Article pubs.acs.org/jnp
Angiogenesis Inhibitors and Anti-Inflammatory Agents from Phoma sp. NTOU4195 Ming-Shian Lee,† Shih-Wei Wang,‡ Guei-Jane Wang,§,◆,∇ Ka-Lai Pang,⊥ Ching-Kuo Lee,∥ Yueh-Hsiung Kuo,#,¶ Hyo-Jung Cha,⊥ Ruo-Kai Lin,*,∥ and Tzong-Huei Lee*,⬡ †
School of Pharmacy and ∥Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 11031, Taiwan Department of Medicine, Mackay Medical College, New Taipei City 25245, Taiwan § School of Medicine, Graduate Institute of Clinical Medical Science, China Medical University, Taichung 40402, Taiwan ⊥ Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 20224, Taiwan # Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 40447, Taiwan ¶ Department of Biotechnology, Asia University, Taichung 41354, Taiwan ⬡ Institute of Fisheries Science, National Taiwan University, Taipei 10617, Taiwan ◆ Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan ∇ Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan ‡
S Supporting Information *
ABSTRACT: Seven new polyketides, phomaketides A−E (1−5) and pseurotins A3 (6) and G (7), along with the known compounds FR-111142, pseurotins A, A1, A2, D, and F2, 14-norpseurotin A, α-carbonylcarbene, tyrosol, cyclo(-LPro-L-Leu), and cyclo(-L-Pro-L-Phe), were purified from the fermentation broth and mycelium of the endophytic fungal strain Phoma sp. NTOU4195 isolated from the marine red alga Pterocladiella capillacea. The structures were established through interpretation of spectroscopic data. The antiangiogenic and anti-inflammatory effects of 1−7 and related analogues were evaluated using human endothelial progenitor cells (EPCs) and lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells, respectively. Of the compounds tested, compound 1 exhibited the most potent antiangiogenic activity by suppressing the tube formation of EPCs with an IC50 of 8.1 μM, and compound 3 showed the most selective inhibitory activity of LPS-induced NO production in RAW264.7 macrophages with an IC50 value of 8.8 μM.
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INTRODUCTION The oceans cover nearly three-quarters of Earth’s surface and contain all types of microbes. Ecological niches, e.g., deep-sea hydrothermal vents, mangrove forests, algae, sponges, and many other marine plants and animals, provide distinct habitats for the isolation of specific microorganisms.1 Marine microorganisms, including bacteria, cyanobacteria, microalgae, and fungi, are an important source of novel bioactive metabolites. Specifically, the secondary metabolites obtained from marinederived fungi have gained considerable attention, as many of them are structurally unique and possess interesting biological and pharmacological properties with the potential to be used for the treatment of human diseases.2−4 Phoma sp. NTOU4195, a marine endophytic fungus, was isolated from the edible red alga Pterocladiella capillacea sampled in the intertidal zone of northern Taiwan. Our preliminary pharmacological evaluation demonstrated that ethyl acetate extracts of the fermentation broth and mycelium of Phoma sp. NTOU4195 displayed significant A549 cell growth inhibition and anti-inflammatory activity. Thus, a series of extraction, separation, and purification procedures were performed on the © XXXX American Chemical Society and American Society of Pharmacognosy
active components of the fermentation broth and mycelium of this fungus, which resulted in the characterization of seven new polyketides (1−7) (Figure 1) and 11 known compounds. Herein, we describe the isolation, structural elucidation, and bioactivities of compounds 1−7.
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RESULTS AND DISCUSSION The ethyl acetate extracts of the fermented broth and mycelium of Phoma sp. NTOU4195 were concentrated to give a brown residue, which was then fractionated through multiple repetitions of Sephadex LH-20 column chromatography and HPLC to yield 18 compounds, including seven previously unreported compounds (1−7) and 11 known compounds. The spectroscopic data for FR-111142, a spiro-epoxide-containing polyketide isolated from Scolecobasidium arenarium and Aspergillus f umigatus, were compatible with the reported data.5,6 Pseurotin A, a spiro-γ-lactam possessing a polyketide, was originally isolated from the fungal strains Pseudeurotium Received: May 7, 2016
A
DOI: 10.1021/acs.jnatprod.6b00407 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Figure 1. Chemical structures of compounds 1−7.
