Isolation and total synthesis of gymnastatin N, a POLO-like kinase 1 active constituent from the fungus Arachniotus punctatus

doi:10.1016/j.tet.2004.09.046

Tetrahedron

Volume 60, Issue 50, 6 December 2004, Pages 11619-11628

https://doi.org/10.1016/j.tet.2004.09.046 Get rights and content

Abstract

A high throughput screen against POLO-like kinase 1 (Plk1), an anti-cancer target, identified an active extract from the fungus Arachniotus punctatus. Bioassay guided fractionation led to the isolation of the new natural product, gymnastatin N (1) and the known compound aranorosinol A (2) with IC₅₀ values of 13 and 118 μM, respectively. A 12′-hydroxy analog of gymnastatin N, 3, was also isolated as a minor component. Gymnastatin N (1) was found to be a 52:48 mixture of (1S,6′R) and (1R,6′R) diastereomers, by synthesis of the four possible diastereomers and comparison of the optical rotation and chiral HPLC profile of each diastereoisomer with the natural product. Analogues of 1 were synthesized and evaluated against the Plk1 assay and these SAR studies suggested that the diene and free carboxylic acid moieties might be responsible for its bioactivity.

Graphical Abstract

Introduction

POLO-like kinases play a pivotal role in various stages of cell division. Recent studies on this novel family of enzymes have unraveled their functions in centrosome maturation and bipolar spindle formation at the onset of mitosis.¹ They are also involved in the activation of cyclin-dependent kinase (Cdk)1-cyclin B.² In addition, they have been implicated in the activation of anaphase-promoting complex (APC) and the inactivation of Cdk1 at the point of mitotic exit.¹ POLO-like kinase 1 (Plk1) is a highly conserved mitotic serine/threonine kinase which has been shown to be commonly overexpressed in cancer cell lines.³ Its expression could reflect the degree of malignancy and proliferation in these cells. Due to its essential roles in cell-cycle regulation, Plk1 could serve as a suitable diagnostic and prognostic marker for tumour progression and as a target for anti-cancer therapy. We have initiated a screening programme of our natural product collection, directed towards the identification of potent Plk1 inhibitors as anti-tumour drugs.

A high throughput screen against Plk1 identified an active extract from the fungus Arachniotus punctatus. Bioassay guided fractionation led to the isolation of the new natural product, gymnastatin N (1) and the known compound aranorosinol A (2) (Fig. 1),⁴ with IC₅₀ values of 13 and 118 μM, respectively. A 12′-hydroxy analog of gymnastatin N, 3, was also isolated as a minor component (Fig. 1). This class of fungal derived natural products contains a 4,6-dimethyl-dodecadien-2E,4E-oic acid unit connected to a tyrosine unit through an amide linkage. The tyrosine unit can have various degrees of oxygenation, halogenation, cyclisation and esterification, as found in aranorosin (4),⁵ aranorosinol A (2), aranorosinol B,⁴ aranochlor A and aranochlor B from Pseudoarachniotus roseus,⁶ gymnastatin A (5) to E,⁷ F to H (6)⁸ and I (7) to M⁹ from Gymnastella dankaliensis (Fig. 1). These compounds have been reported to have antibacterial and anti-tumour activity. The total synthesis of aranorosin (4),10, 11 gymnastatin A (5),¹² gymnastatin H (6)⁸ and gymnastatin I (7),⁹ along with various interconversions between compounds, have enabled the absolute stereochemistry of most compounds in this class to be determined. To date, all of these compounds and gymnasterone A from G. dankaliensis,¹³ have a (6R) configuration at the 4,6-dimethyl-dodecadien-2E,4E-oic acid unit. The occurrence of the (6S) configuration is rare: it has only been reported in the isolation of (6S)-4,6-dimethyl-dodecadien-2E,4E-oic acid and its phomalactone ester derivative, from a Phomopsis sp.¹⁴

Section snippets

Results and discussion

Approximately 300 out of the 90,000 extracts screened showed more than 40% inhibition against the Plk1 assay at a concentration of 250 μg/mL. These extracts were tested against Cdk2, a serine/threonine kinase, to eliminate common serine/threonine kinase inhibitors and the 20 samples that had more than 5 fold potency against Plk1 versus Cdk2 were selected for further progression. One of these samples was the CH₃OH extract of the fungus A. punctatus and bioassay guided fractionation led to the

General experimental procedures

Specific optical rotation was determined using a Jasco DIP-1000 Digital Polarimeter. ¹H and ¹³C NMR spectra of the natural products were acquired on a Bruker Avance DRX-500 NMR spectrometer, using 5 mm inverse (¹H, G-COSY, multiplicity-edited G-HSQC, G-HMBC and G-ROESY spectra) or normal (¹³C spectra) probeheads equipped with z-gradients, while NMR data on the synthetic compounds was acquired using a Bruker AVANCE-400 NMR. Spectra were calibrated to residual protonated solvent signals and all

Acknowledgements

This work was done jointly at the Centre for Natural Product Research (CNPR) and the Medicinal Chemistry group at the Institute of Molecular and Cell Biology (IMCB). We would like to thank Ms. Annetta Leong and Dr. Yicun Huang for the fungal fermentation. We are grateful to Dr. Wong Pui Kwan and Dr. Krishna Gopal Dongol from the Institute of Chemical and Engineering Sciences, Singapore for the use of FT-IR spectrometer. We also express our gratitude to GlaxoSmithKline, the Economic Development

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Gymnastatins are fatty acid amides of unusually oxidized and rearranged tyrosine moiety, first isolated from Gymnascella dankaliensis (Castellani) Currah OUPS-N134 that was separated from the sponge Halichondria japonica [176,177]. Bioassay-guided fractionation of the extracts obtained from fungus Arachniotus punctatus yielded a new member of this series, gymnastatin N (209; IC50 13 mM), and a known compound aranorosinol A (210; 118 mM) as potent POLO-like kinase 1 (Plk1) inhibitors [178]. An attempt was also made to study the SAR of these compounds and found that the diene functionality and free carboxyl of the tyrosine unit are essential for bioactivity [179].
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Isolation and total synthesis of gymnastatin N, a POLO-like kinase 1 active constituent from the fungus Arachniotus punctatus

Abstract

Introduction

Section snippets

Results and discussion

General experimental procedures

Acknowledgements

Proc. Natl. Acad. Sci. U.S.A.

J. Org. Chem.

J. Chromatogr. Sci.

J. Cell Sci.

Genes Dev.

Curr. Opin. Cell Biol.

J. Antibiot.

J. Am. Chem. Soc.

J. Antibiot.

J. Antibiot.