Cytotoxic and antimicrobial drimane meroterpenoids from a fungus of the Stictidaceae (Ostropales, Ascomycota)

Abstract

As part of our ongoing research on bioactive fungal metabolites, two new metabolites were isolated from a fungus of the Stictidaceae (strain MSX62440), dasyscyphins F and G (1 and 3), and the known dasyscyphin C (2). Compound 1 was characterized by HRMS and 1D and 2D NMR data, and its absolute configuration established by ECD spectroscopy. A structural revision of dasyscyphin C (2) was based on NMR data and verified by ECD calculations. Compound 3 was reported previously as a synthetic product, and its identity confirmed by comparison with NMR data in the literature, and its absolute configuration was established by ECD spectroscopy. Compounds 1 and 2 showed moderate cytotoxicity and antimicrobial activity.

In ongoing studies in pursuit of potential drug leads from the Mycosynthetix library of filamentous fungi [1], [2], [3], the organic extract from a fungus of the Stictidaceae (strain MSX62440, Fig. S1) displayed moderate activity against the human cancer cell lines MDA-MB-435 (melanoma), MDA-MB-231 (breast) and OVCAR3 (ovarian) (~65% inhibition of cell growth when tested at 20 μg/mL). Bioactivity-directed fractionation using flash chromatography followed by HPLC resulted in the isolation of two natural products, dasyscyphin F and G (1 and 3), along with the known analogue dasyscyphin C (2) [4] (Fig. 1).

Fig 1.

Compounds isolated from a fungus of the Stictidaceae (strain MSX62440).

Compound 1 [5] was isolated as an optically active powder (${\left[\alpha \right]}_D^{20}=+27$, c = 0.1, CH₃OH). The molecular formula was established as C₂₂H₃₀O₃ by HRESIMS (Fig. S2), indicating an index of hydrogen deficiency of 8. Analysis of the ¹H, ¹³C, and HSQC NMR data (Fig. S3, S4, and Table 1) indicated 22 carbons, consisting of four methyls (δ_H/δ_C 0.49/15.7, 0.80/21.9, 1.25/30.4, and 0.89/33.4), six methylenes (δ_H/δ_C 0.92 and 1.68/41.3, 1.39 and 1.49/18.5, 1.15 and 1.41/42.1, 1.63 and 1.32/19.6, 1.75 and 2.82/32.8, and 2.73 and 2.90/29.4), three methines (δ_H/δ_C 0.98/51.9, 1.75/62.0, and 6.75/116.8), one aldehyde (δ_H/δ_C 9.72/195.9), and eight non-protonated carbons (δ_C 33.4, 37.3, 48.5, 120.1, 140.1, 142.0, 144.6, and 153.0). These data were similar to those reported for dasyscyphin B [4], where the main difference was the lack of a methoxymethyl group attached to the aromatic ring at position C-15 (i.e., δ_H-22/δ_C-22 4.60/74.3 and δ_H/δ_C 3.42/58.0 as reported [4]), which was replaced by an aldehyde (δ_H/δ_C 9.72/195.9), whose position was verified by the HMBC correlations between H-22 with C-14, C-15, and C-16 (Fig. 2). The skeleton for the tricyclic-ring system for the sesquiterpene portion of 1 was confirmed by analysis of the COSY and HMBC experiments (Fig. 2). Specifically, the COSY spectrum (Fig. S5) indicated three isolated spin systems H₂-1/H₂-2/H₂-3, H-5/H₂-6/H₂-7, and H-9/H₂-11 (Fig. 2); key HMBC correlations were used to connect these, particularly H-1 with C-9, H-3 with C-5, H-7 with C-9, H-11 with C-17 and C-12, and H-7 with C-17 (Fig. S6). The relative configuration was assigned through NOESY correlations (Figs. 2 and S7) between H₃-21/H₂-6/H₂-11, H₃-18/H-5/H-9, and H-5/H₃-19; the relative configuration was identical to that of other dasyscyphin analogues [4], [6]. The absolute configuration of 1 was determined by comparison of the experimental and calculated ECD spectra. TDDFT calculations were used to predict the ECD spectrum for the proposed relative configuration, using the B3LYP/G-31+G(d,p) level of theory, and the experimental data matched the calculated spectrum for the 5S,8S,9R,10S configuration (Fig. 3). Compound 1 was ascribed the trivial name dasyscyphin F.