ovalis Stolk and Aspergillus f umigatus, and its absolute stereochemistry was determined from its 12,13-dibromo derivatives using X-ray crystallographic analysis.7−9 Until now, this family of natural products comprises 17 compounds, including pseurotins A, A1, A2, B, C, D, E, F1, and F2, 14-norpseurotin A, synerazol, azaspirene, FD-838, and cephalimysins A−D.7−18 In addition to the two new pseurotin analogues identified in this report, pseurotins A, A1, A2, D, and F2 and 14-norpseurotin A were also found from the same fungus. Moreover, the characterized structures of the afforded α-carbonylcarbene, tyrosol, cyclo(-L-Pro-L-Leu), and cyclo(-L-Pro-L-Phe) were consistent with the reported data.19−22 Compound 1, an amorphous, white powder, was determined to have a molecular formula of C22H35ClO7, as evidenced by its 13 C NMR spectrum and HRESIMS analysis, with an [M + H]+/ [M + 2 + H]+ ratio of approximately 3:1, indicating the presence of a chloride in 1. The IR absorptions at 3431 and 1712 cm−1 revealed the presence of a hydroxy and conjugated ester carbonyl group, respectively, which was further confirmed by a UV λmax at 214 nm. The 1H NMR spectrum (MeOH-d4, 500 MHz) (Table 1) contained conspicuous resonances corresponding to a three-proton doublet at δ 1.18 (3H, d, J = 6.4 Hz, H-6′) connected to a carbinoyl carbon, a three-proton singlet at δ 1.41 (3H, s, H-15), and a methoxyl singlet at δ 3.35 (3H, s, H-16), in addition to two three-proton singlets at δ 1.69 (3H, s, H-14) and 1.75 (3H, s, H-13) for two methyls attached to an olefinic carbon. Additionally, two trans-coupled, downfield-shifted, olefinic methines were observed at δ 6.10 (1H, d, J = 15.9 Hz, H-2′) and 7.09 (1H, dd, J = 15.9, 5.5 Hz, H-3′). The 13 C NMR (MeOH-d4, 125 MHz) (Table 1) in combination with the HSQC spectrum of 1 contained 22 carbon signals corresponding to five methyls at δC 14.4 (C-15), 18.2 (C-14), 19.2 (C-6′), 26.0 (C-13), and 57.1 (C-16); four methylenes at δC 24.7 (C-7), 28.6 (C-10), 30.9 (C-6), and 51.8 (C-1); nine methines at δC 49.8 (C-3), 62.6 (C-9), 68.2 (C-5), 71.4 (C-5′),
76.3 (C-4′), 81.0 (C-4), 119.9 (C-11), 122.5 (C-2′), and 150.0 (C-3′); and four carbons at δC 60.6 (C-8), 76.5 (C-2), 136.1 (C-12), and 167.5 (C-1′). In the HSQC spectrum, the distinctive cross-peaks at δH 6.10 (H-2′)/δC 122.5 (C-2′) and δH 7.09 (H-3′)/δC 150.0 (C-3′) accompanied by an upfield-shifted ester carbonyl signal at δC 167.5 (C-1′) suggested the presence of an α,β-unsaturated ester functionality, which was also reflected in the IR absorption at 1712 cm−1. The double-bond equivalent (DBE) of 1 was calculated to be five, including two olefinic functionalities and one carbonyl group. Two rings then remained. The key COSY spectrum of 1 displayed three sets of contiguous protons as follows: H-2′ → H-3′ → H-4′ → H-5′ → H3-6, H-3 → H-4 → H-5 → H-6 → H2-7, and H-9 → H2-10 → H-11 (Figure 2). The HMBC spectrum contained key crosspeaks of δH 5.56 (H-5)/δC 167.5 (C-1′); δH 3.35 (H3-16)/δC 81.0 (C-4); δH 1.41 (H3-15)/δC 49.8 (C-3), 60.6 (C-8), and 62.6 (C-9); δH 3.46 and 3.81 (H2-1)/δC 24.7 (C-7), 49.8 (C-3), and 76.5 (C-2); and δH 1.75 (H3-13) and 1.69 (H3-14)/δC 119.9 (C-11) and 136.1 (C-12), indicating that the −C-1′−C6′, −O−C-16, −C-8−C-15, and −C-1 side chains were located at C-5, C-4, C-3, and C-2 of the cyclohexyl ring of 1, respectively (Figure 2). Thus, compound 1 was deduced to be the analogue of both FR-111142 and fumagillin.23,24 From comparing the 1H and 13C NMR of 1 with those of FR-111142 and fumagillin,23,24 the conspicuous downfield-shifted H2-1 and C-1 of 1 indicated that the spiro-epoxide attached at C-2 of fumagillin and FR-111142 was opened and that the oxymethylene was converted to a chloride-bearing methylene moiety in 1, based on comparison with previously reported analogues.25 The relative configurations of the −C-1′−C-6′, −O−C-16, −C8−C-15, and −C-1 side chains in 1 were thus determined to be α-axial, α-equatorial, β-equatorial, and α-equatorial, respectively, based on the J values of the mutually coupled H-3/H-4 (J = 11.2 Hz) and H-4/H-5 (J = 2.6 Hz) and the NOESY spectrum, in which key cross-peaks of Hax-4/Hax-6, Hax-3/Hax-7, B
DOI: 10.1021/acs.jnatprod.6b00407 J. Nat. Prod. XXXX, XXX, XXX−XXX
1.78 m
6
C
26.0
1.75 s
1.41 s
3.35 s
14
15
16
4.08 dd (5.5, 5.5)
3.70 dq (6.4, 5.5)
1.18 d (6.4)
4′
5′
6′
a1
19.2
71.4
76.3
1.35 d (5.2)
2.98 dq (5.2, 1.8)
3.27 dd (7.1, 1.8)
6.67 dd (15.6, 7.1)
6.17 d (15.6)
17.8
58.3
58.7
146.9
124.6
1.36 d (5.2)
3.00 dq (5.2, 1.9)
3.27 dd (6.9, 1.9)
6.70 dd (15.6, 6.9)
6.18 d (15.6)
H NMR (500 MHz, MeOH-d4) spectroscopic data [δ in ppm, mult. (J in Hz)].
