Article pubs.acs.org/jnp
Asnovolins A−G, Spiromeroterpenoids Isolated from the Fungus Aspergillus novof umigatus, and Suppression of Fibronectin Expression by Asnovolin E Kazuki Ishikawa,† Fumiaki Sato,† Takeshi Itabashi,† Hiroshi Wachi,† Hisashi Takeda,† Daigo Wakana,† Takashi Yaguchi,‡ Ken-ichi Kawai,† and Tomoo Hosoe*,† †
Department of Organic Chemistry, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo, Japan Medical Mycology Research Center (MMRC), Chiba University, 1-8-1 Inohama, Chuo-ku, Chiba, Japan
‡
S Supporting Information *
ABSTRACT: Seven novel spiromeroterpenoids, asnovolins A−G (1−7), one of which was shown to suppress fibronectin expression, were isolated from Aspergillus novof umigatus CBS117520 along with a known compound, novofumigatonin (8). The structures of asnovolins A−G were elucidated using MS and 2D-NMR data. Asnovolin E (5) suppressed fibronectin expression by normal human neonatal dermal fibroblast cells.
T
using 2D-NMR and MS data and the effects of asnovolins A−E (1−5) on fibronectin expression.
he complex interactions between tumor cells and the extracellular matrix play important roles in mediating and regulating many processes during tumor metastasis, including cell migration, cytoskeletal reorganization, and morphologic transition.1 Fibronectin is a glycoprotein (approximately 250 kDa) component of the extracellular matrix and plays important roles in embryonic development, cell adhesion, migration, tumor invasion, and metastasis.2,3 Fibronectin expression is inversely associated with survival and clinical outcome in cancer patients.4,5 Fibronectin also promotes invasion and metastasis of ovarian cancer and melanoma by interacting with its receptor integrins.6−8 Several studies have shown that fibronectin up-regulates matrix metalloproteinase 2 and matrix metalloproteinase 9 levels, resulting in invasion and metastasis of breast cancer.9,10 Therefore, inhibiting fibronectin expression is a potential strategy for suppressing cancer metastasis. During previous research to identify novel fibronectin expression regulatory factors from fungal secondary metabolites, we isolated novobenzomalvins A−C from the fungus Aspergillus novofumigatus CBS117520.11 These compounds were identified as quinazolinobenzodiazepine derivatives and were shown to up-regulate fibronectin expression in normal human neonatal dermal fibroblast primary cells. Further investigations of methanol (MeOH) extracts of A. novof umigatus CBS117520 led to the discovery of seven novel spiromeroterpenoids, designated asnovolins A−G, along with novofumigatonin (8), isolated as secondary metabolites of A. novof umigatus.12 We tested these compounds for fibronectin expression regulatory activity and found that asnovolin E (5) suppresses fibronectin expression in normal human neonatal dermal fibroblast cells. In this report, we describe the structures of asnovolins A−G as elucidated © 2016 American Chemical Society and American Society of Pharmacognosy
■
RESULTS AND DISCUSSION Structure Elucidation. Compound 1 was isolated as a colorless, amorphous solid, and the molecular formula was determined as C26H38O6 (eight degrees of unsaturation) on the basis of high-resolution electron ionization (HREI)MS. The 1H NMR spectrum of 1 exhibited signals indicative of eight methyl groups, including one methoxy group, five methylene groups, and three methine groups (Table 1). Three signals (δ 193.3, 174.8, and 171.7) in the 13C NMR data revealed the presence of three carbonyl carbons (one ketone and two esters), and two signals (δC 181.0 and 109.0) revealed one olefin. These units account for four degrees of