2. Results and Discussion
The fungus E. maritima BC17 was cultivated in the liquid culture media Czapek-Dox and PDB, and incubated for 14 or 28 days in surface and shaken conditions, as described in Materials and Methods. After extraction and purification by HPLC, in addition to fourteen known compounds (4-17), three new eremophilanes (1-3) were isolated and characterized. In particular, the undescribed eremophilanes 1-3 were only isolated from Czapek-Dox fermentation broth, which showed the highest chemical diversity.
Figure 1.
Eremophilanes (1-17) isolated from the strain Emericellopsis maritima BC17 cultivated in liquid culture media.
Figure 1.
Eremophilanes (1-17) isolated from the strain Emericellopsis maritima BC17 cultivated in liquid culture media.
The molecular formula for compound
1 was established as C
12H
16O
4, deduced from the sodiated molecular ion [M+Na]
+ (
m/z 247.0933 [M+Na]
+, calculated for C
12H
16O
4Na 247.0946) observed in its HRESIMS (
Figure S7). Its IR spectrum showed absorption bands characteristic for hydroxy (3447 cm
−1) and enone carbonyl groups (1637 cm
−1). The
1H NMR data (
Table 1) indicated the presence of two methyl signals at
δH 1.28 (d,
J = 7.0 Hz) and 1.35 (s), and two olefinic signals at
δH 6.17 (d,
J = 1.5 Hz) and 6.30 (s). The
13C NMR data (
Table 1) displayed twelve signals including one carbonyl (
δC 183.9), four olefinic carbons (
δC 124.4, 127.4, 147.9, 171.1), two methyls (
δC 13.2, 22.0), one methylene (
δC 37.5), other three methines, two of which were oxygenated (
δC 43.4, 74.1, 74.6), and another quaternary carbon (
δC 45.0).
Two double bonds and one carbonyl group accounted for three of five degrees of unsaturation, indicating the presence of two rings in the structure. The two-ring system was then established based on
1H−
1H COSY (
Figure S3), HSQC (
Figure S4), and HMBC (
Figure S5) correlations. Inspection of the
1H−
1H COSY and HSQC data led to the assignment of a C(1)H
2−C(2)H−C(3)H−C(4)H−C(15)H
3 unit. The assigned spin system, together with HMBC correlations from H
2-1 to C-3/C-5/C-9/C-10, from H-4 to C-14, and from H-15 to C-3/C-4/C-5, permitted the complete elucidation of ring A. HMBC correlations from H-6 to C-7/C-8/C-10/C-4/C-14 and from H-9 to C-1/C-5/C-7 allowed the elucidation of ring B. Thus, compound
1 was established as a nor-eremophilane sesquiterpene. Its
1H and
13C NMR spectra (
Figures S1 and S2) were very similar to those of compound guignarderemophilane A (
18), previously reported as a metabolite from the endophytic fungus
Guignardia mangiferae [
14], except for the absence of the acetyl group signals at C-3.
The relative configuration of
1 was determined by analysis of 1D NOESY (
Figure S6a-g) correlations and the splitting patterns of related protons (
Figure 2). The NOESY correlations between H-2, H-3 and H-4 revealed that they showed the same orientation. The NOE effect between the equatorial Me-15 and H-6 revealed that C5−C6 was equatorial and that Me-14 was axial. The correlations between Me-14 and Me-15 suggested they were in the
syn conformation. These correlations permitted us to propose the relative configuration of
1 as shown in
Figure 2.
Absolute stereochemistry of compound
1 was established by comparison of the experimental electronic circular dichroism (ECD) spectrum with the one obtained from quantum mechanical time-dependent density functional theory (TD-DFT) calculations for the (2
S,3
R,4
R,5
S) stereoisomer in the 200-400 nm region (
Figure 3) [
15]. Moreover, compound
1 showed an ECD spectrum similar to that of the previously reported acetylated compound guignarderemophilane A (
18) [
14]. Based on these data, the absolute configuration at C-2, -3, -4 and -5 was determined to be
S,
R,
R, and
S, respectively. Therefore, the structure of
1 was characterized and named as 3-deacetyl guignarderemophilane A.
