Article pubs.acs.org/jnp
Prenylated Benzophenones from Triadenum japonicum Atsushi Oya,† Naonobu Tanaka,†,‡ Taishi Kusama,† Sang-Yong Kim,§ Shigeki Hayashi,⊥ Mareshige Kojoma,§ Atsuyuki Hishida,⊥ Nobuo Kawahara,⊥ Kanae Sakai,∥ Tohru Gonoi,∥ and Jun’ichi Kobayashi*,† †
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan § Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Tobetsu 061-0293, Japan ⊥ Hokkaido Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, Nayoro 090-0065, Japan ∥ Medical Mycology Research Center, Chiba University, Chiba 260-8673, Japan ‡
S Supporting Information *
ABSTRACT: Six new prenylated benzophenones, (−)-nemorosonol (1) and trijapins A−E (2−6), were isolated from the aerial parts of Triadenum japonicum. (−)-Nemorosonol (1) and trijapins A−C (2−4) have a common tricyclo[4.3.1.03,7]decane skeleton, while 1 is an enantiomer of (+)-nemorosonol previously isolated from Clusia nemorosa. The absolute configuration of (−)-nemorosonol (1) was assigned by ECD spectroscopy. Trijapins A−C (2−4) are analogues of 1 possessing an additional tetrahydrofuran ring. Trijapins D (5) and E (6) are prenylated benzophenones with a 1,2dioxane moiety and a hydroperoxy group, respectively. (−)-Nemorosonol (1) exhibited antimicrobial activity against Escherichia coli (MIC, 8 μg/mL), Staphylococcus aureus (MIC, 16 μg/mL), Bacillus subtilis (MIC, 16 μg/mL), Micrococcus luteus (MIC, 32 μg/mL), Aspergillus niger (IC50, 16 μg/mL), Trichophyton mentagrophytes (IC50, 8 μg/mL), and Candida albicans (IC50, 32 μg/mL), while trijapin D (5) showed antimicrobial activity against C. albicans (IC50, 8 μg/mL).
P
HPLC to afford (−)-nemorosonol (1, 0.0083%) and trijapins A (2, 0.00025%), B (3, 0.00013%), C (4, 0.000038%), D (5, 0.0020%), and E (6, 0.00017%) (Chart 1). (−)-Nemorosonol (1) was isolated as a colorless, amorphous solid, and the molecular formula, C33H42O4, was established by 13 C NMR spectroscopic and HRESIMS (m/z 525.29684 [M + Na]+, Δ−0.69 mmu) data. The 1H and 13C NMR spectra of 1 were identical to those of (+)-nemorosonol,5 a prenylated benzophenone isolated from Clusia nemorosa (Clusiaceae), whereas the sign of the specific rotation of 1 {[α]D −207 (c 0.7, CHCl3)} was opposite that of (+)-nemorosonol {[α]D +203 (c 0.7, solvent not specified)}.5a These findings suggested 1 to be an enantiomer of (+)-nemorosonol. The electronic circular dichroism (ECD) spectra of the two enantiomers of nemorosonol (1a: 1S, 3R, 5R, 6S, 7R; 1b: 1R, 3S, 5S, 6R, 7S) were calculated by the TDDFT method (Figure 1).6 The experimental ECD spectrum of (−)-nemorosonol (1) agreed well with the calculated spectrum of 1a, suggesting the 1S, 3R, 5R, 6S, 7R configurations for 1. Consequently, the structure of (−)-nemorosonol was assigned as shown in 1 (Chart 1).
lants belonging to the Hypericaceae and Clusiaceae families (APG) have been recognized as a rich source of prenylated acylphloroglucinols (PAPs), which have attracted widespread interest due to their fascinating chemical structures and interesting biological activities.1 PAPs possessing a benzoyl group as the acyl moiety are reported to show a variety of biological activities such as anti-HIV, antimicrobial, and cytotoxic activities.2 The genus Triadenum is a small group within the Hypericaceae, from which flavonoids and stilbenoids have been isolated.3 In a continuing search for structurally interesting natural products from Hypericaceae plants, the isolation of PAPs and meroterpenoids from several plants of the genus Hypericum has been reported.4 As a part of this program, the aerial parts of Triadenum japonicum were investigated and resulted in the isolation of six new prenylated benzophenones, (−)-nemorosonol (1) and trijapins A−E (2−6). Herein, the isolation and structure elucidation of 1−6 are described.