unsaturation, indicating that four rings are present. Detailed 1H−1H COSY and HMBC correlations are shown in Figure 2. The structures of A and B rings in 1 was determined as follows. The A ring was established as a sevenmembered lactone based on 1H−1H COSY correlations between H2-1 [δH 1.80 (t, J = 6.3 Hz)], H2-2 [δH 2.77 (dt, J = 15.6, 6.3 Hz), and δH 2.68 (dt, J = 15.6, 6.3 Hz)] and the HMBC correlations of H2-1 with C-9 (δC 98.5), H2-1, H3-14 [δH 1.46 (s)], and H3-15 [δH 1.47 (s)] with C-5 (δC 51.0), H2-1 and H2-2 with C-3 (δC 174.8), H3-14 and H3-15 with C-4 (δC 85.6), and C-14 (δC 24.4) and C-15 (δC 33.2) methyl groups. The 1H−1H COSY correlations of H-8 [δH 1.88 (m)] with H3-12 [δH 1.09 (d, J = 7.1 Hz)] and HMBC correlations of Received: January 7, 2016 Published: September 14, 2016 2167
DOI: 10.1021/acs.jnatprod.6b00013 J. Nat. Prod. 2016, 79, 2167−2174
1.88 m
2168
41.9
22.2
8.2
171.7
6′
7′
8′
9′
4′, 6′, 9′, 10′
58.3
1.72 s
1.21 s
2′, 3′, 4′
1′, 2′, 6′
172.1
8.4
63.7
2.43 dq (12.1.6.7) 1, 5′, 7, 10′ 41.2
3.25 d (12.1)
(11.9)
(11.6)
3.60 d 1.75 s
(11.6)
3.70 d
2.48 dq (11.9,7.1)
3.66 d
58.2
5′
194.1
111.5
2′, 3′, 4′
1′, 2′, 6′
11
171.4
8.2
22.0
1′, 5′, 7, 9′, 10′ 40.2
4′, 6′, 9′, 10′ 58.0
192.9
107.0
182.1
193.3
175.9
109.0
34.5
32.1
4′
4, 5, 14
4, 5, 15
17.1 22.8
3′
1.47 s
1.45 s
7,9 1, 5, 9, 10
181.0
50.3
33.1
24.3
(7.0)
8, 1′, 7′
2′
4, 5, 14
4, 5, 15
1.11 d 1.32 s
2.00 m
β 1.89 m
s
1.68 s
1.26 s
a
1′, 4′, 6′, 9′, 10′
3
4, 5, 14
4, 5, 15
1, 5, 10
7, 8, 9
8, 9, 10, 1′, 2′, 7′
8, 9,10, 1′, 2′, 7
9
4
4, 9
1, 3
2, 3,10
HMBC
2′, 3′, 4
1′, 2′, 6′
2.52 dq (12.1,6.4) 1′, 5′, 7, 10′
3.25 d (12.1)
3.67
1.37 s
1.33 s
1.28 s
0.83 d (6.2)
β 1.93 d (13.8)
8, 9,10, 1′, 2′, 41.7 α 2.13 d (13.8) 7′
45.1
99.7
38.5
44.1
1.47 s
17.3
β 1.71 m 28.5 α 1.52 m
44.9
33.2
15
1.46 s
7,8,9
1, 5, 9, 10 17.0
1.54 m
2.32 m
β 1.47 m
22.8 α 1.97 m
48.0
74.9
174.1
29.0
1′
24.4
14
1.36 s
1.09 d (7.1)
δH (J in Hz)
asnovolin C (3) 34.2 α 2.24 m
δC
51.8
17.1
13
(13.9)
(13.9)
4,6
3
HMBC
a
16
17.3
12
8, 1′
β 1.69 d
44.2
97.9
39.8
β 1.86 d (13.6)
12
β 1.74 m
29.2 α 1.63 m
β 1.53 m
8, 9. 1′, 2′ 38.0 α 2.34 d
44.2
10
1.88 m
5,12
1.79 m
21.2 α 1.72 m
50.8
85.7
43.5 α 2.09 d (13.6)
98.5
11
40.0
9
β 1.72 m
5
24.4 α 1.61 m
8
7
5
5
6
β 1.52 m
51.0
5
21.4 α 1.72 m
6
1.75 m
85.6
4
β 2.65 ddd (15.9, 8.8, 3.6) 175.1
β 2.68 dt (15.6, 6.3) 3
174.8
3
31.8 α 2.72 ddd (15.9, 8.8, 3.6)
31.4 α 1.78 m
δH (J in Hz)
32.0 α 2.77 dt (15.6, 6.3) 3
3,5,9
δC
2
1.80 t (6.3)
HMBC
asnovolin B (2)
32.0
δH (J in Hz) a
1
position δC
asnovolin A (1)
Table 1. NMR Spectroscopic Data (1H 400 MHz, 13C 100 MHz, CDCl3) for Compounds 1−5
1.56 m
2.35 m
1.99 m
17.2
171.4
8.2
22.0
40.2
58.0
193.0
107.1
182.2
44.1
34.5
31.9
22.0
10
9
1, 3
13
1.70 s
1.27 s
δH (J in Hz)
2.11 m
2.93 dd (8.4, 1, 3 8.3)
β 1.74 m
42.7
98.6
40.4
1.84 m
β 1.70 m
28.5 α 1.60 m
β 1.60 m
24.3 α 1.78 m
49.6
147.2
174.5
30.2
HMBCa
asnovolin E (5) 32.5 α 1.90 m
δC
17.2
193.4
108.0
182.7
44.6
51.9
115.0
23.6
18.3
2′, 3′, 4
1′, 2′, 6′
171.8
8.4
22.0
41.3
(11.9) 4′, 6′, 9′, 10′ 58.3
4, 5,14
4, 5, 15
1, 5, 9, 10
(5.7) 7,9
(13.8) 9, 1′, 6′, 7
2.62 brd (11.9) 1′
3.27 d
1.39 s
1.35 s
1.30 s
0.85 d
β 1.94 d
41.7 α 2.11 d
45.1
99.8
38.6
β 1.67 m
28.5 α 1.56 m
β 1.74 m
22.7 α 1.98 m
48.0
75.1
178.6
29.2
β 1.47 m
HMBC
a
3
5, 14
5,14
4, 5, 15
1, 5, 9, 10
1.73 s
1.24 s
2′, 3′, 4
11, 1′, 2′, 6
2.51 dq (11.9, 1′, 5′, 7 6, 8)
3.27 d (11.9) 4, 6′, 9′, 10′
3.67 s
4.81 s
4.86 s
1.87 s
1.07 s
0.99 d (7.0) 7, 8, 9
β 1.93 d (13.7) 8, 7
(13.8) 9, 1′, 2′, 6′, 7 43.2 α 2.15 d (13.7) 8, 10, 1′, 7′
δH (J in Hz)
34.2 α 2.26 m
δC
asnovolin D (4)
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00013 J. Nat. Prod. 2016, 79, 2167−2174
