HPLC (50% MeOH/H2O) to give compound 4 (15 mg). Fraction EE (15 g) was subjected to silica gel CC (6 × 80 cm), eluted with a gradient of increasing MeOH (0−100%) in CH2Cl2, to give fractions EE1−EE4. EE2 (4.8 g) was chromatographed over RP-C18 silica gel (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0) to give fractions EE2-1 to EE2-3, and EE2-2 (120 mg) was purified by preparative HPLC (40% MeOH/H2O) to obtain compound 27 (70 mg). EE2-3 (195 mg) was separated by preparative HPLC (40% MeOH/H2O) to afford compounds 6 (15 mg), 25 (40 mg), 24 (17 mg), and 8 (35 mg). EE3 (2.8 g) was chromatographed over RP-C18 silica gel column (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0) to give fractions EE3-1 to EE3-5, and EE3-3 (300 mg) was separated by HPLC (30% MeOH/ H2O) to afford compounds 22 (18 mg), 23 (35 mg) and 26 (44 mg). Fraction EE4 (3.6 g) was separated by RP-C18 silica gel CC (2.5 × 30 cm) eluted with MeOH/H2O (30:70, 50:50, 70:30, and 100:0) to give six fractions, and EE4-3 (232 mg) was purified by preparative

Table 3. 1H and 13C NMR Data for Compounds 9−10a 9b

9 position 1 2

δC

δH (J in Hz)

53.5 2.19, td (7.7, 1.7) 26.5 2.00, m 1.90, m 32.4 2.02, m 1.24, m 50.4 2.02, m 83.2 37.3 2.60, dd (17.6, 1.9) 2.99, d (17.6)

3 4 5 6

7 8 9 10 11 12 13 14 15

149.0 149.4 121.5 73.0 129.1 172.3 8.7 16.6 30.1

5.71, s

1.83, s 0.97, d (6.9) 1.29, s

δC

δH (J in Hz)

10 δC

52.9 2.22, m 87.4 26.2 2.40, m 37.75 1.98, m 32.0 2.27, m 37.72 1.31, m 50.0 2.32, m 84.0 82.5 61.3 37.1 2.55, dd 21.9 (17.4, 1.5) 3.15, d (17.4) 148.3 42.8 148.4 203.8 121.7 6.14, s 126.2 71.9 155.2 128.3 24.9 170.7 22.7 9.1 1.81, s 16.9 0.98, d (7.1) 30.8 1.49, s

δH (J in Hz) 1.76, m 2.21, m 1.98, m 2.04, m 1.96, m 1.43, m 1.75, m 2.54, m 5.70, s 1.31, s 1.97, s

a1

H NMR spectra were measured at 600 MHz; 13C NMR spectra were measured at 75 MHz; spectrum of compound 9 was obtained in methanol-d4, and spectrum of 10 was obtained in CDCl3. bObtained in C5D5N.

Table 4. Inhibitory Effects of Compounds 1−28 on NO Production Induced by LPS in Macrophages compound

IC50a (μM)

compound

IC50a (μM)

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

1.5 1.9 57.9 5.9 10.3 12.9 2.9 3.0 30.1 19.6 9.8 4.2 52.2 11.4 4.3

16 17 18 19 20 21 22 23 24 25 26 27 28 indomethacinb hydrocortisoneb

57.4 11.7 13.7 54.2 1.4 16.7 1.3 1.6 1.6 61.8 4.6 87.1 54.2 12.1 43.8

a Inhibitory effects of compounds 1−28 against LPS-induced NO production in RAW 264.7 macrophages. bPositive control.

the MTT assay, and none of the compounds exhibited significant cytotoxicity at their effective concentration for the inhibition of NO production.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an X-4 digital display micromelting point apparatus and are uncorrected. Optical rotations were measured with a PerkinElmer 241 polarimeter. UV spectra were recorded on a Shimadzu UV 2201 spectrophotometer, and IR spectra were recorded on a Bruker IFS 55 spectrometer. CD spectra were determined on a Bio-Logic Science MOS-450 spectrometer. NMR experiments were performed on Bruker ARX-300 and AV-600 spectrometers. Chemical shifts are stated relative E