Table 1.

NMR data for compound 1 in CDCl₃ (400 and 100 MHz for ¹H and ¹³C, respectively).

position	δ C	type	δH, mult (J in Hz)
1	41.3	CH2	1.68, m, 0.92, m
2	18.5	CH2	1.49, m, 1.39, m
3	42.1	CH2	1.15, td (14.2, 13.5, 4.4) 1.41, m
4	33.4	C
5	51.9	CH	0.98 dd (11.4, 4.4)
6	19.6	CH2	1.63, 1.32 m
7	32.8	CH2	2.82 dt (13.8, 5.5) 1.75 m
8	48.5	C
9	62.0	CH	1.75, m
10	37.3	C
11	29.4	CH2	2.90, dd (17.6, 7.9) 2.73, d (17.5)
12	142.0	C
13	153.0	C
14	116.8	CH	6.75, s
15	120.1	C
16	144.6	C
17	140.1	C
18	30.4	CH3	1.25, s
19	33.4	CH3	0.89, s
20	21.9	CH3	0.80, s
21	15.7	CH3	0.49, s
22	195.9	CH	9.72, s
16-OH			10.82, s

Figure 2.

Key COSY (bold lines), HMBC (solid arrows) and NOESY (dashed arrows) correlations of 1.

Figure 3.

Comparison of the ECD spectra of 1 in CH₃OH. Experimental spectrum recorded at 0.2 mg/mL (solid line), and calculated spectra for the two possible enantiomers (dotted and dashed lines).

Compound 2 [7] was isolated as an optically active power (${\left[\alpha \right]}_D^{20}=-28$, c = 0.1, CH₃OH). The HRMS data and the ¹H and ¹³C NMR spectra (Fig. S9) of 2 matched with those reported for dasyscyphin C. In that publication, the structure of 2 corresponded to a meroterpenoid with a 2-cyclohexene-1,2-dione in the fourth ring; however, the ¹³C NMR data did not seem consistent with such an arrangement of the ketones. Indeed, based on the NMR data for several natural products with a 2-cyclohexene-1,2-dione system, the reported chemical shifts for the ketones are typically in the range of 181–192 and 190–197 ppm [8], [9], [10], [11]. On the other hand, in a 2-cyclohexene-1,4-dione system the electron density of the two ketones is more evenly distributed, and thus, the chemical shifts will be more similar to each other, as in the case of the ketone moieties in 2 (i.e., 196.9 and 201.0 ppm for positions 13 and 16) and in many other examples in the literature [9], [12], [13], [14]. Additionally, the HMBC correlations between H-14 with C-13, C-15, C-16, and C-22 supported the 2-cyclohexene-1,4-dione system in 2 (Fig. 4). The ECD spectrum for 2 was used as an orthogonal method to confirm the 2-cyclohexene-1,4-dione moiety. Specifically, the chromophores in 2a and 2b (Fig. 5) are not identical, resulting in different UV absorptions, and thus, different Cotton effects. This was demonstrated by calculating the ECD spectra for the two possible molecules, 2a and 2b, and comparing those with the measured spectrum. As predicted, the calculated ECD spectra for 2a and 2b were not identical. Compound 2a displayed two negative Cotton effects at 210 and 286 nm and two positive Cotton effects at 234 and 340 nm; 2b had a positive Cotton effect at 250 nm and a negative Cotton effect at 290 nm. The experimental ECD data matched with the spectrum calculated for 2a, confirming the 2-cyclohexene-1,4-dione system, serving to refine the structure of dasyscyphin C and establishing its absolute configuration as 5S,8S,9R,10S,12R,17R.

Figure 4.

Key HMBC correlations for compound 2.

Figure 5.

ECD spectrum for 2 and the calculated spectra of 2a and 2b.