9′ 3.77 s
1.01 d (6.8) 1′, 5′, 6′
H3-12 and H3-13 [δH 1.36 (s)] with C-9, H3-12 with C-7 (δC 24.4), H2-6 [δH 1.72 (m) and 1.52 (m)], H2-7 [δH 1.61 (m) and 1.72 (m)], H3-13 with C-5, and H3-13 with C-10 (δC 44.2) indicated that the B ring is a cyclohexane. The structures of the C and D rings were elucidated as a two-ring system from the above data. The C ring was identified as tetrahydrofuran based on HMBC correlations of H2-7′ [δH 1.21 (s)] and H2-11 [δH 2.09 (d, J = 13.6 Hz) and 1.86 (d, J = 13.6 Hz)] with C-1′ (δC 44.9) and of H-11 with C-2′ (δC 181.0) and C-3′ (δC 109.0). The 1H−1H COSY correlations indicated a sequence of H-5′−H6′−H10′. The HMBC correlations of H-5′ [δH 3.25 (d, J = 12.1 Hz)] and H-6′ [δH 2.43 (dq, J = 12.1, 6.7 Hz)] with C-9′ (δC 171.7), H-6′ and H-8′ [δH 1.72 (s)] with C-4′ (δC 193.3), H3-8′ with C-2′ and C-3′ (δC 109.0), H2-7′ with C-2′ and H-6′, and H2-7′ with C-1′ revealed the presence of a D ring having a cyclohexanone unit (Figure 2). Linkage of the B and C rings through a spiro carbon (C-9) was confirmed based on HMBC correlations of H2-11 with C-8 (δC 40.0) and C-9. From the above results, the planar structure of 1 was established as shown in Figure 1. The relative structure of 1 was established from the analysis of NOESY data (Figure 3). Key NOESY correlations of H-2β [δH 2.77 (dt, J = 15.6, 6.3 Hz)]/H3-13 and H3-15, H3-12/H3-13, H-7β [δH 1.72 (m)], and H-6β, and H-2α/H3-14 suggested that the relative configuration of the A/B ring is in the trans-form and that the three methyl groups (H3-12, H3-13, and H3-15) are on the same side, whereas H3-14 and H-5 are on the opposite side. The NOESY correlations of H3-10′ [δH 1.00 (d, J = 6.7 Hz)]/ H-5′ and H3-7′, H3-7′/H-11α, and H-11β/H-6′β suggested that the two methyl groups [C-7′ (δC 22.2) and C-10′ (δC 14.7)] and H-5′ are on the same side in the C and D rings. Moreover, NOESY correlations of H-8 with H-5/H-11α and H2-2/H3-8′ confirmed that the relative configurations of C-5 and C-9 are as shown in Figure 1. Compound 2 was isolated as a colorless, amorphous solid with a determined molecular formula of C26H38O7 (eight degrees of unsaturation) on the basis of HREIMS. The molecular formula of 2 has one additional oxygen atom (16 mass units) compared with that of 1. The 1H and 13C NMR spectra of 2 were very similar to those of 1, except for a downfield-shifted C-7′ signal (2: δC 63.7, 1: δC 22.2) in 13C NMR spectrum and signals indicative of H3-7′ methyl groups in 1 (δH 1.21, s) versus H2-7′ methylene groups in 2 [δH 3.70 (d, J = 11.6 Hz) and δH 3.60 (d, J = 11.6 Hz)] in the 1H NMR spectrum. The above results suggest that 2 is the 7′-oxide of 1. Furthermore, as determined from the results of detailed 2D-NMR analyses shown in Supporting Figures S44 and S45, the relative structure of 2 was determined to be the same as that of 1. Compound 3 was isolated as a colorless, amorphous solid with a determined molecular formula of C27H42O7 (seven degrees of unsaturation) on the basis of HREIMS. Compared with 1, compound 3 had one carbon atom, four additional hydrogen atoms, one additional oxygen atom, and one less degree of unsaturation. The 1H and 13C NMR spectra of 3 were very similar to those of 1, except for an upfield-shifted C-4 signal (4: δC 74.9, 1: δC 85.6) and the presence of methyl groups of C-16 [δH 3.67 (s), δC 51.8] binding to the C-3 carboxylic acid. These results indicate that 3 is an A-ringhydrolyzed methyl ester derivative of 1. Moreover, on the basis of the results of detailed 2D-NMR analyses including NOESY data, the relative configuration of 3 was determined to be the same as that of 1.