8-OMe
7.09 dd (15.9, 5.5)
3′
150.0
122.5
6.10 d (15.9)
2′
166.7
1.35 d (5.1)
3.00 dq (5.1, 1.9)
3.25 dd (7.0, 1.9)
6.68 dd (15.7, 7.0)
6.19 d (15.7)
17.8
58.7
58.3
146.9
124.6
166.7
1.18 d (7.6)
3.68 dq (7.6, 5.5)
4.05 dd (5.5, 5.0)
7.08 dd (15.6, 5.0)
6.11 d (15.6)
19.0
71.2
76.1
149.8
122.4
167.3
3.22 s
C NMR (125 MHz, MeOH-d4) spectroscopic data (δ in ppm).
b13
17.7
58.2
58.6
146.8
124.3
166.6
8.20 t (7.4)
23
167.5
7.47 t (7.4)
1′
7.59 t (7.4)
51.8
130.7
129.5
136.2
129.5
22
5.7
14.6
22.3
137.5
128.8
69.4
72.6
80.6
90.5
170.8
99.1
200.6
115.2
187.9
6b
21
1.73 s
1.02 t (7.6)
2.11−2.26 m
5.62 td (10.8, 7.7)
5.46 dd (10.8, 9.2)
4.70 dd (9.2, 6.5)
4.55 d (6.5)
4.82 s
6a
7.47 t (7.4)
56.8
13.6
18.0
25.8
135.4
120.0
29.3
62.8
61.5
31.3
29.3
68.3
82.1
53.7
146.7
110.8
5b
20
3.39 s
1.32 s
1.73 s
1.67 s
5.24 t (6.9)
2.32 m
2.66 t (9.0)
2.38 m
2.18 m
1.96 m
1.57 m
5.60 m
3.42 dd (10.7, 2.8)
2.20 d (10.7)
4.93 d (1.5)
4.71 d (1.5)
5a
130.7
57.4
16.8
18.2
26.1
133.4
123.6
31.4
80.8
87.0
29.6
26.9
68.8
78.7
53.6
81.4
78.8
4b
8.20 t (7.4)
3.38 s
1.30 s
1.70 s
1.62 s
5.26 t (6.9)
2.34 dt (14.7, 6.9)
2.03 dt (14.7, 6.9)
3.34 t (6.9)
1.66 m
2.14 m
1.87 m
5.65 m
3.75 dd (11.7, 2.9)
2.23 d (11.7)
3.78 d (8.8)
3.63 d (8.8)
4a
19
56.9
14.2
18.1
25.9
136.0
119.8
28.3
62.4
60.5
26.5
30.3
68.3
80.8
49.6
60.6
51.7
3b
134.4
3.41 s
1.20 s
1.76 s
1.67 s
5.24 t (7.1)
2.34 dt (14.7, 7.1)
2.21 dt (14.7, 7.1)
2.68 t (7.1)
2.12 m
1.08 m
1.87 m
5.68 m
3.72 dd (11.2, 2.8)
1.95 d (11.2)
2.98 d (4.1)
2.58 d (4.1)
3a
194.1
57.3
14.3
18.1
26.1
135.9
119.8
28.6
62.5
60.6
24.7
30.9
68.1
79.9
49.8
76.4
51.9
2b
18
3.34 s
1.42 s
1.75 s
1.69 s
5.25 t (7.0)
2.44 dt (14.8, 7.0)
2.18 dt (14.8, 7.0)
3.04 t (7.0)
2.04 m
1.39 m
1.90 m
1.78 m
5.55 m
3.72 dd (11.1, 2.6)
1.97 d (11.1)
3.81 brd (10.8)
3.46 d (10.8)
2a
17
57.1
14.4
18.2
136.1
1.69 s
119.9
13
5.25 t (6.9)
12
11
2.18 dt (14.9, 6.9)
10
2.44 dt (14.9, 6.9)
62.6
3.05 t (6.9)
9
28.6
60.6
24.7
30.9
68.2
8
2.04 m
1.39 m
1.90 m
5.56 m
5
7
3.72 dd (11.2, 2.6)
4
81.0
49.8
1.97 d (11.2)
3
51.8
1b
76.5
3.81 brd (10.9)
3.46 d (10.9)
1a
2
1
position
Table 1. 1H and 13C NMR Data for Compounds 1−7
3.35 s
8.36 t (7.4)
7.50 t (7.4)
7.64 t (7.4)
7.50 t (7.4)
8.36 t (7.4)
1.99 s
1.30 t (7.5)
2.80 m
6.37 d (3.6)
7.18 d (3.6)
4.56 s
7a
52.6
131.9
129.7
135.3
129.7
131.9
135.1
197.4
6.4
12.3
22.7
165.4
109.1
119.4
145.0
76.7
93.7
169.5
94.0
197.6
109.2
173.3
7b
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.6b00407 J. Nat. Prod. XXXX, XXX, XXX−XXX
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configurations of OH-2, H-3, and H3-15 on the furan were determined to be β-, α-, and β-oriented, respectively, based on the key cross-peaks of Hα-1/Hax-3, Hα-1/Hax-7, Hax-3/Hax-7, Hax-4/Hax-6, and H3-15/Hax-4, and no correlation of H-3/H315 was observed in the NOESY spectrum of 4 (Figure 3). The
Figure 2. Key COSY, HMBC, and NOESY correlations of compound 1.