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RESULTS AND DISCUSSION The aerial parts of T. japonicum (366 g, dry) were extracted with MeOH, and the extract was partitioned with n-hexane and H2O. The n-hexane-soluble portions were subjected to silica gel column chromatography repeatedly and purified by ODS © XXXX American Chemical Society and American Society of Pharmacognosy
Received: October 22, 2014
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DOI: 10.1021/np500827h J. Nat. Prod. XXXX, XXX, XXX−XXX
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Chart 1. Structures of (−)-Nemorosonol (1) and Trijapins A−E (2−6)
in 2, forming a tetrahydrofuran ring (C-3, C-7, C-11, and C12). Therefore, the gross structure of 2 was elucidated as shown in Figure 2A. Analysis of the NOESY spectrum of 2 suggested that the relative configuration of the core tricyclic [4.3.1.03,7]decane-2,9-dione moiety in 2 was the same as that of 1 (Figure 2B). NOESY correlations between H-12/H-4a and H-12/H21a revealed the H-5β and H-12β orientations. Thus, the relative configuration of trijapin A (2) was assigned as shown in Figure 2B. Trijapin B (3) has the same molecular formula, C33H42O5, as that of 2. The 1H and 13C NMR spectra of 3 resembled those of 2 (Table 1), indicating that 3 was also an analogue of (−)-nemorosonol (1). The COSY and HMBC spectra revealed that 3 had a tetrahydrofuran ring involving C-1, C-9, C-16, and C-17 (Figure 3A). The β-orientation of H-17 was suggested by a NOESY cross-peak of H-17/H-10a (Figure 3B). Thus, the structure of trijapin B was elucidated to be 3. Trijapin C (4) was isolated as an optically active, colorless, amorphous solid {[α]D +38 (c 0.04, CHCl3)}. The HRESIMS and 13C NMR data revealed a molecular formula of C33H44O6, 18 mass units higher than 3. The signals of the enol moiety (C8 and C-9) for 3 were replaced by resonances of one hemiketal [δH 5.15 (9-OH); δC 104.9] and one sp3 methine (δH 4.19; δC 52.7) in 4 (Table 1). HMBC correlations of H-8, 9-OH, and H2-16 with a carbon signal at δC 104.9 suggested the hemiketal carbon to be C-9, while the sp3 methine was assigned as C-8 via an HMBC correlation of H-8 with C-27. Therefore, the gross structure of 4 was assigned as shown in Figure 4A. The αorientation of the prenyl group at C-5 was implied by a NOESY correlation of 7-OH with H-21a. NOESY correlations for H-8/ H-10b, H-8/H3-26, H-17/H-10b, and H3-19/9-OH suggested β-orientations for H-8 and H-17 as well as an α-orientation for 9-OH. Accordingly, the relative configurations of trijapin C (4) were assigned as shown in Figure 4B. The ECD spectrum of trijapin A (2) showed sequential positive and negative Cotton effects at 336 and 294 nm, respectively, similar to the spectrum of (−)-nemorosonol (1) (Figure 5), implying that the absolute configuration of 2 was the same as that of 1. Although trijapins B (3) and C (4) also gave similar Cotton effects (Figure 5), the absolute
Figure 1. Experimental and calculated ECD spectra of (−)-nemorosonol (1).