52.4
14.7
HMBCa δH (J in Hz)
Article
9′
(6.7) 1′, 5′, 6′ 1.00 d
3.76 s 52.4
14.6
HMBC δH (J in Hz)
9′
1′, 5′, 6′ 1.00 d (6.4)
3.74 s 52.3
14.5
δH (J in Hz)
HMBC correlations are from the proton(s) stated to the indicated carbon. a
9′
1′, 5′, 6′ (7.1) 1.11 d
3.74 s 52.3
15.4 1′, 5′, 6′
9′ 3.77 s 52.4 11′
1.00 d (6.7) 14.7 10′
δH (J in Hz) δH (J in Hz) position δC
asnovolin A (1)
Table 1. continued
HMBC
a
δC
asnovolin B (2)
HMBC
a
δC
asnovolin C (3)
HMBC
a
δC
asnovolin D (4)
a
δC
asnovolin E (5)
Journal of Natural Products
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DOI: 10.1021/acs.jnatprod.6b00013 J. Nat. Prod. 2016, 79, 2167−2174
Journal of Natural Products
Article
Figure 1. Structures of asnovolins A−G (1−7) and novofumigatonin (8).
Figure 2. Key 2D-NMR correlations for asnovolins A, C, and F (1, 3, and 6).
Figure 3. Key NOESY correlations of asnovolins A, C, and F (1, 3, and 6).
(δH 4.86, 4.81)] in 5 instead of the absence of the oxycarbon (δC 75.1) and methyl group in 4. The above results suggest that 5 is a dehydrated derivative of 4. On the basis of the results of detailed 2D-NMR analyses (Supporting Figure S44), the relative structure of 5 was determined to be the same as that of 4 except with respect to C-4 (Figure 1). Compound 6 was isolated as a colorless, amorphous solid with a determined molecular formula of C25H34O8 (nine degrees of unsaturation) on the basis of HR chemical ionization (CI)MS. The 1H NMR spectrum of 6 exhibited signals for seven methyl groups, five methylene groups, and five methine groups (Table 2). The 13C NMR signals (δC 174.6 and 170.6 ppm) and the IR spectrum (1742 and 1637 cm−1) revealed the presence of two carbonyl carbons of esters. These units account for two degrees of unsaturation, indicating that
Compound 4 was isolated as a colorless, amorphous solid with a determined molecular formula of C26H40O7 (seven degrees of unsaturation) on the basis of HREIMS. The 1H and 13C NMR spectra of 4 were very similar to those of 3, except for the absence of methyl groups binding to the C-3 carboxylic acid. On the basis of the results of detailed 2D-NMR analyses, 4 was determined to be a demethylenated derivative of 3, as shown in Supporting Figure S44. Compound 5 was isolated as a colorless, amorphous solid with a determined molecular formula of C27H40O6 (eight degrees of unsaturation) on the basis of HREIMS. Compared with 4, 5 has two fewer hydrogen atoms, one less oxygen atom, and one additional degree of unsaturation. The 1H and 13C NMR spectra of 5 were very similar to those of 4, except for signals indicative of an exomethylene group [δC 147.2 and 115.0 2170
DOI: 10.1021/acs.jnatprod.6b00013 J. Nat. Prod. 2016, 79, 2167−2174
Journal of Natural Products
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Table 2. NMR Spectroscopic Data (1H 400 MHz, 13C 100 MHz, CDCl3) for Asnovolins F and G (6 and 7) asnovolin F (6) position
a
δC
1
75.8
2
35.9
3 4 5 6
170.6 80.8 55.0 17.2
7
30.3
8 9 10 11
42.6 88.4 50.5 49.8
12 13
16.8 65.8
14 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′
31.0 21.7 128.3 107.8 78.7 42.1 39.8 47.7 174.6 22.4 12.3 19.3
asnovolin G (7)
δH (J in Hz)
α β
α β α β
α β α β
δC
HMBCa
4.24
brd
(4.1)
3, 13
27.8
2.89 2.81
dd dd
(15.9, 4.1) (15.9, 4.1)
1, 3, 10 1, 3, 10
29.6
1.57 1.57 1.47 1.57 1.71 2.13
m m brd m m m
2.24 1.85 1.05 4.70 4.49 1.25 1.13
d d d d d s s
4.81 2.71 2.15
d dd m
1.75 1.19 0.90
s d s
175.2 74.8 53.0 21.0
4, 13 (5.3)
δH (J in Hz) α β α β
2.06 2.63 2.56 2.44
m m m m
α β
1.43 1.64 1.38 1.61
m m m m
2.22
brs
2.10 1.92 1.05 4.64 4.45 1.29 1.31
m d d d d s s
4.78 2.68 2.12
d dd m
1.75 1.17 1.03
s d s
28.4
(13.8) (13.8) (7.6) (13.0) (13.0)
(8.6) (8.6, 6.3)
(7.3)
8, 9, 8, 1, 5, 4, 4,
40.2 92.9 45.9 50.2
9, 1′, 10′ 5′, 6′, 10′ 9 3, 5, 10 9, 10 5, 15 5, 14
15.9 35.2 27.1 67.4
5′, 7′, 8′ 2′, 3′, 5′, 6′, 7′ 11, 4′, 6′, 7′, 9′, 10′
2′, 3′ 4′, 5′, 6′ 11, 1′, 5′, 6′
128.8 107.8 78.3 42.2 40.1 48.1 174.8 22.6 12.2 19.2
α β α β
HMBCa
10 3
(13.8) (7.4) (11.6) (11.6)
10, 5′, 6′ 7, 8, 9
4, 5, 15 4, 5, 14
(8.6) (8.6, 6.1)
(7.3)
5′, 7′, 8′ 2′, 3′, 5′, 6′, 7′ 4′, 6′, 9′, 10′
2′, 3′ 4′, 5′, 6′ 11, 1′, 5′, 6′
HMBC correlations are from the proton(s) stated to the indicated carbon.