Compound
2 was obtained as a colourless oil with the molecular formula C
15H
22O
3, as determined by the HRESIMS peak at
m/z 273.1446 [M+Na]
+ (
Figure S14), indicating five degrees of unsaturation. Its IR spectrum showed absorptions corresponding to a hydroxyl group (3496 cm
−1) and an
α,
β-unsaturated keto group (1674 cm
−1). The
13C NMR data (
Table 1) displayed fifteen carbon resonances, some of which could be assigned to one keto group (δ
C 195.2), a C=C double bond (
δC 123.6, 172.4), one 1,2-epoxy group (
δC 64.7, 65.6), one oxygenated sp
3 carbon (
δC 70.8), and four methyl groups in the high-field region (
δC 12.6, 19.1, 21.3, 24.5). These functionalities account for three degrees of unsaturation, thus hinting the bicyclic nature of
2. Analysis of the
1H NMR data (
Table 1) revealed the presence of four methyl groups, including three singlets (
δH 1.30, 1.36, 1.41) and one doublet (
δH 1.15,
J=7.1 Hz) in the high-field region. These data were characteristic of an eremophilane-type sesquiterpene, i.e., a
cis-decaline ring with an isopropyl group (C-11/Me-12/Me-13) and two methyl groups, one connected to a methine group (CH-4) and the other one connected to a quaternary carbon (C-5), resulting in one doublet (Me-15) and one singlet (Me-14) signals. In addition, the signals for one olefinic proton (
δH 5.92) and one oxymethine proton (
δH 3.97) could be seen in the
1H NMR spectrum (
Figure S8), which in combination with the signals observed in the
13C NMR spectrum (
Figure S9) suggested the occurrence of one trisubstituted double bond, and one oxygenated methine group. The positions of these functional groups in the eremophilane skeleton were clarified by analysis of the HMBC spectrum (
Figure S12). The presence of an epoxide moiety with
13C peaks at C-7 (
δC 65.6) and C-11 (
δC 64.7) was confirmed by HMBC correlations from Me-13 (
δH 1.30) to C-7, C-11 and C-12 (
δC 21.3), from H
2-6 (
δH 2.08 and 1.99) to C-7, C-10 (
δC 172.4), C-11, and C-14 (
δC 24.5), and from Me-12 (
δH 1.41) to C-7, C-11, and C-13 (
δC 19.1). Correlation between Me-15 (δ
H 1.15, d) and the oxygenated methine (
δC 70.8) placed the hydroxyl substituent at C-3. The
1H and
13C NMR spectra of
2 (
Figures S8 and S9) were very similar to those of compound 3-acetoxy-7(11)-epoxyeremophil-9-en-8-one (
16), previously reported by our research’s group as an
E. maritima BC17 metabolite from solid culture media [
13], except for the absence of the acetyl group signals at C-3.
The relative configuration of
2 was determined by analysis of 1D NOESY correlations (
Figure S13a-f). The H-1 (
δH 2.90) and Me-14 protons showed strong NOE correlations with each other, indicating that H-1
β and Me-14 were in axial orientations on the same face of ring A. The NOE correlations between H-3 and H-1 (
δH 2.16) and H-4 confirmed that these protons were on the opposite side from H-1
β/Me-14. NOE correlations between Me-14 and Me-15 indicated both methyls were on the same face. The
β-orientation of the epoxide at C-7 and C-11 in
2 was evidenced by the NOE effect from Me-12 to H-6
α (
δH 2.08) (
Figure 2).
The ECD spectrum of
2 was very similar to that obtained for the previously reported compound
16 [
13]. In addition, the ECD curve for the (3
S,4
R,5
R,7
R) stereoisomer, calculated with the TD-DFT theoretical method, matched well with the experimental ECD spectrum of
2 (
Figure 3). As a result, its absolute stereochemistry was assigned as (3
S,4
R,5
R,7
R)-3-hydroxy-7(11)-epoxyeremophil-9-en-8-one (
2).
Compound
3 had a molecular formula of C
15H
20O
2, as deduced from HRESIMS (
Figure S21), which was consistent with six degrees of unsaturation. The
13C NMR data (
Table 1) displayed fifteen signals, including two carbonyl groups (
δC 190.5, 212.1), four olefinic carbons (
δC 127.0, 128.2, 146.1, 162.6), four methyls (
δC 11.0, 22.8, 23.1, 25.1), three methylenes (
δC 29.0, 37.4, 37.5), another methine (
δC 53.6), and an additional quaternary carbon (
δC 42.2). The
1H NMR data (
Table 1) showed four methyl groups, including one singlet (
δH 1.20) and three doublets (
δH 1.10,
J=7.0 Hz; 1.85,
J=1.4 Hz; 2.15,
J=2.0 Hz) in the high-field region.
NMR data (
Table 1) of
3 revealed a great similarity with those of isopetasone (
4) [
16].However, the
1H and
13C NMR spectra (
Figures S15 and S16) of both compounds differed significantly in the shift of the signals corresponding to H-1
β (
δH 2.87 in
3 and 2.72 in
4), H-6
α (
δH 2.49 in
3 and 2.82 in
4), Me-14 (
δC/H 25.1/1.20 in
3 and 18.5/0.96 in
4), and Me-15 (
δC 11.0 in
3 and 7.4 in
4). This suggested a possible difference in the stereochemistry of these methyls. The NOESY correlation between H-1 (
δH 2.87) and Me-14 (
δH 1.20) (
Figures S20a and 20d) revealed that this methyl group was axial, while the correlation from Me-14 to H-4 (
δH 2.60) (
Figure S20d) positioned the latter to the equatorial site, indicating that Me-14 and H-4 shared the same
β-configuration (
Figure 2), in contrast to all eremophilanes described in this work. Hence, compound
3 was elucidated to be 4-
epiisopetasone.