Trijapin A (2) was obtained as an optically active, colorless, amorphous solid {[α]D −150 (c 0.2, MeOH)}. The molecular formula of 2, C33H42O5, was elucidated by 13C NMR spectroscopic and HRESIMS (m/z 541.29260 [M + Na]+, Δ+0.15 mmu) data. Interpretation of the 1H and 13C NMR data (Table 1) indicated that 2 was structurally related to 1, whereas the signals of one oxygenated tertiary carbon, one sp3 oxymethine, one sp3 methylene, and two methyls were observed for 2 in place of the resonances due to the prenyl group at C-3 for 1. The C-3 substituent in 2 was assigned as a 2,3-dihydroxy-2-methylpropyl moiety on the basis of a 1H−1H COSY correlation of H2-11/H-12 and HMBC cross-peaks of H3-14 to C-12, C-13, and C-15 and of H2-11 to C-2, C-3, and C-7 (Figure 2A). The hydrogen deficiency index of 2 implied the presence of an ether linkage between C-7 and C-12 or between C-7 and C-13. Comparison of the chemical shifts of C12 (δC 89.2) and C-13 (δC 69.2) with the corresponding position of related PAPs isolated from Hypericum plants4b,c,f implied the presence of an ether linkage between C-7 and C-12 B
DOI: 10.1021/np500827h J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H and 13C NMR Data for Trijapins A−C (2−4) 2a position
65.0 205.8 65.1 37.9
5 6 7 8 9 10
50.3 51.1 94.7 109.8 197.5 45.9
11
29.1
12 13 14 15 16
89.2 69.2 27.2 24.9 24.5
17 18 19 20 21 22 23 24 25 26 27 28 29, 33 30, 32 31 9-OH a
δC
1 2 3 4
118.7 134.7 18.0 25.8 30.1 123.7 132.5 18.1 26.0 21.5 177.0 135.0 129.9 128.1 131.6
3b δH (J in Hz)
2.12 (1H, dd, 15.7, 9.9) 1.61 (1H, m) 1.96 (1H, m)
1.84 (1H, d, 14.1) 1.60 (1H, d, 14.1) 1.74 (2H, m) 3.83 (1H, dd, 10.2, 5.5) 0.57 1.25 2.69 2.37 5.07
(3H, (3H, (1H, (1H, (1H,
s) s) dd, 14.9, 7.4) dd, 14.9, 7.4) t, 7.4)
1.64 (3H, 1.71 (3H, 2.35, 2.21 5.01 (1H,
s) s) (1H each, m) t, 6.3)
1.63 (3H, s) 1.71 (3H, s) 1.47 (3H, s)
7.86 (2H, d, 7.4) 7.46 (2H, t, 7.4) 7.51 (1H, t, 7.4)
δC
4b δH (J in Hz)
60.2 208.6 60.1 41.5
2.18 (1H, dd, 13.7, 9.0) 1.96 (1H, dd, 13.7, 5.9) 1.63 (1H, m)
48.1 49.8 87.7 105.3 165.9 50.7 31.5 121.8 133.0 17.8 26.5 25.4 94.0 71.3 25.0 25.0 33.5 124.8 132.0 18.2 26.0 19.6 195.0 140.2 129.8 128.0 132.0
1.25 1.21 2.98 2.88 5.48
(1H, (1H, (1H, (1H, (1H,
d, 12.9) d, 12.9) dd, 14.5, 7.0) dd, 14.5, 8.2) t, 7.4)
1.69 1.66 2.78 1.11 3.87
(3H, (3H, (1H, (1H, (1H,
s) s) dd, 13.7, 7.0) dd, 13.7, 8.6) dd, 8.6, 7.0)
0.85 (3H, 0.90 (3H, 2.67, 2.37 5.19 (1H,
s) s) (1H each, m) t, 7.0)
1.74 (3H, s) 1.78 (3H, s) 1.22 (3H, s)
7.93 (2H, d, 6.7) 7.12 (2H, t, 6.7) 7.13 (1H, t, 6.7)
δC 60.1 212.9 62.7 38.8 48.3 46.6 85.0 52.7 104.9 45.7 30.1 121.4 132.9 18.0 26.1 26.0 86.1 71.2 27.7 25.4 33.1 123.6 131.9 18.0 25.8 17.9 201.8 138.5 129.3 127.9 132.9
δH (J in Hz)
1.95 (1H, dd, 13.0, 8.4) 1.63 (1H, m) 1.49 (1H, m)
4.19 (1H, d, 1.5) 1.82 1.22 2.60 2.56 5.58
(1H, (1H, (1H, (1H, (1H,
d, 13.6) d, 13.6) dd, 14.3, 7.6) dd, 14.3, 7.6) t, 7.6)
1.63 1.75 2.49 1.49 4.09
(3H, (3H, (1H, (1H, (1H,
s) s) dd, 13.1, 9.0) m) t, 9.0)
1.18 (3H, 1.10 (3H, 2.19, 2.01 5.01 (1H,
s) s) (1H each, m) t, 7.1)
1.60 (3H, s) 1.68 (3H, s) 1.15 (3H, s)
8.16 7.43 7.53 5.15
(2H, (2H, (1H, (1H,
d, 7.4) t, 7.4) t, 7.4) d, 1.5)
Measured in acetone-d6. bMeasured in CDCl3.