seven rings are present. The 1H−1H COSY correlations indicated three sequences of H-1−H2-2, H2-6−H2-7−H-8−H3-13, and H-3′−H-4′−H-5′−H3-9′, as shown by bold lines in Figure 2. The HMBC correlations of H3-14 [δH 2.77 (s)] and H3-15 [δH 1.13 (s)] with C-4 (δC 80.8) and C-5 (δC 55.0), H-13α [δH 4.70 (d, J = 13.0 Hz)] with C-1 (δC 75.8), C-3 (δC 170.6), C-9 (δC 88.4), and C-10 (δC 50.5), H-1 [δH 4.24 (brd, J = 4.1 Hz)] with C-3 and C-13 (δC 65.8), H2-2 [δH 2.89 (dd, J = 15.9, 4.1 Hz) and 2.81 (dd, J = 15.9, 4.1 Hz)] with C-3 and C-10, and H3-12 [δH 1.05 (d, J = 7.6 Hz)] with C-9 established the presence of A, B, and F rings (Figure 2). The HMBC correlations of H-8′ [δH 1.75 (s)] with C-2′ (δC 107.8) and C-3′ (δC 78.7), H-3′ [δH 4.81 (d, J = 8.6 Hz)] with C-1′ (δC 128.3), C-5′ (δC 39.8), C-7′ (δC 174.6), and C-8′ (δC 22.4), H-4′ [δH 2.71 (dd, J = 8.6, 6.3 Hz)] with C-6′ (δC 47.7) and C-7′, H-5′ [δH 2.15 (m)] with C-6′ and C-10′ (δC 19.3), H3-10′ [δH 0.90 (s)] with C-11 (δC 49.8), C-1′, and C-6′, H-11α [δH 2.24 (d, J = 13.8 Hz)] with C-9, C-1′, and C-10′, and H-11β [δH 1.85 (d, J = 13.8 Hz)] with C-9, C-1′, and C-10′ indicated the presence of C, D, and E rings. The structure of these rings in 6 was found to be identical to the partial structure of the known compound novofumigatonin (8), which was also isolated from A. novofumigatus, and the absolute configuration was determined by vibrational circular dichroism in combination with density functional calculations.12 The planar structure
of 6 indicated that A/B/F rings and C/D/E rings are attached via spiro carbon C-9, based on HMBC correlations of H2-11 with C-8 (δC 42.6), C-9, and C-10. The relative structure of 6 was determined by the detailed analysis of NOESY data (Table 3 and Supporting Figure S44). Key NOESY correlations of H-13α/H-6β [δH 1.47 (brd, J = 5.3 Hz)], H-7β [δH 1.71 (m)], and H3-15, H3-12/H-6β, H-7β, and H2-13, and H-1/H-5 [δH 1.57 (m)] and H-7α [δH 1.57 (m)] indicated that the relative configuration of the A/B ring is in the trans-form, whereas the A/F ring is in the cis-form. Moreover, the relative configuration of the C, D, and E rings, containing three methyl groups (CH3-8′, CH3-9′, and CH3-10′) and two methine groups (CH-3′ and CH-4′) as shown in Figure 1, was determined based on key NOESY correlations: H-3′/H-4′ and H3-8′, H-5′/H-4′ and H-11β, and H3-10′/H-11α. Key NOESY correlations of H-5/H-11α, H-8 [δH 2.13 (m)]/H-11β, and H3-10′/H-2↑β and H-11α confirmed that the relative configuration of 6 is as shown in Figure 1. The relative configuration of spiro carbon C-9 and the above data provided the relative configuration of 6. Compound 7 was isolated as a colorless, amorphous solid with a determined molecular formula of C25H36O8 (eight degrees of unsaturation) on the basis of HRESIMS. The 1H and 13C NMR spectra of 7 were very similar to those of 6, except for the absence of signals indicative of the C-1 methine group of 7 2171
DOI: 10.1021/acs.jnatprod.6b00013 J. Nat. Prod. 2016, 79, 2167−2174
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that 1−5 exhibit the same stereochemical configurations. Moreover, the partial structure of rings A and B in 1−5 was found to be similar to that of the known meroterpenoids simplicissin,13 fumigatonin,14 and novofumigatonin (8).12 These data suggest that the asnovolin biosynthesis pathway (Figure S45, see Supporting Information) is similar to that of these meroterpenoids, which are formed via the common intermediate 3,5-dimethyloesellinate and the terpenoid precursor farnesyl diphosphate.15 On the basis of these data, the absolute configurations of 1−5 (5R, 8S, 9S, 10S, 1′R, 5′R, 6′S) were suggested as shown in Figure 1. The proposed biosynthesis pathway of 6 and 7 is shown in Figure S45 and is based on the similarity of the C, D, and E rings of 6 and 7 and the partial structure of known meroterpenoids fumigatonin and novofumigatonin (8). In accordance with this biosynthesis pathway and the relative structures of 6 and 7 indicated by NOESY spectra (Table 3), the proposed absolute configurations of 6 and 7 are also shown in Figure 1. Biological Activity. The effect of compounds 1−5 on the in vitro expression of fibronectin by normal human neonatal dermal fibroblast cells was examined. The activity of compounds 6 and 7 could not be examined because these compounds were unstable in the assay used. The results are shown in Figure 4. Only compound 5 (25 μM) suppressed fibronectin expression (87% inhibition relative to the DMSO control) (Figure 4a). In addition, the effect of compound 