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

1624, 1383 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Table 3; HRESIMS m/z 287.1259 [M + Na]+ (calcd for C15H20O4Na, 287.1259). Phaeocaulisin J (10): pale yellow oil (MeOH); [α]25D −44.6 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 236 (3.79) nm; CD (MeOH) nm (Δε) 220 (−0.95), 278 (+0.55), 320 (+0.6); IR (KBr) νmax 3264, 2930, 1705, 1651, 1421 cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data, see Table 3; HRESIMS m/z 233.1150 [M + Na]+ (calcd for C12H18O3Na, 233.1148). X-ray Crystallographic Analysis of Phaeocaulisin A (1). Data collection and structural analysis were performed on a Rigaku RAXISRAPID instrument equipped with a narrow-focus, 5.4 kW sealed tube X-ray source (graphite-monochromated Mo Kα radiation, λ = 0.71073 Å). The data were collected at a temperature of 20 ± 2 °C. Data processing was accomplished with the PROCESS-AUTO processing program. Direct methods were used to solve the structure using the SHELXL crystallographic software package. All non-hydrogen atoms were easily found from the difference Fourier map. All hydrogen atoms of the molecule were placed by geometric considerations and were added to the structure factor calculation. All non-hydrogen atoms were refined anisotropically. Crystallographic data for the structures of 1 have been deposited in the Cambridge Crystallographic Data Centre database (deposition number CCDC 922211). Copies of the data can be obtained free of charge from the CCDC at www.ccdc.cam.ac.uk. Crystallographic data for 1: C15H20O5, M = 280.31, colorless block (MeOH), size 0.36 × 0.30 × 0.26 mm3, orthorhombic, space group P212121; a = 7.2956(15) Å, b = 10.729(2) Å, c = 18.181(4) Å, V = 1423.2(5) Å3, T = 298(2) K, Z = 4, ρcalcd = 1.308 g/cm−3, μ = 0.098 mm−1, F(000) = 600, 13 785 reflections in h (−8/9), k (−13/12), l (−23/23), measured in the range 3.01° ≤ θ ≤ 27.44°, completeness θmax = 99.8%, 3246 independent reflections, Rint = 0.0406, 3246 reflections with |F|2 ≥ 2σ|F|2, 187 parameters, 0 restraints, GOF = 1.056. Final R indices: R1 = 0.0465, wR2 = 0.1144. R indices (all data): R1 = 0.0559, wR2 = 0.1193; largest difference peak and hole = 0.178 and −0.205 e Å−3. NO Production Bioassay. The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction. Briefly, RAW 264.7 cells were seeded into 96-well tissue culture plates at a density of 1 × 105 cells/well and stimulated with 1 μg/mL of LPS in the presence or absence of test compounds. After incubation at 37 °C for 24 h, 100 μL of cell-free supernatant was mixed with 100 μL of Griess reagent (mixture of equal volumes of reagent A and reagent B, A: 1% (w/v) sulfanilamide in 5% (w/v) phosphoric acid, B: 0.1% (w/v) N-(1-naphthyl)ethylenediamine). Absorbance was measured in a microplate reader at 540 nm. Nitrite concentrations and the inhibitory rates were calculated by a calibration curve prepared with sodium nitrite standards.1,34