Figure 3. (A) Selected 2D NMR correlations for trijapin B (3) and (B) selected NOESY correlations and the relative configurations for the core unit of 3 (protons of Me-26 are not shown).
Figure 2. (A) Selected 2D NMR correlations for trijapin A (2) and (B) selected NOESY correlations and the relative configurations for the core unit of 2 (protons of Me-26 are not shown).
Trijapin D (5) was obtained as an optically active, pale yellow, amorphous solid {[α]D −58 (c 0.6, MeOH)}. The molecular formula of 5 was assigned as C33H42O8 by the 13C NMR and HRESIMS (m/z 565.28070 [M − H]−, Δ+0.01 mmu) data. The 1H NMR spectrum of 5 in acetone-d6 indicated the presence of a monosubstituted benzene moiety, two trisubstituted olefinic units, and two sp3 methine, five sp3
configurations of 3 and 4 could not been assigned due to a difference of chromophores. On the other hand, based on biosynthesis considerations, the absolute configurations for trijapins B (3) and C (4) are anticipated to be the same as those for 1 and 2. C
DOI: 10.1021/np500827h J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 1H and 13C NMR Data for Trijapins D (5) and E (6) in CDCl3 5 position
Figure 4. (A) Selected 2D NMR correlations for trijapin C (4) and (B) selected NOESY correlations and the relative configurations for the core unit of 4 (protons of Me-26 are not shown).
δC
1 2 3 4
57.9 206.3 62.1 41.5
5 6 7 8 9 10
40.8 82.5 193.5 114.9 191.8 48.3
11
33.4
6
δH (J in Hz)
2.00, 1.72 (1H each, m) 1.74 (1H, m)
2.17 (1H, d, 13.0) 1.46 (1H, brd, 13.0) 2.84 (1H, m)
δC 116.1 172.2 59.8 39.3
42.6 48.0 193.2 78.5 205.0 16.4 30.9
2.66 (1H, m) 12
120.7
4.99 (1H, t, 6.5)
86.6
13 14 15 16
134.6 17.7 25.6 36.5
1.67 (3H, s) 1.68 (3H, s) 2.88 (1H, m)
83.0 19.8 19.9 22.1
2.63 (1H, m)
Figure 5. Experimental ECD spectra of (−)-nemorosonol (1) and trijapins A−C (2−4).
methylene, and seven methyl carbons (Table 2). In contrast, the 1H NMR spectrum in CDCl3 displayed signals characteristic of keto−enol tautomerism via enolic proton signals at δH 17.45 and 16.95 (Supporting Information). These features implied 5 to be a prenylated benzophenone with a diketo enolic moiety. The presence of two prenyl groups (C-11−C-15 and C-16−C-20) was evident from the 1H−1H COSY and HMBC spectra (Figure 6A). The C10 unit (C-4−C-6, C-10, and C-21− C-26) with oxygen functions at C-6, C-22, and C-23 was suggested by 1H−1H COSY cross-peaks of H-4/H-5, H-5/H221, and H2-21/H-22 as well as HMBC correlations of H3-24 to C-22, C-23, and C-25 and of H3-26 to C-5, C-6, and C-10. The phloroglucinol moiety (C-1−C-3 and C-7−C-9) comprised a carbonyl {δC 206.3 (C-2)}, one conjugated carbonyl {δC 193.5 (C-7)}, one β-hydroxyenoyl {δC 191.8 (C-9) and 114.9 (C-8)}, and two sp3 quaternary carbons {δC 62.1 (C-3) and 57.9 (C1)}. HMBC cross-peaks of H2-16 to C-1, C-2, C-9, and C-10 and of H2-11 to C-2, C-3, C-4, and C-7 were indicative of the presence of the prenyl groups at C-1 and C-3 