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Sesquiterpenoids with Anti-MDR Staphylococcus aureus Activities from Ferula ferulioides by Tao Liu a ), Shuangying Wang a ), Lili Xu a ), Wei Fu a ), Simon Gibbons b ), and Qing Mu* a ) a
) School of Pharmacy, Fudan University, 826 Zhangheng Road, Zhangjiang Pudong, Shanghai 201203, P. R. China (phone: þ 86-21-51980109; e-mail: [email protected]) b ) Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, WC1N 1AX, London, UK
Seven new sesquiterpenoids together with 21 known sesquiterpenoid derivatives were isolated from the medicinal plant Ferula ferulioides (Steud.) Korovin. Their structures were elucidated by comprehensive spectroscopic analyses and chemical transformations. The isolated compounds were evaluated for their antibacterial activities against a panel of bacteria including multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA), displaying minimum inhibitory concentration (MIC) values in the range of 0.5 – 128 mg/l.
Introduction. – Methicillin-resistant Staphylococcus aureus (MRSA) is presently the most frequently identified antibiotic-resistant pathogen in many parts of the world [1]. The discovery and development of new antimicrobials together with new technologies are needed to combat bacterial multidrug resistance (MDR) [2]. In the search for bioactive substances, metabolites from plants can play a useful role as bacterial resistance modulators and anti-infective agents [3]. The genus Ferula (Apiaceae) comprises ca. 150 species growing in a vast geographical region ranging from Central Asia to the Mediterranean Basin [4], and 26 species are distributed in P. R. China [5]. Ferula is a rich source of biologically active compounds, such as sesquiterpenes [6 – 9] and sesquiterpene coumarins [10 – 15]. Plant extracts and metabolites from the Ferula genus were reported to possess many biological features including antiviral [16] [17], anti-inflammatory [18], antitumor [19], anticancer [20], and antibacterial [21] activities. The species F. ferulioides (Steud.) Korovin grows in western China. It has been reported to contain a series of sesquiterpenes and derivatives [22 – 27]. In our screening for new antibacterial agents, an extract of F. ferulioides was found to possess antibacterial activity against a panel of bacteria. Apart from a pesticidally active sesquiterpene, guaiol [28], our preliminary research resulted in the isolation of two new antibacterial sesquiterpenoids that inhibit efflux pumps of drug-resistant Staphylococcus aureus strains [29]. In an extensive phytochemical investigation, seven new sesquiterpenoid derivatives, together with 21 known sesquiterpenoid derivatives were isolated. Antibacterial assays were carried out for the isolates against a panel of multidrug-resistant Staphylococcus aureus strains, and the sesquiterpene hydroxyacetophenone derivatives showed antibacterial activities with minimum inhibitory concentration (MIC) values in the range of 0.5 – 128 mg/l (1.2 – 297.7 mm). Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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Results and Discussion. – The powdered roots of F. ferulioides were extracted with 95% EtOH and partitioned between H2O and CH2Cl2 . The CH2Cl2-soluble fraction was subjected to repeated column chromatography (CC) to afford seven new compounds, 13 – 16 and 24 – 26, together with 21 known compounds 1 – 12, 17 – 23, 27, and 28 (Fig. 1). Compound 13 was obtained as yellow resin and showed a peak at m/z 455.24026 ([M þ Na] þ ) in its HR-ESI-MS spectrum, corresponding to the molecular formula C25H36O6 . Its IR spectrum indicated the presence of an intramolecular H-bonded OH group (3405 cm ¢ 1), a benzene ring (1607 cm ¢ 1), and a C¼C bond (1635 cm ¢ 1). UV absorptions at lmax 275.0 and 238.5 nm supported the presence of a characteristic 2,4dihydroxyacetophenone moiety. The 1D-NMR data (Table 1) suggested that compound 13 had a skeleton consisting of a dihydroxyphenyl ethyl ketone connected to a farnesyl moiety. This skeleton was the same as that of 9, previously isolated from the same plant [29]. In comparison with 9, compound 13 had an additional AcO group instead of a OH group at C(8) in 9. Therefore, compound 13 was the 8-AcO- and 9-OHsubstituted derivative of dshamirone (5). This structure was also verified by a careful inspection of the HMBC data of 13 (Table 1 and Fig. 2). The relative configuration of 13 was determined by a ROESY experiment, in which cross-peaks of CH2(3)/Me(15) and H¢C(8)/Me(16) were observed. This result evidenced an (E)-configuration for the C(4)¼C(5) bond, and the relative configurations of the adjacent AcO group at C(8) and of the OH group at C(9) as threo (Table 1). Therefore, compound 13 was identified as depicted in Fig. 1, with yet unknown absolute configuration, and was given the trivial name 8-acetoxy-9-hydroxydshmirone. Compound 14 was obtained as yellow resin whose molecular formula was established as C26H38O6 by HR-ESI-MS (m/z 469.25576 ([M þ Na] þ )). Its UV and IR spectral data were almost the same as those of 13. The 1H- and 13C-NMR spectra of 14 were also highly similar to those of 13, except for the presence of an additional MeO group (d(H) 3.85; d(C) 55.5). The location of the MeO group was confirmed by the difference of the 13C-NMR resonance of their C(4’) (13: d(C) 162.7; 14: d(C) 165.9) and HMBC spectra (Table 1 and Fig. 2). The chemical shifts of the signals of C(7), C(8), and Me(16), and a series of ROESY experiments indicated that 14 had the same relative configuration as 13. The relative configuration of the adjacent AcO group at C(8) and of the vicinal OH groups at C(9) was again threo, combined with an (E)-configuration for the C(4)¼C(5) bond. Therefore, compound 14 was identified as depitced in Fig. 1, and was given the trivial name 8-acetoxy-9-hydroxy-4’-methoxydshmirone. Compound 14 was synthesized from 13 by treatment with Me2SO4 and K2CO3 at room temperature (Scheme 1). The hydrolysis of compound 13 with KOH in MeOH yielded 9. Compound 9 was then converted to 10 by reacting it with TsOH and 2,2dimethoxypropane at room temperature [29]. Chemical transformation from 13 to 14, and 13 to 9 and 10 (Scheme 1) further confirmed the structure elucidation. The relative configurations at C(8) and C(9) of the four compounds were all identified as threo. Compound 15 was obtained as yellow resin whose molecular formula was established as C25H36O7 by HR-ESI-MS (m/z 449.25245 ([M þ H] þ )). The UV, IR, and NMR data (Table 2) for compound 15 suggested a 2,4-dihydroxylated phenone connected to a side chain, which was similar to that in compound 13. Compared with 13, compound 15 possessed an additional O-bearing CH group (d(H) 3.78) instead of an
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Fig. 1. Structures of compounds 1 – 28
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Table 1. NMR Data a ) (in CDCl3 ) of Compounds 13 and 14. d in ppm, J in Hz. Position
13 d( H)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1’ 2’ 3’ 4’ 5’ 6’ 4’-MeO 1’’ 2’’
14 d(C )
204.5 2.91 (t, J ¼ 7.4) 37.8 2.40 – 2.43 (m) 23.2 5.16 (t, J ¼ 6.7) 123.2 135.7 1.94 – 1.96 (m) 36.1 1.67 – 1.69 (m) 27.3 4.82 (dd, J ¼ 10.3, 2.4) 78.9 74.4 1.44 – 1.55 (m) 37.5 2.04 – 2.07 (m) 22.0 5.10 (t, J ¼ 7.0) 124.1 132.2 1.69 (s) 25.7 1.62 (s) 15.9 1.17 (s) 23.5 1.63 (s) 17.7 113.8 165.2 6.37 (overlapped) 107.7 162.7 6.37 (overlapped) 103.5 7.63 (d, J ¼ 9.5) 132.4
2.12 (s)
171.3 21.1
HMBC
d( H)
d(C )
204.5 1, 3, 4 2.92 (t, J ¼ 7.4) 37.9 1, 2, 4, 5-Me 2.42 (dt, J ¼ 7.0, 7.4) 23.1 3, 6, 5-Me 5.18 (t, J ¼ 6.7) 123.1 135.7 4, 5, 7, 8, 5-Me 1.94 – 1.96 (m) 36.1 6, 8 1.66 – 1.69 (m) 27.4 4.82 (d, J ¼ 10.1) 78.9 74.2 8, 9, 11, 12 1.42 – 1.53 (m) 37.5 12 2.03 – 2.07 (m) 22.0 11, 14, 13-Me 5.09 (t, J ¼ 7.0) 124.1 132.1 12, 13, 13-Me 1.68 (s) 25.7 4, 5 1.60 (s) 16.0 8, 9, 10 1.16 (s) 23.5 12, 13, 14 1.62 (s) 17.7 113.5 165.3 1’, 2’, 4’, 5’ 6.42 (overlapped) 107.6 165.9 1’, 2’, 3’, 4’ 6.42 (overlapped) 100.9 1, 2’, 4’, 5’ 7.64 (d, J ¼ 8.6) 131.6 3.85 (s) 55.5 171.0 1’’ 2.08 (s) 21.1
HMBC 1, 3, 4 1, 2, 4, 5 3, 6, 5-Me 4, 5, 7, 8, 5-Me 5, 6, 8
8, 9, 11, 12 12 11, 14, 13-Me 12, 13, 13-Me 4, 5 8, 9, 10 12, 13, 14
1’, 2’, 4’, 5’ 1’, 2’, 3’, 4’ 1, 2’, 4’, 5’ 4’ 1’’
a
) Key ROESY correlations of 13: H¢C(3)/Me(15); H¢C(8)/Me(16). Key ROESY correlations of 14: H¢C(3)/Me(15); H¢C(8)/Me(16).
olefinic H-atom (ca. d(H) 5 ppm) as in 13. The two C-atom signals of a double bond at d(C) 124.1 (CH) and 132.2 (C) in 13 were replaced with two O-bearing C-atom signals at d(C) 84.7 and 71.7 ppm in the side chain of 15. Compound 15 was assumed to be the 12,13-dihydroxy derivative of 13. Comparison of the molecular masses of 15 (m/z 449.2) and 13 (m/z 433.1) indicated the presence of an additional O-atom in the molecule of 15. The characteristic 13C-NMR data, 77.2/84.3 and 84.7/71.7 (Table 2), indicated the presence of an ether moiety in a tetrahydrofuran arrangement [30]. The structure of compound 15 was deduced as depicted in Fig. 1, and this was confirmed by its key HMBC spectral correlations (Fig. 2). The relative configuration of 15 was determined by its ROESY spectrum, in which correlations CH2(3)/Me(15), H¢C(8)/ Me(16), and H¢C(12)/Me(15), were observed, indicating the relative configuration at C(8) and C(9) as erythro, and an (E)-configuration for the C(4)¼C(5) bond. The configuration in the furan ring at C(9) and C(12) was assigned as cis. Compound 16 was obtained as yellow resin, and its molecular formula was deduced as C25H36O7, the same as 15, from HR-ESI-MS (m/z 449.25248 ([M þ H] þ )). The UV,
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Fig. 2. Key HMBCs (H ! C) of compounds 13 – 16 and 24 – 26
IR, NMR, HMQC, and HMBC data for compound 16 indicated a very similar structure to that of 15, with the only difference being the 13C-NMR shifts of C(12) (15: 84.7 ppm; 16: 86.8 ppm) (Table 2). Compound 16 was elucidated as the C(12)-epimer of compound 15. Therefore, the relative configuration of 16 was determined by a ROESY experiment, in which cross-peaks H¢C(12)/Me(16) were present in 15, but absent in 16. The orientation of Me(16) and H¢C(12) at furan ring was trans. Thus, compound 16 was identified as depicted in Fig. 1. The absolute configurations at C(8) and C(9) of compounds 13 – 16 remained unresolved, even though several methodologies were applied. First, MosherÏs ester method was employed. The two OH groups of the phenyl ring were protected by methylation using Me2SO4 , and compound 13a was obtained (Scheme 2). Subsequent-
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Scheme 1. Chemical Transformations of Compound 13 into 9, 10, and 14
a) Acetone, K2CO3 , Me2SO4 , r.t., 4 h; 92%. b) MeOH, KOH, r.t., 1 h; 96%. c) Me2C(OMe)2 , TsOH, r.t., 6 h; 90%.