H2-1/Hax-3, H2-1/Hax-7, and H-11/H3-13 were observed and no correlation of Hax-3/Hax-4 and Hax-6/Hax-7 was found (Figure 2). The relative configuration of the 8,9-epoxide was determined to be the Z-form by comparing the 1H and 13C NMR data with those of fumagillin and FR-111142. The relative configurations of the 4′,5′-diols were deduced to be erythro from comparison of the small mutually coupled 3JH‑4′/H‑5′ = 5.5 Hz to the threo form with 3J = 7.5−8.0 Hz.26 Accordingly, the structure of 1 was deduced as shown in Figure 1, and this compound was named phomaketide A. The physical and NMR data of 2 were in accordance with those of 1, except that the 4′,5′-diol in 1 was substituted by a 4′,5′-epoxide group in 2, as judged from the characteristic resonances at δH 3.27 (1H, dd, J = 7.1, 1.8 Hz, H-4′)/δC 58.7 (C-4′) and δH 2.98 (1H, dq, J = 5.2, 1.8 Hz, H-5′)/δC 58.3 (C-5′) in its 1H (MeOH-d4, 500 MHz) and 13C NMR (MeOH-d4, 125 MHz) spectra (Table 1). In addition, the relative configurations of H-4′ and H-5′ were deduced to both be the E form, as evidenced by the 3JH‑4′/H‑5′ of 1.8 Hz, in contrast with that of the cis form (J = 4.4 Hz).27 The HRESIMS of 2 revealed a pseudomolecular ion at m/z 429.2245, 18 Da less than that of 1, and also confirmed that 2 was a dehydrated analogue of 1. Hence, 2 was assigned as the structure shown in Figure 1 and named phomaketide B. The spectral data of 3 were in accordance with those of FR-111142, except that the 4′,5′-diol in FR-111142 was substituted by a 4′,5′-epoxide group in 3, as corroborated by the characteristic resonances at δH 3.27 (1H, dd, J = 6.9, 1.9 Hz, H-4′)/δC 58.6 (C-4′) and δH 3.00 (1H, dq, J = 5.2, 1.9 Hz, H-5′)/δC 58.2 (C-5′) in its 1H (MeOH-d4, 500 MHz) and 13C NMR (MeOH-d4, 125 MHz) spectra (Table 1). The relative configurations of H-4′ and H-5′ were deduced to be the E form, as evidenced by the 3JH‑4′/H‑5′ of 1.9 Hz, which was the same as that in 2. HRESIMS of 3 revealed a pseudomolecular ion [M + H]+ at m/z 393.2273, 18 Da less than that of FR-111142, and also confirmed that 3 is a dehydrated analogue of FR-111142. Conclusively, 3 was determined to be the structure shown in Figure 1 and named phomaketide C. The 13C NMR spectral data of 4 were in accordance with those of 3 with the exception of four distinctive downfieldshifted signals at δC 78.8 (C-1), 80.8 (C-9), 81.4 (C-2), and 87.0 (C-8) attributable to four oxygenated carbons (Table 1), which were also reflected in the downfield-shifted 1H NMR signals of δH 3.63, 3.78 (H2-1), and 3.34 (H-9) observed from key cross-peaks in the HSQC spectrum of 4. Thus, the 1,2- and 8,9-epoxides in 3 were tentatively inferred to be opened in 4. The HMBC spectrum contained key cross-peaks of δH 3.63 and 3.78 (each 1 H, d, J = 8.8 Hz, H2-1)/δC 29.6 (C-7), 53.6 (C-3), 81.4 (C-2), and 87.0 (C-8), indicating that C-1 was connected to C-8 via an ether linkage to form a tetrahydrofuran. The relative
Figure 3. Key COSY, HMBC, and NOESY correlations of compound 4.