as well as the connectivities between the C10 unit and the phloroglucinol moiety as shown in Figure 6A. Given the molecular formula of 5, the existence of a hydroperoxy group at C-23 and the connectivity of C-6 to C-22 via a peroxy bridge, forming a 1,2dioxane ring, were concluded. Thus, the gross structure was assigned as shown in 5. The syn-relationship of the prenyl groups at C-1 and C-3 was necessitated by the steric restrictions imposed by the phloroglucinol-derived and cycloheptanone (C-1−C-6 and C10) rings. NOESY correlations for H-4b/H3-26 and H-5/H-22
17 18 19 20 21
118.5 135.8 17.5 25.6 28.1
22
84.3
23 24 25 26 27 28 29, 33 30, 32 31 34 35 36 37 38
81.6 21.4 19.9 17.4 198.3 138.2 128.7 128.1 132.9
4.87 (1H, t, 7.3) 1.61 (3H, 1.63 (3H, 1.59 (1H, 1.50 (1H, 4.21 (1H, 2.3)
s) s) m) q, 11.5) dd, 11.5,
1.19 (3H, s) 1.06 (3H, s) 1.25 (3H, s)
7.57 (2H, d, 7.6) 7.45 (2H, t, 7.6) 7.59 (1H, m)
120.3 132.6 17.7 25.8 26.6 122.2 137.2 16.2 39.8 23.1 193.5 136.8 128.1 127.8 132.0 26.5 124.0 131.6 17.7 25.7
δH (J in Hz)
2.14 (1H, dd, 13.4, 4.3) 1.63 (1H, m) 1.73 (1H, m)
1.21 (3H, s) 2.65 (1H, dd, 13.4, 10.6) 1.93 (1H, dd, 13.4, 6.5) 4.95 (1H, dd, 10.6, 6.5) 1.29 (3H, 1.30 (3H, 3.14 (1H, 7.3) 3.04 (1H, 7.3) 5.04 (1H,
s) s) dd, 14.0,
1.63 1.63 2.24 1.78 5.00
s) s) 13.4, 5.3) m) t, 6.7)
(3H, (3H, (1H, (1H, (1H,
dd, 14.0, t, 7.3)
1.58 (3H, s) 2.01 (2H, m) 1.41 (3H, s)
7.46 7.20 7.38 2.07 5.07
(2H, (2H, (1H, (2H, (1H,
d, 7.7) t, 7.7) t, 7.7) m) t, 6.9)
1.60 (3H, s) 1.70 (3H, s)
indicated the trans junction between the cycloheptanone ring and the 1,2-dioxane ring as well as the H-22α orientation. Therefore, the relative configurations of 5 were concluded as shown in Figure 6B. Trijapin E (6) was isolated as an optically active, colorless, amorphous solid {[α]D −25 (c 0.2, MeOH)}. The 1H and 13C NMR data of 6 resembled those of hyperibone G,7 a prenylated benzophenone with a bicyclo[3.3.1]nonane-2,4,9-trione core, except for the signals due to the substituent at C-5 and for the chemical shifts of C-12−C-15. The 2D NMR spectra (Figure 7A) revealed that 6 had a geranyl group at C-5 in place of the D
DOI: 10.1021/np500827h J. Nat. Prod. XXXX, XXX, XXX−XXX
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(−)-Nemorosonol (1) exhibited antimicrobial activity against Escherichia coli (MIC, 8 μg/mL), Staphylococcus aureus (MIC, 16 μg/mL), Bacillus subtilis (MIC, 16 μg/mL), Micrococcus luteus (MIC, 32 μg/mL), Aspergillus niger (IFM62678, IC50, 16 μg/mL), Trichophyton mentagrophytes (IFM62679, IC50, 8 μg/ mL), and Candida albicans (IFM62680, IC50, 32 μg/mL), while trijapin D (5) showed antimicrobial activity against C. albicans (IFM62680, IC50, 8 μg/mL).
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Figure 6. (A) Selected 2D NMR correlations for trijapin D (5) and (B) selected NOESY correlations and the relative configurations for the core unit of 5 (protons of Me-24, Me-25, and Me-26 are not shown).
