Anti-Parkinsonian 4-hydroxy-2-pyridones from an endolichenic fungus, Tolypocladium sp. (strain CNC14)

Abstract

4-Hydroxy-2-pyridone alkaloids have attracted considerable attention because of their intriguing structures and diverse bioactivities. In our previous study, 4-hydroxy-2-pyridone alkaloids were shown to exhibit potent activity against neuron-associated targets. To discover this class of neuroactive compounds, an array of endolichenic fungal extracts was screened by analyzing liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MS) profiles. The screening yielded strain Tolypocladium sp. (strain CNC14), which produced compounds with characteristic Ultraviolet patterns for 4-hydroxy-2-pyridone alkaloids using our in-house library. Based on these findings, we conducted a chemical investigation, which led to the isolation of four new (1-4) and ten known (5-14) compounds. Their structures were elucidated via spectroscopic methods such as nuclear magnetic resonance and mass spectrometry. The stereochemistry of the new compounds (1-4) was established using rotating frame overhauser effect spectroscopy (ROESY), and the electronic circular dichrosim (ECD) was compared with the calculated data. Interestingly, the side chains of 4-hydroxy-2-pyridone in 1 and 2 were cyclized in different directions to form benzopyrano[3,4-b]pyridinol from previously reported compounds, and all the new compounds were predicted to be biosynthesized from reduced tolypyridone C (7) via the hetero-Diels-Alder reaction. Among the isolated compounds, 4 significantly protected neuronal cells against treatment with 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonian neurotoxin, in an in vitro Parkinson's disease model. One-Sentence Summary: Four new neuroprotective 4-hydroxy-2-pyridone alkaloids were discovered from an endolichenic fungus Tolypocladium sp.

Keywords: Tolypocladium sp; 4-Hydroxy-2-pyridone alkaloids; Endolichenic fungus; Parkinson's disease.

Graphical Abstract

Fig. 1.

Structures of compounds isolated from the culture extract of Tolypocladium sp. (strain CNC14).

Fig. 2.

Key ¹H-¹H COSY (bold lines) and HMBC (arrows) correlations for compounds 1–4.

Fig. 3.

Key ROESY correlations for compounds 1–6.

Fig. 4.

(a) Comparison of ECD for 1 with two possible stereoisomers P1 and P2. (b) Comparison of ECD for 2 with two possible stereoisomers P3, and P4.

Fig. 5.

Putative biosynthetic pathway of the isolated compounds.

Fig. 6.

Compound 4 exhibits neuroprotective properties in an in vitro PD model. (a) Of the 11 compounds screened, compound 4 shows a protective effect in an in vitro PD model (** p < 0.01, **** p < 0.0001, one-way ANOVA with Tukey's post hoc test). (b) Pretreatment of compound 4 protects SH-SY5Y cells against MPP⁺-induced cell death (** p < 0.01, **** p < 0.0001, one-way ANOVA with Tukey's post hoc test).

Fig. 7.

Compound 4 shows anti-inflammatory effects in an in vitro PD model. (a) Compound 4 attenuates MPP⁺-induced IL-1β expression in SH-SY5Y cells (*p < 0.05, *** p < 0.001, one-way ANOVA with Tukey's post hoc test). (b) Compound 4 reduces MPP⁺-induced TNF-α expression in SH-SY5Y cells (*p < 0.05, **p < 0.01, one-way ANOVA with Tukey's post hoc test). Data represent the mean ± SEM from three independent experiments.