Table 2. NMR Data a ) (in CDCl3 ) of Compounds 15 and 16. d in ppm, J in Hz. Position
15 d( H )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1’ 2’ 3’ 4’ 5’ 6’ 1’’ 2’’
2.92 (t, J ¼ 7.0) 2.40 – 2.42 (m) 5.19 (t, J ¼ 7.0) 1.94 – 1.98 (m) 1.58 – 1.94 (m) 4.96 (d, J ¼ 10.6) 1.82 – 1.84 (m) 1.56 – 1.74 (m) 3.78 (t, J ¼ 7.5) 1.24 (s) 1.60 (s) 1.21 (s) 1.15 (s)
6.39 (overlapped) 6.39 (overlapped) 7.63 (d, J ¼ 9.4) 2.08 (s)
16 d(C ) 204.6 37.7 23.4 123.4 135.4 35.8 34.0 77.2 84.3 26.5 28.1 84.7 71.7 27.4 15.9 22.8 23.9 113.6 165.1 107.6 163.4 103.4 132.5 171.4 21.3
HMBC
d( H )
1, 3, 4 1, 2, 4, 5 2, 3, 6, 5-Me
2.92 (t, J ¼ 7.4) 2.40 – 2.42 (m) 5.18 (t, J ¼ 7.2)
4, 5, 8 6, 7, 8, 5-Me 6, 7, 9, 9-Me, 1’’
1.94 – 1.98 (m) 1.58 – 1.60 (m) 4.92 (d, J ¼ 10.1)
9, 9-Me 8, 10, 12 11, 14, 13-Me
1.82 – 1.84 (m) 1.56 – 1.74 (m) 3.73
12, 13, 13-Me 4, 5 8, 9, 10 12, 13, 14
1.23 (s) 1.60 (s) 1.21 (s) 1.15 (s)
1’, 2’, 4’, 5’
6.39 (overlapped)
1’, 3’, 4’ 1, 1’, 2’, 4’
6.39 (overlapped) 7.63 (d, J ¼ 9.4)
1’’
2.09 (s)
d(C ) 204.7 37.7 23.4 123.2 135.7 35.9 34.5 77.3 84.1 26.5 28.1 86.8 71.2 27.2 15.9 22.9 23.7 113.6 165.1 107.8 163.4 103.4 132.6 171.2 21.2
HMBC 1, 3, 4 1, 2, 4, 5 2, 3, 6, 5-Me 4, 5, 8 6, 8, 5-Me 6, 7, 9, 9-Me, 1’’ 8, 9, 9-Me 10, 12 11, 14, 13-Me 12, 13, 13-Me 4, 5 8, 9, 10 12, 13, 14
1’, 2’, 4’, 5’ 1’, 3’, 4’ 1, 1’, 2’, 4’ 1’’
a ) Key ROESY correlations of 15: H¢C(3)/Me(15); Me(16)/H¢C(8), H¢C(12). Key ROESY correlations of 16: H¢C(3)/Me(15); Me(16)/H¢C(8).
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ly, the Ac group in 13a was hydrolyzed, and 13b, which contained a secondary OH group at C(8), was obtained. This compound was reacted with MosherÏs reagent under four different reaction conditions, respectively (Scheme 2). TLC Monitoring showed that no reaction occurred. It is possible that when the Ac group was removed and a OH was formed, this could form an intramolecular H-bond with the OH at C(9), rendering it unavailable for reaction. It is also possible that side-chain hindrance may reduce the availability for reaction with the MosherÏs reagent (Fig. 3). We also attempted to predict the absolute configuration of 9 by a quantum-chemical calculation using Guassian 09 [31]. The geometries of 9 and its enantiomer were Scheme 2. An Attempt to Determine the Absolute Configuration of Compound 13
a) Acetone, K2CO3 , Me2SO4 , r.t., 24 h; 83%. b) MeOH, KOH, r.t., 1 h; 95%. c) MosherÏs esterification: 1) (RS)-MTPA ( ¼ a-methoxy-a-(trifluoromethyl)phenylacetic acid), DCC ( ¼ N,N’-dicyclohexylcarbodiimide), DMAP ( ¼ 4-(dimethylamino)pyridine), pyridine; 2) (RS)-MTPA, DCC, DMAP, CH2Cl2 ; 3) (RS)-MTPA-Cl, CH2Cl2 ; 4) (RS)-MTPA-Cl, NaH, DMF.
Fig. 3. ChemBio 3D structure of compound 13
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optimized at the B3LYP/6-31G level of density-functional theory (DFT). The electronic circular dichroism (ECD) spectra was then calculated using the timedependent DFT (TDDFT) method at the B3LYP /6-311 þ þ G** level in MeCN. Unfortunately, the predicted ECD spectra of the enantiomers of 9 showed no differences (Fig. 4), implying that a lack of chromophore at the stereogenic centers C(8) and C(9) led to weak circular dispersion between the enantiomers. Therefore, the absolute configurations of the new compounds 13 – 16 have yet to be elucidated. Compound 24 was obtained as colorless resin whose molecular formula was established as C25H32O4 by HR-ESI-MS (m/z 397.23683 ([M þ H] þ )). The UV and IR data of 24 were almost the same as those of 23 (Fig. 1). The 1H- and 13C-NMR spectra of the two compounds were slightly different at C(3) (23: d(H) 4.88, d(C) 89.6; 24: d(H) 4.62, d(C) 93.0), Me¢C(2) (23: d(H) 1.47, d(C) 15.8; 24: d(H) 1.56, d(C) 13.9), and Me¢C(3) (23: d(H) 1.31, d(C) 19.2; 24: d(H) 1.47, d(C) 23.8) (Table 3). An HMBC experiment suggested that 23 and 24 are diastereoisomers. A series of ROESY experiments was carried out to determine the relative configuration of the furan moiety at C(2) and C(3). Cross-peaks between Me¢C(2) and Me¢C(3) appeared in 23, while their absence in 24 indicated that Me¢C(2) and Me¢C(3) were trans-configured in 24 (Table 3). Accordingly, the relative configurations of the furan moiety at C(2) and C(3) were (2S*) and (3S*). Finally, compound 24 was identified as depicted in Fig. 1.
Fig. 4. Calculated ECD spectra of 9a and its enantiomer 9b. The ECD spectra were calculated using the time-dependent density-functional theory (TDDFT) method at B3LYP/6-311 þ þ G** level, in the presence of MeCN by Gaussian09.
d( H)
7’, 8’, 8’-Me 2, 3, 1’ 2, 3, 3a 3’, 4’, 5’ 7’, 8’, 9’
4’, 6’, 7’ 4’, 5’, 7’, 6’, 9’
2, 3, 2’, 3’ 1’, 3’, 4’ 5’, 4’-Me
6, 7, 9a 5a, 7, 9b
5a, 7, 8, 9a
7’, 8’, 8’-Me 2, 3, 1’ 2, 3, 3a 3’, 4’, 5’ 7’, 8’, 9’ 7
3’, 4’, 6’, 7’, 4’-Me 5’, 7’, 8’ 6’, 8’-Me
2, 3, 2’, 3’, 2-Me 1’, 3’, 4’ 2’, 5’, 4’-Me
6, 7, 9a 5a, 7, 9b
4, 5a, 7, 8, 9a
2, 3a, 9b, 1’, 2-Me, 3-Me
d(C) HMBC
96.8 41.9 103.6 161.3 156.8 6.83 ( d, J ¼ 2.4 ) 100.6 163.1 6.83 (dd, J ¼ 2.4, 7.8) 112.2 7.52 (d, J ¼ 9.0) 123.7 106.2 165.0 1.79 – 1.81 (m) 41.7 2.10 – 2.13 (m) 22.1 5.09 (t, J ¼ 6.9) 123.3 135.8 1.93 – 1.95 (m) 39.9 1.41 – 1.43 (m) 22.5 1.41 – 1.43 (m) 43.4 71.0 1.21 (s) 29.2 1.45 (s) 20.5 1.29 (d, J ¼ 7.4) 14.1 1.55 (s) 15.8 1.20 (s) 29.2 3.86 (s) 55.7
d( H )
26
2, 3a, 9b, 3-Me 3.29 ( d, J ¼ 7.0)
d(C ) HMBC
97.1 3.29 (d, J ¼ 7.0) 41.5 103.2 162.3 156.6 5a, 7, 8, 9a 7.12 (d, J ¼ 2.0) 103.2 161.0 7, 9a 6.83 (dd, J ¼ 2.4, 8.6) 113.3 5a, 7 7.52 ( d, J ¼ 8.6 ) 124.1 105.6 166.0 3’ 1.79 – 1.81 (m) 41.5 4’ 2.10 – 2.13 (m) 22.1 5’, 4’-Me 5.09 (t, J ¼ 6.9) 123.5 135.8 4’, 6’, 4’-Me 1.93 – 1.95 (m) 39.8 4’, 5’, 7’ 1.42 – 1.44 (m) 22.5 5’ 1.40 – 1.42 (m) 43.3 71.4 7’, 8’ 1.21 (s) 29.1 2, 3 1.45 (s) 20.7 2, 3, 3a, 1’ 1.29 (d, J ¼ 7.1) 14.1 3’, 4’, 5’ 1.54 (s) 15.8 7’, 8’, 9’ 1.20 (s) 29.0 7
1’, 3a
d(C ) HMBC
93.0 46.6 106.1 160.7 156.9 6.85 (d, J ¼ 2.4) 100.5 163.1 6.83 (dd, J ¼ 2.4, 6.7) 112.2 7.56 ( d, J ¼ 9.4 ) 124.0 106.5 166.1 1.65 – 1.67 (m) 34.7 1.94 – 1.98 (m) 23.4 5.07 (overlapped) 123.7 135.1 1.91 – 1.93 (m) 39.6 2.02 – 2.04 (m) 26.6 5.07 (overlapped) 124.2 131.3 1.69 (s) 25.7 1.56 (d, J ¼ 6.7) 13.9 1.47 (s) 23.8 1.52 (s) 16.0 1.61 (s) 17.7 3.89 (s) 55.7
d(H ) 4.62 (q, J ¼ 6.7)
25
a ) Key ROESY correlations of 24: H¢C(3’)/CH2(5’); H¢C(2)/Me¢C(3). Key ROESY Correlations of 25: H¢C(3’)/CH2(5’); Me¢C(2)/Me¢C(3). Key ROESY correlations of 26: H¢C(3’)/CH2(1’) and CH2(5’); Me¢C(2)/Me¢C(3).
2 3 3a 4 5a 6 7 8 9 9a 9b 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 2-Me 3-Me 4’-Me 8’-Me 7-MeO
Position 24
Table 3. NMR Data a ) (in CDCl3 ) of Compounds 24 – 26. d in ppm, J in Hz.