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were recorded on a JASCO P-1030 digital polarimeter. IR, UV, and ECD spectra were recorded on JASCO FT/IR-230 and JASCO FT/IR-460 Plus, Shimadzu UV-1600PC, and JASCO J-720 spectrophotometers, respectively. NMR spectra were measured on Bruker AMX-600, JEOL ECX400P, and JEOL ECA500 spectrometers. The resonances of residual CHCl3 (7.26 and 77.0 ppm), benzene (7.20 and 128.0 ppm), and acetone (2.05 and 205.7 ppm) were used as internal references for 1 H and 13C NMR spectra, respectively. HRESIMS, HRAPCIMS, ESIMS, and APCIMS were recorded on a Thermo Scientific Exactive spectrometer. Plant Material. The aerial parts of Triadenum japonicum were collected in Hokkaido, Japan, in July 2012. The plant was identified by one of the authors (S.H.), and a herbarium specimen was deposited at the Experimental Station for Medicinal Plants Studies, Hokkaido University (specimen number: TJA120721). Extraction and Isolation. The aerial parts of T. japonicum (366 g, dry) were extracted with MeOH (3 × 5 L), and the extract (108 g) was partitioned with n-hexane (3 × 1 L) and H2O (1 L) to give the nhexane-soluble materials (20.9 g). A part of the n-hexane-soluble materials (4.4 g) was subjected to silica gel column chromatography (eluent n-hexane/EtOAc, 95:5 → 0:100) to give 16 fractions (frs. 1− 16). Separation of fr. 6 on a silica gel column (CHCl3/acetone, 100:0 → 90:10) gave eight fractions (frs. 6.1−8) including (−)-nemorosonol (1, 30.3 mg, 0.0083%, fr. 6.2). Fr. 6.3 was chromatographed on a silica gel column (n-hexane/acetone, 97:3 → 0:100) to give 14 fractions (frs. 6.3.1−14). Fr. 6.3.7 was passed through a silica gel column (n-hexane/ EtOAc, 90:10 → 0:100) and purified using reversed-phase HPLC (Luna 5u phenyl-hexyl, Phenomenex, Inc., 10 × 250 mm; flow rate 3.0 mL/min; UV detection at 254 nm; eluent MeOH/H2O/TFA, 80:20:0.1) to give trijapin D (5, 7.4 mg, 0.0020%). The other part of the n-hexane-soluble materials (16.5 g) was subjected to silica gel colum chromatography (eluent n-hexane/EtOA, 95:5 → 0:100) to give 16 fractions (frs. 1′−16′). Separation of fr. 7′ on silica gel CC (n-hexane/acetone, 95:5 → 0:100) gave 11 fractions (frs. 7′.1−11). Fr. 7′.4 was purified by silica gel CC (CHCl3/n-hexane/ EtOAc, 5:5:0.1 → 5:5:5) and C18 HPLC (COSMOSIL 5C18 AR-II, Nacalai Tesque, Inc.; 10 × 250 mm; 3.0 mL/min; 254 nm; MeCN/ H2O, 77:23) to afford trijapin A (2, 0.9 mg, 0.00025%). Fr. 7′.6 was subjected to silica gel CC (n-hexane/EtOAc, 80:20 → 60:40) to afford nine fractions (frs. 7′.6.1−9). Separation of fr. 7′.6.3 by C18 HPLC (COSMOSIL 5C18 MS-II, 10 × 250 mm; 3.0 mL/min; 254 nm; MeOH/H2O, 85:15) gave six fractions (frs. 7′.6.3.1−6) including trijapin E (6, 0.6 mg, 0.00017%, fr. 7′.6.3.4). Fr. 7′.6.3.3 was purified by C18 HPLC (COSMOSIL 5C18 MS-II, 10 × 250 mm; 3.0 mL/min; 254 nm; MeCN/H2O, 90:10) to give trijapin C (4, 0.1 mg, 0.000038%). Fr. 7′.6.4 was purified by C18 HPLC (COSMOSIL 5C18 MS-II, 10 × 250 mm; 3.0 mL/min; 254 nm; MeCN/H2O, 74:26) to give trijapin B (3, 0.5 mg, 0.00025%). (−)-Nemorosonol (1): colorless, amorphous solid; [α]24D −207 (c 0.7, CHCl3); UV (MeOH) λmax 242 (ε 6150) and 340 (4990) nm; IR (KBr) νmax 3421, 1717, and 1617 cm−1; ECD (MeOH) Δε (nm) −5.5 (246), +14.0 (298), and −12.4 (334); 1H NMR (600 MHz, benzened6) δ 16.07 (1H, s, 27-OH), 7.54 (2H, m, H-30 and H-32), 7.08 (3H, m, H-29, H-31, and H-33), 5.63 (1H, t, J = 7.4 Hz, H-17), 5.19 (1H, t, J = 7.0 Hz, H-12), 4.99 (1H, t, J = 6.7 Hz, H-22), 2.94 (2H, m, H2-16), 2.65 (1H, dd, J = 14.9, 7.8 Hz, H-11a), 2.21 (1H, dd, J = 14.9, 7.8 Hz, H-11b), 2.13 (1H, m, H-21a), 2.11 (1H, m, H-21b), 2.09 (1H, m, H-
Figure 7. (A) Selected 2D NMR correlations for trijapin E (6) and (B) selected NOESY correlations and the relative configurations for the core unit of 6 (protons of Me-10 and Me-26 are not shown).