Compound 3 [15] was isolated as an optically active powder (${\left[\alpha \right]}_D^{20}=+13$, c = 0.1, CH₃OH). The molecular formula was established as C₂₃H₃₂O₃ based on the protonated molecule observed in the HRESIMS spectrum. Analysis of the ¹H, ¹³C and HSQC NMR spectra (Fig. S13) supported the presence of 23 carbons, including four methyl groups (δ_H/δ_C 0.70/16.0, 0.84/21.5, 0.85/32.9, and 1.25/29.8), seven methylene groups (δ_H/δ_C 0.90 and 1.59/41.8, 1.13 and 1.41/42.3, 1.34 and 1.66/19.0, 1.37 and 1.37 and 1.50/18.5, 1.80 and 2.17/30.8, 2.60 and 2.73/29.4, and 4.27/68.1), three methine groups (δ_H/δ_C 1.00/49.9, 1.64/59.9, and 6.63/131.0), one methoxy (δ_H/δ_C 3.44/59.3), and eight non-protonated carbons, including two ketones (δ_C 33.5, 36.9, 49.6, 146.2, 147.5, 153.6, 186.1, and 187.3); these data agree with the structure of an intermediate in the synthesis of dasyscyphin B [16] (Table S2). The reported relative configuration for 3 is the same as compound 2, and its ECD spectrum showed the same Cotton effects observed in 2 (Fig. S16). This evidence, along with biogenetic considerations, suggests the absolute configuration for 3 as 5S,8S,9R,10S. This is the first-time compound 3 has been isolated as a natural product, and it was ascribed the trivial name dasyscyphin G.

Compounds 1-3 were tested against the human cancer cells lines MDA-MB-435 (melanoma), MDA-MB-231 (breast) and OVCAR3 (ovarian). The results showed that 1 and 2 were moderately active with IC₅₀ values ranging between 4 to 16 μM against the three cell lines (Table 2); compound 3 was effectively inactive. These data were consistent with the literature on related dasyscyphin analogues, where cytotoxic activities ranged between 1 to 8 μM [17].

Table 2.

Cytotoxic activity of compounds 1-3 against MDA-MB-435 (melanoma), MDA-MB-231 (breast) and OVCAR3 (ovarian) cancer cell lines.

	IC50 values (μM)
	MDA-MB-435	MDA-MB-231	OVCAR3
1	4.1	8.2	16.2
2	14.1	12.2	10.4
3	19.7	>25	>25
taxol	0.007	0.002	0.003

Since the dasyscyphins have been studied as antifungal and antibacterial agents [6], [17] we evaluated the antimicrobial activity of 1 and 2 against Escherichia coli, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Mycobacterium smegmatis, Bacillus subtilis, Bacillus anthracis, Saccharomyces cerevisiae, Candida albicans and Aspergillus niger. Compounds 1 and 2 (Table 3) inhibited the growth of P. aeruginosa, Methicillin-resistant Staphylococcus aureus (MRSA), and B. anthracis, and of those, the MIC of 2 against B. anthracis (MIC value of 2 μg/mL) was the most potent.

Table 3.

Antimicrobial activities of compounds 1 and 2 against a suite of microorganisms.

	Minimal inhibitory concentration (μg/mL)
Microorganisms	1	2	Erythromycin
E.coli	>125	>125	8
S. aureus	>125	>125	0.125
MRSA	63	16	>25
P. aeruginosa	125	63	32
M. smegmatis	>125	>125	125
B. subtilis	>125	>125	>125
B. anthracis	31	2	0.25
S. cerevisiae	>125	>125	>125
C. albicans	>125	>125	>125
A. niger	>125	>125	>125

In conclusion, we report the isolation and elucidation of three drimane meroterpenoids (1-3) from a fungus of the Stictidaceae. This includes the description of two natural products, dasyscyphin F (1) and G (3), and a structural revision of dasyscyphin C (2), demonstrating that it has a 2-cyclohexene-1,4-dione ring, instead of a 2-cyclohexene-1,2-dione, as noted by data from analysis of both NMR chemical shifts and ECD calculated spectra. The structural characterizations of 1-3 were established using a combination of conventional spectrometric (HRESIMS) and spectroscopic (NMR) techniques, while their absolute configurations were determined by analysis of measured vs calculated ECD spectra. Moreover, we report that dasyscyphin F and C (1 and 2) had moderate cytotoxic activity, and the potent MIC of compound 2 vs B. anthracis merits further study.