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Compound 25 was obtained as colorless oil with the molecular formula C24H32O5 as deduced from HR-ESI-MS data (m/z 423.21364 ([M þ Na] þ )). The UV absorptions at 318 and 239 nm supported the presence of a substituted 7-hydroxycoumarin. The IR spectrum indicated a C¼O group (1718 cm ¢ 1), a benzene ring (1639 cm ¢ 1), and a double bond of a furocoumarin (1614 cm ¢ 1). The 1H- and 13C-NMR spectra of 25 exhibited signals of a 1,2,4-trisubstituted benzene ring (d(H) 6.83, 7.12, 7.52; d(C) 113.3, 103.2, 124.1), a substituted C¼C bond (d(H) 5.09; d(C) 123.5), and an ester moiety (d(C) 156.6) (Table 3). These spectral data revealed that 25 had the same skeleton as the furocoumarin compound 17, which had been isolated previously from the same plant [25]. However, in comparison with 17, two 13C signals of the C(7’)¼C(8’) bond, at d(C) 124.2 (CH) and 131.5 (C), were replaced by those of a CH2 group (d(C) 43.3) and an OH bearing quaternary C-atom (d(C) 71.4) in compound 25. The signals for two Me groups at 29.0 and 29.1 ppm supported this elucidation (Table 3). Based on the HMBC spectrum (Fig. 2), compound 25 is constructed of a coumarin, a fused dihydrofuran moiety, and a side chain. The configuration of C(3’)¼C(4’) bond was deduced as (E) from its ROESY spectrum with the cross-peaks CH2(1’)/H¢C(3’) and H¢C(3’)/ CH2(5’). The relative configurations within in the dihydrofuran moiety at C(2) and C(3) were identified as (2S*) and (3R*) by a ROESY spectrum with cross-peaks between Me¢C(2) and Me¢C(3) (Table 3). Compound 25 was therefore identified as depicted in Fig. 1. Compound 26 was also obtained as colorless oil with the molecular formula C25H34O5 as deduced from its HR-ESI-MS data (m/z 437.22822 ([M þ Na] þ )). The UV, IR, and NMR data for compound 26 indicated a similar skeleton of a furocoumarin as in 25. The only difference was that the OH group at C(7) of 25 was replaced by a MeO group (d(H) 3.86; d(C) 55.7) in compound 26 (Table 3). We confirmed the structure consisting of a coumarin, a fused dihydrofuran, and a prenyl side chain based on an HMBC spectrum (Table 3 and Fig. 2). The configuration of C(3’)¼C(4’) bond was deduced as (E) from its ROESY spectrum with the cross-peaks CH2(1’)/H¢C(3’) and H¢C(3’)/CH2(5’). The relative configurations of the dihydrofuran moiety, at C(2) and C(3), were identified as (2S*) and (3R*) by its ROESY spectrum with cross-peaks between Me¢C(2) and Me¢C(3) (Table 3). Thus, compound 26 was identified as depicted in Fig. 1. Structures of 21 known sesquiterpenes were identified by comparison of their spectroscopic data with those reported in the literature. They were 2,4-dihydroxyacetophenone (1), 2,4-dihydroxybenzoic acid (3), 2,4-dihydroxy-a-oxobenzeneacetic acid (4) [32], 2-hydroxy-4-methoxyacetophenone (2) [33], dshamirone (5) [24], (4E,8E)-1(2-hydroxy-4-methoxyphenyl)-5,9,13-trimethyltetradeca-4,8,12-trien-1-one (6) [34], (4E,8E)-1-(2,4-dihydroxyphenyl)-2-hydroxy-5,9,13-trimethyltetradeca-4,8,12-trien1-one (7), (6E)-1-(2,4-dihydroxyphenyl)-3,7,11-trimethyl-3-vinyldodeca-6,10-dien-1one (8) [24], 8,9-dihydroxydshamirone (9), 8,9-oxoisopropanyldshamirone (10), ferulaeolactone A [29], 3-(2,4-dihydroxybenzoyl)-4,5-dimethyl-5-[(3E,7E)-4,8-dimethylnona-3,7-dien-1-yl]tetrahydrofuran-2-one (11) [22], (2S*,3R*)-2,3-dihydro-7hydroxy-2,3-dimethyl-2-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (17), (2S*,3R*)-2,3-dihydro-7-methoxy-2,3-dimethyl-2-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (19), (2S*,3R*)-2,3-dihydro-7-hydroxy-2,3-dimethyl-2-[(3E)-4,8dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (18), (2R*,3R*)-2,3-dihydro-7-methoxy-
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2,3-dimethyl-2-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (20) [26], (2S*,3R*)-2,3-dihydro-7-hydroxy-2,3-dimethyl-3-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (21), (2S*,3R*)-2,3-dihydro-7-hydroxy-2,3-dimethyl-3-[(3E)-4,8dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (22), (2S*,3R*)-2,3-dihydro-7-methoxy2,3-dimethyl-3-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (23) [23], ferulin A (27), and ferulin B (28) [35]. All compounds were tested for their ability to inhibit the growth of a panel of bacteria including multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA). Most of the sesquiterpene coumarins had no effect on the bacteria. Only individual coumarin compounds, i.e., 17, 18, 21, and 22, showed inhibitory activities against the tetracycline-resistant strain XU212, which possessed the TetK tetracycline efflux protein with a promising MIC value of 2 mg/l, and those of 27 and 28 were 32 and 16 mg/l, respectively, while the MIC of control norfloxacin was 32 mg/l (Table 4). Most of the acetophenone sesquiterpene derivatives, 5 – 13, possessed activities ranging from 0.5 – 128 mg/l against multidrug-resistant S. aureus. Among the tested strains was SA-1199B, which possesses the NorA efflux protein that confers resistance to certain fluoroquinolones and quaternary ammonium antiseptics. Against this strain, 5 (16 mg/l, 44.9 mm), 7 (2 mg/l, 5.4 mm), 8 (16 mg/l, 44.9 mm), 11 (8 mg/l, 20.0 mm), 12 (8 mg/l, 19.1 mm), and 13 (16 mg/l, 37.0 mm) were more active than the control antibiotic norfloxacin (32 mg/l, 100 mm). For MDR strain XU212, which possesses the TetK efflux transporter, and is resistant to both tetracycline and methicillin, 5 (1 mg/l, 2.8 mm), 6 (1 mg/l, 2.7 mm), 7 (1 mg/l, 2.7 mm), 8 (4 mg/l, 11.2 mm), 10 (0.5 mg/l, 1.2 mm), 11 (2 mg/l, 5.0 mm), and 12 (2 mg/l, 4.8 mm) showed more effective inhibition than the control antibiotic tetracycline (128 mg/l, 288 mm). Six 7-hydroxy-coumarins, 17, 18, 21, 22, 27, and 28, also exhibited selective inhibitory activities against this strain. For erythromycin-resistant strain RN4220 that carries the MsrA macrolide efflux protein, 7 (2 mg/l, 5.4 mm), 8 (4 mg/l, 11.2 mm), 11 (4 mg/l, 10.0 mm) and 12 (16 mg/l, 38.4 mm) showed better inhibitory activities than the control antibiotic erythromycin (64 mg/l, 87.2 mm). For the common methicillin-resistant S. aureus (MRSA) strains, 7 (4 mg/l, 10.8 mm), 11 (2 mg/l, 5.0 mm), and 12 (2 mg/l, 4.8 mm) displayed significant inhibitory activities against strain EMRSA-15, and 5 (1 mg/l, 2.8 mm), 7 (2 mg/l, 5.4 mm), and 10 (0.5 mg/l, 1.2 mm) were active against strain EMRSA-16. Of note is that compound 8,9-oxoisopropanyldshamirone (10), which was previously isolated from this plant [28] and synthesized from 13 (Scheme 1), possessed significant inhibitory activity against XU212 and EMRSA-16 with an MIC value of 0.5 mg/l, while it was inactive against the other tested drug-resistant strains including the standard S. aureus strains ATCC25923. In F. ferulioides, the sesquiterpene (farnesyl) acetophenone derivatives lead to its antibacterial properties (Table 4), whilst some of the sesquiterpene (farnesyl) coumarins possess specific antibacterial activity against the tetracycline resistant strain XU212 (Table 4). Some of the examples detailed here, such as 5, 10, and 13, are noteworthy in terms of potency against bacteria. Further work to determine selectivity against mammalian cells would be of interest, as would a mechanistic study.
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Table 4. Anti-MDR and Anti-MRSA Activities (MIC [mg/l]) of the Compounds 1 – 28 Compounds
SA1199B
XU212
ATCC25923
RN4220
EMRSA-15
EMRSA-16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Norfloxacin Tetracycline Erythromycin Oxacillin Vancomycin
> 128 > 128 > 128 > 128 16 > 128 2 16 64 > 128 8 8 16 > 128 > 128 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 32 0.25 0.25 0.25 0.25
> 128 > 128 > 128 > 128 1 1 1 4 64 0.5 2 2 32 > 128 > 128 > 128 2 2 > 128 > 128 2 2 > 128 > 128 > 128 > 128 32 16 8 128 > 128 128 0.5
> 128 > 128 > 128 > 128 16 > 128 2 16 64 > 128 8 8 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 0.5 0.25 0.25 0.25 0.25
> 128 > 128 > 128 > 128 32 > 128 2 4 64 16 4 16 64 > 128 > 128 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 0.5 0.5 64 0.25 0.5
> 128 > 128 > 128 > 128 16 > 128 4 16 64 > 128 2 2 64 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 0.5 0.25 > 128 > 128 0.25
> 128 > 128 > 128 > 128 1 > 128 2 32 64 0.5 128 16 32 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 128 0.25 > 128 > 128 0.25
Experimental Part General. TLC: Silica gel HGF254 plates (SiO2 ; Qingdao Marine Chemical Plant, Yantai, P. R. China) and RP-18 plates (E. Merck Co., Ltd.); visualization with 20% H2SO4 , followed by heating. Column chromatography (CC): silica gel (SiO2 ; 10 – 40 mm; Qingdao Marine Chemical Plant, Yantai, P. R. China). M.p.: X-4 micro melting-point apparatus. Optical rotations: JASCO P-1020 polarimeter. UV Spectra: HITACHI U-2900 spectropolarimeter; lmax (log e) in nm. IR Spectra: Avatar 360 ESP FT-IR spectrophotometer; ˜n in cm ¢ 1. 1H- and 13C-NMR spectra: Varian Mercury Plus 400- and 100-MHz spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. EI-MS: Agilent 5973N MSD spectrometer; in m/z. HR-EI-MS: IonSpec 4.7 T FTMS; in m/z. Plant Material. The roots of F. ferulioides were collected in Xinjiang Uyghur Autonomous Region, China. A voucher specimen (F001) was deposited with the Natural Medicinal Chemistry Laboratory of the School of Pharmacy, Fudan University. The plant was identified by Mrs. Hui-Qin Xie, Associate Professor in the Department of Plant Protection, Shihezi University, China.