prenyl group in hyperibone G.7 The chemical shifts of C-12 (δC 86.6), C-13 (δC 83.0), C-14 (δC 19.8), and C-15 (δC 19.9) for 6 were similar to the corresponding carbons in 33-deoxy-33hydroperoxyfurohyperforin (δC 86.4, 83.0, 20.2, and 19.8, respectively)8 rather than those of hyperibone G (δC 90.3, 71.3, 26.9, and 24.1, respectively).7 These observations indicated that 6 had a hydroperoxy group at C-13, which was suggested by the molecular formula of 6, C38H51O6, established by the 13C NMR and HRAPCIMS {m/z 603.36846 [M + H]+, Δ+0.44 mmu} data. The relative configurations of 6 were assigned by NOESY analysis as shown in Figure 7B. The chemical shift of C-5 (δC 42.6) and the difference in the chemical shifts of H2-6 (Δδ 0.51) supported that the substituent at C-5 was endo.1d,9 Thus, the structure of trijapin E (6) was concluded as shown in Chart 1. The investigation of the aerial parts of T. japonicum (Hypericaceae) resulted in the isolation of six new prenylated benzophenones, (−)-nemorosonol (1) and trijapins A−E (2− 6). It is noteworthy that (−)-nemorosonol (1), a prenylated benzophenone with a complex cage-like tricyclo[4.3.1.03,7]decane skeleton, is an enantiomer of (+)-nemorosonol, previously isolated from C. nemorosa (Clusiaceae). The absolute configuration of (−)-nemorosonol (1) was assigned via experimental and calculated ECD data. Trijapins A−C (2−4) are analogues of 1 possessing an additional tetrahydrofuran ring in common, while trijapins D (5) and E (6) are prenylated benzophenones with a 1,2-dioxane moiety and a hydroperoxy group, respectively. (−)-Nemorosonol (1) and trijapins A−E (2−6) seem to be derived from a common biogenetic precursor having a benzophenone core with two prenyl groups and one lavandulyl group (C10). E
DOI: 10.1021/np500827h J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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4a), 1.75 (3H, s, H3-20), 1.69 (3H, s, H3-19), 1.68 (3H, s, H3-25), 1.60 (2H, m, H-4b and H-10a), 1.58 (1H, m, H-5), 1.58 (3H, s, H3-15), 1.54 (1H, m, H-10b), 1.52 (3H, s, H3-24), 1.44 (3H, s, H3-14), and 0.94 (3H, s, H3-26); 13C NMR (150 MHz, benzene-d6) δ 209.9 (C-2), 199.6 (C-9), 175.3 (C-27), 135.6 (C-28), 133.8 (C-18), 133.4 (C-13), 131.8 (C-23), 130.4 × 2 (C-30 and C-32), 128.6 × 3 (C-29, C-31, and C-33, overlapped with the signal of benzene-d6), 124.1 (C-22), 120.6 (C-12), 120.5 (C-17), 110.1 (C-8), 84.1 (C-7), 63.7 (C-1), 63.0 (C-3), 48.8 (C-5), 47.5 (C-6), 46.5 (C-10), 33.6 (C-21), 29.9 (C-11), 26.2 (C-20), 26.1 (C-15), 25.8 × 2 (C-16 and C-25), 19.1 (C-26), 18.0 × 2 (C-19 and C-24), and 17.8 (C-14); HRESIMS m/z 525.29684 [M + Na]+ (calcd for C33H42O4Na, 525.29753). Trijapin A (2): colorless, amorphous solid; [α]25D −150 (c 0.2, MeOH); UV (MeOH) λmax 242 (ε 4630), 310 (5730), and 330 (6360) nm; IR (KBr) νmax 3445, 1732, 1607, 1590, and 1568 cm−1; ECD (MeOH) Δε (nm) −8.4 (240), +17.9 (294), and −12.1 (336); 1 H and 13C NMR data (Table 1); HRESIMS m/z 541.29260 [M + Na]+ (calcd for C33H42O5Na, 541.29245). Trijapin B (3): colorless, amorphous solid; [α]25D −200 (c 0.1, MeOH); UV (MeOH) λmax 258 (ε 6380), 325 (4170), and 381 (1210) nm; IR (KBr) νmax 3413, 1731, and 1624 cm−1; ECD (MeOH) Δε (nm) +1.8 (234), +10.4 (288), and −12.1 (334); 1H and 13C NMR data (Table 1); HRESIMS m/z 541.29224 [M + Na]+ (calcd for C33H42O5Na, 541.29245). Trijapin C (4): colorless, amorphous solid; [α]25D +38 (c 0.04, CHCl3); UV (MeOH) λmax 247 (ε 13 320) nm; IR (KBr) νmax 3446, 1717, and 1671 cm−1; ECD (MeOH) Δε (nm) −6.9 (244), +1.5 (284), and −2.8 (336); 1H and 13C NMR data (Table 1); HRESIMS: m/z 559.30322 [M + Na]+ (calcd for C33H44O6Na, 559.30301). Trijapin D (5): pale yellow, amorphous solid; [α]25D −58 (c 0.6, MeOH); UV (MeOH) λmax 249 (ε 9320) and 282 (10 990) nm; IR (KBr) νmax 3568, 1721, 1667, and 1652 cm−1; 1H and 13C NMR data (Table 2); HRESIMS m/z 565.28070 [M − H]− (calcd for C33H41O8, 565.28069). Trijapin E (6): colorless, amorphous solid; [α]25D −25 (c 0.2, MeOH); UV (MeOH) λmax 248 (ε 9070), 275 (7560), and 343 (1370 sh) nm; IR (KBr) νmax 3447, 1732, 1698, and 1625 cm−1; 1H and 13C NMR data (Table 2); HRAPCIMS m/z 603.36846 [M + H]+ (calcd for C38H51O6, 603.36802). Calculation of ECD Spectra of (−)-Nemorosonol. Conformational searches and DFT calculations were carried out on Spartan 1010 and Gaussian 09,11 respectively. The two enantiomers {1S, 3R, 5R, 6S, 7R (1a); 1R, 3S, 5S, 6R, 7S (1b)} were subjected to conformational searches using MMFF94S as the force field. The initial low-energy conformers for 1a and 1b with Boltzmann distributions over 1% (11 and 10 conformers, respectively) were further optimized by DFT calculations at the B3LYP/6-31G(d) level in the presence of MeOH with a polarizable continuum model (PCM). The low-energy conformers for 1a and 1b with Boltzmann distributions over 1% (11 and 9 conformers, respectively) were subjected to TDDFT calculations at the B3LYP/6-31G(d) level in the presence of MeOH with a PCM. The resultant rotatory strengths of the lowest 30 excited states for each conformer were converted into Gaussian-type curves with half-bands (0.4 eV) using SpecDis v1.61.12 The calculated CD spectra were composed after correction based on the Boltzmann distribution of the stable conformers. Antimicrobial Testing. Antimicrobial assays against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, Candida albicans, Cryptococcus neoformans, and Trichophyton mentagrophytes were carried out as described previously.13 Amphotericin B, micafungin, hygromycin B, and kanamycin were used as controls for antimicrobial activities against E. coli (MIC, >31.2, >62.5, 2.0, and 31.2, 0.5, 0.5, and 31.2, 0.5, 2.0, and 31.2, 7.8, 1.0, 1.0 μg/mL, respectively), A. niger (IC50, 1.8,