HPLC (30% MeOH/H2O) to obtain compounds 1 (40 mg), 2 (75 mg), and 3 (35 mg). The cyclohexane extract (170 g) was subjected to silica gel CC (10 × 80 cm) eluted with hexanes/EtOAc (100:1, 40:1, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/v) to obtain fractions Q1−Q8. Fraction Q2 (20 g) was subjected to silica gel CC (6 × 80 cm) eluted with hexanes/EtOAc (from 40:1 to 0:1) to produce seven fractions (Q2.1−Q2.7). Q2.7 (140 mg) was purified by PTLC (CH2Cl2/EtOAc) to obtain 14 (35.1 mg). Fraction Q3 (12 g) was recrystallized to give 11 (7.7 g). Fraction Q4 (40 g) was recrystallized to give 12 (30.5 g). Fraction Q5 (12 g) was chromatographed over a silica gel column (6 × 80 cm) eluted with CH2Cl2/EtOAc (from 40:1 to 0:1) to produce fractions Q5. 1−Q5.5. Fraction Q5.2 (1.2 g) was purified by PTLC (CH2Cl2/EtOAc) to obtain compounds 13 (370 mg) and 16 (230 mg). Fraction Q6 (12 g) was chromatographed over a silica gel column (6 × 80 cm) eluted with CH2Cl2/EtOAc (from 40:1 to 0:1) to produce fractions (Q6.1−Q6.5). Fraction Q6.4 (1.5 g) was purified by PTLC (CH2Cl2/EtOAc) to obtain compounds 15 (95 mg), 17 (29 mg), and 18 (397 mg). Phaeocaulisin A (1): colorless needles (MeOH); mp 244−245 °C; [α]25D +38.4 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 215 (3.93) nm; CD (MeOH) nm (Δε) 225 (+2.2), 250 (−0.43); IR (KBr) νmax 3452, 2971, 1756, 1689, 1458, 1022 cm−1; 1H NMR (600 MHz, CD3OD) and 13 C NMR (75 MHz, CD3OD) data, see Table 1; HRESIMS m/z 281.1387 [M + H]+ (calcd for C15H21O5, 281.1389), 303.1204 [M + Na]+ (calcd for C15H20O5Na, 303.1208). Phaeocaulisin B (2): colorless needles (MeOH); mp 172−173 °C; [α]25D +8.7 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 220 (3.94) nm; CD (MeOH) nm (Δε) 218 (−0.16), 250 (+0.39); IR (KBr) νmax 3447, 2923, 1734, 1650, 1460, 1384, 1116 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Table 1; HRESIMS m/z 267.1598 [M + H]+ (calcd for C15H23O4, 267.1596), 289.1416 [M + Na]+ (calcd for C15H22O4Na, 289.1417). Phaeocaulisin C (3): pale yellow oil; [α]25D +26.9 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 220 (4.04) nm; CD (MeOH) nm (Δε) 228 (+0.81), 248 (−4.76); IR (KBr) νmax 3425, 2922, 1731, 1642, 1449, 1384, 1117, 1209 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Table 1; HRESIMS m/z 289.1409 [M + Na]+ (calcd for C15H22O4Na, 289.1410). Phaeocaulisin D (4): pale yellow oil; [α]25D +26.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (3.88), 323 (3.42) nm; CD (CDCl3) nm (Δε) 310 (−0.31); Rh2(OCOCF3)4-induced CD (CDCl3) nm (Δε) 350 (+0.15); IR (KBr) νmax 3417, 2929, 1703, 1615, 1449, 1382 cm−1; 1 H NMR (600 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Table 1; HRESIMS m/z 271.1302 [M + Na]+ (calcd for C15H20O3Na, 271.1305). Phaeocaulisin E (5): pale yellow oil; [α]25D +26.4 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 252 (3.54) nm; CD (MeOH) nm (Δε) 250 (+1.2), 318 (−0.2); IR (KBr) νmax 3422, 2968, 1669, 1455, 1373, 1121 cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data, see Table 2; HRESIMS m/z 275.1617 [M + Na]+ (calcd for C15H24O3Na, 275.1618). Phaeocaulisin F (6): colorless oil; [α]25D +98.2 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 242 (3.72) nm; CD (MeOH) nm (Δε) 240 (−2.1), 278 (+1.9), 330 (+0.9); IR (KBr) νmax 3395, 2970, 1738, 1654, 1450, 1384 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Table 2; HRESIMS m/z 251.1647 [M + H]+ (calcd for C15H23O3, 251.1642), 273.1463 [M + Na]+ (calcd for C15H22O3Na, 273.1467). Phaeocaulisin G (7): pale yellow oil; [α]25D −46.7 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 274 (4.03) nm; IR (KBr) νmax 3437, 2924, 1745, 1634, 1383, 1118 cm−1; 1H NMR (600 MHz, CD3OD) and 13C NMR (75 MHz, CD3OD) data, see Table 2; HRESIMS m/z 287.1256 [M + Na]+ (calcd for C15H20O4Na, 287.1259). Phaeocaulisin H (8): pale yellow oil; [α]25D +60.9 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 275 (4.01) nm; IR (KBr) νmax 3402, 2943, 1743, 1633, 1384, 1125, 1030 cm−1; 1H NMR (600 MHz, CD3OD) and 13 C NMR (75 MHz, CD3OD) data, see Table 2; HRESIMS m/z 281.1383 [M + H]+ (calcd for C15H21O5, 281.1384). Phaeocaulisin I (9): pale yellow oil; [α]25D −52 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 273 (3.78) nm; IR (KBr) νmax 3415, 2924, 1748,