5 was concentration dependent (Figure 4b). No significant differences in fibronectin expression were observed in normal human neonatal dermal fibroblast cells treated with compounds 1−4. Discussion. We sought to identify novel fibronectin expression regulatory factors from fungal MeOH extracts and isolated seven novel spiromeroterpenoids, designated asnovolins A−G (1−7), from A. novof umigatus CBS117520. Novofumigatonin (8), which has a skeleton similar to asnovolins A−G (1−7), was isolated from the same species, A. novof umigatus. However, rings C, D, and E of 8 are oxidized to a greater extent than those of 1−5, and rings A, B, and F of 6 and 7 are formed via a complex cyclization reaction. Accordingly, it is suggested that the asnovolins and novofumigatonin share a common biosynthetic pathway, as proposed by Simpson et al.,15,16 which includes a common intermediate, 3,5-dimethylosellinate, and the terpenoid precursor farnesyl diphosphate. In the tests of fibronectin expression in normal human neonatal dermal fibroblast cells, only 5 showed activity. With the exception of 2, asnovolins A−E share the same structure in rings C and D, which suggests that the substructure is not involved in the suppression of fibronectin expression. Furthermore, cleavage of the A ring and methyl group of the carbonic acid in 5 is not involved in that activity, in comparison with 1−4. The presence of an exomethylene group only in 5 suggests that this group is involved in the suppression of fibronectin expression. The only previous report of a fibronectin-suppressing factor is that of the synthetic oleanane triterpenoid 2-cyano-3,12dioxooleana-1,9-dien-28-oic acid methyl ester, which was shown to suppress fibronectin expression in a bleomycin model of pulmonary fibrosis17 and has been investigated for other biological activity.18−20 Other than asnovolin E, no other fibronectin expression regulatory factors have been reported. Thus, asnovolin E might prove to be a useful fibronectin expression regulatory factor.
Table 3. NOESY Correlations of Asnovolins Fand G (6 and 7) in CDCl3 position 1a 1b 2a 2b 3 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′
6 5, 7α, 10′
7 11α 5
10′
1, 11α
1b, 11α, 11β
12, 13b 1 12, 13b 11β
12, 13a
5, 10′ 8, 5′ 6β, 7β, 13a, 13b 6β, 7β, 12, 15 12
12
1a, 5, 11β, 9′, 10′ 1a, 5, 11α, 8, 5′, 9′ 6β, 8, 13a 6β, 14, 15
13b
13a 13a
4′, 8′ 3′, 5′ 11β, 4′
4′, 8′ 3′, 5′ 11β, 4′
3′
3′ 11α 11α
1, 2b, 11α
and its replacement with a methylene group in 7. The planar structure of 7 was established as shown in Figure 1, and the relative structure was determined by NOESY data (Table 3 and Supporting Figure S44). The relative configuration of the B/F ring (Figure 1) was determined based on the key NOESY correlations of H-13α [δH 4.64 (d, J = 11.6 Hz)]/H-6β [δH 1.38 (m)], H3-14 [δH 1.29 (s)], and H-15 [δH 1.31 (s)], H-6β/H3-12 [δH 1.05 (d, J = 7.4 Hz)], and H-1α [δH 2.06 (m)]/H-5 [δH 1.43 (m)]. Furthermore, it was estimated that the relative configurations of the three methyl groups (CH3-8′, CH3-9′, and CH3-10′) and two methine groups (CH-3′ and CH-4′) of the C, D, and E rings in 7 are the same as those of 6 based on the key NOESY correlations of H-3′ [δH 4.78 (d, J = 8.6 Hz)]/H3-4′ [δH 2.68 (dd, J = 8.6, 6.1 Hz)] and H-8′ [δH 1.75 (s)], H-5′ [δH 2.12 (m)]/H-4′ and H-11β [δH 1.92 (d, J = 13.8 Hz)], and H-11α [δH 2.10 (m)]/H3-9′ [δH 1.17 (d, J = 7.3 Hz)] and H3-10′ [δH 1.03 (s)]. Moreover, key NOESY correlations of H-5/H-11α and H-8/H-11β indicated the relative relationship between the B ring and the structure consisting of C, D, and E rings. Finally, the relative configuration of 7 was established based on the key NOESY correlations of H-1β [δH 2.63 (m)]/H-11α, H-2α [δH 2.56 (m)]/H3-14 and H3-15, H-2β [δH 2.44 (m)]/H3-10′, and H-13α/H3-14 and H3-15. In all CD spectra of 1−5, negative and positive Cotton effects were observed at around 303 and 270 nm (Figure S43, see Supporting Information), respectively. These results suggest 2172
DOI: 10.1021/acs.jnatprod.6b00013 J. Nat. Prod. 2016, 79, 2167−2174
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Figure 4. Effect of asnovolins on the expression of fibronectin by normal human neonatal dermal fibroblast cells. (a) Normal human neonatal dermal fibroblast cells were incubated with 1−5 (each 25 μM) for 24 h. (b) Normal human neonatal dermal fibroblast cells were incubated with 10, 25, or 50 μM asnovolin D (4) or E (5) for 24 h. Values represent mean ± SD. Results are representative of three independent experiments. ***p < 0.001 vs blank (B, DMSO).
