The EtOAc extract of fermented broths of C. globosum Km1226 was fractionated and purified sequentially by column chromatography on Sephadex LH-20 and semipreparative HPLC to yield undescribed compounds 1–3 as well as eleven known compounds 4–14.
Compound 1 was obtained as white powder, and was deduced to have the molecular formula C13H18O3 as evidenced from a quasi-molecular ion [M + H]+ at m/z 223.1329 (calcd 223.1334 for C13H19O3) in the HRESIMS, supported by analysis of 13C NMR data (Table 1), indicating 5 degrees of unsaturation. Its IR spectrum displayed absorptions at 3357 cm− 1, indicating the presence of a hydroxy group. Analysis of 1H NMR and HSQC spectra of 1 indicated signals for six mutually trans-coupled olefinic protons at δH 6.31 (1H, dd, J = 15.0, 10.2 Hz, H-3), 5.72 (1H, dd, J = 15.0, 7.2 Hz, H-4), 5.61 (1H, dd, J = 14.4, 7.8 Hz, H-8), 6.20 (1H, dd, J = 14.4, 10.2 Hz, H-9), 6.23 (1H, ddt, J = 15.0, 10.2, 1.2 Hz, H-10), and 5.76 (1H, dt, J = 15.0, 5.4 Hz, H-11), two mutually coupled carbinoyl methines at δH 4.06 (1H, m, H-5) and 3.70 (1H, t, J = 7.8 Hz, H-6), two oxygenated methylenes at δH 4.08 (2H, dd, J = 5.4, 1.2 Hz, H-12) and 4.06 (1H, m, Ha-13) and 3.70 (1H, t, J = 7.8 Hz, Hb-13), one exomethylene at δH 5.22 (1H, dd, J = 16.2, 1.8 Hz, Ha-1) and 5.09 (1H, dd, J = 10.2, 1.8 Hz, Hb-1), one olefinic proton at δH 6.36 (1H, dt, J = 16.2, 10.2 Hz, H-2), and one methine at δH 2.83 (1H, quintet, J = 7.8 Hz, H-7) (Table 2). Interpretation of the 13C NMR accompanied by HSQC spectra revealed 13 carbon signals attributable to seven olefinic methines at δC 131.8 (C-10), 132.9 (C-8), 133.0 (C-11), 133.3 (C-4), 133.4 (C-9), 134.2 (C-3), and 137.8 (C-2), three methylenes at δC 63.4 (C-12), 72.1 (C-13), and 118.2 (C-1), and three aliphatic methines at δC 52.6 (C-7), 82.6 (C-6), and 86.5 (C-5). Several distinctive signals at δC 63.4 (C-12), 72.1 (C-13), 82.6 (C-6) and 86.5 (C-5) were assigned to oxygen-bearing carbons. Cross-peaks of H-1/H-2, H-2/H-3, H-3/H-4, H-4/H-5, H-5/H-6, H-6/H-7, H-7/H-8, H-8/H-9, H-9/H-10, H-10/H-11, H-11/H-12, and H-7/H-13 in the COSY spectrum together with key cross-peaks of H-3/C-5, H-4/C-6, H-8/C-6, H-9/C-7, H-8/C-13, and H-13/C-5 in the HMBC spectrum (Fig. 2) established the plain structure of 1 as shown. The NOESY correlation of H-5/H-7 (Fig. 2), J values (7.8 Hz) of mutually coupled H-7/H-6 and H-6/H-5, and no NOESY correlation of H-7/H-6 and H-6/H-5 established the relative configurations of H-5, H-6, and H-7 to be S*, R*, and S*, respectively. The plain structure of 1 was almost compatible with that of (–)-aureonitol (4) except that a terminal CH3-12 in 4 was substituted by a hydroxymethyl in 1. The absolute configuration of 1 was further determined to be the same with that of 4 as evidenced from the consistency of CD spectra of 1 and 4 (Fig. S10) and sign of the optical rotational values of 1 ([α]27 D -9.8) and 4 ([α]25 D -31.3) in the literature (Nakazawa et al. 2013).