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Extraction and Isolation. Air-dried and powdered roots of F. ferulioides (2.0 kg) were extracted at 458 with 95% EtOH (3 5,000 ml, 12 h each). After removal of the solvent under reduced pressure, the residue was dissolved in CH2Cl2 (3 500 ml, 20 min each) to afford the lipophilic-soluble fraction Fr. FF (400.0 g). Fr. FF was submitted to CC (SiO2 (10 – 20 mm, 10 75 cm); petroleum ether (PE)/AcOEt 100 : 0 to 0 : 100; flow rate 30 ml/min) to afford 15 fractions of different polarity. Fr. C (1.5 g) eluted with PE/AcOEt 98 : 2 was separated by CC (SiO2 (30 – 40 mm, 1.8 40 cm); PE/acetone 99 : 1; 200 ml) to give pure 6 (390.0 mg). Fr. F (6.0 g) eluted with PE/AcOEt 95 : 5 was re-chromatographed (SiO2 (30 – 40 mm, 2.8 40 cm); PE/AcOEt 98 : 2 – 92 : 8; flow rate, 4 ml/min) to furnish five fractions, Frs. I – V. Fr. I (720.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.8 40 cm); PE/acetone 98 : 2; 300 ml) to yield compound 2 (240.0 mg). Fr. II (120.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.2 40 cm); PE/ acetone 97 : 3; 300 ml) to afford compound 26 (12.0 mg). Fr. IV was purified by CC (Agilent XDB-C18 (9.4 150 mm)) and HPLC (MeOH/H2O 88 : 12; flow rate, 3.0 ml/min; UV at 254 nm) to yield compounds 23 (tR 16.3 min; 4.0 mg) and 24 (tR 17.8 min; 4.0 mg). Fr. G (23.0 g) eluted with PE/AcOEt 95 : 5 – 90 : 10 was re-chromatographed (SiO2 (30 – 40 mm, 5.0 50 cm); PE/AcOEt 95 : 5 – 85 : 15; flow rate, 10.0 ml/min) to afford seven fractions, Fr. I – VII. Fr. I (1.2 g) was subjected to CC (SiO2 (30 – 40 mm, 1.8 40 cm); PE/acetone 96 : 4; 500 ml) to yield compounds 19 (144.0 mg) and 20 (20.0 mg). Fr. V (7.2 g) was separated by CC (SiO2 (30 – 40 mm, 4.2 50 cm); PE/acetone 95 : 5; 2000 ml) to yield compound 5 (3.5 g). Fr. I (35.0 g) eluted with PE/AcOEt 90 : 10 was re-chromatographed (SiO2 (30 – 40 mm, 5 50 cm); PE/acetone 90 : 10; flow rate, 10 ml/min) to give eleven fractions, Fr. I – XI. Fr. II (420.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.4 40 cm); PE/acetone 90 : 10; 200 ml) to yield compound 7 (14.0 mg). Fr. IV was subsequently purified by CC (Agilent XDB-C18 (9.4 150 mm)) and HPLC (MeOH/H2O 85 : 15; flow rate, 3.0 ml/min; UV at 254 nm) to furnish compounds 21 (tR 15.9 min; 35.0 mg) and 22 (tR 17.0 min; 85.0 mg). Fr. VI (230.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.2 40 cm); CH3Cl3/acetone 99 : 1; 200 ml) to afford pure compound 12 (11.0 mg). Fr. J (22.0 g) eluted with PE/AcOEt 85 : 15 was rechromatographed (SiO2 (30 – 40 mm, 5.0 50 cm); PE/acetone 95 : 5 – 80 : 20; flow rate, 10.0 ml/min) to give six fractions, Fr. I – VI. Fr. III (4.8 g) was subjected to CC (SiO2 (30 – 40 mm, 2.8 40 cm); CHCl3 /acetone 98 : 2; 2000 ml) to furnish five fractions. Fr. III-2 (2.4 g) was subjected to CC (reversed-phase (RP) C18 (E. Merck Co. Ltd., 2.4 40 cm); MeOH/H2O 80 : 20 – 85 : 15; flow rate, 1.0 ml/min) to yield compounds 11 (390.0 mg), 17 (390.0 mg), 18 (330.0 mg), and 25 (200.0 mg). Fr. VI (2.2 g) was subjected to CC (RP/C18 column (E. Merck Co. Ltd., 2.4 40 cm); MeOH/ H2O 85 : 15; flow rate, 1.0 ml/min) to yield compounds 8 (12.0 mg) and 11 (420.0 mg). Fr. K (102.0 g) was submitted to CC (SiO2 (20 – 30 mm, 7.5 65 cm); PE/AcOEt 85 : 15 to 0 : 100; flow rate, 25 ml/min) to afford ten fractions. Fr. I (11.2 g) was subjected to CC (SiO2 (30 – 40 mm, 2.6 40 cm); PE/acetone 85 : 15; 2000 ml) to yield compounds 1 (420.0 mg) and 13 (300.0 mg). Fr. III (6.2 g) was subjected to CC (Sephadex LH-20 (3.6 55 cm, MeOH/CHCl3 8 : 2; flow rate, 5 ml/min) to give three fractions. Fr. III-1 (1.4 g) was separated by CC (SiO2 (30 – 40 mm, 1.8 40 cm); CHCl3 /MeOH 98 : 2 – 85 : 15; flow rate, 5.0 ml/min) to yield compounds 3 (146.0 mg) and 4 (160.0 mg). Fr. II (30.0 mg) was subjected to CC (Agilent XDB-C18 (9.4 150 mm)) and purified by HPLC (MeOH/H2O 69 : 31; flow rate, 3.0 ml/min; UV at 254 nm) to yield compounds 15 (tR 17.5 min; 7.0 mg) and 16 (tR 20.5 min; 8.0 mg). Fr. L (15.0 g) eluted with PE/AcOEt 85 : 15 – 80 : 20 was re-chromatographed on a SiO2 column (30 – 40 mm, 4 50 cm; PE/acetone 90 : 10 – 70 : 30; flow rate 8 ml/min to give five fractions, Frs. I – V. Fr. II (120 mg) was subjected to CC (RP C18 (E. Merck Co. Ltd., 1.2 40 cm); MeOH/H2O 90 : 10; flow rate, 0.5 ml/min) to yield compound 10 (11.0 mg). Fr. M (12.0 g) eluted with PE/AcOEt 70 : 30 was re-chromatographed on a SiO2 column (30 – 40 mm, 4 50 cm; PE/acetone 85 : 15 – 60 : 40; flow rate, 8.0 ml/min) to furnish ten fractions, Frs. I – X. Fr. I (129.0 mg) was subjected to CC (RP C18 (E. Merck Co. Ltd., 1.2 40 cm); MeOH/H2O 90 : 10; flow rate 0.5 ml/min) to yield compound 14 (25.0 mg). Fr. VIII (215.0 mg) was submitted to CC (RP C18 (E. Merck Co. Ltd., 1.4 40 cm); MeOH/H2O 80 : 20 – 85 : 15; flow rate, 0.5 ml/ min) to finally yield compounds 9 (22.0 mg), 27 (5.0 mg), and 28 (6.0 mg). Biological Evaluation. S. aureus standard strain ATCC 25923 and tetracycline-resistant strain XU212, which possesses the TetK tetracycline efflux protein, were provided by Dr. Edet Udo [36]. Strain SA-1199B which over-expresses the NorA gene encoding the NorA MDR efflux pump was the gift of Prof. Glenn W. Kaatz [37]. Strain RN4220, which possesses the MsrA macrolide efflux protein, was
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provided by Dr. Jon Cove [38]. EMRSA-15 and EMRSA-16 that possess mecA protein were popular methicillin-resistant S. aureus (MRSA) strains [39]. All strains were cultured on nutrient agar (Oxoid) and incubated for 24 h at 378 prior to MIC determination. The control antibiotic norfloxacin was obtained from Sigma Chemical Co., and MuellerHinton broth (MHB; Oxoid) was adjusted to contain 20 and 10 mg/l of Ca2 þ and Mg2 þ , resp. An inoculum density of 5 105 cfu of each S. aureus strain was prepared in normal saline (9 g/l) by comparison with a 0.5 McFarland turbidity standard. The inoculum (125 ml) was added to all wells, and the microtitre plate was incubated at 378 for 18 h. For MIC determination, 20 ml of a 5 mg/ml methanolic solution of MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma) was added to each of the wells and incubated for 20 min. Bacterial growth was indicated by a color change from yellow to dark blue. The MIC was recorded as the lowest concentration at which no growth was observed. 8-Acetoxy-9-hydroxydshmirone ( ¼ (4E,8R*)-8-(Acetyloxy)-9-hydroxy-1-(2,4-dihydroxyphenyl)5,9,13-trimethyltetradeca-4,12-dien-1-one; 13). Yellow resin. [a] 21 D ¼ ¢ 34 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 315.5 (0.43), 275.0 (0.77). IR: 3405, 2958, 2924, 1635, 1607, 1384. 1H- and 13C-NMR: see Table 1. ESI-MS: 433.1 ([M þ H] þ ). HR-ESI-MS: 455.24026 ([M þ Na] þ , C25H36NaO þ6 ; calc. 455.24041). 8-Acetoxy-9-hydroxy-4’-methoxydshmirone ( ¼ (4E,8R*)-8-(Acetyloxy)-9-hydroxy-1-(2-hydroxy-4methoxyphenyl)-5,9,13-trimethyltetradeca-4,12-dien-1-one; 14). Yellow resin. [a] 21 D ¼ ¢ 49 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 316.5(0.628), 275.5 (1.149). IR: 3452, 2959, 2924, 2845, 1733, 1632, 1374, 1241, 1211, 1129, 1026. 1H- and 13C-NMR: see Table 1. ESI-MS: 447.2 ([M þ H] þ ). HR-ESI-MS: 469.25576 ([M þ Na] þ , C26H38NaO þ6 ; calc. 469.25606). (4E,8R*)-8-(Acetyloxy)-1-(2,4-dihydroxyphenyl)-5-methyl-8-[(5R*)-tetrahydro-5-(1-hydroxy-1methylethyl)-2-methylfuran-2-yl]oct-4-en-1-one (15). Yellow resin. [a] 22 D ¼ ¢ 37 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 315.0 (0.97), 275.5 (1.79). IR: 3359, 2962, 2921, 2851, 1737, 1633, 1374, 1259, 1130, 1026. 1H- and 13 C-NMR: see Table 2. ESI-MS: 449.2 ([M þ H] þ ). HR-ESI-MS: 449.25245 ([M þ H] þ , C25H37O þ7 ; calc. 449.25338). (4E,8R*)-8-(Acetyloxy)-1-(2,4-dihydroxyphenyl)-5-methyl-8-[(5S)-tetrahydro-5-(1-hydroxy-1-methylethyl)-2-methylfuran-2-yl]oct-4-en-1-one (16). Yellow resin. [a] 22 D ¼ ¢ 20 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 315.0 (0.35), 275.0 (0.65). IR: 3348, 2963, 2927, 2849, 1737, 1633, 1375, 1260, 1094, 1027. 1Hand 13C-NMR: see Table 2. ESI-MS: 449.2 ([M þ H] þ ). HR-ESI-MS: 449.25248 ([M þ H] þ , C25H37O þ7 ; calc. 449.25338). (2S*,3S*)-3-[ ( 3E)-4,8-Dimethylnona-3,7-dien-1-yl]-2,3-dihydro-7-methoxy-2,3-dimethyl-4H-furo[3,2-c] [1]benzopyran-4-one; 24). Colorless resin. [a] 22 D ¼ ¢ 64 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 333.0 (0.95), 318.0 (1.23), 288.5 (0.50). IR: 3421, 2963, 2924, 1718, 1639, 1614, 1448, 1414, 1383. 1H- and 13 C-NMR: see Table 3. ESI-MS: 397.2 ([M þ H] þ ). HR-ESI-MS: 397.23683 ([M þ H] þ , C25H33O þ4 ; calc. 397.23734). (2S*,3R*)-2,3-Dihydro-7-hydroxy-2-[(3E)-8-hydroxy-4,8-dimethylnon-3-en-1-yl]-2,3-dimethyl-4Hfuro[3,2-c] [1]benzopyran-4-one; 25). Colorless resin. [a] 22 D ¼ ¢ 31 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 331.0 (0.68), 316.5 (0.87), 288.0 (0.37). IR: 3418, 2955, 2922, 1636, 1459, 1413, 1384. 1H- and 13C-NMR: see Table 3. ESI-MS: 399.2 ([M ¢ H] ¢ ). HR-ESI-MS: 423.21364 ([M þ Na], C24H32NaO þ5 ; calc. 423.21420). (2S*,3R*)-2,3-Dihydro-2-[(3E)-8-hydroxy-4,8-dimethylnon-3-en-1-yl]-7-methoxy-2,3-dimethyl-4Hfuro[3,2-c] [1]benzopyran-4-one; 26). Colorless resin. [a] 22 D ¼ ¢ 37 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 332.0 (1.16), 317.0 (1.50), 288.5 (0.62). IR: 3430, 2924, 2838, 1721, 1638, 1613, 1413, 1273, 1155, 1026. 1H- and 13 C-NMR: see Table 3. ESI-MS: 415.2 ([M þ H] þ ). HR-ESI-MS: 437.22822 ([M þ Na] þ , C25H34NaO þ5 ; calc. 437.22985). This work was supported by Royal Society International Joint Project (Sino-UK Joint Project, JP091083/NSFC81011130165) and partly supported by the NSFC grant (21172041) of P. R. China. We also acknowledge support by Mindao Project for Medical Graduated Students of Fudan University (MDJH2012010). Supplementary Material. – The NMR spectra of compounds 1 – 28 are available as Supplementary Material.