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along with novofumigatonin (6). Fraction 3 (chloroform/acetone [1:4] eluate) was rechromatographed by MPLC on a silica gel column (benzene/acetone [1:1] to acetone) followed by HPLC to give asnovolin A (1: 5.9 mg) (benzene/acetone [10:1]). Asnovolin A (1): colorless, amorphous solid; [α]27 D −92.0 (c 0.11, MeOH); HREIMS obsd 446.2662, calcd for C26H38O6 (M+) 446.2668; UV λmax (MeOH) nm (log ε) 268 (4.1); IR (KBr) cm−1 3435, 1726, 1631; CD (c = 6.73 × 10−5, MeOH) Δε (nm) 5.3 (204), 8.1 (270), −12.9 (303); for the 1H and 13C NMR, HMBC, and NOESY data, see Table 1 and Supporting Information. Asnovolin B (2): colorless, amorphous solid; [α]27 D −73.5 (c 0.16, MeOH); HREIMS obsd 462.2631, calcd for C26H38O7 (M+) 462.2618; UV λmax (MeOH) nm (log ε) 267 (4.1); IR (KBr) cm−1 3438, 1732, 1633; CD (c = 6.49 × 10−5, MeOH) Δε (nm) 1.7 (239), 14.4 (268), −16.9 (302); for the 1H and 13C NMR, HMBC, and NOE data, see Table 1 and Supporting Information. Asnovolin C (3): colorless, amorphous solid; [α]23 D −103.3 (c 0.41, MeOH); HRCIMS obsd 479.3004, calcd for C27H43O7 (M + H)+ 479.3009; UV λmax (MeOH) nm (log ε) 270 (4.1); IR (KBr) cm−1 3526, 1739, 1632; CD (c = 4.18 × 10−5, MeOH) Δε (nm) 0.7 (239), 11.9 (270), −17.7 (303); for the 1H and 13C NMR, HMBC, and NOE data, see Table 1 and Supporting Information. Asnovolin D (4): colorless, amorphous solid; [α]26 D −233.8 (c 0.01, MeOH); HREIMS obsd 464.2784, calcd for C26H40O7 (M+) 464.2774; UV λmax (MeOH) nm (log ε) 270 (3.8); IR (KBr) cm−1 3441, 1739, 1631; CD (c = 6.47 × 10−5, MeOH) Δε (nm) 0.9 (239), 8.9 (269), −12.5 (303); for the 1H and 13C NMR, HMBC, and NOESY data, see Table 1 and Supporting Information. Asnovolin E (5): colorless, amorphous solid; [α]23 D −80.6 (c 0.11, MeOH); HRESITOFMS obsd 483.27424, calcd for C27H40O6Na (M + Na)+ 483.27226; UV λmax (MeOH) nm (log ε) 269 (4.3); IR (KBr) cm−1 1727, 1631; CD (c = 6.52 × 10−5, MeOH) Δε (nm) −1.3 (238), 11.6 (270), −18.1 (303); for the 1H and 13C NMR, HMBC, and NOESY data, see Table 1 and Supporting Information. Asnovolin F (6): colorless, amorphous solid; [α]24 D −122.0 (c 0.13, MeOH); HRCIMS obsd 463.2342, calcd for C25H35O8 (M + H)+ 463.2332; UV λmax (MeOH) nm (log ε) 268 (3.1); IR (KBr) cm−1 1742, 1637; CD (c = 6.49 × 10−5, MeOH) Δε (nm) −2.9 (213), 0.9 (267), −1.3 (303); for the 1H and 13C NMR, HMBC, and NOESY data, see Table 2 and Supporting Information. Asnovolin G (7): colorless, amorphous solid; [α]32 D −93.3 (c 0.12, MeOH); HREIMS obsd 464.2396, calcd for C 25 H36 O8 (M) + 464.2410; UV λmax (MeOH) nm (log ε) 215 (4.3), 246 (3.9), 279 (3.2); IR (KBr) cm−1 3444, 1747, 1634; CD (c = 6.47 × 10−5, MeOH) Δε (nm) −4.8 (229), 16.5 (249), −0.3 (315); for the 1H and 13 C NMR, HMBC, and NOESY data, see Table 2 and Supporting Information.