HRESIMS of 4 revealed a quasi-molecular ion [M + H]+ at m/z 411.1986 (calcd 411.1999 for C22H35O7), confirming that 4 was an isomer of FR-111142. Conclusively, 4 was determined to possess the structure shown in Figure 1 and named phomaketide D. The spectral data for 5 were almost the same as those of FR-111142, except that the oxymethylene in FR-111142 was substituted by a terminal double bond in 5, as evidenced by the conspicuous resonances at δH 4.71 and 4.93 (each 1 H, d, J = 1.5 Hz, H2-1) and δC 110.8 (C-1) and 146.7 (C-2) in its 1H and 13 C NMR spectra (Table 1), respectively. The molecular formula of 5 was determined by HRESIMS to be C22H34O6, which is 16 Da less than that of FR-111142 and confirmed that 5 was a deoxy analogue of FR-111142. Conclusively, 5 was determined to have the structure shown in Figure 1 and named phomaketide E. Compound 6, an amorphous, white powder, was determined by 13C NMR and HRESIMS to have the molecular formula C22H26NO8. The IR spectrum indicated the presence of hydroxy, amide carbonyl, and α,β-unsaturated ketone groups based on the absorption bands at 3362, 1685, and 1706 cm−1, respectively. In the 1H NMR spectrum of 6 (Table 1), five aromatic protons at δH 7.47 (2H, t, J = 7.4 Hz, H-20, -22), 7.59 (1H, t, J = 7.4 Hz, H-21), and 8.20 (2H, t, J = 7.4 Hz, H-19, -23), as well as two cis olefinic methine protons at δH 5.46 (1H, dd, J = 10.8, 9.2 Hz, H-12) and 5.62 (1H, td, J = 10.8, 7.7 Hz, H-13) in the downfield region, revealed the presence of one benzoyl phenyl group and a cis double bond. Furthermore, three oxygenated methines at δH 4.55 (1H, d, J = 6.5 Hz, H-10), 4.70 (1H, dd, J = 9.2, 6.5 Hz, H-11), and 4.82 (1H, s, H-9); three methyls at δH 1.02 (3H, t, J = 7.6 Hz, H3-15), 1.73 (3H, s, H3-16), and 3.22 (3H, s, OMe-8); and one methylene at δH 2.11−2.26 (2H, m, H2-14) were observed (Table 1). The 13 C NMR data (Table 1) coupled with the HSQC spectrum of 6 showed 22 carbon signals corresponding to three methyls at δC 5.7 (C-16), 14.6 (C-15), and 51.8 (8-OMe); one methylene at δC 22.3 (C-14); 10 methines at δC 69.4 (C-11), 72.6 (C-10), 80.6 (C-9), 128.8 (C-12), 129.5 (C-20, 22), 130.7 (C-19, 23), 136.2 (C-21), and 137.5 (C-13); and five carbons at δC 90.5 (C-8), 99.1 (C-5), 115.2 (C-3), 134.4 (C-18), 170.8 (C-6), 187.9 (C-2), 194.1 (C-17), and 200.6 (C-4). The NMR data for 6 were consistent with those of pseurotin A, except for the D
DOI: 10.1021/acs.jnatprod.6b00407 J. Nat. Prod. XXXX, XXX, XXX−XXX
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distinctive signal shifts at δH 4.82 (H-9) and δC 90.5 (C-8), 99.1 (C-5), and 80.6 (C-9), indicating that 6 was the diastereoisomer of pseurotins A, A1, and A2.28 Key cross-peaks of H-9/OMe-8 in the NOESY spectrum suggested the relative configuration between OH-9 and OMe-8 to be trans. In comparing the circular dichroism (CD) spectrum of 6 with those of pseurotin A and FD-838,28,29 the CD spectra of pseurotin A and 6 showed different Cotton effects at only C-8, and the spectra of FD-838 and 6 displayed different Cotton effects at only C-5. The CD spectrum of 6 displayed positive (Δε227.0 +6.3), negative (Δε251.0 −2.2), positive (Δε310.0 +0.9), and negative (Δε352.0 −0.7) Cotton effects (Figure 4), indicating
age-related macular degeneration (AMD), and various chronic inflammatory diseases.30 Tumor angiogenesis, defined as the formation of blood vessels from an existing vascular network toward a tumor, is crucial for disease progression in various tumor types. Emerging evidence indicates that bone-marrowderived endothelial progenitor cells (EPCs) can contribute to postnatal neovascularization and tumor angiogenesis.31,32 EPCs reportedly mediate early tumor growth and late metastatic progression by intervening with the angiogenic switch.33 These findings establish the role of EPCs in pathological angiogenesis and support the idea that EPC-targeted therapies are an effective strategy against cancer and other angiogenesis-related diseases.34 Previous studies reported that FR-111142 and the analogues of pseurotin A exhibit an antiangiogenesis effect.5,35 The capillary-like tube formation is the most important step in angiogenesis. The antiangiogenic effects of the isolated compounds 1−7 in human EPCs using a tube formation assay were investigated. As shown in Figure 5A and B, phomaketides A (1), C (3), D (4), and E (5) and pseurotin G (7) obviously inhibited tube formation in EPCs. These new polyketides had median inhibitory concentrations (IC50) that were lower than 20 μM, except phomaketide B (2) and pseurotin A3 (6) (Table 2). Of these inhibitory compounds, phomaketide A (1) exhibited the most potent antiangiogenic activity by inducing an inhibitory effect against tube formation (IC50 = 8.1 μM). The potency of phomaketides C (3) and D (4) was similar to the known angiogenesis inhibitor FR-11142. Furthermore, the antiangiogenic activity of pseurotin G (7), with an IC50 value of 16.7 μM, was similar to that of other pseurotin compounds. On the basis of the above data, it was speculated that the chloride and diol at C-1, -4′, and -5′ could possibly play crucial roles in the angiogenic activity. Sorafenib is a multikinase inhibitor approved for the clinical treatment of cancer through its antiangiogenic and pro-apoptotic effects and was used as a positive control for the in vitro antiangiogenesis assay, with an IC50 value of 3.6 μM. Importantly, these new polyketides did not induce significant lactate dehydrogenase (LDH) release in EPCs (Figure 5C). These findings provided evidence that the inhibitory activities of these compounds against angiogenesis were not due to their cytotoxicity. In recent years, angiogenesis inhibitors have been in the spotlight and represent a breakthrough in the treatment of cancer and AMD. Here, we identify phomaketide A (1) as a novel angiogenesis inhibitor from the endophytic fungal strain Phoma sp. NTOU4195. These results suggest that phomaketide A (1) is a promising natural product worthy of further development for the treatment of angiogenesis-related diseases. To assess the anti-inflammatory effects of these polyketides, all the compounds were examined for their inhibition of NO production in lipopolysaccharide (LPS)-activated macrophages. Among the tested compounds, phomaketides A (1), B (2), and C (3) and FR-11142 exerted significant inhibitory activities on NO production compared to two positive controls, aminoguanidine (a specific iNOS inhibitor) and Nω-nitro-L-arginine (a nonselective iNOS inhibitor) (Table 3). In addition, none of the compounds (up to 100 μM) affected the cell viability of RAW264.7 cells. Phomaketide C (3) displayed the most potent inhibition of NO production without any cytotoxicity, and its average maximum inhibition (Emax) and IC50 were 100.0% and 8.8 μM, respectively. In preliminary structure−activity relationship studies, the number of epoxide groups in 1−5 appeared to be proportional to the inhibition of NO production. On the basis of these findings, we suggest that the extracts of and/or
Figure 4. ECD spectra of pseurotins A, A3 (6), and G (7).
