Obionin B: An o-pyranonaphthoquinone decaketide from an unidentified fungus (MSX 63619) from the Order Pleosporales

Abstract

A fungal extract (MSX 63619), from the Mycosynthetix library of over 50,000 fungi, displayed promising cytotoxicity against a human tumor cell panel. Bioactivity-directed fractionation led to the isolation of an o-pyranonaphthoquinone decaketide, which we termed obionin B (1). The structure of 1 was deduced via spectroscopic and spectrometric techniques. The IC₅₀ value of 1 was moderate, ranging from 3 to 13 μM, depending on the cell line tested.

Nature, in general, and fungi, specifically, have and continue to be a valuable source of new drug leads.^1–3 For example, two of the most well known drug classes of the 20^th century, the antibiotics and the cholesterol lowering agents, both originated as fungal secondary metabolites (e.g. penicillin and compactin). More recently, in 2010 the U.S. FDA approved a new treatment for multiple sclerosis, fingolimod (Gilenya), which derived from the fungal secondary metabolite, myriocin.⁴ Yet, despite these successes, it has been postulated that fewer than 10% of the estimated 1 to 1.5 million species of fungi in the world have been identified taxonomically, with possibly an even smaller percentage being studied for bioactive leads.⁵ Thus, given the under explored biodiversity, and the aforementioned successes as inspiration, our research team has been probing the Mycosynthetix library of filamentous fungi, representing over 50,000 isolates, for new anticancer drug leads.⁶

An extract of the filamentous fungus MSX 63619⁷ exhibited promising cytotoxic activity, as evidenced by less than 20% survival of human tumor cells when treated with 20 μg/mL of crude extract. The 1:1 chloroform/methanol extract of the solid fermentation of MSX 63619 was subjected to bioactivity-directed fractionation using flash chromatography on silica gel followed by preparative RP-HPLC on C18.⁸ This resulted in the isolation (>95% purity according to HPLC) and characterization of a new decaketide, which was named obionin B (1). This compound was evaluated against the human tumor panel and a pair of antibacterial assays.

Compound 1,⁹ which was a deep purple color, displayed HRESIMS data of m/z 355.1541 in the negative mode (corresponding to C₂₁H₂₄O₅ - H, calcd for 355.1545 [M - H]⁻), indicating an index of hydrogen deficiency of ten. The ¹H- and COSY-NMR data showed signals consistent with a straight saturated alkyl chain from δ_H 0.8–2.3. The rest of the signals were singlets, starting with a three-proton singlet at δ_H 3.81, suggesting a methoxy group. A two-proton singlet was present at δ_H 5.16, followed by one-proton singlets at δ_H 5.56, 6.26, 6.30, and 12.31, the latter consistent with an intramolecular hydrogen bonded phenol. The ¹³C-NMR data showed 21 peaks, consistent with the HRMS data, including seven peaks upfield of δ_C 35, six of which were methylenes and the seventh a methyl according to the multiplicity edited HSQC experiment. These data indicated that the straight saturated alkyl chain was n-heptyl. The only other signals upfield of δ_C 100 were a methylene at δ_C 63.1 that correlated with the two-proton singlet at δ_H 5.16, and the methoxy signal at δ_C 55.9. The remaining signals were for sp² carbons, with five between δ_C 100–120, five between δ_C 130–170 and two between δ_C 175–180 (see Supporting Information for the ¹H- and ¹³C-NMR spectra and Table S1).

Upon purification of 1, the diode-array detector on the HPLC displayed UV maxima at 243, 297, and 464 nm. A search of the Dictionary of Natural Products for the formula C₂₁H₂₄O₅ and a UV range of 460–468 nm returned a single hit; obionin A.¹⁰ The downfield ¹H- and ¹³C-NMR signals of 1 were in close agreement with the NMR data for the o-pyranonaphthoquinone portion of obionin A (2), except for the H-11 resonance, which was split into two one-proton doublets due to the presence of chiral centers in the side chain of 2; this was a two-proton singlet (δ_H 5.16) in achiral 1. However, the data for the upfield region were significantly different. Obionin A (2) has a branched saturated alkyl chain (Figure 1) instead of the n-heptyl chain of 1. The HMBC spectrum showed that the n-heptyl group of 1 was attached to the o-pyranonaphthoquinone at the same position (C-9) as the branched saturated chain in obionin A (Figure 2). To verify that the quinone was ortho instead of para, key HMBC correlations were observed from H-6 to C-4, and H-4 to both C-6 and C-2 (Figure 2). The remaining HMBC correlations (Figure 2) confirmed the structure of 1, which was termed obionin B in deference to the earlier studies on obionin A.¹⁰

Figure 1.

Structures of obionin A (2) and obionin B (1)

Figure 2.

HMBC correlations of obionin B (1)

Compound 1 was assayed against several human cancer cell lines using methods described previously,¹¹ including the MCF-7 (breast carcinoma), NCI-H460 (large cell lung carcinoma), SF-268 (astrocytoma), HT-29 (colorectal adenocarcinoma), and MDA-MB-435 (melanoma) cell lines. The IC₅₀ values (Table 1) indicated that obionin B was moderately cytotoxic. Compound 1 was inactive (MIC values > 500 μg/mL; data not shown) when tested for antimicrobial activity using methods described previously¹¹ against Escherichia coli and Bacillus subtilis. Previously, obionin A (2) was isolated based on the brine shrimp toxicity of the fungal extract,¹⁰ although the authors stated that 2 did “not account for that activity;” to the best of our knowledge, 2 has not been examined for cytotoxicity. A related pair of o-pyranonaphthoquinones, the laccaridiones, were reported as promising antimycotic leads due to inhibition of Candida albicans adhesion to epithelial and endothelial cells, as well as the ability to reduce the release and inhibit the catalytic activity of secreted aspartic proteases.¹² These effects resulted in reduced virulence properties (e.g. colonization and penetration of host tissues) without being fungistatic or fungicidal.¹² However, only laccaridione B has been investigated for cytotoxicity towards human tumor cells, with IC₅₀ values ranging from 4.5 μM in the K-562 human erythroleukemia cell line to 34 μM in HeLa cervical carcinoma cells.¹³ In comparison, obionin B (1) exhibited broad potency on the lower end of this range (Table 1).

Table 1.

Cytotoxicity of compound 1 against a panel of human tumor cell lines.

	IC50 values in μMa
Compound	MCF-7	H460	SF-268	HT-29	MDA-MB-435
obionin B (1)	11.1	7.6	13.2	3.1	7.3
camptothecinb	0.03	0.005	0.13	nt	nt
silvestrolb	ntc	nt	nt	0.004	0.006

^aIC₅₀ values are determined as the concentration required to reduce cellular staining with sulforhodamine B by 50% relative to untreated controls following 72 h of continuous exposure.¹¹

^bPositive controls.

^cIndicates `not tested'

In conclusion, an o-pyranonaphthoquinone decaketide (obionin B, 1) has been isolated and characterized from a terrestrial fungus from the Mycosynthetix library of filamentous fungi; 1 exhibited moderate cytotoxicity toward a panel of five human tumor cell lines (Table 1). Compound 1 is structurally related to the known nonaketides obionin A,¹⁰ leptosphaerodione,¹⁴ and the laccaridiones.¹³ Interestingly, the former two were both isolated from marine-derived fungi of the same Order (Pleosporales) as the terrestrial fungus (MSX 63619)⁷ investigated herein; this is the second time we have reported a correlation between secondary metabolites isolated from fungi of both terrestrial and marine origin.¹⁵