■

ASSOCIATED CONTENT

* Supporting Information S

MS, 1D NMR, 2D NMR, and CD spectra for new compounds 1−10 and crystallographic data of 1 together with the structures of the known compounds 11−28. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +86 24 23986463. Fax: +86 24 23993994. E-mail: [email protected] (F. Qiu), zhaofeng74@yahoo. com.cn (F. Zhao). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 30973630) and the Liaoning F

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(29) (a) Frelek, J.; Szczepek, W. J. Tetrahedron: Asymmetry 1999, 10, 1507−1520. (b) Frelek, J.; Jagodzinski, J.; Mayer-Figge, H.; Scheldrick, W. S.; Wieteska, E.; Szczepek, W. J. Chirality 2001, 13, 313−321. (30) Chen, M. H.; Gan, L. S.; Lin, S.; Wang, X. L.; Li, L.; Li, Y. H.; Zhu, C. G.; Wang, Y. N.; Jiang, B. Y.; Jiang, J. D.; Yang, Y. C.; Shi, J. G. J. Nat. Prod. 2012, 75, 1167−1176. (31) (a) Snatzke, G. Tetrahedron 1965, 21, 413−419. (b) Snatzke, G. Tetrahedron 1965, 21, 421−438. (c) Snatzke, G. Tetrahedron 1965, 21, 439−448. (32) Masuda, T.; Jitoe, A.; Nakatani, N. Chem. Lett. 1991, 9, 1625− 1628. (33) Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E. J. Am. Chem. Soc. 1968, 90, 5480−5486. (34) Dirsch, V. M.; Stuppner, H.; Vollmar, A. M. Planta Med. 1998, 64, 423−426.

Province Drug Discovery Platform Key Technology Platform under Grant No. 2009ZX09301-012.