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Sesquiterpenoids with Anti-MDR Staphylococcus aureus Activities from Ferula ferulioides by Tao Liu a ), Shuangying Wang a ), Lili Xu a ), Wei Fu a ), Simon Gibbons b ), and Qing Mu* a ) a
) School of Pharmacy, Fudan University, 826 Zhangheng Road, Zhangjiang Pudong, Shanghai 201203, P. R. China (phone: þ 86-21-51980109; e-mail: [email protected]) b ) Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, WC1N 1AX, London, UK
Seven new sesquiterpenoids together with 21 known sesquiterpenoid derivatives were isolated from the medicinal plant Ferula ferulioides (Steud.) Korovin. Their structures were elucidated by comprehensive spectroscopic analyses and chemical transformations. The isolated compounds were evaluated for their antibacterial activities against a panel of bacteria including multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA), displaying minimum inhibitory concentration (MIC) values in the range of 0.5 – 128 mg/l.
Introduction. – Methicillin-resistant Staphylococcus aureus (MRSA) is presently the most frequently identified antibiotic-resistant pathogen in many parts of the world [1]. The discovery and development of new antimicrobials together with new technologies are needed to combat bacterial multidrug resistance (MDR) [2]. In the search for bioactive substances, metabolites from plants can play a useful role as bacterial resistance modulators and anti-infective agents [3]. The genus Ferula (Apiaceae) comprises ca. 150 species growing in a vast geographical region ranging from Central Asia to the Mediterranean Basin [4], and 26 species are distributed in P. R. China [5]. Ferula is a rich source of biologically active compounds, such as sesquiterpenes [6 – 9] and sesquiterpene coumarins [10 – 15]. Plant extracts and metabolites from the Ferula genus were reported to possess many biological features including antiviral [16] [17], anti-inflammatory [18], antitumor [19], anticancer [20], and antibacterial [21] activities. The species F. ferulioides (Steud.) Korovin grows in western China. It has been reported to contain a series of sesquiterpenes and derivatives [22 – 27]. In our screening for new antibacterial agents, an extract of F. ferulioides was found to possess antibacterial activity against a panel of bacteria. Apart from a pesticidally active sesquiterpene, guaiol [28], our preliminary research resulted in the isolation of two new antibacterial sesquiterpenoids that inhibit efflux pumps of drug-resistant Staphylococcus aureus strains [29]. In an extensive phytochemical investigation, seven new sesquiterpenoid derivatives, together with 21 known sesquiterpenoid derivatives were isolated. Antibacterial assays were carried out for the isolates against a panel of multidrug-resistant Staphylococcus aureus strains, and the sesquiterpene hydroxyacetophenone derivatives showed antibacterial activities with minimum inhibitory concentration (MIC) values in the range of 0.5 – 128 mg/l (1.2 – 297.7 mm). Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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Results and Discussion. – The powdered roots of F. ferulioides were extracted with 95% EtOH and partitioned between H2O and CH2Cl2 . The CH2Cl2-soluble fraction was subjected to repeated column chromatography (CC) to afford seven new compounds, 13 – 16 and 24 – 26, together with 21 known compounds 1 – 12, 17 – 23, 27, and 28 (Fig. 1). Compound 13 was obtained as yellow resin and showed a peak at m/z 455.24026 ([M þ Na] þ ) in its HR-ESI-MS spectrum, corresponding to the molecular formula C25H36O6 . Its IR spectrum indicated the presence of an intramolecular H-bonded OH group (3405 cm ¢ 1), a benzene ring (1607 cm ¢ 1), and a C¼C bond (1635 cm ¢ 1). UV absorptions at lmax 275.0 and 238.5 nm supported the presence of a characteristic 2,4dihydroxyacetophenone moiety. The 1D-NMR data (Table 1) suggested that compound 13 had a skeleton consisting of a dihydroxyphenyl ethyl ketone connected to a farnesyl moiety. This skeleton was the same as that of 9, previously isolated from the same plant [29]. In comparison with 9, compound 13 had an additional AcO group instead of a OH group at C(8) in 9. Therefore, compound 13 was the 8-AcO- and 9-OHsubstituted derivative of dshamirone (5). This structure was also verified by a careful inspection of the HMBC data of 13 (Table 1 and Fig. 2). The relative configuration of 13 was determined by a ROESY experiment, in which cross-peaks of CH2(3)/Me(15) and H¢C(8)/Me(16) were observed. This result evidenced an (E)-configuration for the C(4)¼C(5) bond, and the relative configurations of the adjacent AcO group at C(8) and of the OH group at C(9) as threo (Table 1). Therefore, compound 13 was identified as depicted in Fig. 1, with yet unknown absolute configuration, and was given the trivial name 8-acetoxy-9-hydroxydshmirone. Compound 14 was obtained as yellow resin whose molecular formula was established as C26H38O6 by HR-ESI-MS (m/z 469.25576 ([M þ Na] þ )). Its UV and IR spectral data were almost the same as those of 13. The 1H- and 13C-NMR spectra of 14 were also highly similar to those of 13, except for the presence of an additional MeO group (d(H) 3.85; d(C) 55.5). The location of the MeO group was confirmed by the difference of the 13C-NMR resonance of their C(4’) (13: d(C) 162.7; 14: d(C) 165.9) and HMBC spectra (Table 1 and Fig. 2). The chemical shifts of the signals of C(7), C(8), and Me(16), and a series of ROESY experiments indicated that 14 had the same relative configuration as 13. The relative configuration of the adjacent AcO group at C(8) and of the vicinal OH groups at C(9) was again threo, combined with an (E)-configuration for the C(4)¼C(5) bond. Therefore, compound 14 was identified as depitced in Fig. 1, and was given the trivial name 8-acetoxy-9-hydroxy-4’-methoxydshmirone. Compound 14 was synthesized from 13 by treatment with Me2SO4 and K2CO3 at room temperature (Scheme 1). The hydrolysis of compound 13 with KOH in MeOH yielded 9. Compound 9 was then converted to 10 by reacting it with TsOH and 2,2dimethoxypropane at room temperature [29]. Chemical transformation from 13 to 14, and 13 to 9 and 10 (Scheme 1) further confirmed the structure elucidation. The relative configurations at C(8) and C(9) of the four compounds were all identified as threo. Compound 15 was obtained as yellow resin whose molecular formula was established as C25H36O7 by HR-ESI-MS (m/z 449.25245 ([M þ H] þ )). The UV, IR, and NMR data (Table 2) for compound 15 suggested a 2,4-dihydroxylated phenone connected to a side chain, which was similar to that in compound 13. Compared with 13, compound 15 possessed an additional O-bearing CH group (d(H) 3.78) instead of an
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Fig. 1. Structures of compounds 1 – 28
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Table 1. NMR Data a ) (in CDCl3 ) of Compounds 13 and 14. d in ppm, J in Hz. Position
13 d( H)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1’ 2’ 3’ 4’ 5’ 6’ 4’-MeO 1’’ 2’’
14 d(C )
204.5 2.91 (t, J ¼ 7.4) 37.8 2.40 – 2.43 (m) 23.2 5.16 (t, J ¼ 6.7) 123.2 135.7 1.94 – 1.96 (m) 36.1 1.67 – 1.69 (m) 27.3 4.82 (dd, J ¼ 10.3, 2.4) 78.9 74.4 1.44 – 1.55 (m) 37.5 2.04 – 2.07 (m) 22.0 5.10 (t, J ¼ 7.0) 124.1 132.2 1.69 (s) 25.7 1.62 (s) 15.9 1.17 (s) 23.5 1.63 (s) 17.7 113.8 165.2 6.37 (overlapped) 107.7 162.7 6.37 (overlapped) 103.5 7.63 (d, J ¼ 9.5) 132.4
2.12 (s)
171.3 21.1
HMBC
d( H)
d(C )
204.5 1, 3, 4 2.92 (t, J ¼ 7.4) 37.9 1, 2, 4, 5-Me 2.42 (dt, J ¼ 7.0, 7.4) 23.1 3, 6, 5-Me 5.18 (t, J ¼ 6.7) 123.1 135.7 4, 5, 7, 8, 5-Me 1.94 – 1.96 (m) 36.1 6, 8 1.66 – 1.69 (m) 27.4 4.82 (d, J ¼ 10.1) 78.9 74.2 8, 9, 11, 12 1.42 – 1.53 (m) 37.5 12 2.03 – 2.07 (m) 22.0 11, 14, 13-Me 5.09 (t, J ¼ 7.0) 124.1 132.1 12, 13, 13-Me 1.68 (s) 25.7 4, 5 1.60 (s) 16.0 8, 9, 10 1.16 (s) 23.5 12, 13, 14 1.62 (s) 17.7 113.5 165.3 1’, 2’, 4’, 5’ 6.42 (overlapped) 107.6 165.9 1’, 2’, 3’, 4’ 6.42 (overlapped) 100.9 1, 2’, 4’, 5’ 7.64 (d, J ¼ 8.6) 131.6 3.85 (s) 55.5 171.0 1’’ 2.08 (s) 21.1
HMBC 1, 3, 4 1, 2, 4, 5 3, 6, 5-Me 4, 5, 7, 8, 5-Me 5, 6, 8
8, 9, 11, 12 12 11, 14, 13-Me 12, 13, 13-Me 4, 5 8, 9, 10 12, 13, 14
1’, 2’, 4’, 5’ 1’, 2’, 3’, 4’ 1, 2’, 4’, 5’ 4’ 1’’
a
) Key ROESY correlations of 13: H¢C(3)/Me(15); H¢C(8)/Me(16). Key ROESY correlations of 14: H¢C(3)/Me(15); H¢C(8)/Me(16).
olefinic H-atom (ca. d(H) 5 ppm) as in 13. The two C-atom signals of a double bond at d(C) 124.1 (CH) and 132.2 (C) in 13 were replaced with two O-bearing C-atom signals at d(C) 84.7 and 71.7 ppm in the side chain of 15. Compound 15 was assumed to be the 12,13-dihydroxy derivative of 13. Comparison of the molecular masses of 15 (m/z 449.2) and 13 (m/z 433.1) indicated the presence of an additional O-atom in the molecule of 15. The characteristic 13C-NMR data, 77.2/84.3 and 84.7/71.7 (Table 2), indicated the presence of an ether moiety in a tetrahydrofuran arrangement [30]. The structure of compound 15 was deduced as depicted in Fig. 1, and this was confirmed by its key HMBC spectral correlations (Fig. 2). The relative configuration of 15 was determined by its ROESY spectrum, in which correlations CH2(3)/Me(15), H¢C(8)/ Me(16), and H¢C(12)/Me(15), were observed, indicating the relative configuration at C(8) and C(9) as erythro, and an (E)-configuration for the C(4)¼C(5) bond. The configuration in the furan ring at C(9) and C(12) was assigned as cis. Compound 16 was obtained as yellow resin, and its molecular formula was deduced as C25H36O7, the same as 15, from HR-ESI-MS (m/z 449.25248 ([M þ H] þ )). The UV,
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Fig. 2. Key HMBCs (H ! C) of compounds 13 – 16 and 24 – 26
IR, NMR, HMQC, and HMBC data for compound 16 indicated a very similar structure to that of 15, with the only difference being the 13C-NMR shifts of C(12) (15: 84.7 ppm; 16: 86.8 ppm) (Table 2). Compound 16 was elucidated as the C(12)-epimer of compound 15. Therefore, the relative configuration of 16 was determined by a ROESY experiment, in which cross-peaks H¢C(12)/Me(16) were present in 15, but absent in 16. The orientation of Me(16) and H¢C(12) at furan ring was trans. Thus, compound 16 was identified as depicted in Fig. 1. The absolute configurations at C(8) and C(9) of compounds 13 – 16 remained unresolved, even though several methodologies were applied. First, MosherÏs ester method was employed. The two OH groups of the phenyl ring were protected by methylation using Me2SO4 , and compound 13a was obtained (Scheme 2). Subsequent-
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Scheme 1. Chemical Transformations of Compound 13 into 9, 10, and 14
a) Acetone, K2CO3 , Me2SO4 , r.t., 4 h; 92%. b) MeOH, KOH, r.t., 1 h; 96%. c) Me2C(OMe)2 , TsOH, r.t., 6 h; 90%.