that the absolute configurations of C-8 and C-5 are the R and S forms, respectively. Taken together, the absolute configuration of C-9 was deduced to be the R form. Unambiguously, the structure of 6 was established as shown in Figure 1, and this compound was named pseurotin A3. The physical and NMR data of 7 were almost in accordance with those of pseurotin A, except that the C-10−C-15 side chain moiety in pseurotin A was substituted by 10,12-heptadiene10,13-diol in 7, as judged from key HMBC cross-peaks of δH 2.80 (H2-14)/δC 12.3 (C-15), 109.1 (C-12), and 165.4 (C-13) and δH 7.18 (H-11)/δC 109.1 (C-12), 145.0 (C-10), 165.4 (C-13), and 173.3 (C-2). The NOESY spectrum showed no cross-peaks of H-9/OMe-8, suggesting the relative configuration between OH-9 and OMe-8 to be cis. Although 7 showed the same chemical shifts of C-5, -8, and -9 as those of pseurotin A, the CD spectrum of 7 showed positive (Δε227.0 +0.5), negative (Δε251.0 −0.4), negative (Δε310.0 −6.4), and positive (Δε352.0 +2.0) Cotton effects in contrast to the negative (Δε227.0 −1.2), positive (Δε251.0 +3.8), positive (Δε310.0 +2.94), and negative (Δε352.0 −1.2) effects in pseurotin A (Figure 4). However, compound 7 not only had similar chemical shifts of C-5, -8, and -9 but also shared the same Cotton effects of C-5 and -8 in its CD spectrum as those of FD-838.29 Taken together, these data indicate that the absolute configurations of C-8 and C-5 were both the R form. Therefore, the absolute configuration of C-9 was deduced to be the S form. Furthermore, a key cross-peak of H2-14/H-12 was found in the NOESY spectrum of 7, and no correlation between H2-14 and H-11 was observed, revealing that the configurations of Δ10 and Δ12 were both the Z form. Accordingly, the structure of 7 was characterized as that shown in Figure 1, and this compound was named pseurotin G. Angiogenesis is recognized as a common denominator underlying a variety of debilitating human diseases, including cancer, E
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Figure 5. Effects of compounds 1−7, FR-111142, and pseurotins A, A1, and A2 on tube formation and cytotoxicity in human EPCs. (A and B) Cells were plated on Matrigel-coated plates in the presence of the indicated polyketides (10 μM) and sorafenib (10 μM), and tubular morphogenesis was recorded by an inverted phase-contrast microscope. Images are representative of the results from five to seven separate experiments. (C) Cells were treated with the indicated polyketides (20 μM) and sorafenib (20 μM), and the cytotoxicity was then determined using the LDH assay. Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05 compared with the control group. The red frame indicates the most potent antiangiogenic activity in this assay. Cultivation and Fermentation of Phoma sp. Phoma sp. NTOU 4195 was collected, isolated, and identified by one of us (K.L.P.) and then deposited at the Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan. The ITS gene sequence was deposited in NCBI’s GenBank with the accession number KX216732. The mycelium of Phoma sp. NTOU 4195 was inoculated into 5 L serum bottles, each containing 6 g of Bacto peptone, 3 g of yeast extract (Becton, Dickinson and Company, Sparks, USA), 30 g of Difco dextrose, and 20% seawater (3 L). The fermentation was conducted with aeration at 25−30 °C for 10 days. Extraction and Isolation. The filtered fermentation broth (30 L) containing Phoma sp. was partitioned with recycled EtOAc and then concentrated under vacuum to dryness (4.0 g). Subsequently, the crude extract was redissolved in MeOH, applied onto a Sephadex LH-20 column (3 cm i.d. × 70 cm), and eluted with MeOH at a flow rate of 2.08 mL/min. The composition of each collected fraction (25 mL) was determined by TLC using CH2Cl2/MeOH (20:1, v/v) for development and dipping in vanillin/H2SO4 to detect compounds
purified compounds from Phoma sp. NTOU4195 may lead to new anti-inflammatory agents.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations and UV spectra were measured on a JASCO P-2000 polarimeter (Tokyo, Japan) and Thermo UV−visible Helios α spectrophotometer (Bellefonte, CA, USA), respectively. 1H and 13C NMR spectra were acquired on a Bruker DRX-500 SB and AVIII-800 spectrometer (Ettlingen, Germany). High-resolution mass spectra were obtained using a high-definition mass spectrometry system with an ESI interface and TOF analyzer (Waters Corp., Manchester, UK). IR spectra were recorded on a JASCO FT/IR 4100 spectrometer (Tokyo, Japan). Sephadex LH-20 (Amersham Biosciences, Filial Sverige, Sweden) and MCI CHP20P (Mitsubishi Chemical, Tokyo, Japan) were used for open column chromatography. TLC was performed using silica gel 60 F254 plates (0.2 mm, Merck). A reflective index detector (Bischoff, Leonberg, Germany) was used in HPLC purification. F