This stereochemistry was confirmed by comparison of the predicted ECD spectrum from TD-DFT calculations with the experimental one of compound
3. The calculated spectrum reproduced well the experimental data (
Figure 3), confirming the 4
S and 5
R absolute configuration of
3.
In view of these results, it is noteworthy that the new metabolites could only be isolated under surface culture conditions and with Czapek-Dox medium. In particular, compound 3 belongs to a different stereochemical series than all eremophilanes previously isolated from the E. maritima BC17 strain. This indicates that the OSMAC approach is useful for accessing cryptic metabolites that would not be obtained under standard laboratory conditions.
The other isolated compounds
4-
17 were identified by comparing the spectroscopic data with the literature: isopetasone (
4) [
17], (+)-3-
epiisopetasol (
5) [
18], 1
α-hydroxydehydrofukinone (
6) [
19], eremofortin A alcohol (
7) [
20], (+)-aristolochene (
8) [
21], 7-hydroxy-4a,5-dimethyl-3-prop-1-en-2-yl-3,4,5,6,7,8-hexahydronaphthalen-2-one (
9) [
22], warburgiadione (
10) [
17], (1
R,7
S,8a
R)-1,8a-dimethyl-7-(prop-1-en-2-yl)-1,7,8,8a-tetrahydronaphthalene-2,6-dione (
11) [
23], eremofortine B (
12) [
24], 3
β-hydroxy-7
β-eremophil-1(2),9(10),11(12)-trien-8-one (
13) [
25], PR toxin (
14) [
24], PR toxin 3-deacetyl (
15) [
26,
27], (3
S)-acetoxy-7(11)-epoxyeremophil-9-en-8-one (
16) [
13], and eremofortine A (
17) [
24]. Compounds
11,
12, and
15 are reported for the first time as fungal metabolites from
E. maritima BC17 in this work, indicating the usefulness of the OSMAC strategy to increase the chemical diversity of the strain under study.
PR toxin 3-deacetyl (
15) was previously chemically synthesised from PR toxin (
14) by Wei et al [
27]. In order to confirm the configuration at C-3 of
15, this compound was acetylated with acetic anhydride and p-toluenesulfonic acid to give a product whose spectroscopic data coincided with those found in literature for PR toxin (
14) [
24]. This has allowed us to confirm the absolute configuration of compound
15 and report it for the first time as a fungal metabolite.
The undescribed isolated metabolites
1 and
3 and the known compounds
11 and
15 were evaluated for antimicrobial and cytotoxic activities. They were tested in triplicates against a panel of 8 human pathogens, including both Gram-negative and Gram-positive bacteria (
Escherichia coli ATCC25922,
Pseudomonas aeruginosa PAO-1,
Acinetobacter baumannii ATCC19606,
Klebsiella pneumoniae ATCC700603, methicillin-resistant
Staphylococcus aureus MB5393, and sensitive
S. aureus ATCC29213), yeast (
Candida albicans ATCC64124) and fungal (
Aspergillus fumigatus ATCC46645) strains. No antimicrobial activity was detected for these metabolites against the human pathogenic strains assayed, except for compound
15 that exhibited selective activity against methicillin-sensitive
S. aureus ATCC29213 at the highest concentration tested of 128 µg/mL (
Table 2).
Antitumoral activity of these compounds (1, 3, 11, and 15) was also tested against liver (HepG2, ATCC HB-8065), breast (MCF-7, ATCC HTB-22), lung (A549, ATCC CCL-185), skin (A2058, ATCC CRL-3601), and pancreas (Mia PaCa-2, ATCC CRL-1420) human cancer cells using MTT test. Methyl methanesulfonate (MMS) 4mM was used as positive control of cell death and doxorubicin was included as known chemotherapeutic agent.
In previous studies,
in vitro tests of compound
11, isolated from dry root of
Valeriana jatamansi, indicated that it played a restraining effect to human brain malignant glioblastoma U251 cell by inhibiting cell proliferation and inducing cell apoptosis [
23]. However, in this work we did not detect antiproliferative activity of this metabolite, nor of compounds
1 and
3, against the human cancer cells assayed. Among the eremophilanes tested, only compound
15 was active against all of the tested cell lines with IC
50 values ranging from 2.5 to 14.7
µM (
Table 3). In a previous study, we reported that the PR toxin (
14) inhibited the same human cancer cells tested with IC
50 values in the range of 3.75-33.44
µg/mL [
13]. These results confirm that the aldehyde group at C-12 present in both compounds,
14 and
15, is directly related to their biological activity.