■

REFERENCES

(1) Li, J.; Zhao, F.; Li, M. Z.; Chen, L. X.; Qiu, F. J. Nat. Prod. 2010, 73, 1667−1671. (2) Li, J.; Liao, C. R.; Wei, J. Q.; Chen, L. X.; Zhao, F.; Qiu, F. Bioorg. Med. Chem. Lett. 2011, 21, 5363−5369. (3) Lou, Y.; Zhao, F.; He, H.; Peng, K. F.; Zhou, X. H.; Chen, L. X.; Qiu, F. J. Asian Nat. Prod. Res. 2009, 11, 737−747. (4) Lou, Y.; Zhao, F.; Hu, Z. H.; Peng, K. F.; Wei, X. C.; Chen, L. X.; Qiu, F. Helv. Chim. Acta 2009, 92, 1665−1672. (5) Lou, Y.; Zhao, F.; He, H.; Peng, K. F.; Chen, L. X.; Qiu, F. Chem. Biodiversity 2010, 7, 1245−1253. (6) Tohda1, C.; Nakayama, N.; Hatanaka1, F.; Komatsu, K. Evid.-Based Complementary Altern. 2006, 3, 255−260. (7) Masuda, T.; Jitoe, A.; Isobe, J.; Nakatani, N.; Yonemori, S. Phytochemistry 1993, 32, 1557−1560. (8) Chen, X. P.; Pei, L. X.; Zhong, Z. F.; Guo, J. J.; Zhang, Q. W.; Wang, Y. T. Phytomedicine 2011, 18, 1238−1243. (9) Ali, M. S.; Tezuka, Y.; Awale, S.; Banskota, A. H.; Kadota, S. J. Nat. Prod. 2001, 64, 289−293. (10) Li, M.; Zhang, Z.; Hill, D. L.; Wang, H.; Zhang, R. Cancer Res. 2007, 67, 1988−1996. (11) Tao, Q. F.; Xu, Y.; Lam, R. Y. Y.; Schneider, B.; Dou, H.; Leung, P. S.; Shi, S. Y.; Zhou, C. X.; Yang, L. X.; Zhang, R. P.; Xiao, Y. C.; Wu, X.; Stockigt, J.; Zeng, S.; Cheng, C. H. K.; Zhao, Y. J. Nat. Prod. 2008, 71, 12−17. (12) Motterlini, R.; Foresti, R.; Bassi, R.; Green, C. J. Free Radical Biol. Med. 2000, 28, 1303−1312. (13) Matsuda, H.; Morikawa, T.; Ninomiya, K.; Yoshikawa, M. Tetrahedron 2001, 57, 8443−8453. (14) Matsuda, H.; Ninomiya, K.; Morikawa, T.; Yoshikawa, M. Bioorg. Med. Chem. Lett. 1998, 8, 339−344. (15) Matsuda, H.; Morikawa, T.; Ninomiya, K.; Yoshikawa, M. Bioorg. Med. Chem. 2001, 9, 909−916. (16) Dohare, P.; Garg, P.; Sharma, U.; Jagannathan, N. R.; Ray, M. BMC Complementary Altern. Med. 2008, 8, 55−75. (17) Pharmacopoeia Commission of People’s Republic of China. Pharmacopoeia of the People’s Republic of China (Part 1); Chinese Medical Science and Technology Press: Beijing, 2010; pp 257−258. (18) Matsuda, H.; Morikawa, T.; Toguchida, I.; Ninomiya, K.; Yoshikawa, M. Chem. Pharm. Bull. 2001, 49, 1558−1566. (19) Weyerstahl, P.; Marschall, H.; Splittgerber, U.; Wolf, D. Flavour Fragr. J. 2000, 15, 153−173. (20) Yoshikawa, M.; Yamaguchi, S.; Matsuda, H.; Kohda, Y.; Ishikawa, H.; Tanaka, N.; Yamahara, J.; Murakami, N. Chem. Pharm. Bull. 1994, 42, 1813−1816. (21) Firman, K.; Kinoshit, T.; Itai, A.; Sankawa, U. Phytochemistry 1988, 27, 3887−3891. (22) Ohshiro, M.; Kuroyanagi, M.; Ueno, A. Phytochemistry 1990, 29, 2201−2205. (23) Kuroyanagi, M.; Ueno, A.; Ujue, K. Chem. Pharm. Bull. 1987, 35, 53−59. (24) Takano, I.; Yasudam, I.; Takeya, K.; Itokawa, H. Phytochemistry 1995, 40, 1197−1200. (25) Kasahara, K.; Nomura, S.; Subeki, M. H.; Yamasaki, M.; Yamato, O.; Maede, Y.; Katakura, K.; Suzuki, M.; Trimurnigsih; Chairul; Yoshihara, T. Planta Med. 2005, 71, 482−484. (26) Sumikoa, H.; Harinantenaina, L.; Matsunami, K.; Otsuka, H.; Kawahata, M.; Yamaguchi, K. Phytochemistry 2011, 72, 2165−2171. (27) Kuroyanagi, M.; Ueno, A.; Koyama, K.; Natori, S. Chem. Pharm. Bull. 1990, 38, 55−58. (28) (a) Minoru, Y.; Cai, Y.; Cui, Z.; Hirotaka, S.; Yoshihiro, H.; Nobuo, T.; Isao, K. Chem. Pharm. Bull. 1986, 34, 434−437. (b) Kuroyanagi, M.; Shirota, O.; Sekita, S.; Nakane, T. Nat. Prod. Commun. 2012, 7, 441−446. G

dx.doi.org/10.1021/np400202f | J. Nat. Prod. XXXX, XXX, XXX−XXX