Table 2. NMR Data a ) (in CDCl3 ) of Compounds 15 and 16. d in ppm, J in Hz. Position
15 d( H )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1’ 2’ 3’ 4’ 5’ 6’ 1’’ 2’’
2.92 (t, J ¼ 7.0) 2.40 – 2.42 (m) 5.19 (t, J ¼ 7.0) 1.94 – 1.98 (m) 1.58 – 1.94 (m) 4.96 (d, J ¼ 10.6) 1.82 – 1.84 (m) 1.56 – 1.74 (m) 3.78 (t, J ¼ 7.5) 1.24 (s) 1.60 (s) 1.21 (s) 1.15 (s)
6.39 (overlapped) 6.39 (overlapped) 7.63 (d, J ¼ 9.4) 2.08 (s)
16 d(C ) 204.6 37.7 23.4 123.4 135.4 35.8 34.0 77.2 84.3 26.5 28.1 84.7 71.7 27.4 15.9 22.8 23.9 113.6 165.1 107.6 163.4 103.4 132.5 171.4 21.3
HMBC
d( H )
1, 3, 4 1, 2, 4, 5 2, 3, 6, 5-Me
2.92 (t, J ¼ 7.4) 2.40 – 2.42 (m) 5.18 (t, J ¼ 7.2)
4, 5, 8 6, 7, 8, 5-Me 6, 7, 9, 9-Me, 1’’
1.94 – 1.98 (m) 1.58 – 1.60 (m) 4.92 (d, J ¼ 10.1)
9, 9-Me 8, 10, 12 11, 14, 13-Me
1.82 – 1.84 (m) 1.56 – 1.74 (m) 3.73
12, 13, 13-Me 4, 5 8, 9, 10 12, 13, 14
1.23 (s) 1.60 (s) 1.21 (s) 1.15 (s)
1’, 2’, 4’, 5’
6.39 (overlapped)
1’, 3’, 4’ 1, 1’, 2’, 4’
6.39 (overlapped) 7.63 (d, J ¼ 9.4)
1’’
2.09 (s)
d(C ) 204.7 37.7 23.4 123.2 135.7 35.9 34.5 77.3 84.1 26.5 28.1 86.8 71.2 27.2 15.9 22.9 23.7 113.6 165.1 107.8 163.4 103.4 132.6 171.2 21.2
HMBC 1, 3, 4 1, 2, 4, 5 2, 3, 6, 5-Me 4, 5, 8 6, 8, 5-Me 6, 7, 9, 9-Me, 1’’ 8, 9, 9-Me 10, 12 11, 14, 13-Me 12, 13, 13-Me 4, 5 8, 9, 10 12, 13, 14
1’, 2’, 4’, 5’ 1’, 3’, 4’ 1, 1’, 2’, 4’ 1’’
a ) Key ROESY correlations of 15: H¢C(3)/Me(15); Me(16)/H¢C(8), H¢C(12). Key ROESY correlations of 16: H¢C(3)/Me(15); Me(16)/H¢C(8).
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ly, the Ac group in 13a was hydrolyzed, and 13b, which contained a secondary OH group at C(8), was obtained. This compound was reacted with MosherÏs reagent under four different reaction conditions, respectively (Scheme 2). TLC Monitoring showed that no reaction occurred. It is possible that when the Ac group was removed and a OH was formed, this could form an intramolecular H-bond with the OH at C(9), rendering it unavailable for reaction. It is also possible that side-chain hindrance may reduce the availability for reaction with the MosherÏs reagent (Fig. 3). We also attempted to predict the absolute configuration of 9 by a quantum-chemical calculation using Guassian 09 [31]. The geometries of 9 and its enantiomer were Scheme 2. An Attempt to Determine the Absolute Configuration of Compound 13
a) Acetone, K2CO3 , Me2SO4 , r.t., 24 h; 83%. b) MeOH, KOH, r.t., 1 h; 95%. c) MosherÏs esterification: 1) (RS)-MTPA ( ¼ a-methoxy-a-(trifluoromethyl)phenylacetic acid), DCC ( ¼ N,N’-dicyclohexylcarbodiimide), DMAP ( ¼ 4-(dimethylamino)pyridine), pyridine; 2) (RS)-MTPA, DCC, DMAP, CH2Cl2 ; 3) (RS)-MTPA-Cl, CH2Cl2 ; 4) (RS)-MTPA-Cl, NaH, DMF.
Fig. 3. ChemBio 3D structure of compound 13
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optimized at the B3LYP/6-31G level of density-functional theory (DFT). The electronic circular dichroism (ECD) spectra was then calculated using the timedependent DFT (TDDFT) method at the B3LYP /6-311 þ þ G** level in MeCN. Unfortunately, the predicted ECD spectra of the enantiomers of 9 showed no differences (Fig. 4), implying that a lack of chromophore at the stereogenic centers C(8) and C(9) led to weak circular dispersion between the enantiomers. Therefore, the absolute configurations of the new compounds 13 – 16 have yet to be elucidated. Compound 24 was obtained as colorless resin whose molecular formula was established as C25H32O4 by HR-ESI-MS (m/z 397.23683 ([M þ H] þ )). The UV and IR data of 24 were almost the same as those of 23 (Fig. 1). The 1H- and 13C-NMR spectra of the two compounds were slightly different at C(3) (23: d(H) 4.88, d(C) 89.6; 24: d(H) 4.62, d(C) 93.0), Me¢C(2) (23: d(H) 1.47, d(C) 15.8; 24: d(H) 1.56, d(C) 13.9), and Me¢C(3) (23: d(H) 1.31, d(C) 19.2; 24: d(H) 1.47, d(C) 23.8) (Table 3). An HMBC experiment suggested that 23 and 24 are diastereoisomers. A series of ROESY experiments was carried out to determine the relative configuration of the furan moiety at C(2) and C(3). Cross-peaks between Me¢C(2) and Me¢C(3) appeared in 23, while their absence in 24 indicated that Me¢C(2) and Me¢C(3) were trans-configured in 24 (Table 3). Accordingly, the relative configurations of the furan moiety at C(2) and C(3) were (2S*) and (3S*). Finally, compound 24 was identified as depicted in Fig. 1.
Fig. 4. Calculated ECD spectra of 9a and its enantiomer 9b. The ECD spectra were calculated using the time-dependent density-functional theory (TDDFT) method at B3LYP/6-311 þ þ G** level, in the presence of MeCN by Gaussian09.
d( H)
7’, 8’, 8’-Me 2, 3, 1’ 2, 3, 3a 3’, 4’, 5’ 7’, 8’, 9’
4’, 6’, 7’ 4’, 5’, 7’, 6’, 9’
2, 3, 2’, 3’ 1’, 3’, 4’ 5’, 4’-Me
6, 7, 9a 5a, 7, 9b
5a, 7, 8, 9a
7’, 8’, 8’-Me 2, 3, 1’ 2, 3, 3a 3’, 4’, 5’ 7’, 8’, 9’ 7
3’, 4’, 6’, 7’, 4’-Me 5’, 7’, 8’ 6’, 8’-Me
2, 3, 2’, 3’, 2-Me 1’, 3’, 4’ 2’, 5’, 4’-Me
6, 7, 9a 5a, 7, 9b
4, 5a, 7, 8, 9a
2, 3a, 9b, 1’, 2-Me, 3-Me
d(C) HMBC
96.8 41.9 103.6 161.3 156.8 6.83 ( d, J ¼ 2.4 ) 100.6 163.1 6.83 (dd, J ¼ 2.4, 7.8) 112.2 7.52 (d, J ¼ 9.0) 123.7 106.2 165.0 1.79 – 1.81 (m) 41.7 2.10 – 2.13 (m) 22.1 5.09 (t, J ¼ 6.9) 123.3 135.8 1.93 – 1.95 (m) 39.9 1.41 – 1.43 (m) 22.5 1.41 – 1.43 (m) 43.4 71.0 1.21 (s) 29.2 1.45 (s) 20.5 1.29 (d, J ¼ 7.4) 14.1 1.55 (s) 15.8 1.20 (s) 29.2 3.86 (s) 55.7
d( H )
26
2, 3a, 9b, 3-Me 3.29 ( d, J ¼ 7.0)
d(C ) HMBC
97.1 3.29 (d, J ¼ 7.0) 41.5 103.2 162.3 156.6 5a, 7, 8, 9a 7.12 (d, J ¼ 2.0) 103.2 161.0 7, 9a 6.83 (dd, J ¼ 2.4, 8.6) 113.3 5a, 7 7.52 ( d, J ¼ 8.6 ) 124.1 105.6 166.0 3’ 1.79 – 1.81 (m) 41.5 4’ 2.10 – 2.13 (m) 22.1 5’, 4’-Me 5.09 (t, J ¼ 6.9) 123.5 135.8 4’, 6’, 4’-Me 1.93 – 1.95 (m) 39.8 4’, 5’, 7’ 1.42 – 1.44 (m) 22.5 5’ 1.40 – 1.42 (m) 43.3 71.4 7’, 8’ 1.21 (s) 29.1 2, 3 1.45 (s) 20.7 2, 3, 3a, 1’ 1.29 (d, J ¼ 7.1) 14.1 3’, 4’, 5’ 1.54 (s) 15.8 7’, 8’, 9’ 1.20 (s) 29.0 7
1’, 3a
d(C ) HMBC
93.0 46.6 106.1 160.7 156.9 6.85 (d, J ¼ 2.4) 100.5 163.1 6.83 (dd, J ¼ 2.4, 6.7) 112.2 7.56 ( d, J ¼ 9.4 ) 124.0 106.5 166.1 1.65 – 1.67 (m) 34.7 1.94 – 1.98 (m) 23.4 5.07 (overlapped) 123.7 135.1 1.91 – 1.93 (m) 39.6 2.02 – 2.04 (m) 26.6 5.07 (overlapped) 124.2 131.3 1.69 (s) 25.7 1.56 (d, J ¼ 6.7) 13.9 1.47 (s) 23.8 1.52 (s) 16.0 1.61 (s) 17.7 3.89 (s) 55.7
d(H ) 4.62 (q, J ¼ 6.7)
25
a ) Key ROESY correlations of 24: H¢C(3’)/CH2(5’); H¢C(2)/Me¢C(3). Key ROESY Correlations of 25: H¢C(3’)/CH2(5’); Me¢C(2)/Me¢C(3). Key ROESY correlations of 26: H¢C(3’)/CH2(1’) and CH2(5’); Me¢C(2)/Me¢C(3).
2 3 3a 4 5a 6 7 8 9 9a 9b 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 2-Me 3-Me 4’-Me 8’-Me 7-MeO
Position 24
Table 3. NMR Data a ) (in CDCl3 ) of Compounds 24 – 26. d in ppm, J in Hz.