EXPERIMENTAL SECTION
General Experimental Procedures. CI and EIMS data were measured on a JMS-MS 600W spectrometer (JEOL Co. Ltd., Tokyo, Japan). UV and IR spectra were recorded on an Ultrospec 2100 pro UV−visible spectrophotometer (Amersham Biosciences Ltd., UK) and an FT/IR-4100 instrument (JASCO Co. Ltd., Tokyo, Japan), respectively. 1H and 13C NMR spectra were recorded using a Bruker AVANCE-400 spectrometer (400.13 MHz for 1H, 100.61 MHz for 13 C, Bruker Biospin, Billerica, MA, USA) or Lambda-500 (500.00 MHz for 1H, 125.43 MHz for 13C, JEOL Ltd., Tokyo, Japan). Chemical shifts (δ) were measured in ppm using tetramethylsilane as an internal standard. CD curves were determined on a J-820 spectropolarimeter (JASCO Co. Ltd.). Optical rotations were measured with a P-1020 photopolarimeter (JASCO Co. Ltd.). TLC was visualized by UV light at 254 nm and/or by spraying with phosphomolybdic acid (5%)/ceric acid (trace) in 5% H2SO4 and then heating. Column chromatography was performed using a Sephadex LH-20 column (GE Healthcare BioScience AB, Uppsala, Sweden). MPLC was performed using a Chemco Low-Prep 81-M-2 pump (Chemco Scientific Co. Ltd., Osaka, Japan) and an ULTRA PACK SI-40B column (300 × 26 mm, Yamazen Corp., Osaka, Japan). HPLC was performed using a Senshu SSC-3160 pump (flow rate 7 mL/min, Senshu Scientific Co. Ltd., Tokyo, Japan) and a YMCPack PEGASIL silica 60-5 column (300 × 10 mm, YMC Co. Ltd., Kyoto, Japan), equipped with a YRD-883 RI detector (Shimamuratech Ltd., Tokyo, Japan). Fermentation and Isolation of A. novofumigatus CBS117520 Metabolites. Polished rice (Akitakomachi, 24 kg) was soaked in water for 30 min and then sterilized using an autoclave. A. novof umigatus CBS11752021 was cultivated for 21 days in 200 Roux flasks, each containing 140 g of moist rice. The cultivated rice was extracted with MeOH, and the extract was concentrated in vacuo. The resulting residue was suspended in water and extracted with ethyl acetate. The extract of ethyl acetate (52 g) was partitioned between n-hexane and MeOH to yield an acetonitrile-soluble mixture. The acetonitrile extract (29.4 g) was sequentially extracted with n-hexane, benzene, chloroform, ethyl acetate, and MeOH (100 mL each). The benzene extract (18 g) was chromatographed on a Sephadex LH-20 column (solvent system: n-hexane/chloroform [1:4], 200 mL; chloroform/acetone [3:2], 200 mL; chloroform/acetone [1:4], 200 mL; acetone, 200 mL; and then MeOH, 500 mL). Fraction 2 (chloroform/ acetone [3:2] eluate) was rechromatographed by medium-pressure liquid chromatography (MPLC) on a silica gel column (n-hexane/ acetone [2:1] to acetone) followed by HPLC to give asnovolin E (5: 2.1 mg) (benzene/acetone [20:1]), asnovolins D (4: 7.7 mg) and F (7: 2.0 mg) (hexane/acetone [3:1]), asnovolin G (8: 2.0 mg) (chloroform/acetone [20:1]), asnovolin C (3: 2.2 mg) (hexane/acetone [2:1]), and asnovolin B (2: 4.0 mg) (chloroform/acetone [10:1]), 2173
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Immunoblotting. The antibodies used for Western blot analysis included mouse antifibronectin (BD Bioscience, Franklin Lakes, NJ, USA) and anti-GAPDH (Millipore, Bedford, MA, USA). Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG (GE Healthcare, Tokyo, Japan) were used as secondary antibodies. Normal human neonatal dermal fibroblast cells (Takara Bio Inc., Shiga, Japan) were grown in complete medium: Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (Sigma), L-glutamine, and penicillin/streptomycin (Invitrogen Co. Ltd., Carlsbad, CA, USA). Cells were plated at a density of 5 × 105 cells/well in a 24-well plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland). After 24 h of incubation in serum-free Dulbecco’s modified Eagle’s medium, cells were treated with complete medium containing various compounds. After 24 h of incubation, cells were washed with cold phosphate-buffered saline and lysed on ice in lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, and protease inhibitor cocktail [Roche Applied Science, Indianapolis, IN, USA]). Proteins were resolved on 7.5% Tris-glycine polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Bio-Rad Bioscience, Hercules, CA, USA). The membranes were blocked for 1 h in Tris-buffered saline with 0.05% Tween-20 and 5% fat-free milk and then incubated overnight at 4 °C with the primary antibody. The membranes were washed with Tris-buffered saline with 0.05% Tween-20 four times and then incubated for 1 h at room temperature with horseradish peroxidase conjugated anti-mouse IgG. Immunoreactivity was assessed by chemiluminescence.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00013. Additional information (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-3-5498-5788. Fax: +81-3-5498-5788. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. H. Kasai and Dr. M. Ikegami of Hoshi University for technical assistance. This work was supported in part by “Open Research Center” Project funds from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a Grant-in-Aid for Scientific Research (No. 20590017) from the Japan Society for the Promotion of Science. This study was also supported by the Cooperative Research Program of the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University.
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