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spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 447.2145 (calcd 447.2149 for C22H3635ClO7), [M + 2 + H]+ at m/z 449.2256 (calcd 449.2261 for C22H3637ClO7), [M + Na]+ at m/z 469.2167 (calcd 469.2161 for C22H35Na35ClO7), and [M + 2 + Na]+ at m/z 471.2024 (calcd 471.2010 for C22H35Na37ClO7). Phomaketide B (2): colorless oil; [α]25D −123.0 (c 0.4, MeOH); UV (MeOH) λmax (log ε) = 214 (4.14) nm; IR (KBr) νmax 3423, 2928, 1718, 1656, 1445, 1379, and 1264 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 429.2245 (calcd 429.2250 for C22H3435ClO6), [M + 2 + H]+ at m/z 431.2161 (calcd 431.2165 for C22H3437ClO6), [M + Na]+ at m/z 451.2056 (calcd 451.2061 for C22H33Na35ClO6), and [M + 2 + Na]+ at m/z 453.1982 (calcd 453.1990 for C22H33Na37ClO6). Phomaketide C (3): colorless oil; [α]25D −93.0 (c 0.4, MeOH); UV (MeOH) λmax (log ε) = 214 (4.05) nm; IR (KBr) νmax 2947, 1714, 1668, 1450, 1381, and 1259 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 393.2428 (calcd 393.2437 for C22H33O6). Phomaketide D (4): colorless oil; [α]25D −10.7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) = 218 (4.48) nm; IR (KBr) νmax 3412, 2925, 1714, 1653, 1446, 1385, and 1264 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 411.1986 (calcd 411.1999 for C22H35O7). Phomaketide E (5): colorless oil; [α]25D −17.1 (c 0.2, MeOH); UV (MeOH) λmax (log ε) = 221 (4.32) nm; IR (KBr) νmax 3441, 2929, 1711, 1525, 1450, 1376, and 1265 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 395.2006 (calcd 395.2016 for C22H35O6). Pseurotin A3 (6): amorphous, white powder; [α]25D −39.1 (c 0.2, MeOH); UV (MeOH) λmax (log ε) = 250 (4.31) and 282 (4.17) nm; IR (KBr) νmax 3362, 1706, 1685, 1625, 1441, and 1267 cm−1; 1H and 13 C NMR spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 432.1173 (calcd for C22H26NO8, m/z 432.1185). Pseurotin G (7): amorphous, white powder; [α]25D +2.4 (c 0.2, MeOH); UV (MeOH) λmax (log ε) = 252 (4.19), 324 (4.28) and 344 (4.21) nm; IR (KBr) νmax 3392, 1726, 1684, 1612, 1569, 1404, and 1258 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS [M + H]+ at m/z 430.1502 (calcd for C22H24NO8, m/z 430.1510). Isolation and Cultivation of EPCs. Ethical approval was granted by the Institutional Review Board of Mackay Medical College, New Taipei City, Taiwan (reference number: P1000002). Informed consent was obtained from healthy donors before the collection of peripheral blood (80 mL). The peripheral blood mononuclear cells (PBMCs) were fractionated from the other blood components by centrifugation on a Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s instructions. CD34-positive progenitor cells were obtained from the isolated PBMCs using a CD34 MicroBead Kit and MACS cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolation and maintenance of CD34-positive EPCs were performed as described previously.36,37 Briefly, human CD34-positive EPCs were seeded onto 1% gelatincoated plasticware and cultured in MV2 complete medium (PromoCell, Heidelberg, Germany) with 20% defined fetal bovine serum (FBS) (HyClone, Logan, UT). Cells were seeded onto 1% gelatincoated plasticware and cultured in humidified air containing 5% CO2 at 37 °C for further treatment. Capillary Tube Formation Assay. Matrigel (BD Biosciences, Bedford, MA, USA), which was used to promote the differentiation of EPCs into a capillary tube-like structure, was added to 48-well plates. The Matrigel-coated 48-well plates were incubated at 37 °C for 30 min to allow for polymerization. After gel formation, EPCs (6 × 104 cells) were seeded in each well onto the layer of polymerized Matrigel in MV2 complete medium containing 2% FBS with the indicated concentration of the tested compounds, followed by incubation for 24 h at 37 °C. Photomicrographs of capillary tube formation were taken with an inverted phase-contrast microscope. Tube formation was quantified by measuring the long axis of each tube in three random fields per well using Image-Pro Plus software.
Table 2. Antiangiogenic Effects of Compounds 1−7, FR-111142, and Pseurotins A, A1, and A2 in Human EPCsa compound
IC50 (μM)
1 2 3 4 5 6 7 FR-111142 pseurotin A pseurotin A1 pseurotin A2 sorafenib
8.1 >20.0 17.8 16.2 19.2 >20.0 16.7 18.4 >20.0 >20.0 >20.0 3.6
a
Tube formation in EPCs was quantified by measuring the length of the tubes in three random fields per well using Image-Pro Plus software and was calculated against the DMSO control. Data are expressed as the average of five to seven independent experiments.