Table 1
13C NMR spectroscopic data for compounds 1–3 (δ in ppm)
| | 1a | | 2 a | | 3 a |
position | | δC, mult | | δC, mult | | δC, mult |
1 | | 118.2 d | | 13.2 q | | 13.2 q |
2 | | 137.8 d | | 27.7 t | | 27.7 t |
3 | | 134.2 d | | 153.3 d | | 153.4 d |
4 | | 133.3 d | | 126.4 d | | 126.4 d |
5 | | 86.5 d | | 155.9 d | | 155.9 d |
6 | | 82.6 d | | 137.4 s | | 137.5 s |
7 | | 52.6 d | | 71.5 d | | 71.5 d |
8 | | 132.9 d | | 199.1 s | | 198.9 s |
9 | | 133.4 d | | 125.6 d | | 127.5 d |
10 | | 131.8 d | | 148.5 d | | 145.6 d |
11 | | 133.0 d | | 73.1 d | | 83.1 d |
12 | | 63.4 t | | 66.5 t | | 64.8 t |
13 | | 72.1 t | | 194.9 d | | 194.9 d |
11-OCH3 | | | | | | 57.9 q |
a Measured in methanol-d4 (150 MHz). |
Table 2
1H NMR spectroscopic data for compounds 1–3 (δ in ppm, mult., J in Hz)
| | 1 a | | 2 a | | 3 a |
position | | δH, mult (J in Hz) | | δH, mult (J in Hz) | | δH, mult (J in Hz) |
1a | | 5.22 dd (16.2, 1.8) | | 1.09 t (7.2) | | 1.10 t (7.2) |
1b | | 5.09 dd (10.2, 1.8) | | | | |
2 | | 6.36 dt (16.2, 10.2) | | 2.29 qd (7.2, 7.2) | | 2.30 qd (7.2, 7.2) |
| | | | 2.29 qd (7.2, 7.2) | | 2.30 qd (7.2, 7.2) |
3 | | 6.31 dd (15.0, 10.2) | | 6.50 dt (15.0, 7.2) | | 6.51 dt (15.0, 7.2) |
4 | | 5.72 dd (15.0, 7.2) | | 6.76 ddt (15.0, 12.0, 1.2) | | 6.78 ddt (15.0, 10.2, 1.2) |
5 | | 4.06 m | | 7.20 d (12.0) | | 7.22 d (10.2) |
6 | | 3.70 t (7.8) | | | | |
7 | | 2.83 quintet (7.8) | | 5.43 s | | 5.44 s |
8 | | 5.61 dd (14.4, 7.8) | | | | |
9 | | 6.20 dd (14.4, 10.2) | | 6.41 dd (15.6, 1.8) | | 6.38 dd (15.6, 1.2) |
10 | | 6.23 ddt (15.0, 10.2, 1.2) | | 6.98 ddd (15.6, 5.0, 1.8) | | 6.79 (15.6, 6.0) |
11 | | 5.76 dt (15.0, 5.4) | | 4.25 br q (5.0) | | 3.85 qd (6.0, 1.2) |
12a | | 4.08 dd (5.4, 1.2) | | 3.51 dd (12.0, 5.0) | | 3.55 dd (10.2, 6.0) |
12b | | | | 3.48 dd (10.8, 5.0) | | 3.49 dd (10.2, 6.0) |
13a | | 4.06 b | | 9.42 s | | 9.42 s |
13b | | 3.70 t (7.8) | | | | |
11-OCH3 | | | | | | 3.31 s |
a Measured in methanol-d4 (600 MHz). b Signal without multiplicity was overlapped, and was picked up from HSQC experiment. |
The HRESIMS of compound 2 showed a quasi-molecular ion peak at m/z 255.1227 [M + H]+ (calcd 255.1232 for C13H19O5) and a deprotonated molecular ion [M − H]− at m/z 253.1087 (calcd 253.1081 for C13H17O5), supported by analysis of 13C NMR data (Table 1), indicating 5 degrees of unsaturation. Its IR spectrum displayed absorptions at 3365, 1704, and 1672 cm− 1, indicating the presence of a hydroxy, an α,β-unsaturated aldehyde, and an α,β-unsaturated ketone, respectively. The 1H NMR spectrum of 2 showed signals for five mutually-coupled olefinic protons at δH 6.50 (1H, dt, J = 15.0, 6.6 Hz, H-3), 6.76 (1H, ddt, J = 15.0, 12.0, 1.2 Hz, H-4), 7.20 (1H, d, J = 12.0 Hz, H-5), and 6.41 (1H, dd, J = 15.6, 1.8 Hz, H-9), and 6.98 (1H, ddd, J = 15.6, 5.0, 1.8 Hz, H-10), two carbinoyl methines at δH 5.43 (1H, s, H-7) and 4.25 (1H, br q, J = 5.0 Hz, H-11), one aldehydic methine at δH 9.42 (1H, s, H-13), one oxymethylene at δH 3.51 (1H, dd, J = 12.0, 5.0 Hz, Ha-12) and 3.48 (1H, dd, J = 10.8, 5.0 Hz, Hb-12), one methylene at δH 2.29 (2H, qd, J = 7.2, 6.6 Hz, H-2), and one methyl at δH 1.09 (3H, t, J = 7.2, H-1) (Table 2), which were supported by the COSY assignments of 2. Interpretation of the 13C NMR accompanied by HSQC spectrum revealed 13 carbon signals attributable to seven olefinic methines at δC 125.6 (C-9), 126.4 (C-4), 148.5 (C-10), 153.3 (C-3), 155.9 (C-5), and 194.9 (C-13), two aliphatic methylenes at δC 27.7 (C-2) and 66.5 (C-12), two nonprotonated carbons at δC 137.4 (C-6) and 199.1 (C-8), two aliphatic methines at δC 71.5 (C-7) and 73.1 (C-11), and one methyl at δC 13.2 (C-1). Of all the assigned carbon signals, three distinctive signals at δC 66.5 (C-12), 71.5 (C-7), and 73.1 (C-11) were assigned to be oxygenated carbons. Key cross-peaks of H-5, -7, and − 13/C-6, H-5 and − 13/C-7, H-5 and − 7/C-13, and H-7, -9, and − 10/C-8 in the HMBC spectrum (Fig. 1) indicated the gross structure of 2 was as shown. The configurations of ∆3, ∆5, and ∆9 were deduced to be all E forms based on a coupling constant of H-3/H-4 (J = 15.0 Hz), a key NOESY correlation of H-5/H-13 (Fig. 2), and a coupling constant of H-9/H-10 (J = 15.6 Hz), respectively. Due to biogenetic relationship, the relative configuration of C-7 and − 11 in 2 was speculated to be the same with those of mollipilin A (5). The experimental CD spectrum of 2 was compatible with that of 5 (Fig. S28), and the sign of optical rotational value of 2 ([α]27 D + 22.2) was the same with that of 5 ([α]25 D + 39.3) in the literature (Asai et al. 2012). The absolute configurations of C-7 and − 11 of 2 were deduced to be S and R, respectively, as shown in Fig. 1.
The HRESIMS of compound 3 showed protonated molecular ion peaks at m/z 269.1383 [M + H]+ (calcd 269.1389 for C14H21O5) and a deprotonated molecular ion [M − H]− at m/z 267.1242 (calcd 267.1238 for C14H19O5), indicating a molecular formula of C14H20O5 for 3. When comparing the 1H and 13C NMR data of 3 with those of 2, compound 3 almost coincided well with 2 except that the substituent at C-11 in 3 was changed to be a methoxy as judged from the chemical shift of C-11 (δC 73.1) in 2 shifted to δC 83.1 in 3. A key cross-peak of –OCH3/C-11 in the HMBC spectrum corroborated that the additional methoxy group was attached at C-11. The gross structure of 3 was thus determined. Since both the experimental CD spectrum of 3 (Fig. S28) and the sign of optical rotational value of 3 were in compliance with those of 2, the absolute configuration of 3 was determined to be the same as that of 2.