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Compound 25 was obtained as colorless oil with the molecular formula C24H32O5 as deduced from HR-ESI-MS data (m/z 423.21364 ([M þ Na] þ )). The UV absorptions at 318 and 239 nm supported the presence of a substituted 7-hydroxycoumarin. The IR spectrum indicated a C¼O group (1718 cm ¢ 1), a benzene ring (1639 cm ¢ 1), and a double bond of a furocoumarin (1614 cm ¢ 1). The 1H- and 13C-NMR spectra of 25 exhibited signals of a 1,2,4-trisubstituted benzene ring (d(H) 6.83, 7.12, 7.52; d(C) 113.3, 103.2, 124.1), a substituted C¼C bond (d(H) 5.09; d(C) 123.5), and an ester moiety (d(C) 156.6) (Table 3). These spectral data revealed that 25 had the same skeleton as the furocoumarin compound 17, which had been isolated previously from the same plant [25]. However, in comparison with 17, two 13C signals of the C(7’)¼C(8’) bond, at d(C) 124.2 (CH) and 131.5 (C), were replaced by those of a CH2 group (d(C) 43.3) and an OH bearing quaternary C-atom (d(C) 71.4) in compound 25. The signals for two Me groups at 29.0 and 29.1 ppm supported this elucidation (Table 3). Based on the HMBC spectrum (Fig. 2), compound 25 is constructed of a coumarin, a fused dihydrofuran moiety, and a side chain. The configuration of C(3’)¼C(4’) bond was deduced as (E) from its ROESY spectrum with the cross-peaks CH2(1’)/H¢C(3’) and H¢C(3’)/ CH2(5’). The relative configurations within in the dihydrofuran moiety at C(2) and C(3) were identified as (2S*) and (3R*) by a ROESY spectrum with cross-peaks between Me¢C(2) and Me¢C(3) (Table 3). Compound 25 was therefore identified as depicted in Fig. 1. Compound 26 was also obtained as colorless oil with the molecular formula C25H34O5 as deduced from its HR-ESI-MS data (m/z 437.22822 ([M þ Na] þ )). The UV, IR, and NMR data for compound 26 indicated a similar skeleton of a furocoumarin as in 25. The only difference was that the OH group at C(7) of 25 was replaced by a MeO group (d(H) 3.86; d(C) 55.7) in compound 26 (Table 3). We confirmed the structure consisting of a coumarin, a fused dihydrofuran, and a prenyl side chain based on an HMBC spectrum (Table 3 and Fig. 2). The configuration of C(3’)¼C(4’) bond was deduced as (E) from its ROESY spectrum with the cross-peaks CH2(1’)/H¢C(3’) and H¢C(3’)/CH2(5’). The relative configurations of the dihydrofuran moiety, at C(2) and C(3), were identified as (2S*) and (3R*) by its ROESY spectrum with cross-peaks between Me¢C(2) and Me¢C(3) (Table 3). Thus, compound 26 was identified as depicted in Fig. 1. Structures of 21 known sesquiterpenes were identified by comparison of their spectroscopic data with those reported in the literature. They were 2,4-dihydroxyacetophenone (1), 2,4-dihydroxybenzoic acid (3), 2,4-dihydroxy-a-oxobenzeneacetic acid (4) [32], 2-hydroxy-4-methoxyacetophenone (2) [33], dshamirone (5) [24], (4E,8E)-1(2-hydroxy-4-methoxyphenyl)-5,9,13-trimethyltetradeca-4,8,12-trien-1-one (6) [34], (4E,8E)-1-(2,4-dihydroxyphenyl)-2-hydroxy-5,9,13-trimethyltetradeca-4,8,12-trien1-one (7), (6E)-1-(2,4-dihydroxyphenyl)-3,7,11-trimethyl-3-vinyldodeca-6,10-dien-1one (8) [24], 8,9-dihydroxydshamirone (9), 8,9-oxoisopropanyldshamirone (10), ferulaeolactone A [29], 3-(2,4-dihydroxybenzoyl)-4,5-dimethyl-5-[(3E,7E)-4,8-dimethylnona-3,7-dien-1-yl]tetrahydrofuran-2-one (11) [22], (2S*,3R*)-2,3-dihydro-7hydroxy-2,3-dimethyl-2-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (17), (2S*,3R*)-2,3-dihydro-7-methoxy-2,3-dimethyl-2-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (19), (2S*,3R*)-2,3-dihydro-7-hydroxy-2,3-dimethyl-2-[(3E)-4,8dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (18), (2R*,3R*)-2,3-dihydro-7-methoxy-
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2,3-dimethyl-2-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (20) [26], (2S*,3R*)-2,3-dihydro-7-hydroxy-2,3-dimethyl-3-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (21), (2S*,3R*)-2,3-dihydro-7-hydroxy-2,3-dimethyl-3-[(3E)-4,8dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (22), (2S*,3R*)-2,3-dihydro-7-methoxy2,3-dimethyl-3-[(3E)-4,8-dimethylnona-3,7-dienyl]furo[3,2-c]coumarin (23) [23], ferulin A (27), and ferulin B (28) [35]. All compounds were tested for their ability to inhibit the growth of a panel of bacteria including multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA). Most of the sesquiterpene coumarins had no effect on the bacteria. Only individual coumarin compounds, i.e., 17, 18, 21, and 22, showed inhibitory activities against the tetracycline-resistant strain XU212, which possessed the TetK tetracycline efflux protein with a promising MIC value of 2 mg/l, and those of 27 and 28 were 32 and 16 mg/l, respectively, while the MIC of control norfloxacin was 32 mg/l (Table 4). Most of the acetophenone sesquiterpene derivatives, 5 – 13, possessed activities ranging from 0.5 – 128 mg/l against multidrug-resistant S. aureus. Among the tested strains was SA-1199B, which possesses the NorA efflux protein that confers resistance to certain fluoroquinolones and quaternary ammonium antiseptics. Against this strain, 5 (16 mg/l, 44.9 mm), 7 (2 mg/l, 5.4 mm), 8 (16 mg/l, 44.9 mm), 11 (8 mg/l, 20.0 mm), 12 (8 mg/l, 19.1 mm), and 13 (16 mg/l, 37.0 mm) were more active than the control antibiotic norfloxacin (32 mg/l, 100 mm). For MDR strain XU212, which possesses the TetK efflux transporter, and is resistant to both tetracycline and methicillin, 5 (1 mg/l, 2.8 mm), 6 (1 mg/l, 2.7 mm), 7 (1 mg/l, 2.7 mm), 8 (4 mg/l, 11.2 mm), 10 (0.5 mg/l, 1.2 mm), 11 (2 mg/l, 5.0 mm), and 12 (2 mg/l, 4.8 mm) showed more effective inhibition than the control antibiotic tetracycline (128 mg/l, 288 mm). Six 7-hydroxy-coumarins, 17, 18, 21, 22, 27, and 28, also exhibited selective inhibitory activities against this strain. For erythromycin-resistant strain RN4220 that carries the MsrA macrolide efflux protein, 7 (2 mg/l, 5.4 mm), 8 (4 mg/l, 11.2 mm), 11 (4 mg/l, 10.0 mm) and 12 (16 mg/l, 38.4 mm) showed better inhibitory activities than the control antibiotic erythromycin (64 mg/l, 87.2 mm). For the common methicillin-resistant S. aureus (MRSA) strains, 7 (4 mg/l, 10.8 mm), 11 (2 mg/l, 5.0 mm), and 12 (2 mg/l, 4.8 mm) displayed significant inhibitory activities against strain EMRSA-15, and 5 (1 mg/l, 2.8 mm), 7 (2 mg/l, 5.4 mm), and 10 (0.5 mg/l, 1.2 mm) were active against strain EMRSA-16. Of note is that compound 8,9-oxoisopropanyldshamirone (10), which was previously isolated from this plant [28] and synthesized from 13 (Scheme 1), possessed significant inhibitory activity against XU212 and EMRSA-16 with an MIC value of 0.5 mg/l, while it was inactive against the other tested drug-resistant strains including the standard S. aureus strains ATCC25923. In F. ferulioides, the sesquiterpene (farnesyl) acetophenone derivatives lead to its antibacterial properties (Table 4), whilst some of the sesquiterpene (farnesyl) coumarins possess specific antibacterial activity against the tetracycline resistant strain XU212 (Table 4). Some of the examples detailed here, such as 5, 10, and 13, are noteworthy in terms of potency against bacteria. Further work to determine selectivity against mammalian cells would be of interest, as would a mechanistic study.
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Table 4. Anti-MDR and Anti-MRSA Activities (MIC [mg/l]) of the Compounds 1 – 28 Compounds
SA1199B
XU212
ATCC25923
RN4220
EMRSA-15
EMRSA-16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Norfloxacin Tetracycline Erythromycin Oxacillin Vancomycin
> 128 > 128 > 128 > 128 16 > 128 2 16 64 > 128 8 8 16 > 128 > 128 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 32 0.25 0.25 0.25 0.25
> 128 > 128 > 128 > 128 1 1 1 4 64 0.5 2 2 32 > 128 > 128 > 128 2 2 > 128 > 128 2 2 > 128 > 128 > 128 > 128 32 16 8 128 > 128 128 0.5
> 128 > 128 > 128 > 128 16 > 128 2 16 64 > 128 8 8 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 0.5 0.25 0.25 0.25 0.25
> 128 > 128 > 128 > 128 32 > 128 2 4 64 16 4 16 64 > 128 > 128 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 0.5 0.5 64 0.25 0.5
> 128 > 128 > 128 > 128 16 > 128 4 16 64 > 128 2 2 64 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 0.5 0.25 > 128 > 128 0.25
> 128 > 128 > 128 > 128 1 > 128 2 32 64 0.5 128 16 32 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 > 128 128 0.25 > 128 > 128 0.25
Experimental Part General. TLC: Silica gel HGF254 plates (SiO2 ; Qingdao Marine Chemical Plant, Yantai, P. R. China) and RP-18 plates (E. Merck Co., Ltd.); visualization with 20% H2SO4 , followed by heating. Column chromatography (CC): silica gel (SiO2 ; 10 – 40 mm; Qingdao Marine Chemical Plant, Yantai, P. R. China). M.p.: X-4 micro melting-point apparatus. Optical rotations: JASCO P-1020 polarimeter. UV Spectra: HITACHI U-2900 spectropolarimeter; lmax (log e) in nm. IR Spectra: Avatar 360 ESP FT-IR spectrophotometer; ˜n in cm ¢ 1. 1H- and 13C-NMR spectra: Varian Mercury Plus 400- and 100-MHz spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. EI-MS: Agilent 5973N MSD spectrometer; in m/z. HR-EI-MS: IonSpec 4.7 T FTMS; in m/z. Plant Material. The roots of F. ferulioides were collected in Xinjiang Uyghur Autonomous Region, China. A voucher specimen (F001) was deposited with the Natural Medicinal Chemistry Laboratory of the School of Pharmacy, Fudan University. The plant was identified by Mrs. Hui-Qin Xie, Associate Professor in the Department of Plant Protection, Shihezi University, China.