Table 3. Effects of Compounds 1−7, FR-111142, and Pseurotins A, A1, and A2 on NO Production and Cell Viability in RAW 264.7 Cells Stimulated by LPS compound
Emax (%)c
IC50 (μM)d
cell viability (%)
1 2 3 4 5 6 7 FR-111142 pseurotin A pseurotin A1 pseurotin A2
87.9 100.0 100.0 93.4 80.8 79.8 63.1 100.0 38.8 67.2 78.2 83.7 42.1
19.3 13.5 8.8 22.8 35.4 34.5 57.0 20.6 >100.0 62.5 40.6 24.7 144.3
99.2 100.8 98.7 92.2 93.0 99.8 97.6 97.6 102.7 99.3 100.1 99.2 101.7
a b
a
Positive control aminoguanidine (a selective iNOS inhibitor). bPositive control Nω-nitro-L-arginine (a nonselective iNOS inhibitor). cEmax indicates the mean maximum inhibitory effect at a concentration of 100 μM, expressed as the percentage inhibition of NO production induced by LPS (200 ng/mL) in the presence of the vehicle. dIC50, mean concentration producing 50% Emax. n = 4−6 in each group. with similar skeletons. All the fractions were combined into four portions, I−IV. Portion I was purified by HPLC on a semipreparative, reversed-phase column (Thermo BDS 5 μm C18, 10 × 250 mm, Hypersil, CA, USA) with MeCN/H2O (2:8, v/v) as the eluent at 2 mL/min to give α-carbonylcarbene (8.0 mg), tyrosol (10.0 mg), cyclo(-L-Pro-L-Leu) (7.2 mg), and cyclo(-L-Pro-L-Phe) (4.0 mg). Portion II was purified by HPLC on a semipreparative, reversed-phase column (Phenomenex Luna 5 μm PFP, 10 × 250 mm, Torrance, CA, USA) with MeOH/H2O (7:3, v/v) as the eluent at 2 mL/min to afford FR-111142 (21.6 mg) and compounds 1 (35.3 mg), 2 (39.1 mg), 3 (35.1 mg), 4 (12.6 mg), and 5 (10.7 mg). Portion III was purified by HPLC on a semipreparative, reversed-phase column (Phenomenex Luna 5 μm PFP, 10 × 250 mm, Torrance, CA, USA) with MeCN/ H2O (3:7, v/v) as the eluent at 2 mL/min to afford compounds 6 (3.6 mg) and 7 (7.5 mg), pseurotins A (400 mg), A1 (10.3 mg), A2 (8.5 mg), D (5.2 mg), and F2 (2.9 mg), and 14-norpseurotin A (2.4 mg). Phomaketide A (1): colorless oil; [α]25D −77.5 (c 0.4, MeOH); UV (MeOH) λmax (log ε) = 214 (4.01) nm; IR (KBr) νmax 3431, 2970, 1712, 1653, 1447, 1380, and 1268 cm−1; 1H and 13C NMR G
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Cytotoxicity Assay. EPCs were seeded onto 96-well plates at a density of 5 × 103 cells per well. Then, the cells were treated with MV2 complete medium containing 2% FBS in the presence of the tested compounds for 24 h. The percentage of LDH release was calculated from the ratio of LDH activity in the medium to LDH activity in the cell lysate. Measurement of NO Production and Cell Viability. Murine macrophage RAW264.7 cells were purchased from the Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan) and maintained in DMEM medium containing 10% FBS, L-glutamine, penicillin, and streptomycin at 37 °C with 5% CO2. Cell aliquots were grown to confluence on 24-well plates for 24 h. The medium was switched to serum-free DMEM for another 4 h to render the attached cells quiescent. To assess the effects on LPS-induced NO production, all compounds (purity >98% checked by HPLC and 1H NMR) and two positive controls were added before the addition of LPS (200 ng/mL) to the RAW 264.7 cells. The nitrite concentration in the culture medium was determined spectrophotometrically as an index of NO production using the Griess reaction. Additionally, the AlamarBlue assay was used to determine the cell viability of RAW 264.6 cells in the presence of the tested compounds. The results are expressed as the percentage of inhibition, calculated versus the vehicle plus the LPS-treated cells.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00407. 1 H NMR, 13C NMR, COSY, NOESY, HSQC, and HMBC spectra of compounds 1−7 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Phone (R.-K. Lin): +886-2-27361661, ext. 6162. E-mail:
[email protected]. *Phone (T.-H. Lee): +886-2-33661828. E-mail: thlee1@ntu. edu.tw. ORCID
Tzong-Huei Lee: 0000-0001-8036-7563 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Science and Technology. We thank Ms. S.-L. Huang of the Instrumentation Center of the College of Science, National Taiwan University, for the NMR data acquisition and Ms. Y.-C. Wu of the Small Molecule Metabolomics Core Facility at the Institute of Plant and Microbial Biology and of the Academia Sinica Scientific Instrument Center of Academia Sinica for the MS data acquisition.
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REFERENCES
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