(–)-Aureonitol (4), a tetrahydrofuran derivative, has been isolated from C. globosum, and was found to act like a transcriptional regulator for the biosynthesis of other secondary metabolites in that fungal species (Nakazawa et al. 2013). The structure of mollipilin A (5), an epoxide-containing polyketide, was found to exhibit moderate growth inhibitory effects on HCT-116 cells (Asai et al. 2012). Mollipilins E (6) and F (7), two spiro-furan-containing polyketides, accompanied with mollipilin A (5) have been isolated from C. mollipilium (Asai et al. 2012). Chaetoglobosins A, C, and D (8–10), three cytochalasan alkaloids, have been isolated from a Ginkgo biloba-derived fungal strain C. globosum No.04, and were found to exhibit potent anti-fungal effects (Zhang et al. 2019). Aureochaeglobosins B and C (11 and 12), two rare aureonitol derivative-fused chaetoglobosins via [4 + 2] cycloaddition, have been isolated from C. globosum (Yang et al. 2018). Chaetoviridin A (13) and chaetomugilin A (14), two chloro-azaphilones, have been found from C. globosum by using a molecular epigenetic approach (Wang et al. 2017), and showed significant cytotoxicity against cultured P388 cells and HL-60 cells (Yamada et al. 2008).
Compounds 1–14 were tested for anti-inflammatory and anti-angiogenic activities. The anti-inflammatory assay was performed by measuring the amount of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced microgial BV-2 cells. Compounds 1–14 inhibited 42.3%, 50.2%, 51.7%, 68.2%, 111.6%, 44.7%, 48.2%, 92.6%, 71.8%, 45.7%, 99.1%, 90.6%, 72.9% and 45.0% of NO production (Fig. 3A), respectively, at a concentration of 20 µM without any cytotoxicity (Fig. 3B) except compounds 5 and 8. The positive control curcumin exhibited inhibition of NO production 100.4%. Of all the isolates, mollipilin A (5), aureochaeglobosins B (11) and C (12) exhibited significant nitric oxide production inhibitory activity in LPS-induced BV-2 microglial cells with IC50 values of 0.7 ± 0.1, 1.2 ± 0.1 and 1.6 ± 0.2 µM, respectively (Table 3). Eendothelial progenitor cells (EPCs) can dictate tumor angiogenesis and cancer progression by activating the angiogenic switch in tumor microenvironment. Therefore, targeting EPCs to develop anti-angiogenic agents is an attractive therapeutic approach for cancer treatment. In this study, it was found that compounds 5, 8, 9, 10, 11, and 12 showed promising growth-inhibitory effects on EPCs with IC50 values ranging from 1–10 µM, with sorafenib as the positive control. As shown in Table 4, chaetoglobosin D (10) exhibited the most potent anti-angiogenic activity by suppressing EPCs growth (IC50 = 0.8 ± 0.3 µM).
Table 3
IC50 values of 5, 11, and 12 on nitric oxide production inhibitory activities induced by lipopolysaccharide in microglial BV-2 cells
compound | IC50 (µM) a,b |
5 | 0.7 ± 0.1*** |
11 | 1.2 ± 0.1** |
12 | 1.6 ± 0.2** |
Curcumin c | 2.7 ± 0.3 |
a IC50 = concentration that reduces NO production by 50%. |
b Asterisks denote significance compared to curcumin (positive control) according to the two-tailed t-test. **p < 0.01, and ***p < 0.001. |
c Positive control used in this study. |
Table 4
Anti-angiogenic activity of isolated compounds in human EPCs.
compound | IC50 (µM) a |
1 | > 50 |
2 | > 50 |
3 | > 50 |
4 | > 50 |
5 | 5.6 ± 0.1 |
6 | > 50 |
7 | > 50 |
8 | 4.6 ± 0.1 |
9 | 8.1 ± 0.4 |
10 | 0.8 ± 0.3 |
11 | 5.2 ± 0.3 |
12 | 2.9 ± 0.3 |
13 | > 50 |
14 | > 50 |
Sorafenib b | 4.8 ± 0.3 |
a EPCs were treated with the indicated compounds for 48 h. Anti-angiogenic effects were evaluated in a cell growth assay (n = 3). Data are displayed as the mean ± SEM. |
b Positive control used in this study. |