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Extraction and Isolation. Air-dried and powdered roots of F. ferulioides (2.0 kg) were extracted at 458 with 95% EtOH (3 5,000 ml, 12 h each). After removal of the solvent under reduced pressure, the residue was dissolved in CH2Cl2 (3 500 ml, 20 min each) to afford the lipophilic-soluble fraction Fr. FF (400.0 g). Fr. FF was submitted to CC (SiO2 (10 – 20 mm, 10 75 cm); petroleum ether (PE)/AcOEt 100 : 0 to 0 : 100; flow rate 30 ml/min) to afford 15 fractions of different polarity. Fr. C (1.5 g) eluted with PE/AcOEt 98 : 2 was separated by CC (SiO2 (30 – 40 mm, 1.8 40 cm); PE/acetone 99 : 1; 200 ml) to give pure 6 (390.0 mg). Fr. F (6.0 g) eluted with PE/AcOEt 95 : 5 was re-chromatographed (SiO2 (30 – 40 mm, 2.8 40 cm); PE/AcOEt 98 : 2 – 92 : 8; flow rate, 4 ml/min) to furnish five fractions, Frs. I – V. Fr. I (720.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.8 40 cm); PE/acetone 98 : 2; 300 ml) to yield compound 2 (240.0 mg). Fr. II (120.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.2 40 cm); PE/ acetone 97 : 3; 300 ml) to afford compound 26 (12.0 mg). Fr. IV was purified by CC (Agilent XDB-C18 (9.4 150 mm)) and HPLC (MeOH/H2O 88 : 12; flow rate, 3.0 ml/min; UV at 254 nm) to yield compounds 23 (tR 16.3 min; 4.0 mg) and 24 (tR 17.8 min; 4.0 mg). Fr. G (23.0 g) eluted with PE/AcOEt 95 : 5 – 90 : 10 was re-chromatographed (SiO2 (30 – 40 mm, 5.0 50 cm); PE/AcOEt 95 : 5 – 85 : 15; flow rate, 10.0 ml/min) to afford seven fractions, Fr. I – VII. Fr. I (1.2 g) was subjected to CC (SiO2 (30 – 40 mm, 1.8 40 cm); PE/acetone 96 : 4; 500 ml) to yield compounds 19 (144.0 mg) and 20 (20.0 mg). Fr. V (7.2 g) was separated by CC (SiO2 (30 – 40 mm, 4.2 50 cm); PE/acetone 95 : 5; 2000 ml) to yield compound 5 (3.5 g). Fr. I (35.0 g) eluted with PE/AcOEt 90 : 10 was re-chromatographed (SiO2 (30 – 40 mm, 5 50 cm); PE/acetone 90 : 10; flow rate, 10 ml/min) to give eleven fractions, Fr. I – XI. Fr. II (420.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.4 40 cm); PE/acetone 90 : 10; 200 ml) to yield compound 7 (14.0 mg). Fr. IV was subsequently purified by CC (Agilent XDB-C18 (9.4 150 mm)) and HPLC (MeOH/H2O 85 : 15; flow rate, 3.0 ml/min; UV at 254 nm) to furnish compounds 21 (tR 15.9 min; 35.0 mg) and 22 (tR 17.0 min; 85.0 mg). Fr. VI (230.0 mg) was subjected to CC (SiO2 (30 – 40 mm, 1.2 40 cm); CH3Cl3/acetone 99 : 1; 200 ml) to afford pure compound 12 (11.0 mg). Fr. J (22.0 g) eluted with PE/AcOEt 85 : 15 was rechromatographed (SiO2 (30 – 40 mm, 5.0 50 cm); PE/acetone 95 : 5 – 80 : 20; flow rate, 10.0 ml/min) to give six fractions, Fr. I – VI. Fr. III (4.8 g) was subjected to CC (SiO2 (30 – 40 mm, 2.8 40 cm); CHCl3 /acetone 98 : 2; 2000 ml) to furnish five fractions. Fr. III-2 (2.4 g) was subjected to CC (reversed-phase (RP) C18 (E. Merck Co. Ltd., 2.4 40 cm); MeOH/H2O 80 : 20 – 85 : 15; flow rate, 1.0 ml/min) to yield compounds 11 (390.0 mg), 17 (390.0 mg), 18 (330.0 mg), and 25 (200.0 mg). Fr. VI (2.2 g) was subjected to CC (RP/C18 column (E. Merck Co. Ltd., 2.4 40 cm); MeOH/ H2O 85 : 15; flow rate, 1.0 ml/min) to yield compounds 8 (12.0 mg) and 11 (420.0 mg). Fr. K (102.0 g) was submitted to CC (SiO2 (20 – 30 mm, 7.5 65 cm); PE/AcOEt 85 : 15 to 0 : 100; flow rate, 25 ml/min) to afford ten fractions. Fr. I (11.2 g) was subjected to CC (SiO2 (30 – 40 mm, 2.6 40 cm); PE/acetone 85 : 15; 2000 ml) to yield compounds 1 (420.0 mg) and 13 (300.0 mg). Fr. III (6.2 g) was subjected to CC (Sephadex LH-20 (3.6 55 cm, MeOH/CHCl3 8 : 2; flow rate, 5 ml/min) to give three fractions. Fr. III-1 (1.4 g) was separated by CC (SiO2 (30 – 40 mm, 1.8 40 cm); CHCl3 /MeOH 98 : 2 – 85 : 15; flow rate, 5.0 ml/min) to yield compounds 3 (146.0 mg) and 4 (160.0 mg). Fr. II (30.0 mg) was subjected to CC (Agilent XDB-C18 (9.4 150 mm)) and purified by HPLC (MeOH/H2O 69 : 31; flow rate, 3.0 ml/min; UV at 254 nm) to yield compounds 15 (tR 17.5 min; 7.0 mg) and 16 (tR 20.5 min; 8.0 mg). Fr. L (15.0 g) eluted with PE/AcOEt 85 : 15 – 80 : 20 was re-chromatographed on a SiO2 column (30 – 40 mm, 4 50 cm; PE/acetone 90 : 10 – 70 : 30; flow rate 8 ml/min to give five fractions, Frs. I – V. Fr. II (120 mg) was subjected to CC (RP C18 (E. Merck Co. Ltd., 1.2 40 cm); MeOH/H2O 90 : 10; flow rate, 0.5 ml/min) to yield compound 10 (11.0 mg). Fr. M (12.0 g) eluted with PE/AcOEt 70 : 30 was re-chromatographed on a SiO2 column (30 – 40 mm, 4 50 cm; PE/acetone 85 : 15 – 60 : 40; flow rate, 8.0 ml/min) to furnish ten fractions, Frs. I – X. Fr. I (129.0 mg) was subjected to CC (RP C18 (E. Merck Co. Ltd., 1.2 40 cm); MeOH/H2O 90 : 10; flow rate 0.5 ml/min) to yield compound 14 (25.0 mg). Fr. VIII (215.0 mg) was submitted to CC (RP C18 (E. Merck Co. Ltd., 1.4 40 cm); MeOH/H2O 80 : 20 – 85 : 15; flow rate, 0.5 ml/ min) to finally yield compounds 9 (22.0 mg), 27 (5.0 mg), and 28 (6.0 mg). Biological Evaluation. S. aureus standard strain ATCC 25923 and tetracycline-resistant strain XU212, which possesses the TetK tetracycline efflux protein, were provided by Dr. Edet Udo [36]. Strain SA-1199B which over-expresses the NorA gene encoding the NorA MDR efflux pump was the gift of Prof. Glenn W. Kaatz [37]. Strain RN4220, which possesses the MsrA macrolide efflux protein, was
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provided by Dr. Jon Cove [38]. EMRSA-15 and EMRSA-16 that possess mecA protein were popular methicillin-resistant S. aureus (MRSA) strains [39]. All strains were cultured on nutrient agar (Oxoid) and incubated for 24 h at 378 prior to MIC determination. The control antibiotic norfloxacin was obtained from Sigma Chemical Co., and MuellerHinton broth (MHB; Oxoid) was adjusted to contain 20 and 10 mg/l of Ca2 þ and Mg2 þ , resp. An inoculum density of 5 105 cfu of each S. aureus strain was prepared in normal saline (9 g/l) by comparison with a 0.5 McFarland turbidity standard. The inoculum (125 ml) was added to all wells, and the microtitre plate was incubated at 378 for 18 h. For MIC determination, 20 ml of a 5 mg/ml methanolic solution of MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; Sigma) was added to each of the wells and incubated for 20 min. Bacterial growth was indicated by a color change from yellow to dark blue. The MIC was recorded as the lowest concentration at which no growth was observed. 8-Acetoxy-9-hydroxydshmirone ( ¼ (4E,8R*)-8-(Acetyloxy)-9-hydroxy-1-(2,4-dihydroxyphenyl)5,9,13-trimethyltetradeca-4,12-dien-1-one; 13). Yellow resin. [a] 21 D ¼ ¢ 34 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 315.5 (0.43), 275.0 (0.77). IR: 3405, 2958, 2924, 1635, 1607, 1384. 1H- and 13C-NMR: see Table 1. ESI-MS: 433.1 ([M þ H] þ ). HR-ESI-MS: 455.24026 ([M þ Na] þ , C25H36NaO þ6 ; calc. 455.24041). 8-Acetoxy-9-hydroxy-4’-methoxydshmirone ( ¼ (4E,8R*)-8-(Acetyloxy)-9-hydroxy-1-(2-hydroxy-4methoxyphenyl)-5,9,13-trimethyltetradeca-4,12-dien-1-one; 14). Yellow resin. [a] 21 D ¼ ¢ 49 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 316.5(0.628), 275.5 (1.149). IR: 3452, 2959, 2924, 2845, 1733, 1632, 1374, 1241, 1211, 1129, 1026. 1H- and 13C-NMR: see Table 1. ESI-MS: 447.2 ([M þ H] þ ). HR-ESI-MS: 469.25576 ([M þ Na] þ , C26H38NaO þ6 ; calc. 469.25606). (4E,8R*)-8-(Acetyloxy)-1-(2,4-dihydroxyphenyl)-5-methyl-8-[(5R*)-tetrahydro-5-(1-hydroxy-1methylethyl)-2-methylfuran-2-yl]oct-4-en-1-one (15). Yellow resin. [a] 22 D ¼ ¢ 37 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 315.0 (0.97), 275.5 (1.79). IR: 3359, 2962, 2921, 2851, 1737, 1633, 1374, 1259, 1130, 1026. 1H- and 13 C-NMR: see Table 2. ESI-MS: 449.2 ([M þ H] þ ). HR-ESI-MS: 449.25245 ([M þ H] þ , C25H37O þ7 ; calc. 449.25338). (4E,8R*)-8-(Acetyloxy)-1-(2,4-dihydroxyphenyl)-5-methyl-8-[(5S)-tetrahydro-5-(1-hydroxy-1-methylethyl)-2-methylfuran-2-yl]oct-4-en-1-one (16). Yellow resin. [a] 22 D ¼ ¢ 20 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 315.0 (0.35), 275.0 (0.65). IR: 3348, 2963, 2927, 2849, 1737, 1633, 1375, 1260, 1094, 1027. 1Hand 13C-NMR: see Table 2. ESI-MS: 449.2 ([M þ H] þ ). HR-ESI-MS: 449.25248 ([M þ H] þ , C25H37O þ7 ; calc. 449.25338). (2S*,3S*)-3-[ ( 3E)-4,8-Dimethylnona-3,7-dien-1-yl]-2,3-dihydro-7-methoxy-2,3-dimethyl-4H-furo[3,2-c] [1]benzopyran-4-one; 24). Colorless resin. [a] 22 D ¼ ¢ 64 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 333.0 (0.95), 318.0 (1.23), 288.5 (0.50). IR: 3421, 2963, 2924, 1718, 1639, 1614, 1448, 1414, 1383. 1H- and 13 C-NMR: see Table 3. ESI-MS: 397.2 ([M þ H] þ ). HR-ESI-MS: 397.23683 ([M þ H] þ , C25H33O þ4 ; calc. 397.23734). (2S*,3R*)-2,3-Dihydro-7-hydroxy-2-[(3E)-8-hydroxy-4,8-dimethylnon-3-en-1-yl]-2,3-dimethyl-4Hfuro[3,2-c] [1]benzopyran-4-one; 25). Colorless resin. [a] 22 D ¼ ¢ 31 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 331.0 (0.68), 316.5 (0.87), 288.0 (0.37). IR: 3418, 2955, 2922, 1636, 1459, 1413, 1384. 1H- and 13C-NMR: see Table 3. ESI-MS: 399.2 ([M ¢ H] ¢ ). HR-ESI-MS: 423.21364 ([M þ Na], C24H32NaO þ5 ; calc. 423.21420). (2S*,3R*)-2,3-Dihydro-2-[(3E)-8-hydroxy-4,8-dimethylnon-3-en-1-yl]-7-methoxy-2,3-dimethyl-4Hfuro[3,2-c] [1]benzopyran-4-one; 26). Colorless resin. [a] 22 D ¼ ¢ 37 (c ¼ 0.10, CHCl3 ). UV (CHCl3 ): 332.0 (1.16), 317.0 (1.50), 288.5 (0.62). IR: 3430, 2924, 2838, 1721, 1638, 1613, 1413, 1273, 1155, 1026. 1H- and 13 C-NMR: see Table 3. ESI-MS: 415.2 ([M þ H] þ ). HR-ESI-MS: 437.22822 ([M þ Na] þ , C25H34NaO þ5 ; calc. 437.22985). This work was supported by Royal Society International Joint Project (Sino-UK Joint Project, JP091083/NSFC81011130165) and partly supported by the NSFC grant (21172041) of P. R. China. We also acknowledge support by Mindao Project for Medical Graduated Students of Fudan University (MDJH2012010). Supplementary Material. – The NMR spectra of compounds 1 – 28 are available as Supplementary Material.
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